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The use of microbial iron chelators and polyamine analogues as antineoplastics

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The use of microbial iron chelators and polyamine analogues as antineoplastics
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Ingeno, Michael Joseph, 1953-
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vi, 158 leaves : ill. ; 29 cm.

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Cell cycle ( jstor )
Cell growth ( jstor )
Cell lines ( jstor )
Cells ( jstor )
Dosage ( jstor )
Enzymes ( jstor )
Inhibitory concentration 50 ( jstor )
Mitochondrial DNA ( jstor )
Polyamines ( jstor )
Tumors ( jstor )
Antineoplastic Agents ( mesh )
Dissertations, Academic -- Medicinal Chemistry -- UF ( mesh )
Iron Chelates -- therapeutic use ( mesh )
Medicinal Chemistry thesis Ph.D ( mesh )
Polyamines -- therapeutic use ( mesh )
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bibliography ( marcgt )
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Thesis:
Thesis (Ph.D.)--University of Florida, 1988.
Bibliography:
Bibliography: leaves 151-157.
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Typescript.
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Vita.
Statement of Responsibility:
by Michael Joseph Ingeno.

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THE USE OF MICROBIAL IRON CHELATORS AND POLYAMINE
ANALOGUES AS ANTINEOPLASTICS














By

MICHAEL JOSEPH INGENO


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1988




THE USE OF MICROBIAL IRON CHELATORS AND POLYAMINE
ANALOGUES AS ANTINEOPLASTICS
By
MICHAEL JOSEPH INGENO
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1988


This work is dedicated to my family, with love.


ACKNOWLEDGMENTS
I would like to express my sincere appreciation to my research
advisor, Dr. Raymond J. Bergeron, for his generous guidance, expert
advice and friendship over the past four years. His enthusiasm for
science has been an inspiration.
I would also like to thank Dr. Richard R. Streiff for his assist
ance in the completion of the animal studies and Dr. Raul Braylan for
his advice on flow cytometry and cell cycle analysis.
I am especially grateful for the assistance and dedication of
Jack Kramer Austin, Jr., Gabriel Luchetta, Annette Zaytoun, and Dexter
E. Beck whose time and effort have helped make this work possible, and
for the technical assistance of Joe Kayal, Stacia Goldey, B. Wesson
Young, and Rosa Rosado.
I wish to acknowledge the other coworkers, past and present, in
Dr. Bergeron's laboratory who have been helpful in the completion of my
many projects and helped make my learning experience an enjoyable one.
I also wish to acknowledge Irma Smith for her expert advice and
assistance in the preparation of this manuscript.
Finally, I wish to thank my wonderful family: I am eternally
gratefuly to Mr. and Mrs. James Ingeno for a lifetime of love and
encouragement, they were always there when I needed them; I am blessed
with my daughter, Leah, who could make me smile when all else failed;
and I am constantly indebted to my beautiful wife, Deborah, for her
love, tenderness, patience and support throughout the completion of
iii
this work.


TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS iii
ABSTRACT v
CHAPTERS
I. INTRODUCTION 1
Catechol amide Iron Chelators 1
Polyamine Analogues 11
II. CATECHOLAMIDE CHELATORS 21
Materials and Methods 21
Results 30
Discussion 63
III. POLYAMINE ANALOGUES 76
Materials and Methods 76
Results 87
Discussion 127
IV. CONCLUSIONS 145
REFERENCES 151
BIOGRAPHICAL SKETCH 158
iv


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
THE USE OF MICROBIAL IRON CHELATORS AND POLYAMINE
ANALOGUES AS ANTINEOPLASTICS
By
Michael Joseph Ingeno
December 1988
Chairman: Raymond J. Bergeron
Major Department: Medicinal Chemistry
This research focuses on the development of compounds which are
effective in controlling the growth of cancer cells. The two classes
of agents studied are the microbial iron catechol amide chelators
(siderophores) and the N-alkyl polyamine analogues.
The biological activity of the catecholamide chelators, parabac-
tin and vibriobactin, has been assigned, at least in part, to their
ability to chelate iron and inhibit the iron dependent enzyme ribonuc
leotide reductase. These siderophores are potent inhibitors of neo
plastic cell growth, with 50% inhibitory concentrations in the micro
molar range. The ligands have pronounced effects on cell cycle kinet
ics producing a G^-S cell cycle block. On short term exposure to the
chelators, this block in DNA synthesis is reversed simply by washing
the chelator away. The cells remained synchronized for at lease three
cycles after release of the block. The cell cycle block and synchroni
zation induced by the chelators may be exploited to potentially enhance
cytotoxicity when used in combination with other antineoplastics.
v


The biological activity of the N-alkylated polyamine derivatives
has been postulated to be a consequence of their similarity to the
natural intracellular polyamines, spermidine and spermine in terms of
uptake, while functionally they are unable to fulfill or interfere with
important roles of the natural polyamines. Inhibition of cell growth
may result as a consequence of alterations in the polyamine biosyn
thetic network. Preliminary studies of the structural boundary condi
tions required for significant antiproliferative activity of the poly
amine analogues have been carried out. The most active analogues, the
terminally diethylated tetrarnines N* ,N^-diethyl spermi ne and N*,N^-
diethylhomospermine, have been shown to be potent inhibitors of neo
plastic cell growth, with 50% inhibitory concentrations in the micro
molar range in vitro and significant in vivo activity. They have been
found to have only incidental effects on nuclear DNA, while having pro
found effects on mitochondrial DNA.
Results are presented as to the action of these drugs both in
vitro and in vivo. From these studies a great deal has been determined
about the growth inhibitory effects, mechanisms of actions, broadness
of activity against various cell lines, structure activity relation
ships, and effectiveness of these compounds either alone or in combina
tion with other agents.
vi


CHAPTER I
INTRODUCTION
This research focuses on the development of compounds which are
effective in controlling the growth of cancer cells.
Catechol amide Iron Chelators
The first part of the dissertation is concerned with the evalua
tion of catechol amide iron chelators as a means of controlling the pro
liferative processes. Over the past decade there has been a dramatic
increase in the attention given to the crucial role which iron plays in
proliferative processes. Although iron has oxidation states between -2
and +6, the equilibrium between the +2 and +3 oxidation states, an
equilibrium which is very sensitive to both ligation and pH (1), best
characterizes the biological use of this metal. This transition metal
plays a critical role in a variety of biological redox systems within
the cell, e.g., the cytochromes, peroxidases, catalases, and ribonucle
otide reductase (2). Even though virtually all organisms are auxo-
tropic for Fe(III), because of the insolubility of ferric hydroxide
(FeiOH)^), the predominant form of the metal under physiological con-
38 4
ditions (Ksp=10 tv), nature has had to develop rather sophisticated
mechanisms for accessing the metal (3).
Microorganisms produce low-molecular weight ligands (sidero-
phores) which chelate and facilitate transport of iron into the cell
(2). Most of these siderophores fall into two general structural
classes, hydroxamates and catecholamides (4). The catecholamides,
1


2
typified by enterobactin (Fig. 1-la), generally form tighter iron
48 1
complexes (metal-1igand formation constant (Kf=10 M ) (5) than the
31 1
hydroxamates, exemplified by desferrioxamine (Fig. 1-lb) (K^=10 M" )
(6).
Mammalian cells have developed larger, more complicated mole
cules, iron binding proteins for the metal's transport and utilization:
e.g., the iron shuttle proteins, transferrin (7), lactoferrin (8),
uteroferrin (9), and the iron storage protein, ferritin (10). It has
been suggested that the virulence of pathogenic bacteria may be par
tially related to the efficiency with which its siderophore removes
iron from these iron binding proteins (11).
Transferrin, the major iron transport protein in animals, binds
two iron atoms in the +3 oxidation state, each with a slightly differ
ent metal-protein formation constant. This binding is very sensitive
to the oxidation state of the metal and to pH with an apparent iron
28 1
binding constant at physiological pH of Kf=10 M (12). Once the
transferrin is bound to the transferrin receptor on the cell's surface,
it is internalized via endocytotic vesicles. The iron-protein-receptor
complex enters a nonlysosomal acidic vacuole where conditions favor
release of the iron for utilization or storage as an iron-ferritin com
plex. The apotransferrin-receptor complex then returns to the cell
surface where the apotransferrin is released for further use (13).
The number of transferrin receptors on a cell membrane has been
directly linked to the proliferative state of the cell and/or the


3
b.
Figure 1-1. The microbial iron chelators
a. Enterobactin
b. Desferrioxamine


4
availability of iron (14). For example, it has been observed that
highly proliferative cells have significantly greater numbers of trans
ferrin receptors than resting cells (15). Alternatively, if iron is
withheld from cells there is a substantial increase in the number of
transferrin receptors (16). This increase is due to stimulation of
synthesis and translation of the transferrin receptor mRNA, and can be
inhibited by the addition of iron (17). Conversely, if iron is added
to the cells in a utilizable form there is a dramatic decrease in
transferrin receptor expression, and an increase in cellular ferritin
iron as a means of defense against bacterial infection and neoplasms.
It is interesting to observe how the body alters iron metabolism
in response to a bacterial infection. In the course of an infection,
the body attempts to withhold iron from the invading organism by
decreasing gastrointestinal absorption of iron, lowering the serum
transferrin iron saturation, and increasing liver ferritin levels and
iron ferritin storage in reticuloendothelial systems (18). The same
response is seen in febrile states, neoplasia, and on injection of var
ious sham substances (19). It has been demonstrated that mice fed a
low iron diet developed smaller and slower growing tumors than mice
maintained on a normal iron diet (20). Conversely, iatrogenic iron
overload as well as idiopathic hemochromotosis, a genetic disease char
acterized by iron overload, have been associated with increased inci
dence of neoplasia (21,22). These findings suggest continued study of
the role of iron metabolism in malignancy and the possible adverse
effects of iron supplementation in patients with cancer.
There is evidence to indicate that activated macrophage cytotox
icity may be mediated by iron removal from the target cells (23). A


5
selective inhibition of iron dependent DNA replication and mitochondri
al respiration is observed. The authors speculate that a macrophage-
derived iron chelator could be involved, leading to cytostasis. Later
studies (24) found that aconitase, a citric acid cycle enzyme contain
ing iron-sulfur clusters, activity declined simultaneously with arrest
of DNA synthesis. The cytotoxic activated macrophage-induced inhibi
tion of aconitase is shown to be due to loss of iron from the iron-
sulfer cluster. More recently (25), a newly discovered cyclic hydrox-
amate siderophore, bisucaberin, was found to render tumor cells sus
ceptible to cytolysis mediated by macrophages and showed specific
inhibition by addition of ferric iron.
One of iron's more interesting roles in cells is its association
with ribonucleotide reductase (Fig. 1-2), an enzyme which catalyses the
production of deoxyribonucleotides in the rate limiting step of DNA
synthesis (26). In mammalian cells, ribonucleotide reductase consists
of two nonidentical dimer subunits, Ml and M2, analogous to the B1 and
B2 subunits of Escherichia col i (E. col i) (27). The M2 dimer contains
a nonheme iron center. The metal stabilizes a tyrosyl free radical at
the active site and, due to the rapid turnover of the enzyme, a con
stant supply of iron is essential for the enzyme's activity (28).
Ribonucleotide reductase levels are elevated in neoplastic cells and
have been shown to be transformation-linked and progression-1inked in a
series of hepatomas (29).
If the theory that withholding iron as a defense mechanism
against neoplasia is correct, then artificially inducing a state of
iron deprivation by means of the siderophores may prove to be effective
in controlling neoplastic cell growth. Iron chelators could control


6
Substrate
Fiqure 1-2. Model of E. coli ribonucleotide reductase (from
Ref. 26).


7
cellular proliferation by complexing extracellular iron (including that
bound to transferrin), "free" cytoplasmic iron, and/or iron bound to
critical proteins or enzymes.
There are several rationales for the use of the microbial cate
chol amide iron chelators as a means of controlling neoplastic growth.
1. The formation constants for the catecholamide iron complexes
(Kf=10^-10^ M"l) are far greater than those observed for chelators
previously used as antiproliferatives (4). Considering how effectively
the catecholamide siderophores bind iron, one would expect these che
lators to remove iron from several enzyme bound sites, e.g., ribonucle
otide reductase, mitochondrial enzymes, as well as from transferrin.
However, in spite of the thermodynamic binding advantage these ligands
have over various proteins, if the ligand cannot access the protein
bound iron, it obviously cannot remove it. Thus, removal of iron is a
kinetic problem.
2. Previously low biological yields from microorganisms and ted
ious isolation procedures of these compounds limited their use. Chemi
cal schemes have now been devised for the efficient synthesis of the
catecholamide microbial siderophores as well as a number of synthetic
analogues (30-32). The natural products are 2,3-dihydroxybenzoylamide
derivatives of polyamines, e.g., ,N^-bis(2,3-dihydroxybenzoyl)sperm-
idine (compound II) and parabactin, first isolated from cultures of
Paracoccus denitrificans by Tait (33) and vibriobactin, isolated from
Vibrio cholerae by Neilands et al. (34) (Fig. 1-3).
3. It has been postulated that the transferrin receptor is a
potential marker for the identification of proliferating cells (35).


8
a.
H
b. R = H
c. R = CH3
d. R = H
e. R = CH3
Figure 1-3. The catecholamide iron chelators.
a. Compound II
b. Parabactin
c. Tetramethyl parabactin
d. Vibriobactin
e. Permethylated vibriobactin


9
In a study using monoclonal antibodies to the transferrin receptor, it
was found that in all cases of breast carcinomas transferrin receptors
could be detected while in samples of normal breast tissue little or no
evidence of staining was exhibited (36). The evidence that neoplastic
cells, as indicated by their uninhibited proliferation and increased
means of iron acquisition, have higher iron requirements in comparison
to host cells provides a rationale for the potential selectivity of
iron removal by the microbial iron chelators as a target in antineo
plastic strategy.
4. Evidence suggested that the catechol amide chelators could
penetrate cellular membranes. A correlation between antiproliferative
activity and octanol/saline partition coefficients for a series of
siderophores has been established (37). It was found that the ratios
of the ligand partition coefficients between octanol and phosphate
buffered saline for the catechol ami des versus dihydroxybenzoic are
closer to the ratios of the ligand's antileukemic activity than are the
ratios of their binding constants. This suggested that cellular pene
tration as well as iron-chelation potential may determine the chelat
or's antiproliferative activity.
5. Parabactin and compound II have pronounced inhibitory effects
on the growth of L1210 cells in vitro with IC^q values in the micro
molar range (38). These values are in the range of antineoplastics
currently being used and are at concentrations attainable as serum
levels j_n vivo.
6. There is good evidence that the catecholamide siderophores,
unlike previously studied chelators, are relatively nontoxic to the


10
host. It has been observed that compound II, the biological precursor
to parabactin, has an LD^q in mice greater than 800 mg/kg when given as
a single intraperitoneal (i.p.) injection (39). It has been further
observed that compound II and parabactin at concentrations up to 2 mM
(100 times the 50% inhibitory concentration for murine L1210 leukemia
in vitro) appeared to be nontoxic to a confluent monolayer of monkey
kidney cel Is (37).
7. The iron dependent enzyme ribonucleotide reductase is a suit
able target for antineoplastic activity of the catecholamide chelators.
Hydroxyurea, an inhibitor of ribonucleotide reductase (40), has found
clinical usefulness as an anticancer agent. A number of other less
avid iron chelators such as desferrioxamine (41), a-picolinic acid (42)
as well as certain toxic thiosemicarbazones (43) have been shown to
inhibit tumor growth by interfering with ribonucleotide reductase
activity.
Parabactin and compound II have been shown to be potent inhibi
tors of ribonucleotide reductase activity and to block DMA biosynthesis
at the G-pS boundary of the cell cycle. However, RNA and protein syn
thesis were inhibited to a much lesser extent, and DNA polymerase activ
ity was essentially unaffected (38). Alterations in intracellular deox-
ynucleoside triphosphate levels revealed elevated dTTP pool and lower
ing of dATP pool size as is characteristic of other ribonucleotide
reductase inhibitors. Cytidine diphosphate (CDP) reductase activity
was inhibited by >97% in L1210 cells treated for 4 h with 5 yM parabac
tin. The cell cycle block seen in LI210 cells after a 4 h exposure to 5
yM parabactin could be partially reversed by the addition of exogenous


11
Fe(Cl)3 and the bulk of the cells cascade into S-phase 3 h later.
Catechol amide chelators have also demonstrated pronounced effects
against the DNA virus Herpes simplex type I (37), another ribonucleo
tide reductase dependent system, while they were inactive against the
RNA virus, Vesicular stomatitis.
It is clear that there is sufficient evidence to suggest that
iron withholding should be pursued as a means of controlling the pro
liferative process. The microbial catechol amide siderophores and their
synthetic derivatives may represent an important class of pharmacologi
cal agents with potential value in the treatment of proliferative
disorders. They may prove useful in cancer chemotherapy as single
agents or in combination with other agents.
Polyamine Analogues
The second part of this dissertation deals with the evaluation of
the N-alkylated derivatives of the polyamines as potential antiprolif
eratives. The role of polyamines in proliferative processes has been
extensively reviewed in recent years (44-50). The polyamines spermi
dine, spermine, and the diamine putrescine (Fig. 1-4) are present in
all mammalian cells and are required for normal cellular maintenance,
proliferation, and differentiation. These basic amines are extensively
protonated at physiological pH and capable of electrostatic interac
tions with a variety of macromolecules including nucleic acids and pro
teins. Although many roles have been ascribed to the polyamines, their
exact functions in cellular physiology are not yet well understood.
The intracellular concentrations of these polyamines are main
tained through a highly regulated metabolic system (49,50) (Fig. 1-5).


12
a.
H2N
H
N
NH2
b.
H2N
nh2
Figure 1-4. The polyamines.
a. Putrescine
b. Spermidine
c. Spermine


13
ARGININE
DECARBOXYLATED
S-ADENOSYLMETHIONINE
S-ADENOSYL-
METHIONINE
5-METHYLTHIOi
ADENOSINE
DECARBOXYLATED
S-ADENOSYLMETHIONINE
5'-METHYLTHIO
ADENOSINE
UREA
ORNITHINE
CO,
N1-acetylspermidine
6y
SPERMIDINE^
SPERMINE
N1 ACET YLSPERMINE
Figure 1-5. Pathway of polyamine synthesis and intercon
version in mammalian cells. Enzymes involved are: (1) arginase,
(2) S-adenosylmethionine decarboxylase, (4) Spermidine synthase,
(5) Spermine synthase, (6) Spermidine N -acetyltransferase, and (7)
Polyamine oxidase (49).


14
In mammalian cells putrescine is derived from ornithine by the pyridox-
al phosphate-dependent enzyme ornithine decarboxylase (ODC). Ornithine
is available in the plasma or formed from arginine by the action of
arginase. Levels of ODC are normally low in quiescent cells, and its
activity can be increased manyfold within hours after exposure to such
stimuli as hormones, drugs, tissue regeneration and growth factors
(47).
To convert putrescine to spermidine an ami nopropyl group must be
added. The aminopropyl moiety is derived from S-adenosylmethionine
(SAM) which is committed to polyamine biosynthesis as an aminopropyl
donor once it is decarboxylated by S-adenosylmethionine decarboxylase
(AdoMetDC). Levels of AdoMetDC are regulated by the cell's need for
spermidine and the availability of putrescine as a substrate for sperm
idine synthesis. Its activity is also regulated by growth-promoting
stimuli (49).
The transfer of the aminopropyl group from decarboxylated
S-adenosylmethionine to putrescine is catalyzed by the action of the
enzyme spermidine synthase to yield spermidine. In a similar fashion,
a second aminopropyl transfer to spermidine, catalyzed by spermine
synthase, yields spermine. Despite the similarity of these reactions,
spermidine synthase and spermine synthase are substrate specific
enzymes. The two synthases are thought to be regulated by the avail
ability of their substrates as well as factors stimulating cell growth.
The other product of the aminopropyl transfer reaction is 5'-methyl-
thioadenosine (MTA). The MTA produced by polyamine synthesis is


15
rapidly degraded and converted by a salvage mechanism into 5'-AMP and
methionine (50).
Spermine may also be interconverted into spermidine and spermi
dine into putrescine by the action of the enzymes N^-acetyltransferase
and polyamine oxidase. The acetyltransferase uses acetyl-CoA to con
vert spermine into spermidine and spermidine into putrescine. The N"-
acetylated polyamines are good substrates for polyamine oxidase, which
cleaves at the internal nitrogen yielding 3-acetamidopropanal and
putrescine or spermidine. Normally, only small amounts of the acetyl-
ated derivatives are present due to the much greater activity of poly
amine oxidase (49).
An active transport system distinct from those for amino acids is
also present (49,50). It is unclear why cells would maintain a poly
amine transport system since intracellular synthesis is normally used
to provide polyamines and extracellular polyamine levels are normally
low. However, uptake and efflux may be important mechanisms by which
cellular polyamine levels are maintained. The uptake pathways for the
polyamines have not been fully characterized at the biochemical level
and there may be multiple transport mechanisms (50).
Rapidly proliferating tissues have high levels of polyamines and
their biosynthetic enzymes (47), and increased quantitites of poly
amines are found in the urine and serum of humans and animals bearing
tumors (51). These facts suggest the potential of impacting on poly
amine metabolism as a useful target for antiproliferative therapy.
Interruption of the polyarnine biosynthetic network has been par
tially successful as a means of controlling the proliferative process


16
including the growth of cancer cells (52-54). The antineoplastic
drugs, a-difluoromethylornithine (DFMO) and methylglyoxyal-bis(guanyl
hydrazone) (MGBG) (Fig. 1-6), are both potent inhibitors of enzymes
which are critical to polyamine biosynthesis. DFMO is an enzyme-
activated irreversible inhibitor of ODC which causes depletion of
intracellular polyamine pools (55). It is postulated that DFMO is
recognized by ODC as a substrate and that its decarboxylation yields a
highly reactive intermediate which binds irreversibly to the active
site of the enzyme. The action of MGBG, however, is not specific.
MGBG induces spermine/spermidine acyl-transferase, inhibits diamine
oxidase and AdoMetDC, and has other effects (50). Although it is not
clear which of MGBG's roles is most important in inhibiting the growth
of cancer cells, uptake of the drug by the polyamine transport appar
atus seems critical. The drug has been regarded as a structural ana
logue of spermidine, and since its uptake involves an active transport
mechanism cells, can actually concentrate the drug so that millimolar
quantities of MGBG can accumulate intracel1ularly (56).
Although DFMO and MGBG have shown impressive results against
transplantable murine tumor models, the success of both in a clinical
setting, unfortunately, has been somewhat marginal (50,57). The action
of DFMO on most neoplasms is cytostatic, and the rapid rate of synthe
sis of new ODC (half-life of less than 1 h in many cell lines) makes it
essential to maintain high drug concentrations since enzyme levels and
cell growth are restored rapidly once drug is removed (49). MGBG has
produced unacceptable host toxicity in early trials.


17
a.
b.
Figure 1-6. The inhibitors of polyamine biosynthesis.
a. DFMO
b. MGBG


18
The combination of MGBG and DFMO has been reported to have syner
gistic activity (57). It was speculated that the simultaneous inhibi
tion of AdoMetDC by MGBG and reduction of ODC levels by DFMO treatment
would enhance tumor cell kill. Furthermore, DFMO depletion of intra
cellular polyamine pools increases uptake of MGBG by the polyamine
uptake apparatus (58). Since DFMO is relatively nontoxic (53), it was
hoped that potential tumor cell selectivity would be increased while
decreasing MGBG's host toxicity. This does not seem to be the case in
several trials (50). However, the fact that these agents do have anti
cancer activity, albeit limited, supports the aim of impacting on poly
amine metabolism as a rational approach for controlling the prolifera
tive process.
The initial design, synthesis and testing of polyamine deriva
tives which utilize the polyamine uptake apparatus and exhibit antipro
liferative properties was conducted by Porter, Bergeron and Stolowich
(59). There are several properties which may be desirable for the
design of polyamine analogues as antiproliferatives: (1) the analogues
may utilize the polyamine uptake apparatus as a means of delivery, and
such uptake may be enhanced by polyamine depletion, (2) the analogues
may fulfill some but not all of the functions of polyamines and thereby
interfere with normal polyamine metabolism and/or function, and (3) the
analogues may act to regulate the polyamine-biosynthetic pathway lead
ing to depletion of cellular polyamines and their biosynthetic en
zymes.
In a systematic approach, a number of spermidine analogues were
synthesized and studied for their structure/function relationships


19
(59-65) and some were found to meet the above requirements. It was
found that, in terms of uptake, retention of charge is of major import
ance since N-acetylation as opposed to N-alkylation at either the cen
tral or tne terminal nitrogens decreased the ability of the analogue to
compete with spermidine for uptake into the cell (65). While the
N ,N-alkyl derivatives (65) were poorer competitors for spermidine
uptake than N^-alkyl derivatives, they accumulated to high concentra
tions during longer incubations (66). The antileukemic activity of
several N^-alkyl spermidine analogues was enhanced by pretreatment of
cells with DFMO (67). Structural analogues of spermidine differing in
aliphatic chain length separating the amines were also studied (68).
Several of the homologues were found to support cell growth and no more
than a one-carbon extention was tolerated for biological activity.
1 ft
Certain of the N,N -alkyl spermidine derivatives, particularly
,N-diethylspermidine (BESPD), were found to inhibit L1210 cell
growth, diminish ODC activity, reduce AdoMetDC, and deplete intracell
ular polyamine pools (65,66,69). It has been concluded that BESPD
affects polyamine biosynthesis, at least, at one critical point by
regulating ODC at a translational and/or post-translational level in a
manner similar to the natural polyamines. However, the analogue is
apparently incapable of fulfilling certain critical functions of natur
al polyamines necessary for normal cellular function and proliferation
(65). Depletion of intracellular polyamine pools by BESPD may also
occur by displacement of the natural polyamines from their intracell
ular binding sites. In addition, potent induction of the spermidine/
spermine acetyl transferase activity by BESPD (70) could lead to the


20
back-conversion of spermine and spermidine to putrescine and efflux of
the polyamines from the cell.
The effects of these polyamine analogues may vary considerably
from one cell type to another depending on the cellular requirements
for polyamines. Despite inhibition of cell growth, BESPD was not toxic
towards L1210 cells (69). However, BESPD at concentrations of greater
than 5 yM was found to be cytotoxic to human large-cell lung cancer
cells normally quite resistant to DFMO (71).
The design, synthesis, evaluation, and study of the biochemical
and physiological effects of this new class of antiproliferatives is
currently the area of intense investigation.


CHAPTER II
CATECHOLAMIDE CHELATORS
Materials and Methods
The catecholamide chelators and methylated analogues were synthe
sized by methods previously described (30-32,72). Stock solutions (2
mM) of the catecholamide chelators were made in 50% (v/v) ethanol and
water. Stock solutions of Adriamycin and ara-C were prepared in water.
BCNU was prepared in 50% (v/v) ethanol and water. All solutions were
passed through a 0.2 ym filter prior to use. Reagents for cell culture
were obtained from Gibco (Grand Island, HY). Cell culture flasks, 25
and 75 cm^, were purchased from Corning (Corning, NY). Adriamycin
was obtained from Adria (Columbus, OH), carmustine (BCNU) from Bristol
(Syracuse, NY), hydroxyurea from Aldrich Chemical Co. (Milwaukee, WI)
and cytosar (ara-C) from Upjohn (Kalamazoo, MI). Diamidino-phenylin-
dole (DAPI) and propidium iodide (PI) were obtained from Sigma (St.
Louis, MO). Desferrioxamine was provided by Ciba-Geigy (Basel, Swit
zerland). Iron atomic absorption standard solution, 1003 yg/mL Fe in
2% HNO3 (Aldrich) was used as an Fe(III) source. Cremophore RH-40
was provided by BASF-Wyandot Corp. (Parsipany, NJ). Rhodamine-123
(Rh-123) was purchased from (Eastman Kodak Company, Rochester, NY),
[methyl- H]Thymidine was obtained from New England Nuclear (Boston,
MA). DBA/2 mice were obtained from Jackson Laboratories (Bar Harbor,
ME).
21


22
Cell Culture.--Murine L1210 leukemia cells and human Burkitt
lymphoma (Daudi) cells were maintained in exponential growth as
suspension cultures at 0.3 x 10^ to 1.5 x 10^ cells/mL and 1 x 10^ to
2 x 10^ cells/mL, respectively. Chinese hamster ovary (CH0) cells and
murine B-16 melanoma cells were grown as monolayers to near confluency
in 48 h after reseeding at 2.5 x 10^ cells/25 cm^ flask. Human fore
skin fibroblasts (HFF) cells were grown as monolayers to near conflu
ency in 96 h after a 1:4 dilution of cells in fresh medium. Monolayer
cells were lifted as single cell suspensions with 0.05% trypsin and
O
0.02% ethylenediaminetetraacetic acid tetrasodium salt (2 mL/25 cnr)
and diluted with Hanks' balanced salt solution. All cells were grown
in complete medium containing RPMI-1640, 2% 4-(2-hydroxyethyl)-l-piper-
azinethanesulfonic acid/3-(N- morpholino)propanesulfonic acid (HEPES--
M0PS buffer), and 10% fetal bovine serum unless otherwise stated.
p
Cells were grown in 25 cnr tissue culture flasks in a total volume of
10 rnL under a humidified 5% CO2 atmosphere at 37C.
ICc,q Value Determinations.Cultures in logarithmic growth were
treated with compounds of interest at concentrations ranging from 10"^
to 10^ M. Cells were counted by electronic particle analysis (Coulter
Counter, Model ZB1, Coulter Electronics, Hialeah, FL) and confirmed
periodically with hemocytometer measurements. Cell viability was
assessed by trypan blue dye exclusion.
The percentage of control growth was determined as follows:
% of control growth = 100 x [Final treated cell no.-initial
inoculum]/[Final untreated cell no.-initial inoculum]


23
The 50% inhibitory concentration (IC^g) value is defined as the
concentration of compound necessary to reduce cell growth to 50% of
control growth after defined intervals of exposure. The IC^q values
were determined as the chelator concentration necessary to inhibit cell
growth to 50% of the control growth at 48 h for all cell lines except
HFF cells, for which the ICgg values were determined at 96 h.
Preformed chelator-iron chelates were formed by the addition of
equimolar amounts of chelator and iron to the media 30 min prior to the
addition of the cel Is.
A 50% ethanol and water solution in appropriate amounts was used
to treat cells in all control flasks. Addition of iron and ethanol
alone in the concentrations used had no significant effect on growth
studies.
Stock suspensions of L1210 cells were maintained for 48 h, with
normal 10-12 h doubling times, in complete mediums containing 5%, 10%,
and 20% fetal bovine serum. The IC5Q value of parabactin was deter
mined for cells grown in each medium.
Drug Combinations.--L1210 cells in suspension culture were incu
bated in the presence of parabactin and antineoplastics at various con
centrations and combinations. Two types of experiments were performed
for each drug combination.
a. To obtain median effect plots for simultaneous drug combina
tions, cells at 3 x 10^ cells/mL were exposed to a constant ratio of
chelator to antineoplastic, a ratio in which absolute concentrations
were varied. The absolute concentrations of the drugs were chosen
in the neighborhood of their IC^g values. This gave the ratio of


24
parabactin to ara-C of 100:1, of parabactin to Adriamycin of 100:1, and
of parabactin to BCNU of 1:1. The growth was monitored at 48 h, and an
IC^Q value was determined. A separate experiment was run to determine
the IC^Q value of parabactin, ara-C, Adriamycin, and BCNU.
b. In a second experiment, cells at 3 x 10^/mL were first incu
bated with 5 pM parabactin for 5 h at 37C, then washed twice with
fresh medium, resuspended at 3 x 10^ cells/mL, allowed to incubate at
37C for 3 h (allowing the cells to cascade into S phase), and then
treated with varying concentrations of the antineoplastic. Growth was
monitored every 12 h for 96 h.
Additional experiments were carried out with various combinations
of parabactin and Adriamycin, ara-C or BCNU treatments.
c. Cells at 3 x 10^ cells/mL were treated with 5 pM parabactin
for 3 h, and then BCNU was added at varying concentrations. The flasks
were allowed to incubate for an additional 5 h. The cells were next
washed with fresh medium and resuspended at 3 x 10^ cells/mL, then
their growth was monitored for up to 96 h.
d. Cells at 3 x 10^ cells/mL were treated with 5 pM parabactin
for 4 h, and then Adriamycin was added. The flasks were allowed to
incubate for an additional 2 h. The cells were next washed with fresh
medium and resuspended at 1 x 10^ cells/mL, then their growth was mon
itored for 48 h.
e. Cells at 3 x 10^ cells/mL were treated with 2 pM parabactin
for 4 h, and then ara-C was added and the flasks were allowed to
incubate for an additional 12 h. The cells were next washed with fresh
medium and resuspended at 5 x 10^ cells/mL, then their growth was mon
itored for up to 120 h.


25
f. Cells at 3 x 10^ cells/mL were simultaneously treated with 5
yM parabactin and ara-C. The cells were allowed to incubate for 5 h,
washed with fresh medium, resuspended at 5 x 10^ cells/mL, and their
growth was monitored for up to 96 h.
In each case, the drug concentrations and treatment times are
indicated on the figure legends.
Data Analysis for Simultaneous Drug Combi nations.Briefly, this
procedure involves (a) determination of an ICgg value for each drug and
(b) determination of an IC^q value for a constant ratio of two drugs.
The molar ratio of the drugs is maintained while the absolute concen
tration of the drugs is changed. The cell growth data from these
experiments are plotted as log Fa/Fu versus concentrations (log [c]),
where Fa is fraction of cells affected, Fu is fraction of cells unaf
fected, and c is molar concentration. The data from the median effect
plots were analyzed by the method of Chou and Talalay (73) according to
the following equation.
A/Da + B/Dg = Cl (A)
The D values are the ICgg concentrations of each of the drugs alone,
the A and B values are the concentrations of each drug at the IC^q
value of the constant ratio combination, and Cl is the combination in
dex. When the Cl is in excess of 1, the combination is "antagonistic.
When it is less than one, the combination is "synergistic"; and when
the Cl is equal to 1, the combination is "additive."


26
Regrowth Studies.--L1210 cells in suspension culture at approxi
mately 3 x 105 cells/mL were incubated in the presence of chelator for
5 h at 37C. The cells were then washed twice in fresh complete medium,
resuspended at a final concentration of 5 x 10^ cells/mL, and incubated
at 37C. Cell samples during logarithmic growth were counted for up to
60 h after ligand treatment and compared to controls.
Cloning Assay.Cells at 3 x 10^ cells/mL were incubated with 5
yM parabactin for 5 h and washed with fresh medium. Treated cells were
then plated in triplicate 96-well microtiter plates at 0.4 cells/well
with each well containing 100 yM of sample. The plates were incubated
at 37C in a humidified incubator in an atmosphere of 5% CO^ and 95%
air. The plates were examined with an inverted phase microscope at
X100 magnification. The final number of colonies per plate was quanti
tated at 7 days after plating. Groups of 50 or more cells per well were
identified as having been cloned from a single viable cell.
Viability Assay.--Cell viability was assayed by two dye exclusion
methods. In one method cells were diluted 1:1 with 0.4% trypan blue in
phosphate-buffered saline and counted in a hemacytometer using a light
microscope. In the second method 10^ cells were diluted in 1 mL of a
1.12% sodium citrate solution of propidium iodide (50 yg/mL) and count
ed on a fluorescent microscope.
Viability determination by Rh-123 uptake (74) was assayed by add
ing the mitochondrial dye Rh-123 (10 yg/mL) to a aliquot of 10^ cells.
The samples were incubated at 37C for 10 min and washed once with med
ium. Trypan blue was added, prior to counting, to give a final concen
tration of 0.2 yg/mL. Rh-123 and trypan blue stained cultures were


27
observed in a hemocytometer using a Zeiss epifluorescence Axioscop
microscope. The excitation wavelength was 485 nm.
Flow Cytometric Analysis.--Cel 1 analysis was performed with a
RAT-COM flow cytometer (RATCOM, Inc., Miami, FL) interfaced with a
microcomputer (IBM-XT). Cell samples of 106 cells were taken at vari
ous intervals and stained with DAPI (10 yg/mL) in a nuclear isolation
media (NIM-DAPI) (75). DNA distributions were obtained from the fluor
escence and analysis of the DAPI-stained cells.
Alternately, cell analysis was performed with a FACS II flow
cytometer (Becton Dickinson FACS Systems, Sunnyvale, CA) interfaced
with a microcomputer (Hewlett Packard 45B, Fort Collins, CO). Samples
of 10^ cells were removed and stained with PI and then exposed to
RNase. DNA distributions were obtained from analysis of the red fluor
escence from the Pi-stained DNA (76).
Effect of Percent Fetal Bovine Serum in the Medium on Cell Cycle
Kinetics.--Stocks of L1210 cells were maintained for 48 h with normal
10-12 h doubling times in complete mediums containing 5%, 10%, 15% and
20% fetal bovine serum. The time course for the effect of 5 yM para-
bactin on the cell cycle kinetics of cells grown in each of the mediums
was followed for up to 8 h.
Bromodeoxyuridine (BrdUrd) Incorporation Studies.--Cultured LI210
cells were treated with 10 yM vibriobactin, washed, resuspended, and
incubated as in the regrowth studies. At times, 0, 5, 10, 15 and 20 h
of regrowth cell samples were removed for dual parameter flow cytomet
ric measurements of cellular DNA content and amount of BrdUrd incorpor
ated into cellular DNA by methods presented elsewhere (77,78).


28
Radiolabeled Thymidine Incorporation.Cells treated with 5 pM
parabactin for 5 h were washed, resuspended in fresh complete medium,
and incubated at 37C for 30 min with [^H]thymidine (specific activ
ity, 80.9 Ci/mmol; 1 pCi/mL) in triplicate tubes containing 10^
cells in a total volume of 1 mL of complete medium. Labeling was
halted by the addition of 0.5 mL of ice-cold Hanks' balanced salt
solution (HBSS) containing thymidine (1 mg/mL) to each tube. The cells
were washed, resuspended in 1 mL of 10% trichloroacetic acid in HBSS
containing thymidine (1 mg/mL), and allowed to sit on ice for 30 min.
The acid-precipitable material was filtered and the filter washed.
Then filters were air dried and counted in Biofluor (New England
Nuclear) scintillation fluid. Background labeling was evaluated in
O
cell samples pulsed for 30 min with [ H]thymidine at 5C.
% of control incorporation = treated cpm treated background cpm x 100
control cpm control background cpm
Cr^Release Assay. LI210 cells were treated with vibriobactin
(10 pM) for 5 h, washed with fresh media and regrown for 20 h. Approx
imately 2 x 10^ cells were centrifuged and 150 pCi Cr^ added to the
pelleted cells. The pellet was incubated for 45 min at 37C in a 5%
CO2 atmosphere. Sample radiation was counted with an automatic gamma
counter (LKB-Wallac RiaGamma 1274, Wallac Oy, Finland).
A microwell assay using triplicate samples of 200 pL containing
1 x 10^ cells was employed as described elsewhere (79). The percent


29
51
Cr release for the control cells was compared to that for the vibrio-
bactin treated cells.
Animal Studies.--The murine LI210 leukemia cells were maintained
in DBA/2J mice. Cells from a single mouse which was injected i.p. with
1.25 x 10^ cells 7 days earlier were harvested and diluted with cold
saline so that an inoculum of 10^ cells could be administered by a
0.25 mL i.p. injection. In each study, mice were injected with L1210
cells on day 0.
The catechol amide chelators were first solubilized in 20% Cremo-
phor RH-40 in 0.9% saline with sonication and gentle heating (<60C)
and then diluted with an equal volume of 0.9% saline. The chelator was
administered by i.p. injection according to the appropriate dosing
schedule. Concentrations of chelator were adjusted so that the mice
were injected with a volume of 1-2 mL/100 g of animal/dose (i.e.: a 25
g mouse was injected with 0.25-0.5 mL of drug solution).
The in vivo effectiveness of parabactin alone and combinations of
parabactin with ara-C or Adriamycin against the L1210 ascites tumor
were studied. Ara-C and Adriamycin were diluted in 0.9% saline so that
the mice were injected i.p. with a volume of 1 mL/100 g of animal/dose.
Due to the possible toxicity or antitumor activity of Cremophor RH-40,
groups of mice treated with 10% Cremophor RH-40 as well as untreated
mice served as controls. Groups of 6 mice were used in each treatment
schedule.
One parameter used for treatment evaluation was mean animal sur
vival time (percent increased life span, % ILS).


30
% ILS = 100 x [mean survival time treated animals-
mean survival time controls]/[mean survival time controls]
Another parameter used for treatment evaluation was tumor burden.
The L1210 cells were harvested from the animal's peritoneum by two
washings with 5 mL of 0.9% saline. The total number of cells recovered
was determined by counting on a hemocytometer.
The in vivo effect of a single i.p. injection of parabactin (100
mg/kg) on L1210 cell cycle kinetics was studied in mice which had been
injected i.p. with 1 x 10^ L1210 cells 6 days earlier. Mice were sac
rificed by cervical dislocation each hour after injection of the che
lator and the cells were harvested from the peritoneum with HBSS. The
cells were stained witli NIM-DAPI and DNA content measured by flow cyto
metric analysis.
Results
Inhibition of Cell Growth
The effectiveness of various iron chelators as inhibitors of
L1210 cell growth was assessed by comparison of their IC^q values
(Table 2-1). The bidentate compound, dihydroxybenzoic acid, while
being a good iron chelator (Kf = lO^M-*) (5,37) is not very effective
cell growth inhibitor, requiring millimolar concentrations. Hydroxy
urea, which structurally resembles a bidentate hydroxamate chelator,
was only fair at inhibiting cell growth, with an IC^q value of 40 yM.
The hexacoordinate hydroxamate iron chelator, desferrioxainine, and the
tetracoordinate catechol amide chelator, compound II, showed similar
activity against L1210 cell growth with ICgg values of 8 yM and 7 yM
respectively. The most effective of the compounds tested were the
hexacoordinate catecholamide chelators, vibriobactin and parabactin,


31
TABLE 2-1
ICcn VALUES OF VARIOUS CHELATORS AGAINST
CULTURED LI210 LEUKEMIA CELLS
Chelators
IC^q Values at 48 H
Dihydroxybenzoic Acid
2.8 nM
Hydroxyurea
40 pM
Desferrioxanine
8 pM
Compound II
7 pM
Vibriobactin
2 pM
Parabactin
1.5 pM


32
having IC^q values of 2 pM and 1.5 pH respectively. The dose-effect
curves generated in these studies indicate linear inhibitory effects
for parabactin and vibriobactin within a very narrow range of ligand
concentrations (Fig. 2-1). For example, at concentrations of greater
than 5 pM, cells uniformly grow to only about 10% of controls, while
below 1 pM approximately 90% control growth is obtained.
The time dependence of growth inhibition of cultured LI210 cells
by the siderophores is shown in Figures 2-2 and 2-3. The concentration
of 10 pM was selected for both parabactin and vibriobactin as a value
at approximately 5 times their IC^q values. The onset of growth
inhibition by both siderophores was almost immediate. Both chelators
eventually caused a total cessation cell growth. In either case, the
growth inhibition could be prevented with the addition of an equimolar
amount (10 pM) of Fe(III) to the culture medium at the same time as the
chelator.
Methylation of the ligand catechol hydroxyl groups, as in tetra-
methyl parabactin (Fig. l-3c) and permethylated vibriobactin (Fig.
l-3e), greatly reduced the growth inhibitory activity of both chelat
ors. Total methylation of the three catechol groups of vibriobactin
completely eliminated its activity at a concentration 10 pM (Fig. 2-2).
In fact, it showed no inhibitory activity at concentrations of up to 50
pM. Tetramethyl parabactin (10 pM), while having greatly reduced
inhibitory activity, did have some effect on L1210 growth (Fig. 2-3).
In fact, it had an IC5Q value of 30 pM.
The IC^q values for parabactin were determined against L1210
cells grown in culture mediums with varying concentrations of fetal


33
PERCENT CONTROL GROWTH
Figure 2-1. Dose-response curves illustrating the effects of
increasing concentrations of parabactin and vibriobactin on the
growth of L1210 cells.


CELLS/ml
Figure 2-2. Comparison of the effects of 10 yM vibriobactin,
in the presence or absence of Fe(III), and 10 yM permethylated
vibriobactin on the growth of L1210 cells.


CELLS/ml
35
Figure 2-3. Comparison of the effects of 10 yM parabactin,
in the presence or absence of Fe(III), and 10 yM tetramethyl
parabactin on the growth of L1210 cells.


36
bovine serum (Fig. 2-4). Cells grown in 5, 10 and 20% FBS had IC^g
values of 0.8, 1.2, and 1.6 yM respectively.
Parabactin was also found to have potent growth inhibitory ef
fects on cultured B16-melanoma and CHO cells exhibiting a 48 h IC^g
value of 1.5 yM for both cell lines. Both vibriobactin and parabactin
exhibited a 48 h IC^q value of 2 yM for Daudi cells and parabactin was
active, with a 96 h IC^q value of 2 yM, against HFF cells. The IC^q
value for HFF cells was determined at 96 h due to their relatively slow
doubling time (45-50 h) compared to the other cell lines studied (12-24
h).
Treatment Reversibility
When cells were treated for 5 h with 5 yM parabactin, the growth-
inhibitory effects of parabactin were found to be reversible on washing
away the ligand. Viability of parabactin-treated cells, taking cell
samples every 5 h for 35 h after washing away of the ligand, was
verified by dye exclusion and Rh-123 uptake methods. At 10 h after
washing, the viability decreased to a minimum of 88% by the dye
exclusion methods and only 85% by Rh-123, the most sensitive of the
methods used. These results were in agreement with the cloning assay
which indicated 91% of colonies were cloned or single cell after
short-term exposure (5 h) to the chelator compared to single control
cells. The number of cells per colony from the parabactin-treated
cells was consistently less than from the control cells.
Cells having been treated for 5 h with 10 yM vibriobactin showed
similar reversibility of inhibitory effects on washing away of the lig
and and reseeding the cells in fresh medium. A ^Cr release assay per
formed on vibriobactin treated cells at 15 h after removal of the lig
and showed no increase in ^Cr release relative to control cells,


50% INHIBITORY CONCENTRATION
37
% FETAL BOVINE SERUM
Figure 2-4. The effect of varying amounts of fetal bovine
serum in the L1210 growth medium on the 50% inhibitory concentra
tion of parabactin.


38
establishing little, if any, cytotoxicity due to short term (less than
5 h) 10 yM vibriobactin.
However, when cells were treated for an extended period of time
with 5 yM parabactin, cytocidal activity was observed (Table 2-2). For
example, at 60 h, only 30% of the cells were viable as determined by
trypan blue exclusion. Dye exclusion of cells treated with 10 yM vib
riobactin for 48 h indicated only 10% viability, and only 20% viability
of cells exposed to 100 yM desferrioxamine for 60 h. Surprisingly, 300
yM hydroxyurea was the least toxic of the compounds tested with only
25% cytotoxicity after 60 h of treatment. Although in dividing HFF
cells, growth was inhibited by parabactin (IC^q value of 2 yM at 96
h), treatment of a confluent, nondividing, monolayer of HFF cells with
20 yM parabactin for 24 h appeared to be nontoxic by light microscopy.
Also, the treated confluent cells diluted and regrown in drug-free med
ium grew at the same rate as control cells.
Effects of the Catechol amide Chelators
on Cell Cycle Kinetics
Flow cytometric analysis was employed as the method to follow the
effects of the chelators on the cell cycle progression. The time
course for the effect of 10 yM vibriobactin on the DNA content of L1210
cells is seen in Figure 2-5. Flow cytometric analysis of untreated
L1210 cells reveals high S (50%) and G2-M (15%) phase components of
the cell population with about 35% of the cells having phase DNA
content. After only 2 h alterations in the DNA content of the vibrio
bactin treated cells were observed. There was a substantial effect on
the cell cycle kinetics with a clear block at the G^-S border after 3


TABLE 2-2
CYTOCIDAL ACTIVITY OF CHELATORS
39
Ligand
Percent of Activity3
0
12 h
24 h
48 h
60 h
Parabactin (5 pM)
<2
5
15
49
70
Vibriobactin (10 pM)
<2
4
36
90
-
Desferrioxamine (100 pm)
<2
5
20
60
80
Hydroxyurea (300 pM)
<2
-
15
-
25
aThe percentage of L1210 cell kill in cells treated with parabactin,
vibriobactin, desferrioxamine and hydroxyurea with treatment time.
Cell viability determined by trypan blue exclusion.


RELATIVE CELL NUMBER
40
DNA CONTENT
Figure 2-5. Flow cytometric analysis of L1210 cells.
A. Control cells.
B. Cells treated with 10 pM vibriobactin for 3 h.
C. Cells treated with 10 pM vibriobactin for 4 h.
D. Cells treated with 10 pM vibriobactin for 5 h.


41
h. Within 5 h, cells with G£-M phase content were eliminated, and
cells with S phase content were reduced to 30%. The cells were pre
vented from entering S phase and 70% of the cells have phase con
tent. A comparable cell cycle block in DNA synthesis was observed in
the DNA histograms of cells treated with 5 yM parabactin for 5 h (Fig.
2-6). Cells treated for up to 24 h with 5 yM parabactin, however,
showed no greater block in cell cycle kinetics than cells treated for 5
h, and cells treated with less than 4 yM parabactin did not appear to
reduce S phase to the extent that the 5 yM treatment did. For further
studies it was therefore indicated that the minimum concentration and
time for parabactin to effectively eliminate DNA synthesis in L1210
cells was 5 yM for 5 h.
In a study to determine the ability of 5 yM parabactin to cause a
cell cycle block in L1210 cells grown in culture mediums with various
percentages of fetal bovine serum (5%, 10%, 15% and 20%), it was found
that there was no significant alteration in time to reach complete cell
cycle block (5 h) for the cells in any of the different culture medi
ums.
In a comparative study, parabactin was shown to be a far more
effective cell cycle blocking agent than either hydroxyurea or desfer-
rioxamine (Fig. 2-6). For example, to generate cell cycle blocks simi
lar to that produced by 5yM parabactin (IC^q, 1.5 yM) at 5 h, L1210
cells required 100 yM desferrioxamine (IC^q, 7.5 yM) for 5 h or 300 yM
hydroxyurea (IC^q, 50 yM) for 5 h. When comparing the IC^q value of
each of the ligands, it is clear that the compounds with the higher
IC5Q values are also the poorer cycle blocking agents.


RELATIVE CELL NUMBER
42
DNA CONTENT
Figure 2-6. Flow cytometric analysis of L1210 cells.
A. Control cells.
B. Cells treated with 5 yM parabactin for 5 h.
C. Cells treated with 100 yM desferrioxamine for 5 h.
D. Cells treated with 300 yM hydroxyurea for 5 h.
T I 1
420


43
Synchronization Effects
Flow cytometry analysis of the cell cycle kinetics of cells
treated with 10 yM vibriobactin for 5 h indicated a clear block at the
Gj-S border of the cell cycle. Washing and reseeding the treated cells
in fresh complete medium caused pronounced changes in the cell cycle
phases with time (Fig. 2-7). The block in DNA synthesis is released
and at 5 h after removal of the drug the majority of cells have S phase
DNA content. The progression through the cell cycle phases continued
for up to 20 h. Cell cycling was further substantiated by BrdUrd
incorporation into DNA (Table 2-3). The percent BrdUrd incorporation
at various times after removal of the chelator was roughly consistant
with changes in S phase DNA content as determined by flow cytometry.
Cells incubated with 5 yM parabactin for 5 h exhibited a block in
DNA synthesis at the G^-S border (by flow cytometry) with a greatly
decreased S and G phase (Figs. 2-6 and 2-8). Concentrations of 100 yM
desferrioxamine and 300 yM hydroxyurea for 5 h were required to produce
similar blocks. On removal of the chelators by simple washing and
reseeding the cells in drug-free culture medium, the block in DNA syn
thesis was removed and the cells cascaded into S phase. Parabactin-
treated cells maintained a synchronous population of cells for 3 cell
cycles. (Figure 2-8 only includes cells moving into the third cycle.)
However, desferrioxamine- and hydroxyurea-treated cells showed normal
ized DNA histograms after 12 h.
Radiolabeled Thymidine Incorporation.--Incorporation of [ H]thy-
midine into L1210 cells after the cells were treated with 5 yM


RELATIVE CELL NUMBER
44
DNA CONTENT
Figure 2-7. Flow cytometric DNA content analysis of L1210
cells washed and regrown in fresh medium after a 5-h treatment with
10 pM vibriobactin.
Analysis performed after removal of ligand.
A. 0 h
B. 5 h
C. 10 h
D. 15 h
E. 20 h


45
TABLE 2-3
PERCENT BROMODEOXYURIDINE INCORPORATION INTO L1210
CELLS TREATED WITH 10 yM VIBRIOBACTIN FOR 5 H THEN
WASHED AND RESEEDED IN FRESH MEDIUM
Time after Wash
Percent BrdUrd
Incorporation
Control
Treated
0
44
16
5
50
47
10
53
45
15
47
16
20
49
32


Figure 2-8. Cell synchronization of L1210 cells by 5 yM
parabactin. DNA content determined by flow cytometric analysis.
A. Control cells.
B. Cells treated with 5 yM parabactin for 5 h.
Cells were then washed, resuspended in drug-free medium, and
samples removed at the following times.
c.
4 h.
D.
6 h.
E.
10 h.
F.
12 h.
G.
14 h.
H.
16 h.
I.
18 h.
J.
22 h.


NUMBER OF CELLS
47
DNA CONTENT


48
parabactin for 5 h and washed (Fig. 2-9) revealed cycling of incorpor
ation in a time frame consistent with the normal 10 h to 12 h doubling
time of L1210 cells, and with the cycling of DNA content as seen in the
flow cytometric studies (Fig. 2-8).
Drug Combination Studies
Adriamycin-treated Cel Is.--The Adriamycin IC^q under our experi
mental conditions was 0.027 yM at 48 h, while the parabactin IC^g was
1.34 yM at 48 h. However, when L1210 cells were treated with para
bactin and Adriamycin in simultaneous combination, the results indicat
ed that the combination was "anatagonistic" (Fig. 2-10). Following the
method of analysis of Chou and Talalay, the cells were exposed to a
constant molar ratio of parabactin to Adriamycin of 100:1. The micro
molar concentrations at this constant ratio were 2:0.02, 1.75:0.0175,
1.5:0.015, 1.25:0.0125, 1.0:0.01, and 0.5:0.005. The IC^g of the com
bination was 1.42 yM (Cl = 1.57). Chou and Talalay's analysis of the
data (see Materials and Methods) clearly revealed the combination to be
"antagonistic" at the 100:1 ratio.
In a second experiment, cells were treated with 5 yM parabactin
for 5 h, washed free of the ligand, placed in fresh medium to allow the
cells to cascade into the S phase of the cell cycle, and grown with
0.02 yM or 0.03 yM Adriamycin (Fig. 2-11). In this experiment the
parabactin clearly potentiated the activity of Adriamycin (see Discus
sion). Finally, in a third experiment cells were treated with 5 yM
parabactin for 4 h and then Adriamycin 0.2 yM was added for an addi
tional 2 h. Cells were washed, reseeded and grown in fresh drug-free


49
TIME (hours)
Figure 2-9. Time course for the incorporation of [^H]tny-
midine into the acid-precipitable fraction of L1210 cells grown in
fresh medium after treatment with 5 yM parabactin for 5 h.


LOG Fa/Fu
Figure 2-10. Median effect plots for L1210 cells exposured for
48 h to Adriamycin, parabactin, and the simultaneous combination of
parabactin:Adriamycin at a molar ratio of 100:1.
o


51
Figure 2-11. The effects of Adriamycin on the growth of
L1210 cells, and Adriamycin on the growth of cells which have been
pretreated with 5 yM parabactin for 5 h then washed to remove the
block in DNA synthesis. The growth of control cells and cells
exposed to 5 yM parabactin for 5 h and resuspended in drug-free
medium is shown for comparison.


52
medium (Fig. 2-12). The results indicated additivity of the two drug
effects.
Ara-C-treated Cells.--The IC^g of ara-C in our L1210 cell assay
system was 0.033 yM, L1210 cells were treated with parabactin and
ara-C simultaneously at a constant molar ratio of 100:1. The concen
trations at a constant ratio of parabactin to ara-C were 1.75:0.0175,
1.5:0.015, 1.25:0.0125, 1.0:0.01, 0.75:0.0075, and 0.5:0.005. The IC5Q
of the combination was 1.39 yM (Cl = 1.33). Again, Chou and Talalay
analysis of the growth data indicated "antagonism" (Fig. 2-13).
However, when cells were first treated with 5 yM parabactin for 5
h, washed with fresh medium, and followed by treatment with ara-C, as
with the Adriamycin:parabactin combination, the parabactin potentiated
the activity of the ara-C (Fig. 2-14).
When cells were simultaneously treated with 5 yM parabactin and
1 yM or 2 yM ara-C for 5 h (Fig. 2-15), or first treated with 2 yM
parabactin for 4 h then 1 yM or 2 yM ara-C for an additional 12 h, then
washed, reseeded and grown in fresh drug-free medium (Fig. 2-16), the
results indicated additivity of the two drug effects for both combina
tions.
BCNU-treated Cells.--The IC^g for BCNU in our system was 4.13 yM.
When cells were treated simultaneously with parabactin and BCNU at a
molar ratio of 1:1, the ICgg of the combination was 2.28 yM (Cl = 1.1),
indicating additivity (Fig. 2-17). The absolute micromolar drug
concentrations to which cells were exposed were 3:3, 2:2, 1:1, 0.5:0.5,
and 0.25:0.25.


53
Figure 2-12. The effects of 0.2 yM Adriamycin exposure for 2
h, parabactin 5 yM treatment for 6 h, and 5 yM parabactin for 4 h
plus the simultaneous exposure of 0.2 yM Adriamycin for an addi
tional 2 h on the growth of L1210 cells. Treated cells were washed
and resuspended in drug-free medium at time = 0 h.


LOG Fa/Fu
Figure 2-13. Median effect plots for L1210 cells exposed for 48 h
to ara-C, parabactin and the simultaneous combination of parabactin:ara-C
at a molar ratio of 100:1.
<_n
4^


55
TIME (hours)
Figure 2-14. The effects of ara-C on the growth of L1210
cells, and ara-C on the growth of cells which have been pretreated
with 5 pM parabactin for 5 h then washed to remove the block in DNA
synthesis. The growth of control cells and cells exposed to 5 pM
parabactin for 5 h and resuspended in drug-free medium is shown for
comparison.


Figure 2-15. The effects of ara-C exposure for 5 h,
parabactin 5 yM treatment for 5 h, and the simultaneous exposure of
5 yM parabactin plus ara-C for 5 h on the growth of L1210 cells.
Treated cells were washed and resuspended in drug-free medium at
time-0 h.


57
Figure 2-16. The effects of ara-C exposure for 12 h on the
growth of L12110 cells, and 2 yM parabactin treatment for 4 h plus
exposure to ara-C and parabactin for an additional 12 h on the
growth of L1210 cells. The growth of control (untreated) cells and
cells exposed to 2 yM parabactin for 16 h is shown for comparison.
Treated cells were washed and resuspended in drug-free medium at
time-0 h.


A BCNU
Parabactin
Combination (1:1, Parabactin:BCNU)
-1.5 H 1 I 1 1 1 1 1 1 1 1 1 1 1 1 1
- 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
LOG DOSE (uM)
Figure 2-17. Median effect plots for L1210 cells exposed for 48 h
to BCNU, parabactin and the simultaneous combination of parabactin:BCNU
at a molar ratio of 1:1.


59
When cells were first treated with 5 pM parabactin for 5 h,
washed with fresh medium, and treated with BCNU immediately, an
additive effect was again observed (Fig. 2-18).
However, when cells were first exposed to 5 pM parabactin for 3
h, then presented with varying concentrations of BCNU for an additional
5 h, washed, and resuspended in fresh medium, cell growth indicated
parabactin potentiation of BCNU activity (Fig. 2-19).
Animal Studies.--Ascites L1210 tumor cells in DBA/2 mice were
blocked and synchronized by a single i.p. injection of parabactin 100
mg/kg (Fig. 2-20). The DNA histogram of the untreated ascites tumor
shows a DNA content distribution similar to that of the cultured cells.
At seven hours after the parabactin treatment a block in DNA synthesis
at the Gj-S border of the cells is evident by the reduction in G£ phase
content and the broadening of the G^-S content peak. At 8 h cells
which were blocked at the G^-S border begin to enter S phase, and by
9 h after the injection a large majority of the ascites cells have S
phase DNA content indicating a synchronous population of cells.
There was no significant increased life span in mice given
1 x 105 L1210 cells i.p. and then treated with a daily (0D) injection
of parabactin 100 mg/kg on days 1-6 compared to untreated mice or mice
treated with only the 10% Cremophore drug vehicle. However, there was
an 80% reduction in the ascites tumor burden of mice treated with para
bactin 100 mg/kg every 8 h (98 h) for 5 injections beginning on day 5,
compared to untreated mice. This dosage schedule could not be


60
Figure 2-18. The effects of BCNU on the growth of L1210
cells, and BCNU on the growth of cells which have been pretreated
with 5 pM parabactin for 5 h then washed to remove the block in DNA
synthesis. The growth of cells exposed to 5 yM parabactin for 5 h
and resuspended in drug-free medium is shown for comparison.


61
TIME (hours)
Figure 2-19. The effects of BCNU exposure for 5 h,
parabactin 5 pM treatment for 8 h, and 5 pM parabactin for 3h plus
the simultaneous exposure to BCNU for an additional 5 h on the
growth of L1210 cells. Treated cells were washed and resuspended
in drug-free medium at time-0 h.


NUMBER OF CELLS
62
Figure 2-20. Flow cytometric analysis of ascites L1210 cells
from DBA/2 mice after a single i.p. injection of parabactin 100
mg/kg.
A. 0 h
B. 7 h
C. 8 h
D. 9 h


63
continued for more than the 5 doses in the mice due to the observed
toxic effects (constipation and death) of the Cremophore vehicle when
administered alone at concentrations necessary to solubilize this dose
of parabactin.
Various combinations of parabactin with ara-C or Adriamycin in
mice with L1210 tumors (Table 2-4) did not disclose any significant
increases in life span beyond the additive effects of the individual
treatments.
Discussion
Iron chelators have been shown to have profound effects on the
growth properties of various tumor cell lines (37,41-43). The antipro
liferative activity of the siderophores against L1210 cells as
assessed by their IC^q values indicated that, when ranked according to
their ability to sequester iron, the compounds with the higher iron
binding constants generally demonstrated better antileukemic activity
(37). The most active of the compounds tested here (the hexacoordinate
catechol amide chelators, vibriobactin and parabactin) have an enormous
affinity for iron and exhibited the most potent antiproliferative
activity with IC^q values against L1210 cells of 2 yM and 1.5 yM,
respectively. Both compounds were able to cause nearly complete cessa
tion of cell growth in 24 h at concentrations above 5 yM while below 1
yM little growth inhibition was seen.
In an effort to ascertain the broadness of activity of the cate-
cholamide chelators, they were tested against various other cultured
cell lines. Parabactin and vibriobactin were found to be potent growth
inhibitors of various animal and human tumor cells with 48 h IC^g


TABLE 2-4
ACTIVITY OF PARABACTIN, ARA-C AND ADRIAMYCIN, EITHER ALONE OR IN COMBINATION, AGAINST L1210
MURINE LEUKEMIA IN DBA/2 MICE
Experi-
ment
Number
Drug
Treatment
Dosage
Schedule
Mean
Survival
(days) S.D.
% ILS
1.
Parabactin
100 mg/kg
OD day 1
9.5 1.0
3
Ara-C
120 mg/kg
Day 1
12.0 1.9
23
Ara-C/Parabacti na
OD day 1
12.6 1.8
29
NONE
OD
9.7 0.8
0
2.
Parabactin
100 mg/kg
ql2h days 1-2
11.0 2.2
13
and 6-7
Ara-C
40 kg/mg
ql2 h days 1-2
19.5 2.7
100
and 6-7
Ara-C/Parabactinb
Days 1-2 and
15.7 5.9
62
6-7
NONE
9.8 0.8
0
3.
Parabactin
100 mg/kg
OD day 1
10.0 1.0
5
Adrianycin
2 mg/kg
OD day 1
10.5
11
Adriamycin/Parabactinc
OD day 1
11.5 1.2
21
NONE
9.5 0.7
0
4.
Parabactin
100 mg/kg
OD days 2 and 4
10.0 1.0
11
Adriamycin
3 mg/kg
OD days 2 and 4
14.1 5.2
57
Adriamycin/Parabactind
OD days 2 and 4
15.1 3.3
68
NONE
9.0 0.4
0
aAra-C given 7 h after parabactin. NOTE:
bAra-C alternated ql2 h after parabactin.
dAdriamycin given 7 h after parabactin
dAdriamycin given 7 h after parabactin.
DBA/2J mice given 10b L1210 cells i.p. on day 0.
All treatments given by i.p. injection.
OD = Once daily,
ql2h = every 12 h.


65
values of approximately 2 pM for all cell lines tested. Parabactin was
also found to be a potent inhibitor of actively-dividing normal HFF
cell growth with a 96 h IC^g value of 2 pM.
That growth inhibition is due to iron chelation seems apparent
from studies in which iron, added at the same time as the chelators,
prevents the effects. This is consistent with previous findings (37)
in which the antileukemic and antiviral viral activities of compound II
could similarly be prevented. Also, methylation of the ligand's cate
chol groups essentially blocking the siderophore's chelating function
alities (Fig. 1-3) eliminated the antileukemic activity of vibriobactin
and greatly reduced the activity of parabactin (Figs. 2-2 and 2-3).
The residual activity of tetramethyl parabactin is likely due to the
remaining central oxazoline nitrogen and phenolic hydroxyl group's
ability to chelate iron. It has also been shown that parabactin was
able to suppress the growth of a variety of bacteria, while the addi
tion of the tetramethylated parabactin analogue or the preformed
ferric-parabactin chelate was unable to inhibit microbial prolifer
ation (72).
Cytostasis (apparent by dye exclusion, Rh-123 mitochondrial stain
ing, ^Cr release and clonagenic assays) is observed on a short-term
exposure (5 h) of L1210 cells to 5 pM parabactin or 10 pM vibriobactin,
and cidal activity when the cells are exposed for longer periods of
time (Table 2-2). The stasis is reversible by simply washing the lig
and away from the cells within the first 5 h. However, a nondividing
confluent monolayer of HFF cells exposed to 20 pM parabactin (10 times
its IC^q value) for 24 h exhibited no apparent toxic effects.


66
The effects of vibriobactin and parabactin on L1210 cell growth
are even more apparent after examining their impact on cell cycle kin
etics. Both ligands hold the cells at the G^-S border until they are
released by washing the chelator away, at which point cells cascade
into S phase (Figs. 2-7 and 2-8). This synchronous population of cells
can be followed through the cell cycle with time, and in the case of
parabactin a portion of the cells remained synchronized for 3 cell
cycles. When parabactin treated cells are reseeded in fresh medium
there is approximately a 14 h lag in growth relative to untreated con
trols. At this point cells appear to grow normally with a doubling
time of 12 h (Figs. 2-11, 2-14, 2-18.)
Cycling of [^H]thymidine incorporation into DNA (Fig. 2-9) during
the first 12 h after washing parabactin from treated cells, or cycling
of BrdUrd incorporation into DNA after vibriobactin pretreatment (Table
2-3) was consistant with the time frame of changes in S-phase followed
by flow cytometry. However, the percentage of control incorporation
during certain phases in the cell cycle reveals an actual DNA synthetic
rate which does not correspond to the levels expected from the percent
ages of cells with S-phase DNA content by flow cytometry (Fig. 2-8).
For example, flow cytometry indicated that at 8 h after washing the
percentage of cells having S-phase DNA content is approximately twice
that (200%) of control; however, [^H]thymidine incorporation corre
sponds to a DNA synthetic rate of only 120% of control. From these
studies it seems that while a sizeable fraction of the cells moves
synchronously through the cell cycle in 10 to 12 h, there is also a
fraction of cells which are moving more slowly through the cell cycle.
Together, the fraction of cells moving more slowly through the cell


67
cycle along with the decrease in viability of cells after short-term
parabactin treatment may account for the 14 h delay in cell growth
relative to controls.
The phase specificity of the effects of the catechol amides is
similar to that seen with hydroxyurea (80-81) which is believed to
inhibit cell growth through interference with the enzyme ribonucleotide
reductase by a free radical seavaging mechanism (26). The hydroxamate
iron chelator desferrioxamine has also been shown to inhibit DNA syn
thesis through interference with the same enzyme. Interfering with
this enzyme's activity blocks DNA synthesis, and cells accumulate at
the G^-S boundary of the cell cycle (81). It has been demonstrated
that parabactin is a potent inhibitor of ribonucleotide reductase
(38).
It is highly unlikely that the growth inhibition and cell cycle
effects of the catecholamides are a consequence of extracellular iron
chelation. It has been shown that the removal of iron from transferrin
by siderophores occurs at a rate greater than 10% per hour only at high
ligand to transferrin ratios (82), and removal is impeded in the pres
ence of serum proteins (83). Transferrin is supplied to the cell
growth medium along with other growth factors as a 10% serum supple
ment. Unless the removal of iron from the extracellular medium by the
chelator occurred at a rate far in excess of 10% per hour it would not
account for the early onset of inhibitory effects of the siderophores
on DNA synthesis (3 h) (Fig. 2-5) or cell growth (12 h) (Fig. 2-3)
since L1210 cells grown in medium with 5% or 10% serum proliferate at
essentially the same rate. Also, there was not a proportional increase
in IC^Q values or significant change in the time to obtain a complete


68
cell cycle block when parabactin treated cells are grown in 5%, 10% or
20% fetal calf serum. If cell growth inhibition required an estab
lished concentration of chelator to limit extracellular iron, there
should be a proportional response of IC^q values in relation to changes
in serum concentration in the medium. Hence, if deprivation of iron to
the cells caused by chelator-induced deferration of transferrin in the
medium was the mechanism by which the catecholamides inhibit DNA syn
thesis, the times necessary to see a reduction in cell growth (5 yM
parabactin for 12 h) and DNA content (5 yM parabactin for 3 h) would be
expected to be much greater. Finally, there exists a good correlation
between the antiproliferative activity and 1 ipophi1icity of various
catecholamide chelators examined (37). This correlation would not be
expected if extracellular chelation were taking place.
The effects of the parabactin on the cell cycle kinetics of L1210
cells were compared with the effects of desferrioxamine and hydroxyurea
(Fig. 2-6). Cells exposed to 5 yM parabactin experienced a block at
the Gj-S border in 5 h, while desferrioxamine was required to be in
excess of 100 yM for a 5 h exposure. Finally, hydroxyurea needed to be
in excess of 300 yM concentration for the same 5 h exposure time. It
is interesting that the concentration of desferrioxamine required to
block cells at the Gj-S border is in excess of 13 times the 48 h IC^g
value of desferrioxamine and 7.5 times the 48 h IC^q value of hydroxy
urea. The cell cycle blocking ability of parabactin at a concentra
tion of 3.5 times its IC^q value was therefore far superior to that


69
of hydroxyurea or desferrioxamine. The release of the desferrioxamine
(84) and hydroxyurea (85) blocks results in less effective cell cycle
synchronizations, as the DNA histograms are normalized following one
passage through the cell cycle.
As mentioned above, the reversibility of the effects of parabac-
tin is dependent on how long the cells are exposed to the ligand. For
example, when the cells are exposed to 5 yM parabactin for 48 h 59% of
the cells die and 10 yM desferrioxamine kills 60% (Table 2-2).
The potent cell cycle blocking ability of parabactin and its
reversibility suggested its application in combination with other
phase-specific antineoplastic drugs.
Parabactin-treated cells offer two opportunities for potentiation
of growth inhibition.
a. Agents acting at the G^-S border should be more effective
since the cells are held at this phase.
b. Agents acting during S phase should have improved activity
since a larger percentage of the cell population will be synchronized
into the S phase when the parabactin block is released.
When utilized in combination with ara-C, Adriamycin, or 8CNU,
parabactin can produce "antagonistic," "additive," or "synergistic"
(potentiation) effects depending on the time frame during which the
drugs are applied.
Two basic experiments were run in the combination chemotherapy
studies.
a. Parabactin was used in simultaneous combination for 48 h with
BCNU, ara-C, or Adriamycin.


70
b. Cells were first blocked with parabactin and held at the G^-S
border, then the parabactin was washed away allowing cells to cascade
into S phase. The cells were then exposed to BCNU, ara-C, or Adriamy-
ci n.
The latter experiments relied heavily on cell cytometry tech
niques to evaluate the fraction of cells in each phase of the cell
cycle after treatment with the catechol amide chelator.
A third experiment was performed with RCNU. The cells were
treated with parabactin for 3 h and then with BCNU for 5 h. Then,
after both drugs were washed away, cells were resuspended and regrown.
When cells are exposed to parabactin and ara-C simultaneously for
48 h, the effect of the combination is an "antagonistic" one (Fig.
2-13). This is in keeping with the previous observations that ara-C,
an inhibitor of DMA polymerase (86), kills cells best in S phase (87)
and that parabactin holds cells at the G^-S border. This parabactin
block thus diminishes the S-phase component of the cell population
reducing the number of cells most susceptible to ara-C. Finally, the
"antagonism" is apparent from the analysis of drug-induced growth
inhibition. The IC^q value for the ara-C direct combination with para
bactin at a 100:1 ratio was 1.33 yM with a CI>1 indicating an
"antagonistic" effect.
However, when cells were first exposed to 5 yM parabactin for 5
h, washed free of the ligand releasing the block, and then treated with
ara-C, the parabactin potentiates the activity of ara-C particularly at
0.04 yM ara-C (Fig. 2-14). Inspection of Figure 2-14 reveals that the
growth of parabactin-treated cells is approximately 14 h behind the


71
growth of the control cells. Consequently, when comparing the effects
of the parabactin:ara-C-treated cells with ara-C treated cells, the 14
h lag for the parabactin-treated cells must be considered. Therefore,
in evaluating the combination curve relative to the ara-C curve, one
must look at a point on the combination line 14 h ahead of the ara-C
line. Even at this, it is clear that parabactin potentiates the activ
ity of ara-C.
The same experiments were carried out with Adriamycin. The sim
ultaneous drug combination experiment with 100:1 ratio of parabactin to
Adriamycin indicated "antagonism." Applying Equation A to the data
from Figure 2-10 resulted in a Cl value of 1.57 indicating antagonism.
However, again when the cells were treated first with parabactin to
initiate a block at G^-S and then washed free of the ligand and treated
with Adriamycin, the effect of Adriamycin was potentiated particularly
at 0.03 yM (Fig. 2-11). Again, one must consider the 14-h lag time in
growth generated by the parabactin. However, even after accounting for
this, reduction in growth rate is about twice that observed for the
Adriamycin itself. In view of the toxicity of Adriamycin, this may be
of some practical significance. As both Adriamycin (88) and ara-C are
proposed to operate best on cells in the S phase, the observed results
are as expected.
The final drug studied in this evaluation was BCNU. When cells
were exposed to parabactin and BCNU simultaneously for 48 h the results
indicated the combination to be additive with a Cl approximately equal
to 1 (Fig. 2-17).
When the cells were first treated with 5 yM parabactin for 5 h,
and then released and immediately treated with BCNU (Fig. 2-18), the


72
effects were still additive. However, when the cells were treated with
5 yM parabactin for 3 h, then exposed to BCNU for an additional 5 h,
and then washed and resuspended in culture, there is clearly a potenti
ation of the combined drug effects (Fig. 2-19). After accounting for
the parabactin-induced lag the effect of the BCNU was two times as
great as it was in the absence of parabactin. It was interesting to
note that 1 yM BCNU in combination with parabactin was more active than
3 yM BCNU alone. In fact, cells treated with 1 yM BCNU (not shown)
grew at the same rate as controls. The literature suggests that BCNU
operates best at the G^-S border and in G£ of the cell cycle, while
cells in S phase are more resistant (89). The BCNU potentiation
effects of the drug combination were consistent with the observation
that cells treated with parabactin were held at the Gj-S border. It
remains somewhat unclear, however, why we did not observe synergistic
effects when the drugs were used in direct simultaneous combination
without washing.
The above observations also suggest that proper pretreatment of
cells with parabactin could result in a decrease of the cytoxic effects
of phase-specific antineoplastics by reducing the population of cells
in the phase where the chemotherapeutic exerts its greatest cytotoxic
action. In an attempt to demonstrate the possible "protective" effects
of parabactin treatment against phase-specific toxicity, several exper-
ments were devised. Cells were first treated with parabactin to reduce
the S-phase population of cells. Adriamycin (Fig. 2-12) or ara-C
(Fig. 2-16) was then added for an additional amount of time, and the


73
cells were washed and regrown in drug free medium. Additionally, cells
were treated simultaneously with both parabactin and ara-C for 5 h and
the cells were washed and regrown (Fig. 2-15). The results revealed
additive effects of the combinations in all cases, thus failing to
indicate any protection afforded cells out of synchronization with the
phase specificity of ara-C or Adriamycin by parabactin treatment. The
implications of these results may demonstrate, that while ara-C and
Adriamycin are reported to exert their greatest cytotoxic effects on
cells in S phase, they may enter the cell during any phase of the cell
cycle. These results further imply that studies of simultaneous com
binations for these types of drugs deserve further consideration.
Analysis of the median effect plots by the method of Chou and Talalay
(73) is only a mathematical manipulation of the 48-h IC^q values of the
drugs at one specific molar ratio. The Cl may not be a true reflection
of the activity of the simultaneous combination of two compounds with
such different mechanisms of action and the results must be interpreted
with care.
The ability of parabactin to enhance the chemotherapeutic effects
of various antineoplastic drugs was highly dependent on its ability to
block cell cycle kinetics and to effectively reverse this block releas
ing the cells into a synchronized cell cycle. A consideration of the
ICgg values of the drugs is probably relevant when considering the
effectiveness of these compounds as cell cycle blocking agents. At 3.5
times its IC5Q parabactin was an excellent cell blocking agent; it was
better than desferrioxamine at greater than 13 times its IC^g and the
hydroxyurea at 7.5 times its IC^q (Fig. 2-6). Furthermore, one of
the most critical issues was the fact that parabactin-treated cells
maintained partial synchronization for at least three cell cycles while


74
cell synchronization disappeared after one cell cycle in desferriox-
amine- and hydroxyurea-treated cells.
The antiproliferative effects of parabactin were also studied in
vivo. The L1210 cell tumor model has been used as a useful preclinical
screening system for potential antineoplastics (90). The murine L1210
leukemia is a convenient and very reproducible in vivo model system.
The tumor is very aggressive having an in vivo doubling time of about
10-12 h. Mean survival time of animals injected with 10^ cells is
approximately 9.5 days. It has been determined that one viable cell
will kill an animal in about 18 days with a tumor burden of approxi
mately 1010 cells (91). Therefore, it would require a one log reduc
tion in cell number (90% cell kill) to see a one day increase in life
span (ILS), and a two-log reduction in cell number (99% cell kill) to
see a 2-3 day ILS, while even with a 99.99% cell kill the animals would
still die in less than 20 days.
It is, therefore, not very surprising that leukemic mice treated
with parabactin, which requires constant exposure of cells for signifi
cant periods of time to exhibit cytotoxic activity, on a single daily
injection schedule for 6 days, showed no increased life span over
untreated mice. However, leukemic mice treated with parabactin every
6 h for 5 injections did exhibit a reduction in tumor burden of 80%
over controls. Considering the doubling time of this tumor, even this
reduction in cell number would not extend the life of the mice to a
significant degree. A drug having an ILS of greater than 35% in this
system is considered to have significant activity by the National


75
Cancer Institute (92). Extended dosing on this schedule, while exhib
iting promising activity, is prevented by the low solubility of the
catecholamides in most vehicles suitable for parental administration
(the solubility of parabactin in phosphate buffered saline being on the
order of 1.25 x 10"^ M) (37), and the toxicity of the vehicle alone on
frequent administration.
The ability of parabactin to block and synchronize cells has also
been demonstrated in vivo. Ascites L1210 cells were synchronized by a
single i.p. injection of parabactin (Fig. 2-20) in a manner similar to
that seen in culture. Attempts to demonstrate enhanced cytotoxic
effects of ara-C and Adriamycin on a parabactin synchronized in vivo
cell population resulted in no significant increase in life span of
L1210 mice over the additive effects of the individual treatments
(Table 2-4). Considering the optimum conditions studied with cultured
cells, which exhibited a maximum 2-3 fold potentiation of the cytotoxic
effects of the antineoplastics tested, very little increase in life
span would be expected in vivo by a reduction in tumor burden of
50-75%. Further refinements in the drug dosage and multiple dosing
schedules are currently under investigation.


CHAPTER III
POLYAMINE ANALOGUES
Materials and Methods
The polyamine analogues were synthesized by methods previously
described (30,31). Stock solutions (10 mM) of analogues and dilu
tions were made in sterile water, and passed through a 0.2 ym filter
prior to use. RPMI 1640, fetal bovine serum, HEPES, and MOPS were
obtained from Gibco (Grand Island, NY). Cell culture flasks, 25 and
75 cm^, were purchased from Corning (Corning, NY). Diamidino-
phenylindole (DAPI), propidium iodide (PI), actinomycin-D and pro
teinase K were obtained from Sigma (St. Louis, MO). Rhodamine-123
(Rh-123) was purchased from Eastman Kodak Company (Rochester, NY).
RNase T1 was obtained from BRL Scientific (Gaithersburg, MD). DBA/2J
and C57B1/6J mice were obtained from Jackson Laboratories (Bar Har
bor, ME). Albino CD-I mice were obtained from Harlan Sprague-Dawley
(Indianapolis, IN). Sprague-Dawley rats were obtained from Charles
River (Wilmington, MA).
Cell Culture
All cell culture lines were grown as previously described in
Chapter II. Several additional cell lines utilized in these studies
were maintained in complete medium as before except where noted.
O
Chinese hamster lung (DC3F) cells were grown as monolayers in 25 crrr
flasks, seeded at 2 x 10^ total cells and grown to confluency in
in approximately 96 h. Actinomycin-D resistant Chinese hamster lung
76


77
(DC3F/ADX) cells were similarly maintained with actinomycin-D 10
yg/mL present in the medium. The Chinese Hamster Lung cell lines,
DC3F and DC3F/ADX, were kindly donated by Dr. June Biedler of
Memorial SIoan-Kettering Cancer Center, NY.
Resistant L1210 cell Lines
L1210 cell lines resistant to DESPM (LI210/DES-10) and DEHSPM
(L1210/HDES-1) were selected out by maintaining the cells in increas
ing concentrations of polyamine analogues over an extended period of
time. Initially, L1210 cells were exposed to either 1 yM DESPM or
0.1 yM DEHSPM for seven days and maintained between 1 x 10 and
1 x 10 cells/mL. Cells were then washed free of the drug and incu
bated in fresh medium until cell doubling time returned to normal (12
h). These cloned cells were then again exposed to a higher concen
tration of the analogue until normal cell doubling time resumed.
Analogue concentrations were gradually increased over a period of
approximately one month until cells could be maintained at a concen
tration of 10 yM DESPM and 1 yM DEHSPM. Resistant cells maintained
at these concentrations of analogue for over six months had normal
L1210 morphology and doubling time.
An Adriamycin resistant L1210 cell line (LI210/D0X-0.6) was
similarly selected out by initially exposing cells to 0.01 yM Adria
mycin. When approximate normal doubling time resumed, the Adriamycin
concentration was increased until over a period of three months the
cells could be maintained in the presence of 0.6 yM Adriamycin. The
L1210/D0X-0.6 cells maintained for over six months differed from the
normal L1210 cells in that they had irregular shapes and longer doub
ling times of 14-16 h.


78
IC^q Determinations
The cells were treated while iri logarithmic growth (LI210
cells, 3 x 10^ cells/mL; Daudi and HL-60, 1 x 10^ cells/mL) with the
polyamine derivatives diluted in sterile water and filtered through a
0.2 yM filter immediately prior to use. Following a 48 h incubation
with L1210 cells and a 72 h incubation with Daudi or HL-60 cells,
cells were reseeded (L1210 cells, 3 x 10^ cells/mL; Daudi and HL-60
cells, 1 x 10^ cells/mL) and incubated in the presence of the poly
amine derivative for an additional 48 h or 72 h, respectively.
Determination of IC$q values for the most active compounds
(IC50 ^ vm at ^6 11) was a^so Perfrmed in the presence of 1 mM
aminoguanidine, a serum diamine oxidase inhibitor (93).
Chinese hamster ovary (CH0) cells and murine B-16 melanoma
cells were seeded at 2 x 10^ cells/25 cm^ flask and allowed to attach
for 4 h. At this time the cells were exposed to the polyamine ana
logue for 48 h. Monolayer cells were not reseeded for additional
exposure times due to problems in plating efficiency after the ini
tial 48 h treatment.
Cell samples were removed at the indicated time periods for
counting. Cell number was determined by electronic particle analysis
(Coulter Counter, Model ZB1, Coulter Electronics, Hialeah, FL) and
confirmed periodically with hemocytometer measurements.


79
The percentage of control growth was determined as follows:
% of control growth = 100 x [Final treated cell no.-
initial inoculum]/[Final untreated cell no.-initial inoculum]
The IC^Q is defined as the concentration of compound necessary
to reduce cell growth to 50% of control growth after defined inter
vals of exposure.
Analysis of Drug Effects on Mitochondrial DNA
The LI210 cells (in complete media at 1 x 10^ cells/mL) were
incubated at 37C in the presence of the compound to be tested.
Every 24 h, for 144 h, cell samples were removed for counting, and
the cells were reseeded in fresh medium and drug at 1 x 10^ cells/mL
Cells were assayed daily for mitochondrial DNA (mtDNA) content.
Mitochondrial DNA assays were performed by R. Bortell and L. Raynor
of Dr. A. Neims' laboratory, Department of Pharmacology and Thera
peutics, College of Medicine, University of Florida.
Because recovery of the organelles might vary with drug treat
ment, a dot blot procedure for assay of mtDNA was developed which
involves analysis of cell lysates rather than preparations of mito
chondria (94). Cells (5 x 10^ to 2 x 10^/mL) were lysed in 2% SDS i
the presence of proteinase K (5 mg) and RNase T1 (100 units). The
total cell lysate was applied to nitrocellulose paper with use of a
96-well, vacuum operated dot-blot apparatus. The blots were hybrid-
ized to a S-labeled dATP nick translated probe made by inserting
full-length mouse mtDNA into pSP64 vector at the Sacl site. Dot
blots were visualized by autoradiography and cut out, and radioactiv
ity was determined by scintillation counting.


80
Flow Cytometric Analysis
Flow cytometric analysis of nuclear DNA (nDNA) content was
performed with the RATCOM flow cytometer (RATCOM Inc., Miami, FL)
interfaced with a microcomputer (IBM-XT). Cultured LI210 cells in
log-phase growth were incubated at 37C with various polyamine ana
logues and samples removed at 0 h, 48 h, 96 h and 144 h were analyzed
for nDNA content distributions after staining with diamidinophenyl in
dole in a nuclear isolation media (NIM-DAPI) (75).
Alternately, cell analysis was performed with a FACS II flow
cytometer (Becton Dickinson FACS Systems, Sunnyvale, CA) interfaced
with a microcomputer (Hewlett Packard 45B, Fort Collins, CO).
Samples of 10^ cells were removed and stained with propidium iodide
(PI) (Calbiochem, San Diego, CA) and then exposed to RNase. DNA
distributions were obtained from analysis of the red fluorescence
from the Pi-stained DNA (76).
Cloning Assay
L1210 cells maintained between 105 and 10^ cells/mL were incu
bated with 10 pM DESPM for 96 h. At 24 h intervals treated cells
were washed (2 x 10 mL) and diluted in fresh complete media and
plated in triplicate 96-well microtiter plates at 0.4 cells/well with
each well containing 100 pL of sample. The plates were incubated at
37C in a humidified incubator in an atmosphere of 5% C0£ and 95%
air. The plates were examined with an inverse phase microscope at
100X magnification. The final number of colonies per plate was quan
titated seven days after plating. Groups of 50 or more cells/well
were identified as having been cloned from a single viable cell.


81
Regrowth Studies
L1210 cells maintained between 10^ and 10^ cells/mL were incu
bated with 10 yM DESPM, 30 yM DENSPM or DEHSPM 0.6 yM for 96 h. At
each 24 h interval, treated cell samples were washed and the cells
resuspended in fresh media at 1 x 10^ cells/mL in duplicate 10 mL
flasks. The regrowth of the treated cells was followed for up to
144 h.
Cell Size
Cell size was determined directly by the method of Schwartz, et
al. (95). In brief, uniform polymeric microspheres ranging from
4.72-10.2 yM in diameter (Polysciences, Warrington, PA) were diluted
in Hematall (Fisher Scientific Co.). Electronic size was measured on
the FACS Analyzer (Becton Dickinson, Sunnyvale, CA) with the ampli
fier in the log mode. The peak channel number for each size micro
bead was plotted against the correspond'ng calibrated diameter and
calculated volume to obtain a calibration curve. L1210 cells were
treated with 10 yM DESPM, 30 yM DENSPM or DEHSPM 0.6 yM for 0-144 h
and samples of 10^ cells were removed at 24 h intervals and pelleted.
The cells were resuspended in 0.5 mL of Hematall and analyzed. The
peak channel number of the treated cells was plotted on the
calibration curve to obtain the approximate cell size directly.
Viability Assay
The viability of cells which had been treated with 10 yM DESPM
or 0.3 yM DEHSPM and or 30 yM DENSPM maintained between 5 x 10^ and


82
1.2 x 106 cells/mL was assayed at appropriate times using two dif
ferent methods: trypan blue dye exclusion and Rh-123 mitochondrial
staining (74).
Briefly, 1 x 106 cells were pelleted by centrifugation and
resuspended in 1 mL of complete medium containing Rh-123 10 yg/mL,
incubated at 37C for 10 min, and washed once with medium. Trypan
blue was added prior to counting to give a final concentration of 0.2
yg/mL. Rh-123 and trypan blue stained cultures were observed in a
hemocytometer using a Zeiss epifluorescence Axioscop microscope at
630X magnification. The excitation wavelength was 485 nm. Viable
cells show mitochondrial specific uptake of the Rh-123 dye while
dying cells showed diffuse cytoplasmic staining and dead cells did
not fluoresce at all. Cells which did not fluoresce did not exclude
trypan blue. Both methods gave comparable results as to cell death,
however, trypan blue exclusion does not reflect dying cells.
Animal Studies
The polyamine analogue was diluted in sterile 0.9% saline
within 24 h of use and the unused portion stored at 5C. Concentra
tions of the spermine analogue tetrahydrochlorides, at each dose were
adjusted so that the mice were injected with a volume of 1 mL/100 g
(i.e. a 25 g mouse was injected with 0.25 mL of drug solution).
Untreated mice served as controls.
The compounds were administered by i.p. injection according to
the appropriate dosing schedule. The murine L1210 leukemia cells
were maintained in DBA/2J mice. Cells from a single mouse which
was injected i.p. with 1.25 x 10^ cells seven days earlier were


83
harvested and diluted with cold saline so that an inoculum of 10^ or
10^ cells could be administered by a 0.25 mL i.p. injection. In
each study mice were injected i.p. with the appropriate number of
L1210 cells on day 0.
Alternately, Alzet mini-osmotic pumps, model 2001 (Alza, Palo
Alto, CA) designed to deliver 1 yL/h for 7 days, were filled with
DEHSPM so that the pump would deliver 20 mg/kg/day. The filled pumps
were placed in sterile saline 0.9% and incubated at 37C for 8 h
prior to placement in the mice. The pumps were implanted, on day 1,
subcutaneously (s.q.) in the dorsal side of the mice through an
incision behind the right flank, and the incision closed with a wound
clip. After 6 days the pumps were removed and the wound closed. The
parameter used for treatment evaluation of activity against L1210
leukemia was mean survival time (percent increased life span, % ILS).
%ILS = 100 x [mean survival time treated animals-
mean survival time controls]/[mean survival time controls]
The murine B-16 melanoma was maintained in C57B1/6J mice. The
solid tumor was excised from a single mouse, which was injected sub
cutaneously (s.q.) 14 days earlier, and minced with 9 volumes of
Hanks' balanced salt solution (10:1 brei). The brei (0.1 mL) was
administered by s.q. injection in the right flank of the animal on
day 0.
The murine Lewis lung carcinoma was maintained in C57B1/6J
mice. The solid tumor was excised from a single mouse which was
injected s.q. 14 days earlier, and the tumor divided into equal


34
fragments 2-4 mm in size. Each mouse was injected s.q. with a frag
ment in the sternum region using a 14 g trochar needle on day 0.
The parameter measured for treatment evaluation was median
tumor weight change based on length and width measurements in milli
meters (92). The tumor weights (mg) were calculated from tumor
dimensions (mm x mm) following the formula for volume of a prolate
el 1ipsoid:
L x where L is the longer of the two
2 measurements recorded
Median tumor weights were calculated for test (T) and control (C)
groups on days 10, 14 and 17 after inoculation of the tumor.
The activity of the analogue was reported as T/C% on the
measurement day giving the lowest value.
Rates of Uptake of Polyamine Analogues in L1210 Cells
L1210 cells maintained in mid-log phase growth were exposed to
the polyamine analogues for 0, 2, 6, 12 and 24 h, and samples of 10^
cells were removed. The cells were washed with fresh medium (2 x 10
mL) and centrifuged to pellet the cells. The supernatant was care
fully removed and the cells resuspended in 0.6 N perchloric acid
(107 cells/mL). Cells were freeze-fractured by four successive
freeze-thawings using liquid nitrogen. Cell lysates were stored at
-20C until HPLC analysis. For HPLC analysis an aliquot of the solu
bilized polyamine perchlorates was reacted with dansyl chloride, sod
ium carbonate and diaminohexane as an internal standard. After con
centration of the reaction products on a C18 chromatographic plug,
the dansylated polyamines were analyzed on reversed phase analytical


85
C18 column and quantitated using fluorescence detection and elec
tronic integration. Analysis of HPLC samples were performed by
J.R.T. Vinson, B. Jennings, and V. Andaloro of Dr. R.J. Bergeron's
laboratory, Department of Medicinal Chemistry, College of Pharmacy,
University of Florida.
Pharmacokinetic Studies
Preliminary pharmacokinetic studies to determine half-life of
the analogues in the serum were studied in mice. Blood samples were
collected by exsanguination of CD-I mice into nonheparinized tubes at
appropriate times after a single i.p. injection of DESPM (25 mg/kg).
The serum was separated from the cells by centrifugation and diluted
with an equal volume of 1.2 N perchloric acid. Analogue levels in
the serum were determined by HPLC analysis by the above method.
The excretion of DEHSPM (25 mg/kg) after a single i.p. injec
tion was followed in the urine and bile of Sprague-Dawley rats util
izing bile duct cannulation model. Rat bile duct cannulations were
performed by K. Crist and E. LaGraves of Dr. R.J. Bergeron's labora
tory, Department of Medicinal Chemistry, College of Pharmacy, Uni
versity of Florida. The bile duct was cannulated with a segment of
22 ga polyethylene tubing and the bile directed from the rat to frac
tion collector by a fluid swivel mounted above a metabolic cage.
Normal urine flow was also diverted with a segment of 5 mn tubing
from the bottom of the metabolic cage to the fraction collector.
This system allows the animal to move freely in the cage while
continuous bile and urine samples were being collected in 3 h frac
tions for 24 h. Sample volumes were measured and diluted with an


86
equal volume of 1.2 N perchloric acid. Analogue levels in each
fraction were determined by HPLC analysis by the above method.
Toxicity Studies
Both single dose and five-day daily-dose toxicity in CD-I white
mice were determined for DESPM and DEHSPM. Initially, small groups
of 2-3 mice were used to determine the approximate range of toxicity
for the compound. More accurate determinations of toxicity were made
using groups of greater than 6 mice at each dose of analogue. Dos
ages ranged so that at the lowest dosage 10% or less of the animals
die and at the highest dosage >90% of the animals die. At least 3
toxicity dosages between these two extremes were determined.
The polyamine analogue was diluted in sterile 0.9% saline with
in 24 h of use and the unused portion stored at 5C. Concentrations
of the spermine analogue tetrahydrochlorides at each dose were
adjusted so that the mice were injected i.p. with a volume of 1
mL/100 g.
For the single dose toxicity studies each group of mice
received a single i.p. injection of the analogue at an appropriate
dosage. For the 5-day daily-dose toxicity studies each group of mice
received a single i.p. injection of the analogue at an appropriate
dosage daily for 5 days. Animal deaths were checked daily for 30
days. The number of deaths was expressed as percent lethality
[100 x (number of deaths per group in 30 days/number of mice per
group)].


87
Results
In Vitro Activity
The polyamines which were evaluated for their antiproliferative
activity include the spermidine analogues (Fig. 3-1);
a. N,n'-bis(ethyl)-l ,8-diami nooctane (BEDAO),
b. ,N^-bis(propyl)spermidine (BPSPD),
c. N1 ,N-bis(ethyl)-N^-(p-nitrophenyl)spermidine (BEPNPSPD),
d. ,N^-bis(ethyl)norspermidine (BENSPD),
e. ,N^-bis(ethyl)spermidine (BESPD),
and the spermine analogues (Fig. 3-2);
f. N,N-diethyl-l,12-diaminododecane (DEDAD),
g. ,N^-dibenzylspermine (DBSPM),
h. N* ,N^-di (2,2,2-tri fl uoroethyl )spermine (FDESPM),
i. l,4-bis-[3-(ethylamino)propyl]piperazine (DEPIP),
j. N^,N^-diethyl spermine (IDESPM),
k. N^,N^,N^,N^-tetraethylspermine (TESPM),
and (Fig. 3-3)
l. N* ,N^-diethylnorspermine (DENSPM),
m. ,N^ ,N^ ,N^-tetraethyl spermi ne (NTESPM),
n. N* ,N^-dimethyl spermine (DMSPM),
o. l,20-bis(N-ethylamino)-4,8,13,17-tetraazaeicosane (YANK),
p. N*,N^-di propyl spermine (DPSPM),
q. N*,N^-diethylspermine (DESPM),
r. N* ,N^-diethylhomospermine (DEHSPM).
The compounds described in this study are all in some way
related to the natural products spermidine and spermine. They were


88
BEDAO
48 h 96 h
>100 yM >100 yM
BPSPD
> 30 yM > 30 yM
> 10 yM > 10 yM
BESPD
>100 yM 10 yM
50 yM 10 yM
Figure 3-1. Structure and IC^q values of the spermidine
analogues.


89
48 h 96 h
DEDAD
N
H
Cytotoxic > 25 yM
No inhibition < 12.5 yM
Cytotoxic > 10 yM
No inhibition < 3 yM
FDESPM
N CF3
H
>100 yM
>100 yM
DEPIP
> 50 yM > 50 yM
> 25 yM > 25 yM
> 50 yM
5 yM
Figure 3-2. Structure and ICcn values of the less active
spermine analogues.


90
48 h
96 h
H
N.
H
.N.
H
.N,
DENSPM
H
.N.
.NU
y
{
N
NTESPM
H
,N,
'N'
H
>100 yM 4 yM
100 yM 3 yM
H
.N.
H
-N.
N
DMSPM H
>100 yM 0.75 yM
H
,N,
H
,N,
H
.N>
N
YANK H
N'
H
'N'
H
50 yM 0.5 yM
DPSPM
3 yM 0.2 yM
DESPM
N
H
N
H
10 yM 0.1 yM
0.5 yM 0.05 yM
Figure 3-3. Structure and IC^g values of the most active
spermine analogues.


91
designed to evaluate relationships which might exist between the
structure of various alkylated polaymines and their activity against
tumor cells in culture. The relative antiproliferative activities of
the analogues were assessed by determining their 48-h and 96-h IC^g
values. In comparing the spermine compounds with their spermidine
counterparts (Figs. 3-1 to 3-3), it is clear that the spermine com
pounds are substantially more active against L1210 cells.
Of the spermine analogues tested, DESPM and DEHSPM were the
most effective inhibitors of growth of L1210 cells. DESPM had 48 h
and 96 h IC^g values of 10 yM and 0.1 yM, respectively. DEHSPM
demonstrated superior activity and had 48 h and 96 h IC^q values of
0.5 yM and 0.05 yM, respectively. The IC^g values for the most
active analogues (ICgg value <5 yM at 96 h) determined in the
presence of 100 yM arninoguanidine, an inhibitor of diamine oxidase,
remained the same.
The activity of DESPM against Daudi cells and HL-60 cells is
comparable to that against L1210 cells. Because of the differences
in doubling times between L1210 cells (ca. 10-12 h) and Daudi or
HL-60 (ca. 25 h), the IC^qS of DESPM at 48 h and 96 h against L1210
cells are best compared with the IC^qS at 72 h and 144 h against
HL-60 (10 yM and 0.3 yM) and Daudi cells (>40 yM and 0.5 yM). Addi
tionally, DEHSPM was shown to be active against CH0 and B-16 melanoma
cells (doubling time for both lines 14-16 h) with IC^q values of
about 0.1 yM at 48 h for both cell lines.
LI210 cells which were selected for their ability to grow
in the presence of Adriamycin (L1210/D0X-0.6 cells) demonstrated
an approximate 50-fold increase in resistance to Adriamycin over the
parental L1210 cell line. The LI210/D0X-0.6 cells were demonstrated
by immunoblotting to have a large increase in the membrane


92
glycoproteins (P150-170 glycoproteins) and considered to be multidrug
resistant cells (96). Both the parental cell line, as well as the
resistant cell line, were colaterally sensitive to DEHSPM with IC^g
values of 0.5 yM and 0.05 yM at 48 h and 96 h respectively.
The antiproliferative activity of DEHSPM was tested against
DC3F cells and a proven multidrug-resistant subline, DC3F/ADX cells
(97-99). Both cell lines were determined to be equally sensitive to
DEHSPM having identical 96-h IC^g values of 0.1 yM.
L1210 cells which were developed for their resistance to the
polyamine analogues L1210/DES-10 and L1210/HDES-1 did not demonstrate
an overproduction of the P150-170 glycoproteins (96), indicating that
they had not developed multidrug resistance; both were sensitive to
Adriamycin to the same extent (IC^g value of 0.02 yM at 48 h). Both
cell lines did, however, exhibit cross-resistance to other polyanine
analogues. Both of the resistant cell lines were not inhibited by
treatment with 100 yM DENSPM, and were able to grow to 70% of control
in the presence of either 100 yM DESPM or 100 yM DEHSPM.
The role of the methylene backbone in the biological activity
of the polyamine analogues was studied by comparing DENSPM, DESPM and
DEHSPM. Reduction in the length of the polyamine backbone, decreased
the activity of the analogue. The activity of DENSPM against L1210
cells (IC^g value of >100 yM at 48 h, and 4 yM at 96 h) is relatively
poor when compared to that of DESPM and DEHSPM (Figs. 3-4 and 3-5).


CONCENTRATION (uM)
93
% CONTROL GROWTH
Figure 3-4. Concentration dependence of L1210 cell growth
inhibition by DENSPM, DESPM and DEHSPM at 48 h.


Full Text
THE USE OF MICROBIAL IRON CHELATORS AND POLYAMINE
ANALOGUES AS ANTINEOPLASTICS
By
MICHAEL JOSEPH INGENO
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1988

This work is dedicated to my family, with love.

ACKNOWLEDGMENTS
I would like to express my sincere appreciation to my research
advisor, Dr. Raymond J. Bergeron, for his generous guidance, expert
advice and friendship over the past four years. His enthusiasm for
science has been an inspiration.
I would also like to thank Dr. Richard R. Streiff for his assist¬
ance in the completion of the animal studies and Dr. Raul Braylan for
his advice on flow cytometry and cell cycle analysis.
I am especially grateful for the assistance and dedication of
Jack Kramer Austin, Jr., Gabriel Luchetta, Annette Zaytoun, and Dexter
E. Beck whose time and effort have helped make this work possible, and
for the technical assistance of Joe Kayal, Stacia Goldey, B. Wesson
Young, and Rosa Rosado.
I wish to acknowledge the other coworkers, past and present, in
Dr. Bergeron's laboratory who have been helpful in the completion of my
many projects and helped make my learning experience an enjoyable one.
I also wish to acknowledge Irma Smith for her expert advice and
assistance in the preparation of this manuscript.
Finally, I wish to thank my wonderful family: I am eternally
gratefuly to Mr. and Mrs. James Ingeno for a lifetime of love and
encouragement, they were always there when I needed them; I am blessed
with my daughter, Leah, who could make me smile when all else failed;
and I am constantly indebted to my beautiful wife, Deborah, for her
love, tenderness, patience and support throughout the completion of
iii
this work.

TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS iii
ABSTRACT v
CHAPTERS
I. INTRODUCTION 1
Catechol amide Iron Chelators 1
Polyamine Analogues 11
II. CATECHOLAMIDE CHELATORS 21
Materials and Methods 21
Results 30
Discussion 63
III. POLYAMINE ANALOGUES 76
Materials and Methods 76
Results 87
Discussion 127
IV. CONCLUSIONS 145
REFERENCES 151
BIOGRAPHICAL SKETCH 158
iv

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
THE USE OF MICROBIAL IRON CHELATORS AND POLYAMINE
ANALOGUES AS ANTINEOPLASTICS
By
Michael Joseph Ingeno
December 1988
Chairman: Raymond J. Bergeron
Major Department: Medicinal Chemistry
This research focuses on the development of compounds which are
effective in controlling the growth of cancer cells. The two classes
of agents studied are the microbial iron catechol amide chelators
(siderophores) and the N-alkyl polyamine analogues.
The biological activity of the catecholamide chelators, parabac-
tin and vibriobactin, has been assigned, at least in part, to their
ability to chelate iron and inhibit the iron dependent enzyme ribonuc¬
leotide reductase. These siderophores are potent inhibitors of neo¬
plastic cell growth, with 50% inhibitory concentrations in the micro¬
molar range. The ligands have pronounced effects on cell cycle kinet¬
ics producing a G^-S cell cycle block. On short term exposure to the
chelators, this block in DNA synthesis is reversed simply by washing
the chelator away. The cells remained synchronized for at lease three
cycles after release of the block. The cell cycle block and synchroni¬
zation induced by the chelators may be exploited to potentially enhance
cytotoxicity when used in combination with other antineoplastics.
v

The biological activity of the N-alkylated polyamine derivatives
has been postulated to be a consequence of their similarity to the
natural intracellular polyamines, spermidine and spermine in terms of
uptake, while functionally they are unable to fulfill or interfere with
important roles of the natural polyamines. Inhibition of cell growth
may result as a consequence of alterations in the polyamine biosyn¬
thetic network. Preliminary studies of the structural boundary condi¬
tions required for significant antiproliferative activity of the poly¬
amine analogues have been carried out. The most active analogues, the
terminally diethylated tetrarnines ,N^-diethyl spermi ne and N*,N^-
diethylhomospermine, have been shown to be potent inhibitors of neo¬
plastic cell growth, with 50% inhibitory concentrations in the micro¬
molar range in vitro and significant in vivo activity. They have been
found to have only incidental effects on nuclear DNA, while having pro¬
found effects on mitochondrial DNA.
Results are presented as to the action of these drugs both in
vitro and in vivo. From these studies a great deal has been determined
about the growth inhibitory effects, mechanisms of actions, broadness
of activity against various cell lines, structure activity relation¬
ships, and effectiveness of these compounds either alone or in combina¬
tion with other agents.
vi

CHAPTER I
INTRODUCTION
This research focuses on the development of compounds which are
effective in controlling the growth of cancer cells.
Catechol amide Iron Chelators
The first part of the dissertation is concerned with the evalua¬
tion of catechol amide iron chelators as a means of controlling the pro¬
liferative processes. Over the past decade there has been a dramatic
increase in the attention given to the crucial role which iron plays in
proliferative processes. Although iron has oxidation states between -2
and +6, the equilibrium between the +2 and +3 oxidation states, an
equilibrium which is very sensitive to both ligation and pH (1), best
characterizes the biological use of this metal. This transition metal
plays a critical role in a variety of biological redox systems within
the cell, e.g., the cytochromes, peroxidases, catalases, and ribonucle¬
otide reductase (2). Even though virtually all organisms are auxo-
tropic for Fe(III), because of the insolubility of ferric hydroxide
(FeiOH)^), the predominant form of the metal under physiological con-
38 4
ditions (Ksp=10“ tv), nature has had to develop rather sophisticated
mechanisms for accessing the metal (3).
Microorganisms produce low-molecular weight ligands (sidero-
phores) which chelate and facilitate transport of iron into the cell
(2). Most of these siderophores fall into two general structural
classes, hydroxamates and catecholamides (4). The catecholamides,
1

2
typified by enterobactin (Fig. 1-la), generally form tighter iron
48 1
complexes (metal-1igand formation constant (Kf=10 M ) (5) than the
31 1
hydroxamates, exemplified by desferrioxamine (Fig. 1-lb) (K^=10 M" )
(6).
Mammalian cells have developed larger, more complicated mole¬
cules, iron binding proteins for the metal's transport and utilization:
e.g., the iron shuttle proteins, transferrin (7), lactoferrin (8),
uteroferrin (9), and the iron storage protein, ferritin (10). It has
been suggested that the virulence of pathogenic bacteria may be par¬
tially related to the efficiency with which its siderophore removes
iron from these iron binding proteins (11).
Transferrin, the major iron transport protein in animals, binds
two iron atoms in the +3 oxidation state, each with a slightly differ¬
ent metal-protein formation constant. This binding is very sensitive
to the oxidation state of the metal and to pH with an apparent iron
28 1
binding constant at physiological pH of Kf=10 M“ (12). Once the
transferrin is bound to the transferrin receptor on the cell's surface,
it is internalized via endocytotic vesicles. The iron-protein-receptor
complex enters a nonlysosomal acidic vacuole where conditions favor
release of the iron for utilization or storage as an iron-ferritin com¬
plex. The apotransferrin-receptor complex then returns to the cell
surface where the apotransferrin is released for further use (13).
The number of transferrin receptors on a cell membrane has been
directly linked to the proliferative state of the cell and/or the

3
b.
Figure 1-1. The microbial iron chelators
a. Enterobactin
b. Desferrioxamine

4
availability of iron (14). For example, it has been observed that
highly proliferative cells have significantly greater numbers of trans
ferrin receptors than resting cells (15). Alternatively, if iron is
withheld from cells there is a substantial increase in the number of
transferrin receptors (16). This increase is due to stimulation of
synthesis and translation of the transferrin receptor mRNA, and can be
inhibited by the addition of iron (17). Conversely, if iron is added
to the cells in a utilizable form there is a dramatic decrease in
transferrin receptor expression, and an increase in cellular ferritin
iron as a means of defense against bacterial infection and neoplasms.
It is interesting to observe how the body alters iron metabolism
in response to a bacterial infection. In the course of an infection,
the body attempts to withhold iron from the invading organism by
decreasing gastrointestinal absorption of iron, lowering the serum
transferrin iron saturation, and increasing liver ferritin levels and
iron ferritin storage in reticuloendothelial systems (18). The same
response is seen in febrile states, neoplasia, and on injection of var
ious sham substances (19). It has been demonstrated that mice fed a
low iron diet developed smaller and slower growing tumors than mice
maintained on a normal iron diet (20). Conversely, iatrogenic iron
overload as well as idiopathic hemochromotosis, a genetic disease char
acterized by iron overload, have been associated with increased inci¬
dence of neoplasia (21,22). These findings suggest continued study of
the role of iron metabolism in malignancy and the possible adverse
effects of iron supplementation in patients with cancer.
There is evidence to indicate that activated macrophage cytotox¬
icity may be mediated by iron removal from the target cells (23). A

5
selective inhibition of iron dependent DNA replication and mitochondri¬
al respiration is observed. The authors speculate that a macrophage-
derived iron chelator could be involved, leading to cytostasis. Later
studies (24) found that aconitase, a citric acid cycle enzyme contain¬
ing iron-sulfur clusters, activity declined simultaneously with arrest
of DNA synthesis. The cytotoxic activated macrophage-induced inhibi¬
tion of aconitase is shown to be due to loss of iron from the iron-
sulfer cluster. More recently (25), a newly discovered cyclic hydrox-
amate siderophore, bisucaberin, was found to render tumor cells sus¬
ceptible to cytolysis mediated by macrophages and showed specific
inhibition by addition of ferric iron.
One of iron's more interesting roles in cells is its association
with ribonucleotide reductase (Fig. 1-2), an enzyme which catalyses the
production of deoxyribonucleotides in the rate limiting step of DNA
synthesis (26). In mammalian cells, ribonucleotide reductase consists
of two nonidentical dimer subunits, Ml and M2, analogous to the B1 and
B2 subunits of Escherichia col i (_E. col i) (27). The M2 dimer contains
a nonheme iron center. The metal stabilizes a tyrosyl free radical at
the active site and, due to the rapid turnover of the enzyme, a con¬
stant supply of iron is essential for the enzyme's activity (28).
Ribonucleotide reductase levels are elevated in neoplastic cells and
have been shown to be transformation-linked and progression-1inked in a
series of hepatomas (29).
If the theory that withholding iron as a defense mechanism
against neoplasia is correct, then artificially inducing a state of
iron deprivation by means of the siderophores may prove to be effective
in controlling neoplastic cell growth. Iron chelators could control

6
Substrate
Fiqure 1-2. Model of E. coli ribonucleotide reductase (from
Ref. 26).

7
cellular proliferation by complexing extracellular iron (including that
bound to transferrin), "free" cytoplasmic iron, and/or iron bound to
critical proteins or enzymes.
There are several rationales for the use of the microbial cate¬
chol amide iron chelators as a means of controlling neoplastic growth.
1. The formation constants for the catecholamide iron complexes
(Kf=10^-10^ M-*) are far greater than those observed for chelators
previously used as antiproliferatives (4). Considering how effectively
the catecholamide siderophores bind iron, one would expect these che¬
lators to remove iron from several enzyme bound sites, e.g., ribonucle¬
otide reductase, mitochondrial enzymes, as well as from transferrin.
However, in spite of the thermodynamic binding advantage these ligands
have over various proteins, if the ligand cannot access the protein
bound iron, it obviously cannot remove it. Thus, removal of iron is a
kinetic problem.
2. Previously low biological yields from microorganisms and ted¬
ious isolation procedures of these compounds limited their use. Chemi¬
cal schemes have now been devised for the efficient synthesis of the
catecholamide microbial siderophores as well as a number of synthetic
analogues (30-32). The natural products are 2,3-dihydroxybenzoylamide
derivatives of polyamines, e.g., ,N^-bis(2,3-dihydroxybenzoyl)sperm-
idine (compound II) and parabactin, first isolated from cultures of
Paracoccus denitrificans by Tait (33) and vibriobactin, isolated from
Vibrio cholerae by Neilands et al. (34) (Fig. 1-3).
3. It has been postulated that the transferrin receptor is a
potential marker for the identification of proliferating cells (35).

8
a.
H
b. R = H
c. R = CH3
d. R = H
e. R = CH3
Figure 1-3. The catecholamide iron chelators.
a. Compound II
b. Parabactin
c. Tetramethyl parabactin
d. Vibriobactin
e. Permethylated vibriobactin

9
In a study using monoclonal antibodies to the transferrin receptor, it
was found that in all cases of breast carcinomas transferrin receptors
could be detected while in samples of normal breast tissue little or no
evidence of staining was exhibited (36). The evidence that neoplastic
cells, as indicated by their uninhibited proliferation and increased
means of iron acquisition, have higher iron requirements in comparison
to host cells provides a rationale for the potential selectivity of
iron removal by the microbial iron chelators as a target in antineo¬
plastic strategy.
4. Evidence suggested that the catechol amide chelators could
penetrate cellular membranes. A correlation between antiproliferative
activity and octanol/saline partition coefficients for a series of
siderophores has been established (37). It was found that the ratios
of the ligand partition coefficients between octanol and phosphate
buffered saline for the catechol ami des versus dihydroxybenzoic are
closer to the ratios of the ligand's antileukemic activity than are the
ratios of their binding constants. This suggested that cellular pene¬
tration as well as iron-chelation potential may determine the chelat¬
or's antiproliferative activity.
5. Parabactin and compound II have pronounced inhibitory effects
on the growth of L1210 cells in vitro with IC^q values in the micro¬
molar range (38). These values are in the range of antineoplastics
currently being used and are at concentrations attainable as serum
levels j_n vivo.
6. There is good evidence that the catecholamide siderophores,
unlike previously studied chelators, are relatively nontoxic to the

10
host. It has been observed that compound II, the biological precursor
to parabactin, has an LD^q in mice greater than 800 mg/kg when given as
a single intraperitoneal (i.p.) injection (39). It has been further
observed that compound II and parabactin at concentrations up to 2 mM
(100 times the 50% inhibitory concentration for murine L1210 leukemia
in vitro) appeared to be nontoxic to a confluent monolayer of monkey
kidney cel Is (37).
7. The iron dependent enzyme ribonucleotide reductase is a suit¬
able target for antineoplastic activity of the catecholamide chelators.
Hydroxyurea, an inhibitor of ribonucleotide reductase (40), has found
clinical usefulness as an anticancer agent. A number of other less
avid iron chelators such as desferrioxamine (41), a-picolinic acid (42)
as well as certain toxic thiosemicarbazones (43) have been shown to
inhibit tumor growth by interfering with ribonucleotide reductase
activity.
Parabactin and compound II have been shown to be potent inhibi¬
tors of ribonucleotide reductase activity and to block DMA biosynthesis
at the G-pS boundary of the cell cycle. However, RNA and protein syn¬
thesis were inhibited to a much lesser extent, and DNA polymerase activ¬
ity was essentially unaffected (38). Alterations in intracellular deox-
ynucleoside triphosphate levels revealed elevated dTTP pool and lower¬
ing of dATP pool size as is characteristic of other ribonucleotide
reductase inhibitors. Cytidine diphosphate (CDP) reductase activity
was inhibited by >97% in L1210 cells treated for 4 h with 5 yM parabac¬
tin. The cell cycle block seen in LI210 cells after a 4 h exposure to 5
yM parabactin could be partially reversed by the addition of exogenous

11
Fe(Cl)3 and the bulk of the cells cascade into S-phase 3 h later.
Catechol amide chelators have also demonstrated pronounced effects
against the DNA virus Herpes simplex type I (37), another ribonucleo¬
tide reductase dependent system, while they were inactive against the
RNA virus, Vesicular stomatitis.
It is clear that there is sufficient evidence to suggest that
iron withholding should be pursued as a means of controlling the pro¬
liferative process. The microbial catechol amide siderophores and their
synthetic derivatives may represent an important class of pharmacologi¬
cal agents with potential value in the treatment of proliferative
disorders. They may prove useful in cancer chemotherapy as single
agents or in combination with other agents.
Polyamine Analogues
The second part of this dissertation deals with the evaluation of
the N-alkylated derivatives of the polyamines as potential antiprolif¬
eratives. The role of polyamines in proliferative processes has been
extensively reviewed in recent years (44-50). The polyamines spermi¬
dine, spermine, and the diamine putrescine (Fig. 1-4) are present in
all mammalian cells and are required for normal cellular maintenance,
proliferation, and differentiation. These basic amines are extensively
protonated at physiological pH and capable of electrostatic interac¬
tions with a variety of macromolecules including nucleic acids and pro¬
teins. Although many roles have been ascribed to the polyamines, their
exact functions in cellular physiology are not yet well understood.
The intracellular concentrations of these polyamines are main¬
tained through a highly regulated metabolic system (49,50) (Fig. 1-5).

12
a.
H2N
H
N
NH2
b.
H2N
nh2
Figure 1-4. The polyamines.
a. Putrescine
b. Spermidine
c. Spermine

13
ARGININE
DECARBOXYLATED
S-ADENOSYLMETHIONINE
S-ADENOSYL-
METHIONINE
5-METHYLTHIOi
ADENOSINE
DECARBOXYLATED
S-ADENOSYLMETHIONINE
5'-METHYLTHIO
ADENOSINE
UREA
ORNITHINE
CO,
N1-acetylspermidine
6y
SPERMIDINE>
SPERMINE
N1 - ACET YLSPERMINE
Figure 1-5. Pathway of polyamine synthesis and intercon¬
version in mammalian cells. Enzymes involved are: (1) arginase,
(2) S-adenosylmethionine decarboxylase, (4) Spermidine synthase,
(5) Spermine synthase, (6) Spermidine N -acetyltransferase, and (7)
Polyamine oxidase (49).

14
In mammalian cells putrescine is derived from ornithine by the pyridox-
al phosphate-dependent enzyme ornithine decarboxylase (ODC). Ornithine
is available in the plasma or formed from arginine by the action of
arginase. Levels of ODC are normally low in quiescent cells, and its
activity can be increased manyfold within hours after exposure to such
stimuli as hormones, drugs, tissue regeneration and growth factors
(47).
To convert putrescine to spermidine an ami nopropyl group must be
added. The aminopropyl moiety is derived from S-adenosylmethionine
(SAM) which is committed to polyamine biosynthesis as an aminopropyl
donor once it is decarboxylated by S-adenosylmethionine decarboxylase
(AdoMetDC). Levels of AdoMetDC are regulated by the cell's need for
spermidine and the availability of putrescine as a substrate for sperm¬
idine synthesis. Its activity is also regulated by growth-promoting
stimuli (49).
The transfer of the aminopropyl group from decarboxylated
S-adenosylmethionine to putrescine is catalyzed by the action of the
enzyme spermidine synthase to yield spermidine. In a similar fashion,
a second aminopropyl transfer to spermidine, catalyzed by spermine
synthase, yields spermine. Despite the similarity of these reactions,
spermidine synthase and spermine synthase are substrate specific
enzymes. The two synthases are thought to be regulated by the avail¬
ability of their substrates as well as factors stimulating cell growth.
The other product of the aminopropyl transfer reaction is 5'-methyl-
thioadenosine (MTA). The MTA produced by polyamine synthesis is

15
rapidly degraded and converted by a salvage mechanism into 5'-AMP and
methionine (50).
Spermine may also be interconverted into spermidine and spermi¬
dine into putrescine by the action of the enzymes N^-acetyltransferase
and polyamine oxidase. The acetyltransferase uses acetyl-CoA to con¬
vert spermine into spermidine and spermidine into putrescine. The N"-
acetylated polyamines are good substrates for polyamine oxidase, which
cleaves at the internal nitrogen yielding 3-acetamidopropanal and
putrescine or spermidine. Normally, only small amounts of the acetyl-
ated derivatives are present due to the much greater activity of poly¬
amine oxidase (49).
An active transport system distinct from those for amino acids is
also present (49,50). It is unclear why cells would maintain a poly¬
amine transport system since intracellular synthesis is normally used
to provide polyamines and extracellular polyamine levels are normally
low. However, uptake and efflux may be important mechanisms by which
cellular polyamine levels are maintained. The uptake pathways for the
polyamines have not been fully characterized at the biochemical level
and there may be multiple transport mechanisms (50).
Rapidly proliferating tissues have high levels of polyamines and
their biosynthetic enzymes (47), and increased quantitites of poly¬
amines are found in the urine and serum of humans and animals bearing
tumors (51). These facts suggest the potential of impacting on poly¬
amine metabolism as a useful target for antiproliferative therapy.
Interruption of the polyamine biosynthetic network has been par¬
tially successful as a means of controlling the proliferative process

16
including the growth of cancer cells (52-54). The antineoplastic
drugs, a-difluoromethylornithine (DFMO) and methylglyoxyal-bis(guanyl
hydrazone) (MGBG) (Fig. 1-6), are both potent inhibitors of enzymes
which are critical to polyamine biosynthesis. DFMO is an enzyme-
activated irreversible inhibitor of ODC which causes depletion of
intracellular polyamine pools (55). It is postulated that DFMO is
recognized by ODC as a substrate and that its decarboxylation yields a
highly reactive intermediate which binds irreversibly to the active
site of the enzyme. The action of MGBG, however, is not specific.
MGBG induces spermine/spermidine acyl-transferase, inhibits diamine
oxidase and AdoMetDC, and has other effects (50). Although it is not
clear which of MGBG's roles is most important in inhibiting the growth
of cancer cells, uptake of the drug by the polyamine transport appar¬
atus seems critical. The drug has been regarded as a structural ana¬
logue of spermidine, and since its uptake involves an active transport
mechanism cells, can actually concentrate the drug so that millimolar
quantities of MGBG can accumulate intracel1ularly (56).
Although DFMO and MGBG have shown impressive results against
transplantable murine tumor models, the success of both in a clinical
setting, unfortunately, has been somewhat marginal (50,57). The action
of DFMO on most neoplasms is cytostatic, and the rapid rate of synthe¬
sis of new ODC (half-life of less than 1 h in many cell lines) makes it
essential to maintain high drug concentrations since enzyme levels and
cell growth are restored rapidly once drug is removed (49). MGBG has
produced unacceptable host toxicity in early trials.

17
a.
b.
Figure 1-6. The inhibitors of polyamine biosynthesis.
a. DFMO
b. MGBG

18
The combination of MGBG and DFMO has been reported to have syner¬
gistic activity (57). It was speculated that the simultaneous inhibi¬
tion of AdoMetDC by MGBG and reduction of ODC levels by DFMO treatment
would enhance tumor cell kill. Furthermore, DFMO depletion of intra¬
cellular polyamine pools increases uptake of MGBG by the polyamine
uptake apparatus (58). Since DFMO is relatively nontoxic (53), it was
hoped that potential tumor cell selectivity would be increased while
decreasing MGBG's host toxicity. This does not seem to be the case in
several trials (50). However, the fact that these agents do have anti¬
cancer activity, albeit limited, supports the aim of impacting on poly¬
amine metabolism as a rational approach for controlling the prolifera¬
tive process.
The initial design, synthesis and testing of polyamine deriva¬
tives which utilize the polyamine uptake apparatus and exhibit antipro¬
liferative properties was conducted by Porter, Bergeron and Stolowich
(59). There are several properties which may be desirable for the
design of polyamine analogues as antiproliferatives: (1) the analogues
may utilize the polyamine uptake apparatus as a means of delivery, and
such uptake may be enhanced by polyamine depletion, (2) the analogues
may fulfill some but not all of the functions of polyamines and thereby
interfere with normal polyamine metabolism and/or function, and (3) the
analogues may act to regulate the polyamine-biosynthetic pathway lead¬
ing to depletion of cellular polyamines and their biosynthetic en¬
zymes.
In a systematic approach, a number of spermidine analogues were
synthesized and studied for their structure/function relationships

19
(59-65) and some were found to meet the above requirements. It was
found that, in terms of uptake, retention of charge is of major import¬
ance since N-acetylation as opposed to N-alkylation at either the cen¬
tral or tne terminal nitrogens decreased the ability of the analogue to
compete with spermidine for uptake into the cell (65). While the
N ,N°-alkyl derivatives (65) were poorer competitors for spermidine
uptake than N^-alkyl derivatives, they accumulated to high concentra¬
tions during longer incubations (66). The antileukemic activity of
several N^-alkyl spermidine analogues was enhanced by pretreatment of
cells with DFMO (67). Structural analogues of spermidine differing in
aliphatic chain length separating the amines were also studied (68).
Several of the homologues were found to support cell growth and no more
than a one-carbon extention was tolerated for biological activity.
1 ft
Certain of the N,N -alkyl spermidine derivatives, particularly
,N®-diethylspermidine (BESPD), were found to inhibit L1210 cell
growth, diminish ODC activity, reduce AdoMetDC, and deplete intracell¬
ular polyamine pools (65,66,69). It has been concluded that BESPD
affects polyamine biosynthesis, at least, at one critical point by
regulating ODC at a translational and/or post-translational level in a
manner similar to the natural polyamines. However, the analogue is
apparently incapable of fulfilling certain critical functions of natur¬
al polyamines necessary for normal cellular function and proliferation
(65). Depletion of intracellular polyamine pools by BESPD may also
occur by displacement of the natural polyamines from their intracell¬
ular binding sites. In addition, potent induction of the spermidine/
spermine acetyl transferase activity by BESPD (70) could lead to the

20
back-conversion of spermine and spermidine to putrescine and efflux of
the polyamines from the cell.
The effects of these polyamine analogues may vary considerably
from one cell type to another depending on the cellular requirements
for polyamines. Despite inhibition of cell growth, BESPD was not toxic
towards L1210 cells (69). However, BESPD at concentrations of greater
than 5 yM was found to be cytotoxic to human large-cell lung cancer
cells normally quite resistant to DFMO (71).
The design, synthesis, evaluation, and study of the biochemical
and physiological effects of this new class of antiproliferatives is
currently the area of intense investigation.

CHAPTER II
CATECHOLAMIDE CHELATORS
Materials and Methods
The catechol amide chelators and methylated analogues were synthe¬
sized by methods previously described (30-32,72). Stock solutions (2
mM) of the catechol amide chelators were made in 50% (v/v) ethanol and
water. Stock solutions of Adriamycin and ara-C were prepared in water.
BCNU was prepared in 50% (v/v) ethanol and water. All solutions were
passed through a 0.2 um filter prior to use. Reagents for cell culture
were obtained from Gibco (Grand Island, HY). Cell culture flasks, 25
and 75 cm^, were purchased from Corning (Corning, NY). Adriamycin
was obtained from Adria (Columbus, OH), carmustine (BCNU) from Bristol
(Syracuse, NY), hydroxyurea from Aldrich Chemical Co. (Milwaukee, WI)
and cytosar (ara-C) from Upjohn (Kalamazoo, MI). Diamidino-phenylin-
dole (DAPI) and propidium iodide (PI) were obtained from Sigma (St.
Louis, MO). Desferrioxamine was provided by Ciba-Geigy (Basel, Swit¬
zerland). Iron atomic absorption standard solution, 1003 yg/mL Fe in
2% HNO3 (Aldrich) was used as an Fe(III) source. Cremophore RH-40
was provided by BASF-Wyandot Corp. (Parsipany, NJ). Rhodamine-123
(Rh-123) was purchased from (Eastman Kodak Company, Rochester, NY),
[methyl- H]Thymidine was obtained from New England Nuclear (Boston,
MA). DBA/2 mice were obtained from Jackson Laboratories (Bar Harbor,
ME).
21

22
Cell Culture.--Murine L1210 leukemia cells and human Burkitt
lymphoma (Daudi) cells were maintained in exponential growth as
suspension cultures at 0.3 x 10^ to 1.5 x 10^ cells/mL and 1 x 10^ to
2 x 10^ cells/mL, respectively. Chinese hamster ovary (CH0) cells and
murine B-16 melanoma cells were grown as monolayers to near confluency
in 48 h after reseeding at 2.5 x 10^ cells/25 cm^ flask. Human fore¬
skin fibroblasts (HFF) cells were grown as monolayers to near conflu¬
ency in 96 h after a 1:4 dilution of cells in fresh medium. Monolayer
cells were lifted as single cell suspensions with 0.05% trypsin and
O
0.02% ethylenediaminetetraacetic acid tetrasodium salt (2 mL/25 cnr)
and diluted with Hanks' balanced salt solution. All cells were grown
in complete medium containing RPMI-1640, 2% 4-(2-hydroxyethyl)-l-piper-
azinethanesulfonic acid/3-(N- morpholino)propanesulfonic acid (HEPES--
M0PS buffer), and 10% fetal bovine serum unless otherwise stated.
p
Cells were grown in 25 cnr tissue culture flasks in a total volume of
10 rnL under a humidified 5% CO2 atmosphere at 37°C.
ICe,q Value Determinations.—Cultures in logarithmic growth were
treated with compounds of interest at concentrations ranging from 10"^
to 10“^ M. Cells were counted by electronic particle analysis (Coulter
Counter, Model ZB1, Coulter Electronics, Hialeah, FL) and confirmed
periodically with hemocytometer measurements. Cell viability was
assessed by trypan blue dye exclusion.
The percentage of control growth was determined as follows:
% of control growth = 100 x [Final treated cell no.-initial
inoculum]/[Final untreated cell no.-initial inoculum]

23
The 50% inhibitory concentration (IC^g) value is defined as the
concentration of compound necessary to reduce cell growth to 50% of
control growth after defined intervals of exposure. The IC^q values
were determined as the chelator concentration necessary to inhibit cell
growth to 50% of the control growth at 48 h for all cell lines except
HFF cells, for which the ICgg values were determined at 96 h.
Preformed chelator-iron chelates were formed by the addition of
equimolar amounts of chelator and iron to the media 30 min prior to the
addition of the cel Is.
A 50% ethanol and water solution in appropriate amounts was used
to treat cells in all control flasks. Addition of iron and ethanol
alone in the concentrations used had no significant effect on growth
studies.
Stock suspensions of L1210 cells were maintained for 48 h, with
normal 10-12 h doubling times, in complete mediums containing 5%, 10%,
and 20% fetal bovine serum. The IC5Q value of parabactin was deter¬
mined for cells grown in each medium.
Drug Combinations.--L1210 cells in suspension culture were incu¬
bated in the presence of parabactin and antineoplastics at various con¬
centrations and combinations. Two types of experiments were performed
for each drug combination.
a. To obtain median effect plots for simultaneous drug combina¬
tions, cells at 3 x 10^ cells/mL were exposed to a constant ratio of
chelator to antineoplastic, a ratio in which absolute concentrations
were varied. The absolute concentrations of the drugs were chosen
in the neighborhood of their IC^g values. This gave the ratio of

24
parabactin to ara-C of 100:1, of parabactin to Adriamycin of 100:1, and
of parabactin to BCNU of 1:1. The growth was monitored at 48 h, and an
IC50 value was determined. A separate experiment was run to determine
the IC^Q value of parabactin, ara-C, Adriamycin, and BCNU.
b. In a second experiment, cells at 3 x 10^/mL were first incu¬
bated with 5 pM parabactin for 5 h at 37°C, then washed twice with
fresh medium, resuspended at 3 x 10^ cells/mL, allowed to incubate at
37°C for 3 h (allowing the cells to cascade into S phase), and then
treated with varying concentrations of the antineoplastic. Growth was
monitored every 12 h for 96 h.
Additional experiments were carried out with various combinations
of parabactin and Adriamycin, ara-C or BCNU treatments.
c. Cells at 3 x 10^ cells/mL were treated with 5 pM parabactin
for 3 h, and then BCNU was added at varying concentrations. The flasks
were allowed to incubate for an additional 5 h. The cells were next
washed with fresh medium and resuspended at 3 x 10^ cells/mL, then
their growth was monitored for up to 96 h.
d. Cells at 3 x 10^ cells/mL were treated with 5 pM parabactin
for 4 h, and then Adriamycin was added. The flasks were allowed to
incubate for an additional 2 h. The cells were next washed with fresh
medium and resuspended at 1 x 10^ cells/mL, then their growth was mon¬
itored for 48 h.
e. Cells at 3 x 10^ cells/mL were treated with 2 pM parabactin
for 4 h, and then ara-C was added and the flasks were allowed to
incubate for an additional 12 h. The cells were next washed with fresh
medium and resuspended at 5 x 10^ cells/mL, then their growth was mon¬
itored for up to 120 h.

25
f. Cells at 3 x 10^ cells/mL were simultaneously treated with 5
yM parabactin and ara-C. The cells were allowed to incubate for 5 h,
washed with fresh medium, resuspended at 5 x 10^ cells/mL, and their
growth was monitored for up to 96 h.
In each case, the drug concentrations and treatment times are
indicated on the figure legends.
Data Analysis for Simultaneous Drug Combi nations.—Briefly, this
procedure involves (a) determination of an ICgg value for each drug and
(b) determination of an IC^q value for a constant ratio of two drugs.
The molar ratio of the drugs is maintained while the absolute concen¬
tration of the drugs is changed. The cell growth data from these
experiments are plotted as log Fa/Fu versus concentrations (log [c]),
where Fa is fraction of cells affected, Fu is fraction of cells unaf¬
fected, and c is molar concentration. The data from the median effect
plots were analyzed by the method of Chou and Talalay (73) according to
the following equation.
A/Da + B/Dg = Cl (A)
The D values are the ICgg concentrations of each of the drugs alone,
the A and B values are the concentrations of each drug at the IC^q
value of the constant ratio combination, and Cl is the combination in¬
dex. When the Cl is in excess of 1, the combination is "antagonistic.
When it is less than one, the combination is "synergistic"; and when
the Cl is equal to 1, the combination is "additive."

26
Regrowth Studies.--L1210 cells in suspension culture at approxi¬
mately 3 x 105 cells/mL were incubated in the presence of chelator for
5 h at 37°C. The cells were then washed twice in fresh complete medium,
resuspended at a final concentration of 5 x 10^ cells/mL, and incubated
at 37°C. Cell samples during logarithmic growth were counted for up to
60 h after ligand treatment and compared to controls.
Cloning Assay.—Cells at 3 x 10^ cells/mL were incubated with 5
yM parabactin for 5 h and washed with fresh medium. Treated cells were
then plated in triplicate 96-well microtiter plates at 0.4 cells/well
with each well containing 100 yM of sample. The plates were incubated
at 37°C in a humidified incubator in an atmosphere of 5% CO^ and 95%
air. The plates were examined with an inverted phase microscope at
X100 magnification. The final number of colonies per plate was quanti¬
tated at 7 days after plating. Groups of 50 or more cells per well were
identified as having been cloned from a single viable cell.
Viability Assay.--Cell viability was assayed by two dye exclusion
methods. In one method cells were diluted 1:1 with 0.4% trypan blue in
phosphate-buffered saline and counted in a hemacytometer using a light
microscope. In the second method 10^ cells were diluted in 1 mL of a
1.12% sodium citrate solution of propidium iodide (50 yg/mL) and count¬
ed on a fluorescent microscope.
Viability determination by Rh-123 uptake (74) was assayed by add¬
ing the mitochondrial dye Rh-123 (10 yg/mL) to a aliquot of 10^ cells.
The samples were incubated at 37°C for 10 min and washed once with med¬
ium. Trypan blue was added, prior to counting, to give a final concen¬
tration of 0.2 yg/mL. Rh-123 and trypan blue stained cultures were

27
observed in a hemocytometer using a Zeiss epifluorescence Axioscop
microscope. The excitation wavelength was 485 nm.
Flow Cytometric Analysis.--Cel 1 analysis was performed with a
RAT-COM flow cytometer (RATCOM, Inc., Miami, FL) interfaced with a
microcomputer (IBM-XT). Cell samples of 10® cells were taken at vari¬
ous intervals and stained with DAPI (10 yg/mL) in a nuclear isolation
media (NIM-DAPI) (75). DNA distributions were obtained from the fluor¬
escence and analysis of the DAPI-stained cells.
Alternately, cell analysis was performed with a FACS II flow
cytometer (Becton Dickinson FACS Systems, Sunnyvale, CA) interfaced
with a microcomputer (Hewlett Packard 45B, Fort Collins, CO). Samples
of 10® cells were removed and stained with PI and then exposed to
RNase. DNA distributions were obtained from analysis of the red fluor¬
escence from the Pi-stained DNA (76).
Effect of Percent Fetal Bovine Serum in the Medium on Cell Cycle
Kinetics.--Stocks of L1210 cells were maintained for 48 h with normal
10-12 h doubling times in complete mediums containing 5%, 10%, 15% and
20% fetal bovine serum. The time course for the effect of 5 yM para-
bactin on the cell cycle kinetics of cells grown in each of the mediums
was followed for up to 8 h.
Bromodeoxyuridine (BrdUrd) Incorporation Studies.--Cultured LI210
cells were treated with 10 yM vibriobactin, washed, resuspended, and
incubated as in the regrowth studies. At times, 0, 5, 10, 15 and 20 h
of regrowth cell samples were removed for dual parameter flow cytomet¬
ric measurements of cellular DNA content and amount of BrdUrd incorpor¬
ated into cellular DNA by methods presented elsewhere (77,78).

28
Radiolabeled Thymidine Incorporation.--Cel Is treated with 5 yM
parabactin for 5 h were washed, resuspended in fresh complete medium,
and incubated at 37°C for 30 min with [^H]thymidine (specific activ¬
ity, 80.9 Ci/mmol; 1 yCi/mL) in triplicate tubes containing 10^
cells in a total volume of 1 mL of complete medium. Labeling was
halted by the addition of 0.5 mL of ice-cold Hanks' balanced salt
solution (HBSS) containing thymidine (1 mg/mL) to each tube. The cells
were washed, resuspended in 1 mL of 10% trichloroacetic acid in HBSS
containing thymidine (1 mg/mL), and allowed to sit on ice for 30 min.
The acid-precipitable material was filtered and the filter washed.
Then filters were air dried and counted in Biofluor (New England
Nuclear) scintillation fluid. Background labeling was evaluated in
O
cell samples pulsed for 30 min with [ HJthymidine at 5°C.
% of control incorporation = treated cpm - treated background cpm x 100
control cpm - control background cpm
Cr^Release Assay. —LI210 cells were treated with vibriobactin
(10 yM) for 5 h, washed with fresh media and regrown for 20 h. Approx¬
imately 2 x 10^ cells were centrifuged and 150 yCi Cr^ added to the
pelleted cells. The pellet was incubated for 45 min at 37°C in a 5%
CO2 atmosphere. Sample radiation was counted with an automatic gamma
counter (LKB-Wallac RiaGamma 1274, Wallac Oy, Finland).
A microwell assay using triplicate samples of 200 yL containing
1 x 10^ cells was employed as described elsewhere (79). The percent

29
51
Cr release for the control cells was compared to that for the vibrio-
bactin treated cells.
Animal Studies.--The murine LI210 leukemia cells were maintained
in DBA/2J mice. Cells from a single mouse which was injected i.p. with
1.25 x 10^ cells 7 days earlier were harvested and diluted with cold
saline so that an inoculum of 10^ cells could be administered by a
0.25 mL i.p. injection. In each study, mice were injected with L1210
cells on day 0.
The catechol amide chelators were first solubilized in 20% Cremo-
phor RH-40 in 0.9% saline with sonication and gentle heating (<60°C)
and then diluted with an equal volume of 0.9% saline. The chelator was
administered by i.p. injection according to the appropriate dosing
schedule. Concentrations of chelator were adjusted so that the mice
were injected with a volume of 1-2 mL/100 g of animal/dose (i.e.: a 25
g mouse was injected with 0.25-0.5 mL of drug solution).
The in vivo effectiveness of parabactin alone and combinations of
parabactin with ara-C or Adriamycin against the L1210 ascites tumor
were studied. Ara-C and Adriamycin were diluted in 0.9% saline so that
the mice were injected i.p. with a volume of 1 mL/100 g of animal/dose.
Due to the possible toxicity or antitumor activity of Cremophor RH-40,
groups of mice treated with 10% Cremophor RH-40 as well as untreated
mice served as controls. Groups of 6 mice were used in each treatment
schedule.
One parameter used for treatment evaluation was mean animal sur¬
vival time (percent increased life span, % ILS).

30
% ILS = 100 x [mean survival time treated animals-
mean survival time controls]/[mean survival time controls]
Another parameter used for treatment evaluation was tumor burden.
The L1210 cells were harvested from the animal's peritoneum by two
washings with 5 mL of 0.9% saline. The total number of cells recovered
was determined by counting on a hemocytometer.
The in vivo effect of a single i.p. injection of parabactin (100
mg/kg) on L1210 cell cycle kinetics was studied in mice which had been
injected i.p. with 1 x 10^ L1210 cells 6 days earlier. Mice were sac¬
rificed by cervical dislocation each hour after injection of the che¬
lator and the cells were harvested from the peritoneum with HBSS. The
cells were stained witli NIM-DAPI and DNA content measured by flow cyto¬
metric analysis.
Results
Inhibition of Cell Growth
The effectiveness of various iron chelators as inhibitors of
L1210 cell growth was assessed by comparison of their IC^q values
(Table 2-1). The bidentate compound, dihydroxybenzoic acid, while
being a good iron chelator (Kf = lO^M-*) (5,37) is not very effective
cell growth inhibitor, requiring millimolar concentrations. Hydroxy¬
urea, which structurally resembles a bidentate hydroxamate chelator,
was only fair at inhibiting cell growth, with an IC^q value of 40 yM.
The hexacoordinate hydroxamate iron chelator, desferrioxainine, and the
tetracoordinate catechol amide chelator, compound II, showed similar
activity against L1210 cell growth with ICgQ values of 8 yM and 7 yM
respectively. The most effective of the compounds tested were the
hexacoordinate catecholamide chelators, vibriobactin and parabactin,

31
TABLE 2-1
ICcn VALUES OF VARIOUS CHELATORS AGAINST
CULTURED LI210 LEUKEMIA CELLS
Chelators
IC^q Values at 48 H
Dihydroxybenzoic Acid
2.8 nM
Hydroxyurea
40 pM
Desferrioxanine
8 pM
Compound II
7 pM
Vibriobactin
2 pM
Parabactin
1.5 pM

32
having ICgQ values of 2 pM and 1.5 pH respectively. The dose-effect
curves generated in these studies indicate linear inhibitory effects
for parabactin and vibriobactin within a very narrow range of ligand
concentrations (Fig. 2-1). For example, at concentrations of greater
than 5 pM, cells uniformly grow to only about 10% of controls, while
below 1 pM approximately 90% control growth is obtained.
The time dependence of growth inhibition of cultured LI210 cells
by the siderophores is shown in Figures 2-2 and 2-3. The concentration
of 10 pM was selected for both parabactin and vibriobactin as a value
at approximately 5 times their IC^q values. The onset of growth
inhibition by both siderophores was almost immediate. Both chelators
eventually caused a total cessation cell growth. In either case, the
growth inhibition could be prevented with the addition of an equimolar
amount (10 pM) of Fe(III) to the culture medium at the same time as the
chelator.
Methylation of the ligand catechol hydroxyl groups, as in tetra-
methyl parabactin (Fig. l-3c) and permethylated vibriobactin (Fig.
l-3e), greatly reduced the growth inhibitory activity of both chelat¬
ors. Total methylation of the three catechol groups of vibriobactin
completely eliminated its activity at a concentration 10 pM (Fig. 2-2).
In fact, it showed no inhibitory activity at concentrations of up to 50
pM. Tetramethyl parabactin (10 pM), while having greatly reduced
inhibitory activity, did have some effect on L1210 growth (Fig. 2-3).
In fact, it had an IC5Q value of 30 pM.
The IC5Q values for parabactin were determined against L1210
cells grown in culture mediums with varying concentrations of fetal

33
PERCENT CONTROL GROWTH
Figure 2-1. Dose-response curves illustrating the effects of
increasing concentrations of parabactin and vibriobactin on the
growth of L1210 cells.

CELLS/ml
Figure 2-2. Comparison of the effects of 10 yM vibriobactin,
in the presence or absence of Fe(III), and 10 yM permethylated
vibriobactin on the growth of L1210 cells.

CELLS/ml
35
Figure 2-3. Comparison of the effects of 10 yM parabactin,
in the presence or absence of Fe(III), and 10 yM tetramethyl
parabactin on the growth of L1210 cells.

36
bovine serum (Fig. 2-4). Cells grown in 5, 10 and 20% FBS had IC^g
values of 0.8, 1.2, and 1.6 yM respectively.
Parabactin was also found to have potent growth inhibitory ef¬
fects on cultured B16-melanoma and CHO cells exhibiting a 48 h IC^g
value of 1.5 yM for both cell lines. Both vibriobactin and parabactin
exhibited a 48 h IC^q value of 2 yM for Daudi cells and parabactin was
active, with a 96 h IC^q value of 2 yM, against HFF cells. The IC^q
value for HFF cells was determined at 96 h due to their relatively slow
doubling time (45-50 h) compared to the other cell lines studied (12-24
h).
Treatment Reversibility
When cells were treated for 5 h with 5 yM parabactin, the growth-
inhibitory effects of parabactin were found to be reversible on washing
away the ligand. Viability of parabactin-treated cells, taking cell
samples every 5 h for 35 h after washing away of the ligand, was
verified by dye exclusion and Rh-123 uptake methods. At 10 h after
washing, the viability decreased to a minimum of 88% by the dye
exclusion methods and only 85% by Rh-123, the most sensitive of the
methods used. These results were in agreement with the cloning assay
which indicated 91% of colonies were cloned or single cell after
short-term exposure (5 h) to the chelator compared to single control
cells. The number of cells per colony from the parabactin-treated
cells was consistently less than from the control cells.
Cells having been treated for 5 h with 10 yM vibriobactin showed
similar reversibility of inhibitory effects on washing away of the lig¬
and and reseeding the cells in fresh medium. A ^Cr release assay per¬
formed on vibriobactin treated cells at 15 h after removal of the lig¬
and showed no increase in ^Cr release relative to control cells,

50% INHIBITORY CONCENTRATION
37
% FETAL BOVINE SERUM
Figure 2-4. The effect of varying amounts of fetal bovine
serum in the L1210 growth medium on the 50% inhibitory concentra¬
tion of parabactin.

38
establishing little, if any, cytotoxicity due to short term (less than
5 h) 10 yM vibriobactin.
However, when cells were treated for an extended period of time
with 5 yM parabactin, cytocidal activity was observed (Table 2-2). For
example, at 60 h, only 30% of the cells were viable as determined by
trypan blue exclusion. Dye exclusion of cells treated with 10 yM vib¬
riobactin for 48 h indicated only 10% viability, and only 20% viability
of cells exposed to 100 yM desferrioxamine for 60 h. Surprisingly, 300
yM hydroxyurea was the least toxic of the compounds tested with only
25% cytotoxicity after 60 h of treatment. Although in dividing HFF
cells, growth was inhibited by parabactin (IC^q value of 2 yM at 96
h), treatment of a confluent, nondividing, monolayer of HFF cells with
20 yM parabactin for 24 h appeared to be nontoxic by light microscopy.
Also, the treated confluent cells diluted and regrown in drug-free med¬
ium grew at the same rate as control cells.
Effects of the Catechol amide Chelators
on Cell Cycle Kinetics
Flow cytometric analysis was employed as the method to follow the
effects of the chelators on the cell cycle progression. The time
course for the effect of 10 yM vibriobactin on the DNA content of L1210
cells is seen in Figure 2-5. Flow cytometric analysis of untreated
L1210 cells reveals high S (50%) and G2-M (15%) phase components of
the cell population with about 35% of the cells having phase DNA
content. After only 2 h alterations in the DNA content of the vibrio¬
bactin treated cells were observed. There was a substantial effect on
the cell cycle kinetics with a clear block at the G^-S border after 3

TABLE 2-2
CYTOCIDAL ACTIVITY OF CHELATORS
39
Ligand
Percent of Activity3
0
12 h
24 h
48 h
60 h
Parabactin (5 pM)
<2
5
15
49
70
Vibriobactin (10 pM)
<2
4
36
90
-
Desferrioxamine (100 pm)
<2
5
20
60
80
Hydroxyurea (300 pM)
<2
-
15
-
25
aThe percentage of L1210 cell kill in cells treated with parabactin,
vibriobactin, desferrioxamine and hydroxyurea with treatment time.
Cell viability determined by trypan blue exclusion.

RELATIVE CELL NUMBER
40
DNA CONTENT
Figure 2-5. Flow cytometric analysis of L1210 cells.
A. Control cells.
B. Cells treated with 10 pM vibriobactin for 3 h.
C. Cells treated with 10 pM vibriobactin for 4 h.
D. Cells treated with 10 pM vibriobactin for 5 h.

41
h. Within 5 h, cells with G2-M phase content were eliminated, and
cells with S phase content were reduced to 30%. The cells were pre¬
vented from entering S phase and 70% of the cells have phase con¬
tent. A comparable cell cycle block in DNA synthesis was observed in
the DNA histograms of cells treated with 5 yM parabactin for 5 h (Fig.
2-6). Cells treated for up to 24 h with 5 yM parabactin, however,
showed no greater block in cell cycle kinetics than cells treated for 5
h, and cells treated with less than 4 yM parabactin did not appear to
reduce S phase to the extent that the 5 yM treatment did. For further
studies it was therefore indicated that the minimum concentration and
time for parabactin to effectively eliminate DNA synthesis in L1210
cells was 5 yM for 5 h.
In a study to determine the ability of 5 yM parabactin to cause a
cell cycle block in L1210 cells grown in culture mediums with various
percentages of fetal bovine serum (5%, 10%, 15% and 20%), it was found
that there was no significant alteration in time to reach complete cell
cycle block (5 h) for the cells in any of the different culture medi¬
ums.
In a comparative study, parabactin was shown to be a far more
effective cell cycle blocking agent than either hydroxyurea or desfer-
rioxamine (Fig. 2-6). For example, to generate cell cycle blocks simi¬
lar to that produced by 5yM parabactin (IC^q, 1.5 yM) at 5 h, L1210
cells required 100 yM desferrioxamine (IC^q, 7.5 yM) for 5 h or 300 yM
hydroxyurea (IC^q, 50 yM) for 5 h. When comparing the IC^q value of
each of the ligands, it is clear that the compounds with the higher
IC5Q values are also the poorer cycle blocking agents.

RELATIVE CELL NUMBER
42
DNA CONTENT
Figure 2-6. Flow cytometric analysis of L1210 cells.
A. Control cells.
B. Cells treated with 5 yM parabactin for 5 h.
C. Cells treated with 100 yM desferrioxamine for 5 h.
D. Cells treated with 300 yM hydroxyurea for 5 h.
T I 1
420

43
Synchronization Effects
Flow cytometry analysis of the cell cycle kinetics of cells
treated with 10 yM vibriobactin for 5 h indicated a clear block at the
Gj-S border of the cell cycle. Washing and reseeding the treated cells
in fresh complete medium caused pronounced changes in the cell cycle
phases with time (Fig. 2-7). The block in DNA synthesis is released
and at 5 h after removal of the drug the majority of cells have S phase
DNA content. The progression through the cell cycle phases continued
for up to 20 h. Cell cycling was further substantiated by BrdUrd
incorporation into DNA (Table 2-3). The percent BrdUrd incorporation
at various times after removal of the chelator was roughly consistant
with changes in S phase DNA content as determined by flow cytometry.
Cells incubated with 5 yM parabactin for 5 h exhibited a block in
DNA synthesis at the G^-S border (by flow cytometry) with a greatly
decreased S and G¿ phase (Figs. 2-6 and 2-8). Concentrations of 100 yM
desferrioxamine and 300 yM hydroxyurea for 5 h were required to produce
similar blocks. On removal of the chelators by simple washing and
reseeding the cells in drug-free culture medium, the block in DNA syn¬
thesis was removed and the cells cascaded into S phase. Parabactin-
treated cells maintained a synchronous population of cells for 3 cell
cycles. (Figure 2-8 only includes cells moving into the third cycle.)
However, desferrioxamine- and hydroxyurea-treated cells showed normal¬
ized DNA histograms after 12 h.
Radiolabeled Thymidine Incorporation.--Incorporation of [ H]thy-
midine into L1210 cells after the cells were treated with 5 yM

RELATIVE CELL NUMBER
44
DNA CONTENT
Figure 2-7. Flow cytometric DNA content analysis of L1210
cells washed and regrown in fresh medium after a 5-h treatment with
10 viM vibriobactin.
Analysis performed after removal of ligand.
A. 0 h
B. 5 h
C. 10 h
D. 15 h
E. 20 h

45
TABLE 2-3
PERCENT BROMODEOXYURIDINE INCORPORATION INTO L1210
CELLS TREATED WITH 10 yM VIBRIOBACTIN FOR 5 H THEN
WASHED AND RESEEDED IN FRESH MEDIUM
Time after Wash
Percent BrdUrd
Incorporation
Control
Treated
0
44
16
5
50
47
10
53
45
15
47
16
20
49
32

Figure 2-8. Cell synchronization of L1210 cells by 5 yM
parabactin. DNA content determined by flow cytometric analysis.
A. Control cells.
B. Cells treated with 5 yM parabactin for 5 h.
Cells were then washed, resuspended in drug-free medium, and
samples removed at the following times.
c.
4 h.
D.
6 h.
E.
10 h.
F.
12 h.
G.
14 h.
H.
16 h.
I.
18 h.
J.
22 h.

NUMBER OF CELLS
47
DNA CONTENT

48
parabactin for 5 h and washed (Fig. 2-9) revealed cycling of incorpor¬
ation in a time frame consistent with the normal 10 h to 12 h doubling
time of L1210 cells, and with the cycling of DNA content as seen in the
flow cytometric studies (Fig. 2-8).
Drug Combination Studies
Adriamycin-treated Cel Is.--The Adriamycin IC^q under our experi¬
mental conditions was 0.027 yM at 48 h, while the parabactin IC^g was
1.34 yM at 48 h. However, when L1210 cells were treated with para¬
bactin and Adriamycin in simultaneous combination, the results indicat¬
ed that the combination was "anatagonistic" (Fig. 2-10). Following the
method of analysis of Chou and Talalay, the cells were exposed to a
constant molar ratio of parabactin to Adriamycin of 100:1. The micro¬
molar concentrations at this constant ratio were 2:0.02, 1.75:0.0175,
1.5:0.015, 1.25:0.0125, 1.0:0.01, and 0.5:0.005. The IC^g of the com¬
bination was 1.42 yM (Cl = 1.57). Chou and Talalay's analysis of the
data (see Materials and Methods) clearly revealed the combination to be
"antagonistic" at the 100:1 ratio.
In a second experiment, cells were treated with 5 yM parabactin
for 5 h, washed free of the ligand, placed in fresh medium to allow the
cells to cascade into the S phase of the cell cycle, and grown with
0.02 yM or 0.03 yM Adriamycin (Fig. 2-11). In this experiment the
parabactin clearly potentiated the activity of Adriamycin (see Discus¬
sion). Finally, in a third experiment cells were treated with 5 yM
parabactin for 4 h and then Adriamycin 0.2 yM was added for an addi¬
tional 2 h. Cells were washed, reseeded and grown in fresh drug-free

49
TIME (hours)
Figure 2-9. Time course for the incorporation of [^H]tny-
midine into the acid-precipitable fraction of L1210 cells grown in
fresh medium after treatment with 5 yM parabactin for 5 h.

LOG Fa/Fu
Figure 2-10. Median effect plots for L1210 cells exposured for
48 h to Adriamycin, parabactin, and the simultaneous combination of
parabactin:Adriamycin at a molar ratio of 100:1.
c_n
o

51
Figure 2-11. The effects of Adriamycin on the growth of
L1210 cells, and Adriamycin on the growth of cells which have been
pretreated with 5 yM parabactin for 5 h then washed to remove the
block in DNA synthesis. The growth of control cells and cells
exposed to 5 yM parabactin for 5 h and resuspended in drug-free
medium is shown for comparison.

52
medium (Fig. 2-12). The results indicated additivity of the two drug
effects.
Ara-C-treated Cells.--The IC^g of ara-C in our L1210 cell assay
system was 0.033 yM, L1210 cells were treated with parabactin and
ara-C simultaneously at a constant molar ratio of 100:1. The concen¬
trations at a constant ratio of parabactin to ara-C were 1.75:0.0175,
1.5:0.015, 1.25:0.0125, 1.0:0.01, 0.75:0.0075, and 0.5:0.005. The IC5Q
of the combination was 1.39 yM (Cl = 1.33). Again, Chou and Talalay
analysis of the growth data indicated "antagonism" (Fig. 2-13).
However, when cells were first treated with 5 yM parabactin for 5
h, washed with fresh medium, and followed by treatment with ara-C, as
with the Adriamycin:parabactin combination, the parabactin potentiated
the activity of the ara-C (Fig. 2-14).
When cells were simultaneously treated with 5 yM parabactin and
1 yM or 2 yM ara-C for 5 h (Fig. 2-15), or first treated with 2 yM
parabactin for 4 h then 1 yM or 2 yM ara-C for an additional 12 h, then
washed, reseeded and grown in fresh drug-free medium (Fig. 2-16), the
results indicated additivity of the two drug effects for both combina¬
tions.
BCNU-treated Cells.--The IC^g for BCNU in our system was 4.13 yM.
When cells were treated simultaneously with parabactin and BCNU at a
molar ratio of 1:1, the ICgg of the combination was 2.28 yM (Cl = 1.1),
indicating additivity (Fig. 2-17). The absolute micromolar drug
concentrations to which cells were exposed were 3:3, 2:2, 1:1, 0.5:0.5,
and 0.25:0.25.

53
Figure 2-12. The effects of 0.2 yM Adriamycin exposure for 2
h, parabactin 5 yM treatment for 6 h, and 5 yM parabactin for 4 h
plus the simultaneous exposure of 0.2 yM Adriamycin for an addi¬
tional 2 h on the growth of L1210 cells. Treated cells were washed
and resuspended in drug-free medium at time = 0 h.

LOG Fa/Fu
Figure 2-13. Median effect plots for L1210 cells exposed for 48 h
to ara-C, parabactin and the simultaneous combination of parabactin:ara-C
at a molar ratio of 100:1.
<_n
4^

55
TIME (hours)
Figure 2-14. The effects of ara-C on the growth of L1210
cells, and ara-C on the growth of cells which have been pretreated
with 5 pM parabactin for 5 h then washed to remove the block in DNA
synthesis. The growth of control cells and cells exposed to 5 pM
parabactin for 5 h and resuspended in drug-free medium is shown for
comparison.

Figure 2-15. The effects of ara-C exposure for 5 h,
parabactin 5 yM treatment for 5 h, and the simultaneous exposure of
5 yM parabactin plus ara-C for 5 h on the growth of L1210 cells.
Treated cells were washed and resuspended in drug-free medium at
time-0 h.

57
Figure 2-16. The effects of ara-C exposure for 12 h on the
growth of L12110 cells, and 2 yM parabactin treatment for 4 h plus
exposure to ara-C and parabactin for an additional 12 h on the
growth of L1210 cells. The growth of control (untreated) cells and
cells exposed to 2 yM parabactin for 16 h is shown for comparison.
Treated cells were washed and resuspended in drug-free medium at
time-0 h.

A BCNU
• Parabactin
â–¡ Combination (1:1, Parabactin:BCNU)
-1.5 H 1 i < 1 —i 1 1 1 1 > 1 1 1 ■ 1 ■
- 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
LOG DOSE (uM)
Figure 2-17. Median effect plots for L1210 cells exposed for 48 h
to BCN1J, parabactin and the simultaneous combination of parabactin:BCNU
at a molar ratio of 1:1.

59
When cells were first treated with 5 pM parabactin for 5 h,
washed with fresh medium, and treated with BCNU immediately, an
additive effect was again observed (Fig. 2-18).
However, when cells were first exposed to 5 pM parabactin for 3
h, then presented with varying concentrations of BCNU for an additional
5 h, washed, and resuspended in fresh medium, cell growth indicated
parabactin potentiation of BCNU activity (Fig. 2-19).
Animal Studies.--Ascites L1210 tumor cells in DBA/2 mice were
blocked and synchronized by a single i.p. injection of parabactin 100
mg/kg (Fig. 2-20). The DNA histogram of the untreated ascites tumor
shows a DNA content distribution similar to that of the cultured cells.
At seven hours after the parabactin treatment a block in DNA synthesis
at the Gj-S border of the cells is evident by the reduction in G£ phase
content and the broadening of the G^-S content peak. At 8 h cells
which were blocked at the G^-S border begin to enter S phase, and by
9 h after the injection a large majority of the ascites cells have S
phase DNA content indicating a synchronous population of cells.
There was no significant increased life span in mice given
1 x 105 L1210 cells i.p. and then treated with a daily (0D) injection
of parabactin 100 mg/kg on days 1-6 compared to untreated mice or mice
treated with only the 10% Cremophore drug vehicle. However, there was
an 80% reduction in the ascites tumor burden of mice treated with para¬
bactin 100 mg/kg every 8 h (98 h) for 5 injections beginning on day 5,
compared to untreated mice. This dosage schedule could not be

60
Figure 2-18. The effects of BCNU on the growth of L1210
cells, and BCNU on the growth of cells which have been pretreated
with 5 pM parabactin for 5 h then washed to remove the block in DNA
synthesis. The growth of cells exposed to 5 yM parabactin for 5 h
and resuspended in drug-free medium is shown for comparison.

61
TIME (hours)
Figure 2-19. The effects of BCNU exposure for 5 h,
parabactin 5 pM treatment for 8 h, and 5 pM parabactin for 3h plus
the simultaneous exposure to BCNU for an additional 5 h on the
growth of L1210 cells. Treated cells were washed and resuspended
in drug-free medium at time-0 h.

NUMBER OF CELLS
62
Figure 2-20. Flow cytometric analysis of ascites L1210 cells
from DBA/2 mice after a single i.p. injection of parabactin 100
mg/kg.
A. 0 h
B. 7 h
C. 8 h
D. 9 h

63
continued for more than the 5 doses in the mice due to the observed
toxic effects (constipation and death) of the Cremophore vehicle when
administered alone at concentrations necessary to solubilize this dose
of parabactin.
Various combinations of parabactin with ara-C or Adriamycin in
mice with L1210 tumors (Table 2-4) did not disclose any significant
increases in life span beyond the additive effects of the individual
treatments.
Discussion
Iron chelators have been shown to have profound effects on the
growth properties of various tumor cell lines (37,41-43). The antipro¬
liferative activity of the siderophores against L1210 cells as
assessed by their IC^q values indicated that, when ranked according to
their ability to sequester iron, the compounds with the higher iron
binding constants generally demonstrated better antileukemic activity
(37). The most active of the compounds tested here (the hexacoordinate
catechol amide chelators, vibriobactin and parabactin) have an enormous
affinity for iron and exhibited the most potent antiproliterative
activity with IC^q values against LI210 cells of 2 yM and 1.5 yM,
respectively. Both compounds were able to cause nearly complete cessa¬
tion of cell growth in 24 h at concentrations above 5 yM while below 1
yM little growth inhibition was seen.
In an effort to ascertain the broadness of activity of the cate-
cholamide chelators, they were tested against various other cultured
cell lines. Parabactin and vibriobactin were found to be potent growth
inhibitors of various animal and human tumor cells with 48 h IC^g

TABLE 2-4
ACTIVITY OF PARABACTIN, ARA-C AND ADRIAMYCIN, EITHER ALONE OR IN COMBINATION, AGAINST L1210
MURINE LEUKEMIA IN DBA/2 MICE
Experi-
ment
Number
Drug
Treatment
Dosage
Schedule
Mean
Survival
(days) ± S.D.
% ILS
1.
Parabactin
100 mg/kg
OD day 1
9.5 ± 1.0
3
Ara-C
120 mg/kg
Day 1
12.0 ± 1.9
23
Ara-C/Parabacti na
OD day 1
12.6 ± 1.8
29
NONE
OD
9.7 ± 0.8
0
2.
Parabactin
100 mg/kg
ql2h days 1-2
11.0 ± 2.2
13
and 6-7
Ara-C
40 kg/mg
ql2 h days 1-2
19.5 ± 2.7
100
and 6-7
Ara-C/Parabactinb
Days 1-2 and
15.7 ± 5.9
62
6-7
NONE
9.8 ± 0.8
0
3.
Parabactin
100 mg/kg
OD day 1
10.0 ± 1.0
5
Adrianycin
2 mg/kg
OD day 1
10.5
11
Adriamycin/Parabactinc
OD day 1
11.5 ± 1.2
21
NONE
9.5 ± 0.7
0
4.
Parabactin
100 mg/kg
OD days 2 and 4
10.0 ± 1.0
11
Adriamycin
3 mg/kg
OD days 2 and 4
14.1 ± 5.2
57
Adriamycin/Parabactind
OD days 2 and 4
15.1 ± 3.3
68
NONE
9.0 ± 0.4
0
aAra-C given 7 h after parabactin. NOTE:
bAra-C alternated ql2 h after parabactin.
dAdriamycin given 7 h after parabactin
dAdriamycin given 7 h after parabactin.
DBA/2J mice given 10b L1210 cells i.p. on day 0.
All treatments given by i.p. injection.
OD = Once daily.
q!2h = every 12 h.

65
values of approximately 2 pM for all cell lines tested. Parabactin was
also found to be a potent inhibitor of actively-dividing normal HFF
cell growth with a 96 h IC^g value of 2 pM.
That growth inhibition is due to iron chelation seems apparent
from studies in which iron, added at the same time as the chelators,
prevents the effects. This is consistent with previous findings (37)
in which the antileukemic and antiviral viral activities of compound II
could similarly be prevented. Also, methylation of the ligand's cate¬
chol groups essentially blocking the siderophore's chelating function¬
alities (Fig. 1-3) eliminated the antileukemic activity of vibriobactin
and greatly reduced the activity of parabactin (Figs. 2-2 and 2-3).
The residual activity of tetramethyl parabactin is likely due to the
remaining central oxazoline nitrogen and phenolic hydroxyl group's
ability to chelate iron. It has also been shown that parabactin was
able to suppress the growth of a variety of bacteria, while the addi¬
tion of the tetramethylated parabactin analogue or the preformed
ferric-parabactin chelate was unable to inhibit microbial prolifer¬
ation (72).
Cytostasis (apparent by dye exclusion, Rh-123 mitochondrial stain¬
ing, ^Cr release and clonagenic assays) is observed on a short-term
exposure (5 h) of L1210 cells to 5 pM parabactin or 10 pM vibriobactin,
and cidal activity when the cells are exposed for longer periods of
time (Table 2-2). The stasis is reversible by simply washing the lig¬
and away from the cells within the first 5 h. However, a nondividing
confluent monolayer of HFF cells exposed to 20 pM parabactin (10 times
its IC^q value) for 24 h exhibited no apparent toxic effects.

66
The effects of vibriobactin and parabactin on L1210 cell growth
are even more apparent after examining their impact on cell cycle kin¬
etics. Both ligands hold the cells at the G^-S border until they are
released by washing the chelator away, at which point cells cascade
into S phase (Figs. 2-7 and 2-8). This synchronous population of cells
can be followed through the cell cycle with time, and in the case of
parabactin a portion of the cells remained synchronized for 3 cell
cycles. When parabactin treated cells are reseeded in fresh medium
there is approximately a 14 h lag in growth relative to untreated con¬
trols. At this point cells appear to grow normally with a doubling
time of 12 h (Figs. 2-11, 2-14, 2-18.)
Cycling of [^H]thymidine incorporation into DNA (Fig. 2-9) during
the first 12 h after washing parabactin from treated cells, or cycling
of BrdUrd incorporation into DNA after vibriobactin pretreatment (Table
2-3) was consistant with the time frame of changes in S-phase followed
by flow cytometry. However, the percentage of control incorporation
during certain phases in the cell cycle reveals an actual DNA synthetic
rate which does not correspond to the levels expected from the percent¬
ages of cells with S-phase DNA content by flow cytometry (Fig. 2-8).
For example, flow cytometry indicated that at 8 h after washing the
percentage of cells having S-phase DNA content is approximately twice
that (200%) of control; however, [^H]thymidine incorporation corre¬
sponds to a DNA synthetic rate of only 120% of control. From these
studies it seems that while a sizeable fraction of the cells moves
synchronously through the cell cycle in 10 to 12 h, there is also a
fraction of cells which are moving more slowly through the cell cycle.
Together, the fraction of cells moving more slowly through the cell

67
cycle along with the decrease in viability of cells after short-term
parabactin treatment may account for the 14 h delay in cell growth
relative to controls.
The phase specificity of the effects of the catechol amides is
similar to that seen with hydroxyurea (80-81) which is believed to
inhibit cell growth through interference with the enzyme ribonucleotide
reductase by a free radical seavaging mechanism (26). The hydroxamate
iron chelator desferrioxamine has also been shown to inhibit DNA syn¬
thesis through interference with the same enzyme. Interfering with
this enzyme's activity blocks DNA synthesis, and cells accumulate at
the G^-S boundary of the cell cycle (81). It has been demonstrated
that parabactin is a potent inhibitor of ribonucleotide reductase
(38).
It is highly unlikely that the growth inhibition and cell cycle
effects of the catecholamides are a consequence of extracellular iron
chelation. It has been shown that the removal of iron from transferrin
by siderophores occurs at a rate greater than 10% per hour only at high
ligand to transferrin ratios (82), and removal is impeded in the pres¬
ence of serum proteins (83). Transferrin is supplied to the cell
growth medium along with other growth factors as a 10% serum supple¬
ment. Unless the removal of iron from the extracellular medium by the
chelator occurred at a rate far in excess of 10% per hour it would not
account for the early onset of inhibitory effects of the siderophores
on DNA synthesis (3 h) (Fig. 2-5) or cell growth (12 h) (Fig. 2-3)
since L1210 cells grown in medium with 5% or 10% serum proliferate at
essentially the same rate. Also, there was not a proportional increase
in IC^Q values or significant change in the time to obtain a complete

68
cell cycle block when parabactin treated cells are grown in 5%, 10% or
20% fetal calf serum. If cell growth inhibition required an estab¬
lished concentration of chelator to limit extracellular iron, there
should be a proportional response of IC^q values in relation to changes
in serum concentration in the medium. Hence, if deprivation of iron to
the cells caused by chelator-induced deferration of transferrin in the
medium was the mechanism by which the catecholamides inhibit DNA syn¬
thesis, the times necessary to see a reduction in cell growth (5 yM
parabactin for 12 h) and DNA content (5 yM parabactin for 3 h) would be
expected to be much greater. Finally, there exists a good correlation
between the antiproliferative activity and 1 ipophi1icity of various
catecholamide chelators examined (37). This correlation would not be
expected if extracellular chelation were taking place.
The effects of the parabactin on the cell cycle kinetics of L1210
cells were compared with the effects of desferrioxamine and hydroxyurea
(Fig. 2-6). Cells exposed to 5 yM parabactin experienced a block at
the Gj-S border in 5 h, while desferrioxamine was required to be in
excess of 100 yM for a 5 h exposure. Finally, hydroxyurea needed to be
in excess of 300 yM concentration for the same 5 h exposure time. It
is interesting that the concentration of desferrioxamine required to
block cells at the Gj-S border is in excess of 13 times the 48 h IC^g
value of desferrioxamine and 7.5 times the 48 h IC^q value of hydroxy¬
urea. The cell cycle blocking ability of parabactin at a concentra¬
tion of 3.5 times its IC^q value was therefore far superior to that

69
of hydroxyurea or desferrioxamine. The release of the desferrioxamine
(84) and hydroxyurea (85) blocks results in less effective cell cycle
synchronizations, as the DNA histograms are normalized following one
passage through the cell cycle.
As mentioned above, the reversibility of the effects of parabac-
tin is dependent on how long the cells are exposed to the ligand. For
example, when the cells are exposed to 5 yM parabactin for 48 h 59% of
the cells die and 10 yM desferrioxamine kills 60% (Table 2-2).
The potent cell cycle blocking ability of parabactin and its
reversibility suggested its application in combination with other
phase-specific antineoplastic drugs.
Parabactin-treated cells offer two opportunities for potentiation
of growth inhibition.
a. Agents acting at the G^-S border should be more effective
since the cells are held at this phase.
b. Agents acting during S phase should have improved activity
since a larger percentage of the cell population will be synchronized
into the S phase when the parabactin block is released.
When utilized in combination with ara-C, Adriamycin, or BCNU,
parabactin can produce "antagonistic," "additive," or "synergistic"
(potentiation) effects depending on the time frame during which the
drugs are applied.
Two basic experiments were run in the combination chemotherapy
studies.
a. Parabactin was used in simultaneous combination for 48 h with
BCNU, ara-C, or Adriamycin.

70
b. Cells were first blocked with parabactin and held at the G^-S
border, then the parabactin was washed away allowing cells to cascade
into S phase. The cells were then exposed to BCNU, ara-C, or Adriamy-
ci n.
The latter experiments relied heavily on cell cytometry tech¬
niques to evaluate the fraction of cells in each phase of the cell
cycle after treatment with the catechol amide chelator.
A third experiment was performed with RCNU. The cells were
treated with parabactin for 3 h and then with BCNU for 5 h. Then,
after both drugs were washed away, cells were resuspended and regrown.
When cells are exposed to parabactin and ara-C simultaneously for
48 h, the effect of the combination is an "antagonistic" one (Fig.
2-13). This is in keeping with the previous observations that ara-C,
an inhibitor of DMA polymerase (86), kills cells best in S phase (87)
and that parabactin holds cells at the G^-S border. This parabactin
block thus diminishes the S-phase component of the cell population
reducing the number of cells most susceptible to ara-C. Finally, the
"antagonism" is apparent from the analysis of drug-induced growth
inhibition. The IC^q value for the ara-C direct combination with para¬
bactin at a 100:1 ratio was 1.33 yM with a CI>1 indicating an
"antagonistic" effect.
However, when cells were first exposed to 5 yM parabactin for 5
h, washed free of the ligand releasing the block, and then treated with
ara-C, the parabactin potentiates the activity of ara-C particularly at
0.04 yM ara-C (Fig. 2-14). Inspection of Figure 2-14 reveals that the
growth of parabactin-treated cells is approximately 14 h behind the

71
growth of the control cells. Consequently, when comparing the effects
of the parabactin:ara-C-treated cells with ara-C treated cells, the 14
h lag for the parabactin-treated cells must be considered. Therefore,
in evaluating the combination curve relative to the ara-C curve, one
must look at a point on the combination line 14 h ahead of the ara-C
line. Even at this, it is clear that parabactin potentiates the activ¬
ity of ara-C.
The same experiments were carried out with Adriamycin. The sim¬
ultaneous drug combination experiment with 100:1 ratio of parabactin to
Adriamycin indicated "antagonism." Applying Equation A to the data
from Figure 2-10 resulted in a Cl value of 1.57 indicating antagonism.
However, again when the cells were treated first with parabactin to
initiate a block at G^-S and then washed free of the ligand and treated
with Adriamycin, the effect of Adriamycin was potentiated particularly
at 0.03 yM (Fig. 2-11). Again, one must consider the 14-h lag time in
growth generated by the parabactin. However, even after accounting for
this, reduction in growth rate is about twice that observed for the
Adriamycin itself. In view of the toxicity of Adriamycin, this may be
of some practical significance. As both Adriamycin (88) and ara-C are
proposed to operate best on cells in the S phase, the observed results
are as expected.
The final drug studied in this evaluation was BCNU. When cells
were exposed to parabactin and BCNU simultaneously for 48 h the results
indicated the combination to be additive with a Cl approximately equal
to 1 (Fig. 2-17).
When the cells were first treated with 5 yM parabactin for 5 h,
and then released and immediately treated with BCNU (Fig. 2-18), the

72
effects were still additive. However, when the cells were treated with
5 yM parabactin for 3 h, then exposed to BCNU for an additional 5 h,
and then washed and resuspended in culture, there is clearly a potenti¬
ation of the combined drug effects (Fig. 2-19). After accounting for
the parabactin-induced lag the effect of the BCNU was two times as
great as it was in the absence of parabactin. It was interesting to
note that 1 yM BCNU in combination with parabactin was more active than
3 yM BCNU alone. In fact, cells treated with 1 yM BCNU (not shown)
grew at the same rate as controls. The literature suggests that BCNU
operates best at the G^-S border and in G£ of the cell cycle, while
cells in S phase are more resistant (89). The BCNU potentiation
effects of the drug combination were consistent with the observation
that cells treated with parabactin were held at the Gj-S border. It
remains somewhat unclear, however, why we did not observe synergistic
effects when the drugs were used in direct simultaneous combination
without washing.
The above observations also suggest that proper pretreatment of
cells with parabactin could result in a decrease of the cytoxic effects
of phase-specific antineoplastics by reducing the population of cells
in the phase where the chemotherapeutic exerts its greatest cytotoxic
action. In an attempt to demonstrate the possible "protective" effects
of parabactin treatment against phase-specific toxicity, several exper-
ments were devised. Cells were first treated with parabactin to reduce
the S-phase population of cells. Adriamycin (Fig. 2-12) or ara-C
(Fig. 2-16) was then added for an additional amount of time, and the

73
cells were washed and regrown in drug free medium. Additionally, cells
were treated simultaneously with both parabactin and ara-C for 5 h and
the cells were washed and regrown (Fig. 2-15). The results revealed
additive effects of the combinations in all cases, thus failing to
indicate any protection afforded cells out of synchronization with the
phase specificity of ara-C or Adriamycin by parabactin treatment. The
implications of these results may demonstrate, that while ara-C and
Adriamycin are reported to exert their greatest cytotoxic effects on
cells in S phase, they may enter the cell during any phase of the cell
cycle. These results further imply that studies of simultaneous com¬
binations for these types of drugs deserve further consideration.
Analysis of the median effect plots by the method of Chou and Talalay
(73) is only a mathematical manipulation of the 48-h IC^q values of the
drugs at one specific molar ratio. The Cl may not be a true reflection
of the activity of the simultaneous combination of two compounds with
such different mechanisms of action and the results must be interpreted
with care.
The ability of parabactin to enhance the chemotherapeutic effects
of various antineoplastic drugs was highly dependent on its ability to
block cell cycle kinetics and to effectively reverse this block releas¬
ing the cells into a synchronized cell cycle. A consideration of the
IC^q values of the drugs is probably relevant when considering the
effectiveness of these compounds as cell cycle blocking agents. At 3.5
times its IC5Q parabactin was an excellent cell blocking agent; it was
better than desferrioxamine at greater than 13 times its IC^g and the
hydroxyurea at 7.5 times its IC^q (Fig. 2-6). Furthermore, one of
the most critical issues was the fact that parabactin-treated cells
maintained partial synchronization for at least three cell cycles while

74
cell synchronization disappeared after one cell cycle in desferriox-
amine- and hydroxyurea-treated cells.
The antiproliferative effects of parabactin were also studied in
vivo. The L1210 cell tumor model has been used as a useful preclinical
screening system for potential antineoplastics (90). The murine L1210
leukemia is a convenient and very reproducible in vivo model system.
The tumor is very aggressive having an in vivo doubling time of about
10-12 h. Mean survival time of animals injected with 10^ cells is
approximately 9.5 days. It has been determined that one viable cell
will kill an animal in about 18 days with a tumor burden of approxi¬
mately 1010 cells (91). Therefore, it would require a one log reduc¬
tion in cell number (90% cell kill) to see a one day increase in life
span (ILS), and a two-log reduction in cell number (99% cell kill) to
see a 2-3 day ILS, while even with a 99.99% cell kill the animals would
still die in less than 20 days.
It is, therefore, not very surprising that leukemic mice treated
with parabactin, which requires constant exposure of cells for signifi¬
cant periods of time to exhibit cytotoxic activity, on a single daily
injection schedule for 6 days, showed no increased life span over
untreated mice. However, leukemic mice treated with parabactin every
6 h for 5 injections did exhibit a reduction in tumor burden of 80%
over controls. Considering the doubling time of this tumor, even this
reduction in cell number would not extend the life of the mice to a
significant degree. A drug having an ILS of greater than 35% in this
system is considered to have significant activity by the National

75
Cancer Institute (92). Extended dosing on this schedule, while exhib¬
iting promising activity, is prevented by the low solubility of the
catecholamides in most vehicles suitable for parental administration
(the solubility of parabactin in phosphate buffered saline being on the
order of 1.25 x 10"^ M) (37), and the toxicity of the vehicle alone on
frequent administration.
The ability of parabactin to block and synchronize cells has also
been demonstrated in vivo. Ascites L1210 cells were synchronized by a
single i.p. injection of parabactin (Fig. 2-20) in a manner similar to
that seen in culture. Attempts to demonstrate enhanced cytotoxic
effects of ara-C and Adriamycin on a parabactin synchronized in vivo
cell population resulted in no significant increase in life span of
L1210 mice over the additive effects of the individual treatments
(Table 2-4). Considering the optimum conditions studied with cultured
cells, which exhibited a maximum 2-3 fold potentiation of the cytotoxic
effects of the antineoplastics tested, very little increase in life
span would be expected in vivo by a reduction in tumor burden of
50-75%. Further refinements in the drug dosage and multiple dosing
schedules are currently under investigation.

CHAPTER III
POLYAMINE ANALOGUES
Materials and Methods
The polyamine analogues were synthesized by methods previously
described (30,31). Stock solutions (10 mM) of analogues and dilu¬
tions were made in sterile water, and passed through a 0.2 ym filter
prior to use. RPMI 1640, fetal bovine serum, HEPES, and MOPS were
obtained from Gibco (Grand Island, NY). Cell culture flasks, 25 and
75 cm^, were purchased from Corning (Corning, NY). Diamidino-
phenylindole (DAPI), propidium iodide (PI), actinomycin-D and pro¬
teinase K were obtained from Sigma (St. Louis, MO). Rhodamine-123
(Rh-123) was purchased from Eastman Kodak Company (Rochester, NY).
RNase T1 was obtained from BRL Scientific (Gaithersburg, MD). DBA/2J
and C57B1/6J mice were obtained from Jackson Laboratories (Bar Har¬
bor, ME). Albino CD-I mice were obtained from Harlan Sprague-Dawley
(Indianapolis, IN). Sprague-Dawley rats were obtained from Charles
River (Wilmington, MA).
Cell Culture
All cell culture lines were grown as previously described in
Chapter II. Several additional cell lines utilized in these studies
were maintained in complete medium as before except where noted.
O
Chinese hamster lung (DC3F) cells were grown as monolayers in 25 crrr
flasks, seeded at 2 x 10^ total cells and grown to confluency in
in approximately 96 h. Actinomycin-D resistant Chinese hamster lung
76

77
(DC3F/ADX) cells were similarly maintained with actinomycin-D 10
yg/mL present in the medium. The Chinese Hamster Lung cell lines,
DC3F and DC3F/ADX, were kindly donated by Dr. June Biedler of
Memorial SIoan-Kettering Cancer Center, NY.
Resistant L1210 cell Lines
L1210 cell lines resistant to DESPM (LI210/DES-10) and DF.HSPM
(L1210/HDES-1) were selected out by maintaining the cells in increas¬
ing concentrations of polyamine analogues over an extended period of
time. Initially, L1210 cells were exposed to either 1 yM DESPM or
0.1 yM DEHSPM for seven days and maintained between 1 x 10® and
1 x 10® cells/mL. Cells were then washed free of the drug and incu¬
bated in fresh medium until cell doubling time returned to normal (12
h). These cloned cells were then again exposed to a higher concen¬
tration of the analogue until normal cell doubling time resumed.
Analogue concentrations were gradually increased over a period of
approximately one month until cells could be maintained at a concen¬
tration of 10 yM DESPM and 1 yM DEHSPM. Resistant cells maintained
at these concentrations of analogue for over six months had normal
L1210 morphology and doubling time.
An Adriamycin resistant L1210 cell line (LI210/D0X-0.6) was
similarly selected out by initially exposing cells to 0.01 yM Adria¬
mycin. When approximate normal doubling time resumed, the Adriamycin
concentration was increased until over a period of three months the
cells could be maintained in the presence of 0.6 yM Adriamycin. The
L1210/D0X-0.6 cells maintained for over six months differed from the
normal L1210 cells in that they had irregular shapes and longer doub¬
ling times of 14-16 h.

78
IC^q Determinations
The cells were treated while iri logarithmic growth (LI210
cells, 3 x 10^ cells/mL; Daudi and HL-60, 1 x 10^ cells/mL) with the
polyamine derivatives diluted in sterile water and filtered through a
0.2 yM filter immediately prior to use. Following a 48 h incubation
with L1210 cells and a 72 h incubation with Daudi or HL-60 cells,
cells were reseeded (L1210 cells, 3 x 10^ cells/mL; Daudi and HL-60
cells, 1 x 10^ cells/mL) and incubated in the presence of the poly¬
amine derivative for an additional 48 h or 72 h, respectively.
Determination of IC$q values for the most active compounds
(IC50 ^ v‘m at ^6 11) was a^so Per^ormecl in the presence of 1 mM
aminoguanidine, a serum diamine oxidase inhibitor (93).
Chinese hamster ovary (CH0) cells and murine B-16 melanoma
cells were seeded at 2 x 10^ cells/25 cm^ flask and allowed to attach
for 4 h. At this time the cells were exposed to the polyamine ana¬
logue for 48 h. Monolayer cells were not reseeded for additional
exposure times due to problems in plating efficiency after the ini¬
tial 48 h treatment.
Cell samples were removed at the indicated time periods for
counting. Cell number was determined by electronic particle analysis
(Coulter Counter, Model ZB1, Coulter Electronics, Hialeah, FL) and
confirmed periodically with hemocytometer measurements.

79
The percentage of control growth was determined as follows:
% of control growth = 100 x [Final treated cell no.-
initial inoculum]/[Final untreated cell no.-initial inoculum]
The IC^Q is defined as the concentration of compound necessary
to reduce cell growth to 50% of control growth after defined inter¬
vals of exposure.
Analysis of Drug Effects on Mitochondrial DNA
The LI210 cells (in complete media at 1 x 10^ cells/mL) were
incubated at 37°C in the presence of the compound to be tested.
Every 24 h, for 144 h, cell samples were removed for counting, and
the cells were reseeded in fresh medium and drug at 1 x 10^ cells/mL
Cells were assayed daily for mitochondrial DNA (mtDNA) content.
Mitochondrial DNA assays were performed by R. Bortell and L. Raynor
of Dr. A. Neims' laboratory, Department of Pharmacology and Thera¬
peutics, College of Medicine, University of Florida.
Because recovery of the organelles might vary with drug treat¬
ment, a dot blot procedure for assay of mtDNA was developed which
involves analysis of cell lysates rather than preparations of mito¬
chondria (94). Cells (5 x 10^ to 2 x 10^/mL) were lysed in 2% SDS i
the presence of proteinase K (5 mg) and RNase T1 (100 units). The
total cell lysate was applied to nitrocellulose paper with use of a
96-well, vacuum operated dot-blot apparatus. The blots were hybrid-
oc
ized to a S-labeled dATP nick translated probe made by inserting
full-length mouse mtDNA into pSP64 vector at the Sacl site. Dot
blots were visualized by autoradiography and cut out, and radioactiv
ity was determined by scintillation counting.

80
Flow Cytometric Analysis
Flow cytometric analysis of nuclear DNA (nDNA) content was
performed with the RATCOM flow cytometer (RATCOM Inc., Miami, FL)
interfaced with a microcomputer (IBM-XT). Cultured LI210 cells in
log-phase growth were incubated at 37°C with various polyamine ana¬
logues and samples removed at 0 h, 48 h, 96 h and 144 h were analyzed
for nDNA content distributions after staining with diamidinophenyl in¬
dole in a nuclear isolation media (NIM-DAPI) (75).
Alternately, cell analysis was performed with a FACS II flow
cytometer (Becton Dickinson FACS Systems, Sunnyvale, CA) interfaced
with a microcomputer (Hewlett Packard 45B, Fort Collins, CO).
Samples of 10^ cells were removed and stained with propidium iodide
(PI) (Calbiochem, San Diego, CA) and then exposed to RNase. DNA
distributions were obtained from analysis of the red fluorescence
from the Pi-stained DNA (76).
Cloning Assay
L1210 cells maintained between 105 and 10^ cells/mL were incu¬
bated with 10 pM DESPM for 96 h. At 24 h intervals treated cells
were washed (2 x 10 mL) and diluted in fresh complete media and
plated in triplicate 96-well microtiter plates at 0.4 cells/well with
each well containing 100 pL of sample. The plates were incubated at
37°C in a humidified incubator in an atmosphere of 5% C0£ and 95%
air. The plates were examined with an inverse phase microscope at
100X magnification. The final number of colonies per plate was quan¬
titated seven days after plating. Groups of 50 or more cells/well
were identified as having been cloned from a single viable cell.

81
Regrowth Studies
L1210 cells maintained between 10^ and 10^ cells/mL were incu¬
bated with 10 yM DESPM, 30 yM DENSPM or DEHSPM 0.6 yM for 96 h. At
each 24 h interval, treated cell samples were washed and the cells
resuspended in fresh media at 1 x 10^ cells/mL in duplicate 10 mL
flasks. The regrowth of the treated cells was followed for up to
144 h.
Cell Size
Cell size was determined directly by the method of Schwartz, et
al. (95). In brief, uniform polymeric microspheres ranging from
4.72-10.2 yM in diameter (Polysciences, Warrington, PA) were diluted
in Hematall (Fisher Scientific Co.). Electronic size was measured on
the FACS Analyzer (Becton Dickinson, Sunnyvale, CA) with the ampli¬
fier in the log mode. The peak channel number for each size micro¬
bead was plotted against the correspondí'ng calibrated diameter and
calculated volume to obtain a calibration curve. L1210 cells were
treated with 10 yM DESPM, 30 yM DENSPM or DEHSPM 0.6 yM for 0-144 h
and samples of 10^ cells were removed at 24 h intervals and pelleted.
The cells were resuspended in 0.5 mL of Hematall and analyzed. The
peak channel number of the treated cells was plotted on the
calibration curve to obtain the approximate cell size directly.
Viability Assay
The viability of cells which had been treated with 10 yM DESPM
or 0.3 yM DEHSPM and or 30 yM DENSPM maintained between 5 x 10^ and

82
1.2 x 106 cells/mL was assayed at appropriate times using two dif¬
ferent methods: trypan blue dye exclusion and Rh-123 mitochondrial
staining (74).
Briefly, 1 x 106 cells were pelleted by centrifugation and
resuspended in 1 mL of complete medium containing Rh-123 10 yg/mL,
incubated at 37°C for 10 min, and washed once with medium. Trypan
blue was added prior to counting to give a final concentration of 0.2
yg/mL. Rh-123 and trypan blue stained cultures were observed in a
hemocytometer using a Zeiss epifluorescence Axioscop microscope at
630X magnification. The excitation wavelength was 485 nm. Viable
cells show mitochondrial specific uptake of the Rh-123 dye while
dying cells showed diffuse cytoplasmic staining and dead cells did
not fluoresce at all. Cells which did not fluoresce did not exclude
trypan blue. Both methods gave comparable results as to cell death,
however, trypan blue exclusion does not reflect dying cells.
Animal Studies
The polyamine analogue was diluted in sterile 0.9% saline
within 24 h of use and the unused portion stored at 5°C. Concentra¬
tions of the spermine analogue tetrahydrochlorides, at each dose were
adjusted so that the mice were injected with a volume of 1 mL/100 g
(i.e. a 25 g mouse was injected with 0.25 mL of drug solution).
Untreated mice served as controls.
The compounds were administered by i.p. injection according to
the appropriate dosing schedule. The murine L1210 leukemia cells
were maintained in DBA/2J mice. Cells from a single mouse which
was injected i.p. with 1.25 x 10^ cells seven days earlier were

83
harvested and diluted with cold saline so that an inoculum of 10^ or
10^ cells could be administered by a 0.25 mL i.p. injection. In
each study mice were injected i.p. with the appropriate number of
L1210 cells on day 0.
Alternately, Alzet mini-osmotic pumps, model 2001 (Alza, Palo
Alto, CA) designed to deliver 1 yL/h for 7 days, were filled with
DEHSPM so that the pump would deliver 20 mg/kg/day. The filled pumps
were placed in sterile saline 0.9% and incubated at 37°C for 8 h
prior to placement in the mice. The pumps were implanted, on day 1,
subcutaneously (s.q.) in the dorsal side of the mice through an
incision behind the right flank, and the incision closed with a wound
clip. After 6 days the pumps were removed and the wound closed. The
parameter used for treatment evaluation of activity against L1210
leukemia was mean survival time (percent increased life span, % ILS).
%ILS = 100 x [mean survival time treated animals-
mean survival time controls]/[mean survival time controls]
The murine B-16 melanoma was maintained in C57B1/6J mice. The
solid tumor was excised from a single mouse, which was injected sub¬
cutaneously (s.q.) 14 days earlier, and minced with 9 volumes of
Hanks' balanced salt solution (10:1 brei). The brei (0.1 mL) was
administered by s.q. injection in the right flank of the animal on
day 0.
The murine Lewis lung carcinoma was maintained in C57B1/6J
mice. The solid tumor was excised from a single mouse which was
injected s.q. 14 days earlier, and the tumor divided into equal

34
fragments 2-4 mm in size. Each mouse was injected s.q. with a frag¬
ment in the sternum region using a 14 g trochar needle on day 0.
The parameter measured for treatment evaluation was median
tumor weight change based on length and width measurements in milli¬
meters (92). The tumor weights (mg) were calculated from tumor
dimensions (mm x mm) following the formula for volume of a prolate
el 1ipsoid:
L x where L is the longer of the two
2 measurements recorded
Median tumor weights were calculated for test (T) and control (C)
groups on days 10, 14 and 17 after inoculation of the tumor.
The activity of the analogue was reported as T/C56 on the
measurement day giving the lowest value.
Rates of Uptake of Polyamine Analogues in L1210 Cells
L1210 cells maintained in mid-log phase growth were exposed to
the polyamine analogues for 0, 2, 6, 12 and 24 h, and samples of 10^
cells were removed. The cells were washed with fresh medium (2 x 10
mL) and centrifuged to pellet the cells. The supernatant was care¬
fully removed and the cells resuspended in 0.6 N perchloric acid
(107 cells/mL). Cells were freeze-fractured by four successive
freeze-thawings using liquid nitrogen. Cell lysates were stored at
-20°C until HPLC analysis. For HPLC analysis an aliquot of the solu
bilized polyamine perchlorates was reacted with dansyl chloride, sod
ium carbonate and diaminohexane as an internal standard. After con¬
centration of the reaction products on a C18 chromatographic plug,
the dansylated polyamines were analyzed on reversed phase analytical

85
C18 column and quantitated using fluorescence detection and elec¬
tronic integration. Analysis of HPLC samples were performed by
J.R.T. Vinson, B. Jennings, and V. Andaloro of Dr. R.J. Bergeron's
laboratory, Department of Medicinal Chemistry, College of Pharmacy,
University of Florida.
Pharmacokinetic Studies
Preliminary pharmacokinetic studies to determine half-life of
the analogues in the serum were studied in mice. Blood samples were
collected by exsanguination of CD-I mice into nonheparinized tubes at
appropriate times after a single i.p. injection of DESPM (25 mg/kg).
The serum was separated from the cells by centrifugation and diluted
with an equal volume of 1.2 N perchloric acid. Analogue levels in
the serum were determined by HPLC analysis by the above method.
The excretion of DEHSPM (25 mg/kg) after a single i.p. injec¬
tion was followed in the urine and bile of Sprague-Dawley rats util¬
izing bile duct cannulation model. Rat bile duct cannulations were
performed by K. Crist and E. LaGraves of Dr. R.J. Bergeron's labora¬
tory, Department of Medicinal Chemistry, College of Pharmacy, Uni¬
versity of Florida. The bile duct was cannulated with a segment of
22 ga polyethylene tubing and the bile directed from the rat to frac¬
tion collector by a fluid swivel mounted above a metabolic cage.
Normal urine flow was also diverted with a segment of 5 urn tubing
from the bottom of the metabolic cage to the fraction collector.
This system allows the animal to move freely in the cage while
continuous bile and urine samples were being collected in 3 h frac¬
tions for 24 h. Sample volumes were measured and diluted with an

86
equal volume of 1.2 N perchloric acid. Analogue levels in each
fraction were determined by HPLC analysis by the above method.
Toxicity Studies
Both single dose and five-day daily-dose toxicity in CD-I white
mice were determined for DESPM and DEHSPM. Initially, small groups
of 2-3 mice were used to determine the approximate range of toxicity
for the compound. More accurate determinations of toxicity were made
using groups of greater than 6 mice at each dose of analogue. Dos¬
ages ranged so that at the lowest dosage 10% or less of the animals
die and at the highest dosage >90% of the animals die. At least 3
toxicity dosages between these two extremes were determined.
The polyamine analogue was diluted in sterile 0.9% saline with¬
in 24 h of use and the unused portion stored at 5°C. Concentrations
of the spermine analogue tetrahydrochlorides at each dose were
adjusted so that the mice were injected i.p. with a volume of 1
mL/100 g.
For the single dose toxicity studies each group of mice
received a single i.p. injection of the analogue at an appropriate
dosage. For the 5-day daily-dose toxicity studies each group of mice
received a single i.p. injection of the analogue at an appropriate
dosage daily for 5 days. Animal deaths were checked daily for 30
days. The number of deaths was expressed as percent lethality
[100 x (number of deaths per group in 30 days/number of mice per
group)].

87
Results
In Vitro Activity
The polyamines which were evaluated for their antiproliferative
activity include the spermidine analogues (Fig. 3-1);
a. N,n'-bis(ethyl)-l ,8-diami nooctane (BEDAO),
b. ,N^-bis(propyl)spermidine (BPSPD),
c. N1 ,N®-bis(ethyl)-N^-(p-nitrophenyl)spermidine (BEPNPSPD),
d. ,N^-bis(ethyl)norspermidine (BENSPD),
e. ,N^-bis(ethyl)spermidine (BESPD),
and the spermine analogues (Fig. 3-2);
f. N,N'-diethyl-l,12-diaminododecane (DEDAD),
g. N* ,N^-dibenzylspermine (DBSPM),
h. ,N^-di (2,2,2-tri fl uoroethyl )spermine (FDESPM),
i. l,4-bis-[3-(ethylamino)propyl]piperazine (DEPIP),
j. N^,N^-diethyl spermine (IDESPM),
k. N^,N^,N^,N^-tetraethylspermine (TESPM),
and (Fig. 3-3)
l. N* ,N^-diethylnorspermine (DENSPM),
m. N* ,N^ ,N^-tetraethyl spermi ne (NTESPM),
n. N* ,N^-dimethyl spermi ne (DMSPM),
o. l,20-bis(N-ethylamino)-4,8,13,17-tetraazaeicosane (YANK),
p. N* ,N^-di propyl spermine (DPSPM),
q. ,N^-diethylspermine (DESPM),
r. N* ,N^-diethylhomospermine (DEHSPM).
The compounds described in this study are all in some way
related to the natural products spermidine and spermine. They were

88
BEDAO
48 h 96 h
>100 yM >100 yM
BPSPD
> 30 yM > 30 yM
> 10 yM > 10 yM
BESPD
>100 yM 10 yM
50 yM 10 yM
Figure 3-1. Structure and IC^q values of the spermidine
analogues.

89
48 h 96 h
DEDAD
N
H
Cytotoxic > 25 yM
No inhibition < 12.5 yM
Cytotoxic > 10 yM
No inhibition < 3 yM
FDESPM
N CF3
H
>100 yM
>100 yM
DEPIP
> 50 yM > 50 yM
> 25 yM > 25 yM
> 50 yM
5 yM
Figure 3-2. Structure and ICcn values of the less active
spermine analogues.

90
48 h
96 h
H
• N.
H
.N.
H
.N,
DENSPM
H
.N.
.NU
y
{
N
NTESPM
H
,N,
'N'
H
>100 yM 4 yM
100 yM 3 yM
H
.N.
H
-N.
N
DMSPM H
>100 yM 0.75 yM
H
,N,
H
,N,
H
.N.
N
YANK H
50 yM 0.5 yM
H H
DPSPM
3 yM 0.2 yM
DESPM
N
H
N
H
10 yM 0.1 yM
0.5 yM 0.05 yM
Figure 3-3. Structure and IC^g values of the most active
spermine analogues.

91
designed to evaluate relationships which might exist between the
structure of various alkylated polaymines and their activity against
tumor cells in culture. The relative antiproliferative activities of
the analogues were assessed by determining their 48-h and 96-h IC^g
values. In comparing the spermine compounds with their spermidine
counterparts (Figs. 3-1 to 3-3), it is clear that the spermine com¬
pounds are substantially more active against L1210 cells.
Of the spermine analogues tested, DESPM and DEHSPM were the
most effective inhibitors of growth of L1210 cells. DESPM had 48 h
and 96 h IC^g values of 10 yM and 0.1 yM, respectively. DEHSPM
demonstrated superior activity and had 48 h and 96 h IC^q values of
0.5 yM and 0.05 yM, respectively. The IC^g values for the most
active analogues (ICgg value <5 yM at 96 h) determined in the
presence of 100 yM arninoguanidine, an inhibitor of diamine oxidase,
remained the same.
The activity of DESPM against Daudi cells and HL-60 cells is
comparable to that against L1210 cells. Because of the differences
in doubling times between L1210 cells (ca. 10-12 h) and Daudi or
HL-60 (ca. 25 h), the IC^qS of DESPM at 48 h and 96 h against L1210
cells are best compared with the IC^qS at 72 h and 144 h against
HL-60 (10 yM and 0.3 yM) and Daudi cells (>40 yM and 0.5 yM). Addi¬
tionally, DEHSPM was shown to be active against CH0 and B-16 melanoma
cells (doubling time for both lines 14-16 h) with IC^q values of
about 0.1 yM at 48 h for both cell lines.
LI210 cells which were selected for their ability to grow
in the presence of Adriamycin (L1210/D0X-0.6 cells) demonstrated
an approximate 50-fold increase in resistance to Adriamycin over the
parental L1210 cell line. The LI210/D0X-0.6 cells were demonstrated
by immunoblotting to have a large increase in the membrane

92
glycoproteins (P150-170 glycoproteins) and considered to be multidrug
resistant cells (96). Both the parental cell line, as well as the
resistant cell line, were colaterally sensitive to DEHSPM with IC^g
values of 0.5 pM and 0.05 pM at 48 h and 96 h respectively.
The antiproliferative activity of DEHSPM was tested against
DC3F cells and a proven multidrug-resistant subline, DC3F/ADX cells
(97-99). Both cell lines were determined to be equally sensitive to
DEHSPM having identical 96-h IC^g values of 0.1 pM.
L1210 cells which were developed for their resistance to the
polyamine analogues L1210/DES-10 and L1210/HDES-1 did not demonstrate
an overproduction of the P150-170 glycoproteins (96), indicating that
they had not developed multidrug resistance; both were sensitive to
Adriamycin to the same extent (IC^g value of 0.02 pM at 48 h). Both
cell lines did, however, exhibit cross-resistance to other polyanine
analogues. Both of the resistant cell lines were not inhibited by
treatment with 100 pM DENSPM, and were able to grow to 70% of control
in the presence of either 100 pM DESPM or 100 pM DEHSPM.
The role of the methylene backbone in the biological activity
of the polyamine analogues was studied by comparing DENSPM, DESPM and
DEHSPM. Reduction in the length of the polyamine backbone, decreased
the activity of the analogue. The activity of DENSPM against L1210
cells (IC^g value of >100 pM at 48 h, and 4 pM at 96 h) is relatively
poor when compared to that of DESPM and DEHSPM (Figs. 3-4 and 3-5).

CONCENTRATION (uM)
93
% CONTROL GROWTH
Figure 3-4. Concentration dependence of L1210 cell growth
inhibition by DENSPM, DESPM and DEHSPM at 48 h.

CONCENTRATION (uM)
94
% CONTROL GROWTH
Figure 3-5. Concentration dependence of LI210 cell growth
inhibition by DENSPM, DESPM and DEHSPM at 96 h.

95
The time course of growth inhibition of various concentrations
of DENSPM (Fig. 3-6), DESPM (Fig. 3-7) or DEHSPM (Fig. 3-8) indicated
that except at the lower concentrations studied (<0.1 yM), DEHSPM was
a consistently and significantly more active antiproliferative. For
example, 1 yM DEHSPM was 10 times as active as DESPM and at least 100
times more active than DENSPM at 144 h. The plot of the percent
control growth vs. the log of drug concentration (IC^g determination)
for DESPM (Fig. 3-4) exhibited a 48 h curve which approached the 50%
control growth line asymptotically running almost parallel to the
line from 1-100 yM and suggesting cytostasis and not cytotoxicity.
The behavior of DENSPM is very similar to that of DESPM, however the
curve never crosses the 50% control growth line. DEHSPM, however,
does cross the 48 h-50% control growth line at 0.5 yM. A comparison
of the growth curves reflects this behavior quite clearly (Figs. 3-6,
3-7, 3-8). For example, in comparing the growth curves of cells
treated with DEHSPM and DESPM, the growth inhibitory behavior seen
with 0.3 yM DEHSPM is similar to that seen with 1.0 yM, 10 yM or
even 100 yM DESPM. In fact, DEHSPM at a concentration of 1.0 yM was
more effective than DESPM at any concentration studied. The activity
of DENSPM is not very impressive. For example, 30 yM DENSPM is com¬
parable with 1 yM DESPM.
Cytocidal Activity
A comparison of the analogue's impact on cell viability vs.
time (by trypan blue exclusion) further emphasizes their differences
in behavior (Fig. 3-9). When comparing 0.3 yM DEHSPM with 10 yM

CELLS/m I
96
10000
.1 H ' 1 ■ 1 ' 1 ■ 1 ' 1 ' 1—
0 24 48 72 96 120 144
TIME (hours)
Figure 3-6. Time dependence of L1210 cell growth inhibition by
various concentrations of DENSPM.
** * *

CELLS/ml
Figure 3-7. Time dependence of L1210 cell growth inhibition
various concentrations of DESPM.

CELLS/ml
98
Figure 3-8. Time dependence of L1210 cell growth inhibition by
various concentrations of DEHSPM.

VIABILITY
99
Figure 3-9. Trypan blue viability of L1210 cells treated with
30 pM DENSPM, 10 pM DESPM or 0.3 pM DEHSPM.

100
DESPM (their approximate 48 h IC^q values), cell death does not begin
to occur until after 96 h of treatment. However, DEHSPM exhibited
much greater cytotoxicity by 144 h than DESPM, while DENSPM wxhibited
almost no cytotoxicity at this time. The difference in cytotoxicity
between the compounds was further substantiated by measuring the
ability of treated cells to incorporate Rh-123 into their mitochon¬
dria. This measurement provides information regarding the viability
of the cells (74). Based on the nature of mitochondrial staining,
cells can be separated into three populations: viable, dying and
dead, each of which can be plotted as a function of time (Figs.
3-10, 3-11, 3-12). Although both measurements indicate the differ¬
ences in cytotoxicity between the two drugs, the Rh-123 data are more
informative.
The Rh-123 data clearly indicate that the events which impact
on the cell's growth begin earlier in the process than the trypan
blue exclusion data suggest. The data from the Rh-123 uptake method
are known to be a more sensitive and earlier indicator of loss of
cell viability with other antineoplastics (74). The effects of 30 yM
DENSPM on cell viability as measured by both the methods suggests the
compound has little activity relative to DESPM and DEHSPM. Trypan
blue exclusion methods indicated 92% viability at 144 h while the
RH-123 indicate 14% dead, 18% dying and 68% viable cells at 144 h.
Treatment Reversibility
The extent of treatment reversibility on cell growth upon
removal of the drug depends on the duration of the treatment. The

101
CO
UJ
o
TIME (hours)
Figure 3-10. Percentage of L1210 cells viable, dying and dead
after treatment with 30 pM DENSPM as determined by Rh-123 staining.

102
CO
ID
O
TIME (hours)
Figure 3-11. Percentage of L1210 cells viable, dying and dead
after treatment with 10 yM DESPM as determined by Rh-123 staining.

103
C/5
111
O
TIME (hours)
Figure 3-12. Percentage of L1210 cells viable, dying and dead
after treatment with 0.3 uM DEHSPM as determined by Rh-123 staining.

104
longer the cells are exposed to the analogue, the slower they grew
after transferring them into fresh media without the analogue. The
growth inhibitory effects of 1 yM DESPM did not become apparent for
approximately 48 h, and after 96 h there was little cell growth.
Though cell division had nearly ceased by 144 h more than 90% of the
cells remain viable by trypan blue exclusion. However, the fraction
of clonogenic cells decreased significantly during the treatment
(Table 3-1).
When cells were exposed to 0.6 yM DEHSPM, 10 yM DESPM or 30 yM
DENSPM for 24, 48, 72 and 96 h, were washed and regrown in complete
medium, the cytotoxicity of DEHSPM relative to DESPM and DENSPM
became obvious (Fig. 3-13, 3-14, 3-15). A 72 h exposure of the cells
to DEHSPM and DESPM essentially prevented cell regrowth. Growth
inhibition of the cells treated with DENSPM, however, was reversible
even after 144 h.
Cell Size
It was observed during these studies that cells substantially
decreased in size while exposed to the analogues. Utilizing elec¬
tronic particle sizing, exposure of L1210 cells to 10 yM DESPM, 0.6
yM DEHSPM or 30 yM DENSPM resulted in a progressive decrease in cell
diameter from 12.5 ym for control L1210 cells to 9.5 ym for L1210
cells after exposure to the drug for 144 h. This corresponds to a
decrease in cell volume of approximately 60% (1020 ym^ to 450 ym^).
The time course of this phenomenon is presented in Figure 3-16.

105
TABLE 3-1
PERCENT OF CONTROL CLONAGENIC L1210 CELLS AFTER TREATMENT WITH
1 yM DESPM FOR UP TO 96 H
DESPM 1 yM
Treatment
Percent of Clones Relative to Controls
24
h
89
48
h
75
72
h
21
96
h
2

CELLS/ml
106
Figure 3-13. Growth of L1210 cells in fresh medium after
treatment with 30 yM DENSPM for 0 (control), 24, 48, 72 and 96 h.

CELLS/m I
107
Figure 3-14. Growth of L1210 cells in fresh medium after
treatment with 10 uM DESPM for 0 (control), 24, 48, 72 and 96 h.

CELLS/ml
108
TIME (hours)
Figure 3-15. Growth of L1210 cells in fresh medium after
treatment with 0.6 yM DEHSPM for 0 (control), 24, 43, 72 and 96 h.

CELL VOLUME (cubic micrometers)
109
Figure 3-16. Time course of changes in L1210 cell volume after
treatment with 30 yM DENSPM, 10 yM DESPM or 0.6 yM DEHSPM.

no
Impact on Nuclear DNA Content
Because of the unusual growth properties induced in the L1210
cells by DESPM, the nDNA histograms of the treated cells were evalu¬
ated (Fig. 3-17). Flow cytometric analysis of nDNA content of L1210
cells after treatment with 10 yM DESPM for 48 h and 96 h showed only
a slightly decreased S-phase and G2~phase cell populations compared
with controls. This was not consistent with a decrease in cell
growth caused by blocking progression of cells through any particular
phase of the cell cycle. A significant number of cells accumulating
at the Gj/S border was seen only after 144 h of treatment. LI210
cells were exposed to DEHSPM and DENSPM at approximately their 48 h
IC^g concentrations. As with LI210 cells exposed to 10 yM DESPM
there were no significant changes in nuclear DNA content (reduced S
and G2/M phase DNA content) of the L1210 cells until after more than
96 h of treatment with 0.6 yM DEHSPM or 30 yM DENSPM.
Impact on Mitochondrial DNA Content
There was a striking decrease in mtDNA content per L1210 cell
after treatment with DESPM (Fig. 3-18). This decrease in mtDNA con¬
tent is compatible with an almost immediate cessation of mtDNA syn¬
thesis coupled with dilution due to cell division for three or four
doublings (Fig. 3-19). Control LI210 cells grew in log-linear fash¬
ion with a 10-12 h doubling time. Cells treated with DESPM showed no
significant growth inhibition at 24 h. By 48 h, L1210 cells treated
with DESPM contained about 20% of the normal amount mtDNA and the
rate of cell doublings had just begun to decrease. The mtDNA content
of treated cells had decreased to about 10% of control by 96 h at

RELATIVE CELL NUMBER
ill
DNA CONTENT
Figure 3-17. Flow cytometric analysis of nDNA content of L1210
cells after treatment with 10 pM DESPM.
A. 0 h
B. 48 h
C. 96 h
D. 144 h

112
Figure 3-18. Time course for the effect of DESPM on the
percent of control mtDNA in L1210 cells. The amount of mtDNA in
untreated controls was assumed 100% at each time point.

113
Figure 3-19. Kinetic study of accumulated L1210 cell growth
(based on number of doublings) of untreated (control) LI210 cells and
cells treated with DESPM. The time course of mtDNA accumulation
(accumulated cell counts X percent control mtDNA/cell) for untreated
cells follows the untreated cell growth curve, while mtDNA
accumulation for cells treated with DESPM was consistent with lack of
mtDNA synthesis coupled with cell division.

114
which point cell doubling time nearly tripled (30-35 h). Cells
treated with DESPM remained constant at 10% of control mtDNA content
up to 144 h at which time cell division had essentially ceased.
Exposure of L1210 cells to 10 yM DESPM rather than 1 yM DESPM had
little, if any, significant difference in effects on cell growth or
mtDNA content, a finding consistent with the almost complete inhibi¬
tion of mtDNA accumulation caused by the lower concentration of
drug.
Exposure of L1210 cells to 30 yM or 100 yM BESPD for 72 h
decreased mtDNA content to 65% of control. It is clear that the less
active spermidine analogue, BESPD (IC ^value of 100 yM at 48 h, and
10 yM at 96 h), even at 10 times the concentration of DESPM, had less
of an impact on mtDNA content. In a comparative study, L1210 cells
were treated in log-phase growth with 0.1, 1.0 and 10 yM DMSPM, DESPM
or DPSPM for 72 h and analyzed for mtDNA content. The dose-effect
curves generated (Fig. 3-20) further demonstrated a positive correla¬
tion between the antiproliferative and antimitochondrial activity of
the polyamine analogues.
Incubation of L1210 cells for 72 h with 100 yM or 400 yM DFM0
(IC50 va^ue °f 1 mM at 48 h, and 100 yM at 96 h), a known inhibitor
of polyamine biosynthesis, had no significant effect on mtDNA con¬
tent.
Polyamine Analogue Uptake and Impact on Polyamine Pools
L1210 cells in complete medium exposed to 100 yM DESPM absorbed
the analogue to concentrations of 3150 pmole/mil1 ion cells in 24 h

% CONTROL mtDNA
115
CONCENTRATION (uM)
Figure 3-20. Concentration dependence of percent control mtDNA
in L1210 cells treated with DENSPM, DESPM or DEHSPM for 72 h.

116
(Fig. 3-21). Assuming that the total volume of a single L1210 cell
is approximately 1000 ym , the cellular concentration of DESPM is
about 3 mM. What is most interesting is that the total concentration
of polyamines, including the analogue, remains approximately the
same while the concentrations of the native polyamines spermine,
spermidine and putrescine decrease. The greatest relative decrease
in polyamine pool occurs in the putrescine level followed next by a
decrease in spermidine concentration with the least impact on sperm¬
ine pool which decreased by approximately 40% in 24 h. It is notable
that the impact on polyamine concentrations begins almost immediate¬
ly. For example, the spermidine pools which have been diminished by
85% at 24 h have already been reduced by 39% at 2 h.
In Vivo Activity
The spermine analogue, DESPM was screened for its ability to
inhibit the growth of L1210 ascites tumor growth in DBA/2 mice.
Since DESPM is a highly charged, very water soluble compound, it
would be expected to be excreted quickly from the body. Therefore it
was given in a split dosing schedule over several days. The mice
were given the tumor on day 0, and treatment begun 24 h later. The
results (Table 3-2) clearly demonstrated a dose-response relation¬
ship, with i.p. administration of 15 mg/kg every 8 h for 6 days hav¬
ing the optimum effect of the schedules tested. At this dose animals
had an average ILS of greater than 376%, with 60% of the test animals
still alive after 60 day and no sign of tumor. Although the 17.5
mg/kg dosing schedule gave a greater ILS and percentage of long term

117
6000 -i
0 2 6 12 24
TIME (hours)
Figure 3-21. Time course for the effect of 100 yM DESPM on
polyamine levels in L1210 cells over 24 h.

TABLE 3-2
Activity of N ,N -Diethyl spermine *4 HC1 Against L1210 Murine Leukemia in DBA/2 Mice
Experi- Number of Mean
ment Cells Inocu- Survival
Number Treatment3 Scheduleb (Route) lated (Site) Days of Death0 (days) ± S.D. % ILSd LTSe
1.
5 mg/kg
q8h days 1-6 (IP)
103
(IP)
9.5, 10.5, 11.5, 12, 30.5,
13(2), 16.5
12.3
+
2.0
25
0/8
NONE
8.5, 9(2) , 10(2) , 10.5, 11(2)
9.9
+
0.9
2.
10 mg/kg
q8h days 1-6 (IP)
105
(IP)
13(2), 17(2), 20, 22, 23,
23.5, 24
34.7
+
17.5
269
5/20
NONE
8.5(3), 9(7), 9.5(2), 10(5),
10.5(2)
9.4
±
0.7
3.
15 mg/kg
ql2h days 1-6 (IP)
105
(IP)
14(2), 14.5, 15(2), 17
14.9
+
1.2
55
0/6
NONE
8.5, 9.5(3), 10(3)
9.6
+
0.5
4.
15 mg/kg
q8h days 1-6 (IP)
105
(IP)
8, 20, 22, 22.5, 24.5,
28.5, 40, 60(9)
46.6
+
17.6
376
9/16
NONE
8.5, 9(3), 9.5(2), 10(6),
10.5(2), 11
9.8
±
0.7
5.
17.5 mg/kg
q8h days 1-6 (IP)
105
(IP)
8.5, 9.5, 34, 40, 60(4)
52.3
±
11.0
439
4/8
NONE
9(3), 9.5, 10(4), 11
9.7
±
0.6
6.
45 mg/kg
OD days 1-6 (IP)
105
(IP)
7, 7.5, 8, 8.5, 14(2)
9.8
±
3.3
0
0/6
NONE
9(5), 10, 11(4)
9.9
±
1.0
7.
20 mg/kg
q8h days 1-3 (IP)
106
(IP)
13.5, 14(2), 14.5
14.0
±
0.4
57
0/4
NONE
8.5(2), 9, 10
9.0
±
0.6

8.
20 mg/kg
q8h days 1-4 (IP)
106 (IP)
14(2), 15(2), 16, 17, 18,
20, 21, 31
18.1 ±
4.9 103
0/10
NONE
8(2), 9(6), 10
8.9 ±
0.6
9.
20 mg/kg
q8h days 1-5 (IP)
106 (IP)
8, 9, 16(3), 17
13.6 ±
4.0 72
0/6
NONE
7(3), 8(2), 10.5
7.9 ±
1.3
aControl animals were yiven no treatment.
bMi ce given tumor on day 0.
cThe experiment was ended at 60 days. Animal survival was evaluated on this day, however, animals surviving
until this time had no sign of tumor.
^Percent increase in life span (%ILS).
eLong term survivors, >60 days (LTS).

120
survivors, there was some significant drug toxicity as indicated by a
number of early deaths and an average weight loss of greater than
10%. The animals which recovered, however, appeared normal 4-5 days
after the drug treatment was completed. In going from a dose of 5
mg/kg every 8 h for 6 days (a total dose of 90 mg/kg) to 15 mg/kg
every 8 h for 6 days (a total dose of 270 mg/kg) we observed an
increase from 25% to 376% in life span. The drug dose was only
increased 3 times while the life span increased at least 15 times.
The dosing schedule was critical. When the animals received
DESPM 20 mg/kg every 8 h for 4 days (a total dose of 240 mg/kg) there
was a 103% ILS, while those treated with 10 mg/kg every 8 h for 6
days (a total dose of 180 mg/kg) exhibited a 269% ILS. This is in
keeping with observations in cell culture. Irreversibility of cell
growth inhibition in culture required that the cells be exposed to
the drug for a minimum of 96 h, suggesting that in the short-term
dosing experiments the cells were not exposed to the drug long enough
to irreversibly inhibit their proliferation.
The initial dosages and treatment schedules of DEHSPM in leu¬
kemic mice were based on the original in vivo studies with DESPM, and
a consideration of the four-fold greater in vitro antileukemic activ¬
ity of DEHSPM compared to DESPM at 96 h. The mice were given ascites
tumor on day 0. The results with DEHSPM (Table 3-3) clearly demon¬
strated a dose-response relationship, with i.p. administration of 5
mg/kg every 8 h on days 1-6 having the optimum effect of the

TABLE 3-3
Activity of N ,N -Diethylhomospermine *4 HC1 Against L1210 Murine Leukemia in DBA/2 Mice
Experi- Number of Mean
ment Cells Inocu- Survival
Number Treatment3 Schedule^ (Route) lated (Site) Days of Deathc (days) ± S.D. %ILSd LTSe
1.
5 mg/kg
NONE
OD days 1-6 (IP)
105
(IP)
2.
10 mg/kg
NONE
OD days 1-6 (IP)
105
(IP)
3.
15 mg/kg
NONE
OD days 1-6 (IP)
105
(IP)
4.
20 mg/kg
NONE
OD days 1-6 (IP)
105
(IP)
5.
5 mg/kg
NONE
q8h days 1-4 (IP)
105
(IP)
6.
5 mg/kg
NONE
q8h days 1-5 (IP)
105
(IP)
7.
5 mg/kg
NONE
ql2h days 1-6 (IP)
105
(IP)
8.
10 mg/kg
NONE
ql2h days 1-6 (IP)
105
(IP)
9.
1.25 mg/kg
NONE
q8h days 1-6 (IP)
105
(IP)
10.
2.5 mg/kg
NONE
q8h days 1-6 (IP)
105
(IP)
9, 10, 10.5, 12(3)
10.9
+
1.3
20
0/6
8, 9(3), 9.5, 10
9.1
+
0.7
12, 17, 24(2), 27
20.8
±
6.1
115
0/5
9(3), 9.5, 10
9.3
±
0.4
21, 27, 60(3)
45.6
±
19.8
390
3/5
9(2), 9.5(3)
9.3
±
0.3
15, 28, 29.5, 32, 45, 60
34.9
±
15.6
301
1/6
8, 8.5(4), 9(5)
8.7
±
0.4
17, 18, 20, 22(2), 60
26.5
±
16.5
160
1/6
9(4), 10, 11(3), 11.5(2)
10.2
±
1.1
15, 18(2), 23, 31, 39
24.0
±
9.2
135
0/6
9(4), 10, 11(3), 11.5(2)
10.2
±
1.1
26.5, 33, 35, 60(3)
45.7
±
15.8
381
3/6
9(4), 10, 11
9.5
±
0.8
31, 60(5)
55.2
±
11.8
441
5/6
9(4), 10, 11(3), 11.5(2)
10.2
±
1.1
10, 12.5, 13(2)
12.1
±
1.44
20
0/4
9, 9.5, 10.5, 11.5
10.1
±
1.1
20.5, 22, 23, 32, 60
31.5
±
16.6
242
1/5
9(3), 9.5(2)
9.2
±
0.3

11.
5 mg/kg
NONE
q8h days 1-6 (IP)
105
(IP)
12.
5 mg/kg
NONE
q8h days 3-8 (IP)
104
(IP)
13.
5 mg/kg
NONE
q8h days 4-9 (IP)
105
(IP)
14.
12.5 mg/kg
NONE
ql2h days 1-6 (IP)
106
(SQ)
15.
20 mg/kg
NONE
OD days 1-6 (IP)
105
(SQ)
16.
10 mg/kg
NONE
ql2h days 1-6 (SQ)
105
(IP)
17.
20 mg/kg
NONE
OD days 1-6 (SQ)
105
(IP)
18.
20 mg/kg/day
Days 1-6 (SQ PUMP)
105
(IP)
NONE
^Control animals were given no treatment.
^Mice given tumor on day 0.
cThe experiment was ended at 60 days. Animal survival
until this time had no sign of tumor.
dPercent increase in life span (% ILS).
eLong term survivors, >60 days (LTS).
22.5(2), 25, 26, 47, 60(41)
8(3), 8.5(5), 5, 9(6),
9.5(11), 10(4), 10.5(6)
11
29(2), 39, 60(3)
9.5(3), 10.0, 10.5, 11.5
11(2), 11.5(3), 32
8.5, 9, 9.5(2), 11(2)
29, 32.5, 33, 60(3)
8(4), 9, 10
12, 13, 14, 21, 39, 58
10(3), 11(2), 20.5
19.5, 22(2), 60(9)
8.5(4), 9(4), 9.5(2), 10
21, 60(5)
9(4), 9.,5, 10(5)
7, 13, 14.5, 17.5
7, 9.5(3)
56.6
+
10.4
502
41/46
9.4 ^
-
0.8
46.1
+
15.5
361
3/6
10.0
+
0.8
14.8
±
8.4
51
0/6
9.8
±
1.0
45.8
±
15.7
429
3/6
8.5
±
0.8
26.2
±
18.6
116
0/6
12.1
±
4.2
50.3
±
15.9
458
9/12
9.0
±
0.5
53.5
±
15.9
457
5/6
9.6
±
0.5
13.0
±
4.4
46
0/4
8.9
±
1.2
was evaluated on this day, however, animals surviving

123
schedules tested. At this dose animals had an average ILS of greater
than 500%, with 90% of the test animals alive after 60 days with no
sign of tumor. In going from a dose of 1.25 mg/kg every 8 h on days
1-6 (a total dose of 22.5 mg/kg) to 5 mg/kg every 8 h for 6 days (a
total dose of 90 mg/kg) we observed an increase from 20% to 500% in
life span. The drug dose was only increased four times while the
life span increased at least 25 times. It was found that DEHSPM
could be administered every 12 h. At an i.p. dosage every 12 h on
days 1-6, 5 mg/kg gave a 381% ILS with half the animals being long
term survivors (>60 days), and 10 mg/kg exhibited a 441% ILS with 5/6
of the animals surviving >60 days. Even more surprising was that
DEHSPM could be given on a once daily schedule. At the optimum
single daily dosage of 15 mg/kg on days 1-6, there were 3/5 long term
survivors and a 390% ILS. Administration of DEHSPM by a constant
s.c. osmotic pump infusion of 20 mg/kg/day gave poor results (46%
ILS) when compared to a single i.p. injection of the same daily dose
on days 1-6 (301% ILS).
As with DESPM, the length of treatment time was clearly criti¬
cal. Animals treated i.p. with 5 mg/kg every 8 h on days 1-4 or 1-5
exhibited only about a 150% ILS, while animals given 2.5 mg/kg every
8 h on days 1-6 had a 242% ILS.
Studies employing a remote site of injection from the tumor
site also gave excellent results. When the tumor was inoculated in a
s.q. site and DEHSPM administered 12.5 mg/kg on days 1-6 by i.p.
injection, there was a 429% ILS and half the mice survived 60 days.

124
Also, when mice were given i.p. tumor and treated by s.q. injection
on days 1-6 with either 10 mg/kg every 12 h or 20 mg/kg once daily,
there was a 457% ILS and 75% of the animals living >60 days.
Administration of DEHSPM, 5 mg/kg or 7.5 mg/kg every 8 h for
days 1-8, was only moderately effective at inhibiting the growth of
the solid B-16 melanoma tumor in C57B1/6 mice. The mean tumor mass
in treated mice at both dosages was 26% of the mass in control mice
(26% T/C). The analogue was also shown to be effective at inhibiting
the growth of the solid Lewis lung carcinoma in C57B1/6 mice, exhib¬
iting a 20% T/C at a dose of 5 mg/kg every 8 h for days 1-8.
Serum Levels in Mice
Serum levels of DESPM in CD-I mice after a single i.p. injec¬
tion of 25 mg/kg (62 ymoles/kg), indicated rapid adsorption of the
drug from the peritoneum reaching peak serum levels of 48 yg/mL (120
yM) in 15 min (Fig. 3-22). Elimination from the serum was very rapid
with a serum half-life of about 10 min and levels below detection
after 120 min.
Excretion Studies in Rats
In the canulated rat model, DEHSPM was excreted from the body
mainly by the kidneys after a single i.p. injection of DEHSP (25
mg/kg) (Fig. 3-23). The drug decreased the urine and bile flow dur¬
ing the first 3 h after injection as compared to control animals so
that appreciable sample volumes were not collected until the 6-h
fraction and the majority of the analogue was excreted in the 9- and
12-h fractions of the urine. As a dose of 25 mg/kg (58 ymoles/kg)

125
TIME (minutes)
Figure 3-22. Serum levels of DESPM in CD-I mice after a single
i.p. injection of 25 mg/kg. Analysis was performed on serum
samples from duplicate animals at each time point.

126
TIME (hours)
Figure 3-23. Excretion of DEHSPM in rat urine and bile,
collected in 3 h fractions, after a single i.p. injection of 25 mg/kg
(58 umoles/kg). Analysis was performed on urine and bile samples from
duplicate animals at each time point.
URINE EXCRETION (umoles/kg/sample)

127
was administered and the mean cumulative excretion in the urine and
bile totaled about 5.1 ymoles/kg (Fig. 3-24), less than 10% of the
dose administered was excreted unchanged from the body in 24 h.
Toxicity Studies
Acute single-dose i.p. toxicity in CD-I mice for DESPM (Fig.
3-25) and DEHSPM (Fig. 3-26) was very similar. The LDjg, LDgg and
LDgg doses for DESPM were determined to be 200, 385 and 475 mg/kg,
respectively, and for DEHSPM, 250, 330 and 475 mg/kg, respectively.
Animal deaths, at acute toxic doses for both compounds, occurred by
what appeared to be respiratory paralysis within 10 min after
administration, and were unrelated to the gastrointestinal toxicity
and weight loss seen on chronic administration in the in vivo
activity studies.
In chronic 5-day single-dose toxicity studies the i.p. LC^g,
LD^g and LDgg doses for DESPM (Fig. 3-27) were determined to be 72,
83 and 100 mg/kg, respectively, and for DEHSPM (Fig. 3-28), 33, 37
and 52 mg/kg, respectively. Severe weight loss (>20% of original
weight) was observed on the fifth day of treatment in all animals at
toxic doses, and animals at lethal doses either recovered or died
within one week of the final injection.
Piscussion
The compounds described in this study are all in some way
related to the natural products spermidine and spermine. They were
designed to evaluate relationships which might exist between the

128
TIME (hours)
Figure 3-24. Cumulative excretion of DEHSPM in rat urine and
bile after a single i.p. injection of 25 mg/kg (58 ymoles/kg).
CUMULATIVE URINE EXCRETION (umoles/kg)

129
DOSE (mg/kg)
Figure 3-25. Toxicity of DESPM in CD-I mice after a single
i.p. injection (number of deaths/total number of animals).

130
DOSE (mg/kg)
Figure 3-26. Toxicity of DEHSPM in CD-I mice after a single
i.p. injection (number of deaths/total number of animals).

131
Figure 3-27. Toxicity of DESPM in CD-I mice after a single
daily i.p. injection for 5 consecutive days (number of deaths/total
number of animals).

132
DOSE (mg/kg/day)
Figure 3-28. Toxicity of DEHSPM in CD-I mice after a single
daily i.p. injection for 5 consecutive days (number of deaths/total
number of animals).

133
structure of various alkylated polyamine analogues and their activity
against tumor cells in culture.
Although several of the analogues had 48-h 50% inhibitory con¬
centrations (IC^qs) of less than 50 yM, the polyamine analogues gen¬
erally took a significant period of time to exhibit their maximal
effects on cell proliferation. Therefore, the 96 h IC^qS were
selected for comparison and observed the general activity order:
spermine analogues > YANK > spermidine analogues.
In a series of terminally N-alkylated spermidine analogues
(Fig. 3-1), it is obvious that the presence of 3 nitrogens which are
capable of being protonated at physiological pH, are necessary for
activity. When the central nitrogen of the active BESPD is replaced
by a methylene unit (BEDAO) or the pKa of the central nitrogen is
decreased by the addition of a N^-p-nitrophenyl group (BEPNPSPD) the
antileukemic activity of the analogue is destroyed. These results
are consistant with findings which demonstrated that N-acylated
spermidine analogues were poor inhibiters of LI210 cell growth
(59,65). The effect of N-alkyl chain length on activity showed
BESPD > BPSPD. Additionally, the effect of methylene backbone length
demonstrated the greater activity of BESPD over BENSPD. N^,N®-Di-
ethylspermidine was the most active of the spermidine analogues
tested against cultured LI210 cells, and was previously shown to
decrease the intracellular concentrations of ornithine decarboxylase
and spermidine and putrescine (65,66,69).

134
In comparing the spermine compounds (Figs. 3-2 and 3-3) with
their spermidine counterparts, it is clear that the spermine com¬
pounds are substantially more active against LI210 cells. This is in
keeping with the findings that DESPM had a far greater impact on
polyamine biosynthesis and the polyamine biosynthetic enzymes than
the spermidine analogues (100). The spermine compound reduced the
level of intracellular polyamine pools as well as decreasing both ODC
and AdoMetDC levels.
The results with L1210 cells clearly suggest that monoalkyl¬
ation of both of the terminal primary nitrogens of spermine is
important for antiproliferative activity. The terminally alkylated
polyamines DMSPM, DESPM, and DPSPM are all more active than either
the tetraalkylated spermine, TESPM, or the internally dialkylated
spermine, IDESPM.
The effect of the N-alkyl chain length on the activity of
spermine analogues showed ethyl > propyl >> methyl; and the dibenzyl
analogue, while cytotoxic at higher concentrations, was not active at
lower concentrations. As was observed in the spermidine derivatives,
when the basicity of the terminal nitrogens is reduced by the addi¬
tion of trifluoroethyl groups (FDESPM), there is no substantial
activity. Furthermore, when the two central nitrogens of spermine
are replaced by methylenes, while each of the terminal nitrogens
maintains its alkyl group (DEDAD), there is a substantial loss in
activity against L1210 cells even though the distance between the
terminal nitrogens is approximately maintained. When the distance

135
between the two central nitrogens of DESRM is shortened by incorpor¬
ating the nitrogens in a 1,4 piperazine system, the compound's anti¬
proliferative activity essentially disappears despite the fact that
an analogue with internal tertiary nitrogen atoms, NTESPM, does
retain discernible activity. Extension of the DESPM backbone result¬
ing in the hexaamine, YANK, demonstrated LI210 cell growth inhibitory
activity similar to that of DMSPM, DESPM, and DPSPM. Finally, the
role of the methylene backbone in the antiproliferative activity of
the spermine analogues demonstrates DEHSPM > DESPM > DENSPM.
The most active of all compounds tested were the spermine ana¬
logues, DEHSPM and DESPM. Both of these polyamine analogues demon¬
strated a broad spectrum of activity in the range of 0.1 to 1.0 yM
against several animal and human cultured cell lines, and appeared to
be related to the doubling time of the cells.
The development of drug resistance is often a limiting factor
in cancer chemotherapy and is compounded by the fact that drug-
resistant tumor cells often show cross resistance to structurally
unrelated drugs (101). These multidrug resistant (Mdr) cell lines
apparently utilize an energy dependent system to actively expelí the
drug from the cell (102,103). Several of the Mdr cell lines have
been characterized by the increased amount of a 150-170 kd membrane
glycoprotein (P150-170 glycoprotein) and a 140 kb region of amplified
DNA whose level of amplification correlates with the level of drug
resistance (97-99). The Mdr cell lines DC3F/ADX and L1210/D0X-0.6

136
(96) showed colateral sensitivity with their parental cell lines to
DESPM and DEHSPM. The ability of LI210 cells to develop resistance
to the polyamine analogues is, apparently, unrelated to the Mdr
phenomena as L1210/DES-10 and L1210/HDES-1 cells showed no increase
in P140-180 glycoprotein or related amplified DNA regions (96) and
were equally sensitive as their parental lines to Adriamycin. This
lack of cross-resistance between the polyamine analogues and Mdr-
cells may be of some possible clinical significance.
The incubation of 1 mM aminoguanidine, a serum diamine oxidase
inhibitor (93), along with DESPM in culture with L1210 cells had no
significant effects on cell growth or mtDNA content. It is important
that the activity of the polyamine analogues was not decreased by an
inhibitor of diamine oxidase. This enzyme catalyzes the catabolism
of acetylated spermidine and spermine to oxidation products which
have been postulated (104) to be responsible for the antiprolifera¬
tive activity of the natural polyamines. Presumably, the analogues
are not substrates for the enzyme so that the toxic end products,
3-acetamidopropanal and acrolein, were not produced.
Unlike cytotoxic drugs, the dose-response curves for the con¬
centration dependence of growth inhibition at 48 h (Fig. 3-4) are un¬
usually flat over a large concentration range for DESPM and DENSPM.
The cytostatic nature of the growth inhibition of the two compounds
is seen in trypan blue exclusion (Fig. 3-9) and Rh-123 mitochondrial
uptake studies (Figs. 3-10 and 3-11). At the approximate 48-h IC^q
values, DESPM treatment does not exhibit significant cell death until
after 96 h, while exposure to DENSPM is not cytotoxic until 144 h.

137
Even though DEHSPM is much more active over a wider range of concen¬
trations and demonstrates greatly reduced cell viability at 144 h,
significant cytotoxicity is not seen until after 96 h of treatment.
Although the analogues did not appear to demonstrate direct
cytotoxicity, cells exposed to DESPM and DEHSPM at their 48-h IC^gS
for greater than 72 h were unable to proliferate when incubated in
drug-free medium (Figs. 3-14 and 3-15). This may indicate some ear¬
lier lethal event caused by the analogue from which the cells cannot
recover.
Flow cytometric analysis of L1210 cells after treatment with 10
yM DESPM for 48 h and 96 h showed cell populations with only slightly
decreased S-phase and G2-phase nDNA content compared with controls
(Fig. 3-17). Cells exposed to 30 yM DENSPM or 0.6 yM DEHSPM showed
similar changes in nDNA with time. This was not consistent with a
decrease in cell growth caused by blocking progression of cells
through any particular phase of the cell cycle. It appeared that the
cells were slowing throughout the entire cell cycle, as if they no
longer had sufficient energy to synthesize DNA and divide. A signif¬
icant number of cells accumulating at the Gi/S border was seen only
after 144 h of treatment.
The relationship between cell doubling and mtDNA content, seen
in Figure 3-19, is consistent with a mechanism in which DESPM inhibi¬
tion of mtDNA replication causes a depletion in mtDNA content with
cell division until a quantity insufficient to support continued cell
growth remains. Similar results have been obtained in studies of the

138
action of MGBG on L1210 cells in vitro (105). The resultant decrease
in mtDNA due to the polyamine analogues undoubtedly causes secondary
effects on cellular energy production. These results suggest that
although the cells are alive, they simply do not have sufficient
energy to support normal growth and division, although other explana¬
tions are certainly feasible. It is possible that secondary effects
on energy production contribute to the decrease in cell size with
treatment time of the polyamine analogues. It is interesting that
the effect on mtDNA occurs while nDNA synthesis is unaffected. Simi¬
lar differentiated effects on mtDNA have been observed with MGBG
(106).
The antiproliferative activity of the polyamine analogues may
be related to their ability to inhibit mtDNA, and the analogue's
antimitochondrial action may be a better means of assessing the com¬
pound's antiproliferative action than cell growth studies. The rela¬
tive ranking of the IC^qS of DMSPM < DESPM < DPSPM is reflected in
their antimitochondrial effects as shown in Figure 3-20. It would
require approximately 5 times the concentration of DPSPM to see the
same reduction in mtDNA as 1 yM DESPM in a 72-h exposure, while DMSPM
has little effect at concentrations of up to 10 yM. At 100 yM the
less active spermidine analogue, BESPD reduced mtDNA content to only
66% of control L1210 cells after a 72-h incubation.
The striking effects of the polyamine analogues on the mito¬
chondria and growth of the LI210 cells may not be exclusively ac¬
counted for by depletion of intracellular polyamine pools and poly¬
amine biosynthetic enzymes. Depletion of intracellular polyamine

139
pools by DFMO, a specific inhibitor of polyamine biosynthesis, is
shown to be apparently unrelated to inhibition of mtDNA synthesis.
In addition to being an inhibitor of polyamine biosynthesis, MGBG has
been shown to inhibit mtDNA synthesis. This effect may be directly
correlated in a dose-dependent manner with selective mitochondrial
ultrastructural damage (106), and its antimitochondrial activity, not
alterations in polyamine pools, has been postulated as the major
antiproliferative effect of MGBG (107).
Incubation of L1210 cells for 48 h with N*,N^-bis(ethyl)putre-
scine (BEP), BESPD and DESPM were found to cause reduced levels of
intracellular putrescine, spermidine and spermine (100). The time
course for the effect of 100 yM DESPM on cells (Fig. 3-21), however,
demonstrates that while the concentrations of the native polyamines
spermine, spermidine and putrescine decrease the total concentration
of polyamines, including the analogue, remains approximately the same
in the 24 h experiment.
The possibility must be considered that the inhibitory effects
of the analogues could be due to interference at critical binding
sites normally occupied by the native polyamines. It is interesting
to speculate on the nature of these binding sites. Numerous electro¬
static interactions of polyamines have been reported with macromole¬
cules, including nucleic acids and certain proteins (108,109). Along
with the histones, polyamines appear to be present in the nucleus in
quantities sufficient to neutralize 15-30% of the nDNA anionic
charges (110). Histone acetyltransferases also possess polyamine
acetylation activity, and it has been suggested that histone and

140
polyamine acetylation function in concert to lower the stability and
change the conformation of the nucleosome core, thus facilitating
replication and transcription (111). Areas of histone acetylation
have been correlated with active areas of nDNA replication and tran¬
scription (112). Neutralization of cationic charges by acetylation
of the natural polyamines may function to lower the stability and
allow changes in the conformation of certain critical binding sites,
facilitating their normal activity and function. The terminally
N-alkylated polyamines, however, are evidently not substrates for the
acetyltransferases, since there is no evidence of acetylation of the
secondary amines of spermine or spermidine (108). Thus, while poly¬
amine analogues may be able to enter the cell and replace the natural
polyamines at their binding sites, they are not acetylated. In this
way the polyamine analogues would prevent any structural modifica¬
tions necessary for normal activity and function of these critical
areas, and in a sense, the polyamine analogue would "overstabilize"
the site.
It is interesting that BESPD (70) as well as MGBG (113) (which
has also been proposed to replace natural polyamines at intracellular
binding sites (114) and also would not be a substrate for the acetyl¬
transferases) cause several hundred-fold induction of the N^-acetyl-
transferase. It may be proposed that the induction is due to the
cell's inability to acetyl ate and displace the analogue from the
critical "overstabilized" site. This increase in acetylase enzyme
may also help to deplete intracellular polyamine pools by intercon¬
version pathways of acetylated spermine and spermidine to putrescine
and elimination of putrescine from the cell (100).
While nDNA activity does not appear to be affected by the ana¬
logues at an early point, the mitochondria may be one site at which

141
these compounds act to regulate cell growth. Mitochondrial DNA,
being circular and without histones (115), may require polyamines to
a greater degree than nuclear chromatin for its stabilization, and
therefore, may be more sensitive to the effects of the polyamine
analogues.
Despite the apparent similarities in cellular effects, the
polyamine analogues differ from DFMO and MGBG in important ways.
Namely, the analogues do not result in compensatory increases in Ado-
MetDC activity as seen with DFMO or increases in ODC activity as is
the case with MGBG. Preliminary studies suggest that BESPD (66) and
DESPM (116) do not act to inhibit AdoMetDC or ODC activity directly
but rather regulate the enzymes at a translational and/or post-
translational level by the same mechanisms as spermidine. These
points of enzyme regulation may indicate additional sites where
natural polyamines may be replaced by the analogues.
The analogues, DESPM and DEHSPM were shown to have significant
activity against L1210 ascites tumor growth in DBA/2 mice. The
results with DESPM (Table 3-2) clearly demonstrated a dose-response
relationship, with i.p. administration of 15 mg/kg every 8 h for 6
days having the optimum effect of the schedules tested. At this dose
animals had an average increase in ILS of greater than 376% with 60%
long term survivors.
The results with DEHSPM (Table 3-3) clearly demonstrated a
dose-response relationship, with i.p. administration of 5 mg/kg

142
every 8 h on days 1-6 having the optimum effect of the schedules
tested. At this dose animals had an average ILS of greater than
500%, with 90% of the test animals alive after 60 days and no sign of
tumor.
The dosing schedule was critical and possibly related to cell
doubling time. This is in keeping with our observations in cell cul¬
ture. Irreversibility of cell growth inhibition in culture required
that the cells be exposed to the drug for a minimum of 96 h. This
suggesting that in the short-term dosing animal experiments the cells
were not exposed to the drug long enough to irreversibly inhibit
their proliferation.
Studies employing a remote site of injection from the tumor
site also gave excellent results. The s.q. treatment of i.p. L1210
tumor, and the i.p. treatment of s.q. L1210 tumor were equally
effective as i.p. tumor treated by i.p. injection of an equivalent
dose of DEHSPM. In addition, DEHSPM was shown to be moderately
effective at inhibiting the growth of the solid B-16 melanoma tumor
and Lewis lung carcinoma in C57B1/6 mice.
Not only was the in vivo activity of DEHSPM shown to be sub¬
stantially greater than that of DESPM (on a milligram basis) but
DEHSPM was shown to be a better and safer drug. At dosages of 2.5 to
7.5 mg/kg every 8 h for 6 days, DEHSPM had good activity without
significant toxicity. This demonstrated a wider dosage range of
activity than DESPM, which was active only in the dose range of 10-15
mg/kg every 8 h for 6 days, while 17.5 mg/kg every 8 h for 6 days

143
showed signs of toxicity. Also, at a constant total dose of DESPM,
decreasing the dosing schedule from 3 times daily to twice daily
greatly reduced the effectiveness. However, DEHSPM showed signifi¬
cant activity at a constant daily dose whether given on a one, two,
or three times daily schedule. While DEHSPM was shown to be more
toxic (on a milligram basis) it has a better therapeutic index (toxic
dose/active dose) than DESPM. The total dose necessary for the LD^g
(for the 5-day toxicity) was divided by the total dose of the ana¬
logue necessary to produce a significant ILS as a quick indicator of
therapeutic index. For example, to produce a 269% ILS required
DEHSPM 10 mg/kg every 8 h for 6 days (180 mg total dose) and the
total dose for the 5-day LD5q was 415 mg, yielding a value of 2.3
as the index for DESPM. To produce a 242% ILS it required DEHSPM 2.5
mg/kg every 8 h for 6 days (45 mg total dose) and the total dose for
the 5-day LD^g was 185 mg, yielding an index of 4.1. At doses neces¬
sary to yield a comparable ILS against the LI210 tumor, DEHSPM had
1.75-2 times better therapeutic index than DESPM.
Preliminary studies have shown that DESPM is rapidly adsorbed
from the peritoneal cavity of the mouse, reaching peak serum levels
in 15 min. As expected, the water soluble highly charged analogue is
rapidly cleared from the serum, exhibiting a serum half-life of
approximately 10 min. With these kinetics it was surprising, even
though animals were dosed 3 times daily, that the compounds were

144
active. The excretion studies demonstrate, however, that while some
of the drug is eliminated from the body at an early time point
(mainly in the urine) it is probably highly tissue bound and a large
fraction of the dose administered remains in the animal. Apparent
decreased excretion of the analogue due to metabolism to an unknown
product is very unlikely in light of present knowledge of polyamine
metabolism (108). Further pharmacokinetic, metabolic and organ
distribution studies are needed to optimize the dosage schedule of
the analogues to maximize effectiveness and minimize toxicity.

CHAPTER IV
CONCLUSION
The microbial iron catechol amide chelators (siderophores) and
the N-alkyl polyamine analogues have been shown to be effective
agents in controlling the growth of cancer cells.
The hexacoordinate catecholamide chelators, parabactin and vib-
riobactin, were the most active of the ligands tested. When L1210
and Daudi cells were exposed to the siderophores, parabactin and
vibriobactin, cell growth was essentially halted. Both these sidero¬
phores have ICgQ values of less than 2 yM, with reversible cytostatic
activity on short-term exposure and cidal activity on long-term
exposure.
The siderophores are also shown to be potent antiproliferatives
with activity in the micromolar range against a broad range of other
cultured cell lines, e.g., murine B-16 melanoma, CHO cells, HL-60
cells and HFF cells.
The catecholamide siderophores demonstrated their ability to
control cellular proliferation by inhibiting normal iron utilization
by cells. Preliminary studies with the catechol ami des had demon¬
strated their ability to block DNA synthesis through the inhibition
of the iron dependent enzyme ribonucleotide reductase. The anti¬
proliferative activity of the chelators has now been clearly asso¬
ciated with their ability to chelate iron. Methylation of the
145

146
catechol hydroxyl groups of parabactin and vibriobactin, or addition
of equalmolar amounts of Fe(III) to the medium along with the chelat¬
ors resulted in a loss of their cell growth inhibitory activity.
Others have shown that the rate of removal of iron from serum
transferrin by iron chelators is relatively slow when compared to the
very early inhibitory effects of the catechol amide chelators on L1210
cell growth and DNA synthesis. The ligand's antiproliferative activ¬
ity and DNA synthesis inhibition, however, is little affected by
serum concentrations in the growth medium. These studies provide
strong arguments for catechol amide interference with iron utilization
by chelation at an intracellular site rather than the extracellular
removal of iron from transferrin in the medium.
Flow cytometric analysis reveals that the catecholamide chelat¬
ors, parabactin and vibriobactin, block DNA synthesis with cells
accumulating at the G^-S border of the cell cycle. On removal of the
chelator, the cell cycle block is released resulting in a cell cycle
synchronization. Parabactin, a relatively nontoxic microbial iron
chelator, was a more potent cell cycle synchronization agent than
either hydroxyurea or desferrioxamine, with a portion of the cell
population remaining synchronized for 3 cell cycles. Parabactin pro¬
duces a similar cell cycle block and synchronization in mice with
ascites L1210 tumor.
The catechol amides act as potent cell blocking and cell cycle
synchronization agents and in proper combination may enhance the

147
effects of phase-specific antineoplastics such as ara-C, Adriamycin,
or BCNU. However, the time frame for the drug combination is criti¬
cal. This enhancement effect, while offering some potential advant¬
age in the design of combination chemotherapy protocols, has not been
demonstrated in vivo. The search for drug combinations which give
better differential activity between the anti neoplastic alone and
antineoplastic in combination with the chelators, as well as the syn¬
thesis of a potent water soluble chelator is currently under investi¬
gation.
The terminally bis-alkylated polyamines evaluated in this study
were highly effective in controlling the growth of rapidly prolifer¬
ating cells. It is clear that the spermine compounds are substan¬
tially more active against LI210 cells than the corresponding sperm¬
idine derivatives. DESMP and DEHSPM were the most potent compounds
with regard to inhibition of L1210 cells; they were also active
against a variety of human and animal tumor cell lines in vitro with
IC^qS of less than 1 ym in all cases.
The Mdr cell lines showed collateral sensitivity with their
parental cell lines to DESPM and DEHSPM. Resistance to the polyamine
analogues is, apparently, unrelated to the Mdr phenomena and resist¬
ant cells were equally sensitive as their parenteral lines to Adria¬
mycin. This lack of cross-resistance between the polyamine analogues
and Mdr-cells may be of some possible clinical significance.
Although the analogues did not appear to demonstrate direct
cytotoxicity, the ability of treated cells to proliferate when

148
incubated in drug-free medium was dependent on time of exposure to
the drug. This may indicate some earlier lethal event caused by the
analogue from which the cells cannot recover.
Changes in nDNA content of polyamine treated cells were not
consistent with a decrease in cell growth caused by blocking progres¬
sion of cells through any particular phase of the cell cycle.
On exposure of L1210 cells to the active polyamine analogues,
mtDNA was depleted. The decrease was consistent with a lack of syn¬
thesis coupled with continued cell division in the first few days
after treatment. These results suggest that although the cells are
alive, they simply do not have sufficient energy to support normal
division, although other explanations are certainly feasible. The
striking effects of the polyamine analogues on the mitochondria of
the L1210 cells may be independent of depletion of intracellular
polyamine pools and polyamine biosynthetic enzymes.
Exposure of L1210 cells to polyamine analogues, at concentra¬
tions which cause growth inhibition, decreased cell size substantial¬
ly, a decrease that continued with exposure time. This phenomena may
be due to secondary energy effects caused by mtDNA inhibition.
Small changes in the structure of the polyamine analogues, e.g.
DENSPM vs. DESPM vs. DEHSPM, and DMSPM vs. DESPM vs. DPSPM, have pro¬
nounced effects on the ability of the analogues to control the growth
of LI210 cells. An understanding of the differences in activity
among the various polyamine analogues is not only likely to help
reveal the mechanism of the drug's action, but also to provide

149
valuable information as to the biochemical role of polyamines in
cellular function.
In evaluating the effects of the polyamine analogues on poly¬
amine pools, although the concentrations of the polyamines spermi¬
dine, spermine and putrescine are altered over a 24 h exposure, there
is an approximate conservation in total cellular polyamine content
(spermidine, spermine, putrescine and analogue). Conservation of the
total intracellular polyamines is consistant with polyamine analogue
replacement of the natural polyamines at intracellular sites.
Furthermore, these compounds were highly effective in extending
the life span of DBA/2 mice with murine L1210 leukemia. The ana¬
logues, DESPM and DEHSPM, were shown to have significant activity
against L1210 ascites tumor growth in DBA/2 mice. The results
clearly demonstrated a dose-response relationship in activity. At
optimum dosage and treatment schedules both analogues demonstrated
significant increases in life span with a large percentage of
"cures." Similar activity was seen when the analogue was injected at
a site remote from the tumor. DEHSPM was also moderately effective
at inhibiting the growth of the solid tumors, B-16 melanoma and Lewis
lung carcinoma, in vivo.
Not only was the in vivo activity of DEHSPM shown to be sub¬
stantially greater than that of DESPM but DEHSPM was also shown to be
a better and safer drug. DEHSPM demonstrated a wider dosage range of
activity than DESPM, and had significant activity when given as a
one, two, or three times daily dosing schedule. While DEHSPM was

150
shown to be more toxic on a per milligram basis, it has a better
therapeutic index than DESPM.
Preliminary studies indicate that the polyamine analogues are
rapidly adsorbed from the peritoneal cavity of the mouse and rapidly
cleared from the serum. The drugs are eliminated from the body,
mainly in the urine, however, they are presumably highly tissue
bound.
The activity against mouse tumor models and moderate toxicity
of the N-alkylated polyamine analogues qualify them for further study
as to the mechanism of action, pharmacokinetics, metabolism and
activity against human tumors in mice, and for eventual human clin¬
ical testing as antineoplastics.
The catecholamide iron chelators and N-alkyl polyamine ana¬
logues have both been shown to be potent agents for the control of
neoplastic cell growth. Both classes of agents are currently under
investigation for their ability to control growth in other prolifer¬
ative processes.

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26: 3643-3649 (1987).
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York, 1988.

BIOGRAPHICAL SKETCH
Michael J. Ingeno was born in Upper Darby, Pennsylvania, in 1953.
After receiving his B.Sc. in chemistry from the Philadelphia College of
Pharmacy and Science, he went on to receive his M.Sc. at the Institute
of Paper Chemistry in Appleton, Wisconsin. He obtained his B.Sc in
pharmacy from Philadelphia College of Pharmacy in Philadelphia, where
he practiced as a staff pharmacist for four years and was a member of
the nutritional support team.
Among the awards he has received are the Freshman and Junior
chemistry awards, and the American Chemical Society Philadelphia Sec¬
tion's Scholarship Achievement Award while graduating with "meritorious
honors." During his graduate studies he was a finalist in the Excel¬
lence in Graduate Research Award and has numerous publications.
After completion of his Ph.D. in medicinal chemistry from the
University of Florida, Michael J. Ingeno and family will reside in
Basel, Switzerland, while he is holding a postdoctoral position with
Ciba-Geigy Ltd.
158

I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
aymor
Professor of
n, üíair
icinal Chemistry
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
Richard R. Streiff
Professor of Medicina
Chemistry
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
Margaret 0/ James
AssociateTrofessor of Medicinal
Chemistry
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
Associate Professor of Medicinal
Chemistry

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Professor of Biochemistry and
Molecular Biology
This dissertation was submitted to the Graduate Faculty of the
College of Pharmacy and to the Graduate School and was accepted as
partial fulfillment of the requirements for the degree of Doctor of
Philosophy.
December 1988
/
liM*
Dean, College of Pharmacy
Ls
Dean, Graduate School

UNIVERSITY OF FLORIDA
3 1262 08554 3923




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