The binding of the nonsteroidal antiinflammatory drugs benoxaprofen and fenbufen to human serum albumin

The binding of the nonsteroidal antiinflammatory drugs benoxaprofen and fenbufen to human serum albumin


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The binding of the nonsteroidal antiinflammatory drugs benoxaprofen and fenbufen to human serum albumin
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vii, 116 leaves : ill. ; 29 cm.
Fleitman, Jeffrey Scott, 1955-
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Propionates   ( mesh )
Anti-Inflammatory Agents   ( mesh )
Serum Albumin   ( mesh )
Pharmacy thesis Ph.D   ( mesh )
Dissertations, Academic -- Pharmacy -- UF   ( mesh )
bibliography   ( marcgt )
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Thesis (Ph.D.)--University of Florida, 1981.
Bibliography: leaves 110-115.
Statement of Responsibility:
by Jeffrey Scott Fleitman.
General Note:
General Note:

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University of Florida
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Full Text







To my parents, for their unwavering support and encouragement, I

dedicate this with all of my love.

To Dr. John Perrin and Dr. Stephen Schulman for being not only

teachers but friends I will never forget. To Dr. Stephen Curry and

Dr. Federico Vilallonga for always having the time to talk.


ACKNOWLEDGMENTS... ........................................ ii

ABSTRACT..................................................... v


Introduction..... ....................................... 1
Equilibrium Dialysis ................................... 3
Circular Dichroism....................................... 7
Microcalorimetry ........................................ 11
Fluorescence Spectrometry.......................... ..... 12
Investigations Into the Binding of Phenprocoumon to
Albumin Using Fluorescence Spectroscopy.................. 13


BENOXAPROFEN IN PLASMA.................................... 29

Introduction ............................................ 29
Materials ................................................ 29
Procedures .. ............................................. 31
Results .................................................. 32
Discussion .. ............................................. 32


Experimental ............................ ................. 38
Binding of Benoxaprofen to Hemodialyzed Plasma............. 45


Basic Structure......................................... 48
Polymeric Forms......................................... 50
N-F Transition........................................... .. 51
N-B Transition........................................... .. 52


Introduction ............................................ 56
Materials and Methods................................... 56
Results and Discussion................................... 57


Introduction ............................................ 77
Materials and Methods................................ ... .. 78
Results and Discussion................................ ... 79

BOLITES.................................................. 89

Introduction .. ........................................... 89
Materials .................................................. 89
Chromatographic Conditions................................ 92
Analytical Procedure................... ................. .... 92
Results and Discussion.............................. ....... 93

ALBUMIN: DRUG DISPLACEMENT STUDIES ........................ 99

Introduction ............................................. 99
Materials and Methods............................. ... ..... 99
Results and Discussion........................... .... .. 100

BIBLIOGRAPHY............................................... 110

BIOGRAPHICAL SKETCH............................. ........... 116

Abstract of Dissertation Presented to the
Graduate Council of the University of Florida
in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy




JUNE 1981
Chairman: John H. Perrin, Ph.D.
Major Department: Pharmacy

Equilibrium dialysis, microcalorimetry and circular dichroism are

used to study the binding of benoxaprofen and fenbufen to human serum

albumin (HSA). The theoretical basis for these techniques is examined

and then they are used as analytical tools in characterizing these drug-

albumin interactions.

Benoxaprofen has been found to be very highly bound to HSA (>99%)

with an affinity constant for the first binding site (K1) equal to 6.02 x

106 Z/mole. The effects of hemodialysis of benoxaprofen binding in vitro

havealso been studied.

A pH induced conformational change (N-B) in HSA is described and the

effects of the albumin conformation on benoxaprofen binding are elucidated.

It was found that chloride and calcium ions also alter the N-B transition.

Calcium ions alter the albumin conformation such that the binding capacity

for many drugs is increased. However, this study describes an additional

effect in which they compete with the carboxylate residue of benoxaprofen

for binding to the imidazole residue on the amino acid histidine. This

results in a lower binding affinity for benoxaprofen.

Characterization of the binding site on HSA for benoxaprofen is accom-

plished by drug displacement studies. Two high affinity sites of the

binding of anionic drugs are described and the interactions of benoxapro-

fen with drugs specific to these sites is quantitated.

It was found that the binding of fenbufen to HSA was much more diffi-

cult to characterize. Rather than displace drugs common to its binding

site, fenbufen induces a conformational change in the albumin which results

in a cooperativity effect. Fenbufen is described as directly influencing

the N-B transition.

High performance liquid chromatography was used as a method of detec-

tion in the equilibrium dialysis experiments and assays for benoxaprofen

and fenbufen are presented.

A rapid high performance liquid chromatographic assay for the deter-

mination of benoxaprofen in human plasma, is described. Plasma samples

of 1.0 ml, to which benoxaprofen, and warfarin as an internal standard,

had been added, were extracted with ether under acidic conditions. The

samples were analyzed on a MicroPak CN-10 column using 25% acetonitrile

in water (pH 2.5 with phosphoric acid). Detection was made on a variable

wavelength UV absorbance detector at 309 nm.

Samples containing 0.5-10 pg benoxaprofen gave a mean extraction

recovery from control plasma of 90.6 6.8% (n=18). Stability tests

have shown that benoxaprofen in plasma is stable for at least two weeks

after freezing.

A high performance liquid chromatographic assay for the determination

of fenbufen and its two serum metabolites is described. The overall

retention time for the chromatographic procedure is 9 minutes compared


to 22 minutes for the existing method. The compounds were extracted from

a 1.0 ml plasma sample using cyclohexane-ether (7:3) under acidic condi-

tions. Analyses were made on a MicroPak CN-10 column using water-isopro-

panol-acetonitrile-phosphoric acid (84.5:10:5:0.5). Detection was made

on a variable wavelength UV detector at 265 nm. The lower limit of detec-

tion for all compounds was 0.5 ug/ml.



Techniques for the study of drug-albumin interactions can be broadly

classified into two categories: those which separate the bound from

unbound ligand and those which do not. Methods of separation include gel-

filtration, ultrafiltration, ultracentrifugation and equilibrium dialysis.

Methods which do not involve separation are primarily techniques such as

UV, visible wavelength, fluorescence, NMR, ESR, as well as optical rota-

tory dispersion (ORD) and circular dichroism (CD). Flow microcalorimetry

also does not involve separation.

Choice of technique depends largely upon the physical characteristics

of the drug being studied. If it had poor spectral properties due to

lack of aromaticity or conjugation, the most convenient methods of UV-

visible and/or fluorescence spectroscopy may be useless. Photodegradation

problems may also limit these techniques. In such cases, microcalori-

metry is a useful alternative.

The separation methods permit measurements of free and bound drug

at equilibrium to be made. Once the free drug is separated from the bound

complex it can be analyzed by any one of the standard techniques such as

UV, visible wavelength or fluorescence spectroscopy, high performance

liquid chromatography (HPLC) or gas chromatography (GC). Again, if the

drug has poor spectral properties, GC or HPLC with electrochemical

detection might be used. Using a radiolabelled drug with a liquid scin-

tillation counter as a mode of detection can be a great advantage over

the other analytical procedures. There would be no need for sample

extraction as is usually the case with chromatographic procedures and it

may be more specific than direct spectrophotometric analysis.

The separation techniques allow us to calculate the most important

binding parameters such as the binding constant (K) and the number of

binding sites (n). If the binding study is done at different tempera-

tures, the thermodynamics of the binding reaction can be determined using

the Van't Hoff equation. Spectroscopic techniques can also be used to

determine these parameters if distinctions between the optical properties

of the bound and unbound drug can be made. However, by being able to

directly measure the formation of the drug-albumin complex the spectro-

scopic techniques have the advantage of allowing the researcher to char-

acterize the complex between the drug and albumin. For example, the

observance of an isosbestic point or sigmoidal character of a titration

curve can lead one to make direct inferences as to the number of mole-

cules bound per albumin molecule.

These methods should be thought of as complimentary and not competi-

tive. A disadvantage of the separation methods is that they tend to

involve a perturbation of the protein system. Ultracentrifugation and

ultrafiltration may cause changes in protein concentration during an

experiment which may give rise to inaccurate binding parameters. This

problem tends to be minimized when equilibrium dialysis is used.

Four of the techniques mentioned above, equilibrium dialysis, fluor-

escence spectroscopy, circular dichroism and microcalorimetry have been

found to be most useful in studying drug-albumin interactions in our

laboratory. The theoretical and practical aspects of these methods will

be presented in greater detail.

Equilibrium Dialysis
Basically, the principal objectives of an equilibrium dialysis experi-

ment are to determine the free and bound concentration of a ligand in a

system where it has an affinity for a macromolecule. A typical equili-

brium dialysis experiment involves the separation of a solution of a low

molecular weight ligand by a semipermeable membrane from a solution con-

taining a high molecular weight macromolecule. Eventually, equilibrium

will be reached where part of the ligand is bound to the macromolecule

and the free ligand concentration is equal on both sides of the membrane.

Diffusion of the ligand through the membrane obeys Fick's First Law.

This expression states that the amount of a substance diffusing in time

across a plane of area is directly proportional to the change of concen-

tration with distance travelled. An ideal membrane would therefore allow

maximum diffusion of the ligand and completely retain the macromolecule.

Several authors have used equilibrium dialysis to study the binding

of ligands to macromolecules. Original work was first done by Davis (1943)

and Klotz (1946) with improvements subsequently made on these methods.

Technical innovations which decreased dialysis time and increased preci-

sion have also been accomplished (Weder et al. 1971).

The dialysis apparatus used in all of the experiments in this work

is known as the Dianorm equilibrium dialysis system (Diachema AG, Zurich).

It consists of two teflon dialysis cells separated by a semipermeable

membrane. Teflon cells are chosen as they allow for minimum drug absorp-

tion onto the surface. All experiments were done in cells filled to 4.5

ml. The semipermeable membrane is a hydrated cellulose (Diachema type

10.16) and has a molecular weight cutoff of 10,000. Albumin in the

appropriate buffer is injected into one cell and drug in buffer into

the other cell. Each solution should be identical in pH and ionic
strength. The cells are then rotated in a water bath for 12 hr at 250C.
With this apparatus, equilibrium is reached well within this time
and the free ligand concentration CL(f) is the same on both sides of the
membrane. Since CL(o),the starting initial concentration of ligand is
known, we can determine CL(b), the bound ligand concentration, using the
following relationship

CL(b) = CL(o)- 2 CL(f) (1)
This relationship might be upset if some ligand binds to the membrane,
in which case

CL(b) = CL(o) 2 CL(f) CL(m) (2)
The concentration or amount bound to the membrane CL(m) is determined in
a series of experiments without any macromolecule. In the concentration
range of ligand which would be used if macromolecule were present in a
binding experiment, equation 2 becomes

CL(m) = CL(o) 2 CL(f) (3)
The only alternative to this approach is to assay both sides of the mem-
brane for ligand. An efficient extraction procedure which will determine
ligand concentration on the bound side is then a necessity.
Another problem which might arise in drug-albumin studies involves
the Gibbs-Donnan effect, which may effect the equilibrium when charged
species are involved in the experiment. This phenomenon can be better
illustrated by the following example where a negatively charged macro-
molecule solution such as albumin (R-) is placed on one side of a semi-
permeable membrane and NaCl is placed in solution on the other side. If
equilibrium is established, the system can be represented by the diagram
in figure 1.





-----semipermeable membrane

Figure 1. Sodium chloride
of albumin (R~)

in equilibrium in the presence
at physiological pH.



At equilibrium the sodium chloride concentration (in dilute solutions)
must be the same on both sides of the membrane and the total charges on
both sides of the membrane must be neutral. Equations (4), (5) and (6)
represent these conditions

[Na]o [C1-]o = [Na+]i [Cl-]i (4)

[Na+]o = [Cl-] (5)

[Na+]i = [R-]i + [Cl-]i (6)
Substituting equations (5) and (6) into (4) gives

[CI-]o2 = ([Cl-]i + [R-]i)[Cl-]i
=[Cl- (1]i2 (+ (7)

[Cl]o 1 + [R-]i
0 ( = +[C] ) (8)
[C1-]i [C1-ji

When the macromolecule concentration inside the membrane [R-]i is very
large it tends to push the anion [Cl-]i out through the membrane. In
this case, the ratio would be approximately equal to [R-]i. If [Cl']i
is much larger than [R~]i then the ratio in (8) would approach unity and
the salt concentration on both sides of the membrane is the same.
All equilibrium dialysis experiments in this work are done in buffer
solutions of 0.1 M so as to negate the effects of the Donnan Membrane
SWith these considerations realized, we can then use the equilibrium
dialysis apparatus to obtain the drug-albumin binding parameters. The
free fraction f of drug is given as

f L(f) (9)
CL(b) + CL(f)

which in actuality is the concentration of drug analyzed for on the free

side divided by the total drug concentration of the bound side of the


The bound fraction b is

b = 1-f (10)
As the total protein concentration will be known it is convenient to

define an experimentally determinable quantity r, as the average number

of ligand molecules bound per molecule of protein

r = CL(b) ()
Plotting r/CL(f) vs. r will result in a Scatchard plot (Scatchard, 1949)

from which the binding constants Ki, K2, etc. and nl, n2, the average num-

ber of ligands bound to each class of sites (if more than one site exists).

Physiological concentrations of albumin are usually used in dialysis
experiments. The binding parameters obtained from dialysis are lower than
those obtained from the spectrophotometric methods. The lower albumin

concentrations used in fluorescence or CD (sometimes 100X less) tend to

give rise to higher affinity constants. This may be a result of dimeri-

zation of the albumin at high concentrations.

Reviews of methods of handling dialysis data have been done by Weder
et al. (1974), Peters and Pringoud (1979) and Vallner (1974).

Circular Dichroism
Observations about differences in absorption of the components of
circularly polarized light, circular dichroism, were first reported by

Haidinger (1847). Since that time it was used by various authors to study

organic chemical problems but they were very limited by technical diffi-
culties. It wasn't until the 1950's that photoelectric spectropolarime-

ters became available and convenient circular dichroism studies were made

possible (Mitchell, 1957).

Basically, in order to get information from the use of circular

dichroism, one has to have a compound with an optically active chromophore

in the spectral range under examination. This is usually the UV-visible

light absorption region. A molecule may absorb light and not be optically

active so it is necessary to describe what constitutes optical activity.

The Pasteur principle states that in order to be optically active

a molecule must be dissymmetric; the molecule and its mirror image are

not superimposable (Pasteur, 1848). Dissymmetry does not imply the absence

of all elements of symmetry but only the absence of a plane or center of

symmetry. The two structures shown below exhibit optical activity due

to dissymmetry arising from different conditions.

Br- C C1 C3H7 C C2H5
I C4H9
(I) (II)

Structure (I) exhibits dissymmetry arising from the differences in elec-

tronegativity of the atoms surrounding the asymmetric carbon atom. The

optical activity of structure II has dissymmetry which arises from the

spatial arrangements of the attached substituents. It is possible for a

symmetric chromophore to exhibit optical activity if it is placed in the

environment of a dissymmetric moiety. Drug-albumin interactions tend to

exhibit optical activity by this phenomenon.

When one wavelength from a beam of light is sent through a polarizer,

the emitted wave of plane polarized light (e) is made up of two vectors.

One vector corresponds to right circularly polarized wave ER and one to

left circularly polarized wave eL. To be optically active, a medium has

to show different indices of refraction for the left and right circularly

polarized light (nL and nR). This is known as circular birefringence, as

the left and right handed wave components will have different speeds due

to the inequality of nL and nR. The lengths of eL and ER remain equal

and plane polarized. If this phenomenon occurs in a region where there

is an optically active chromophore which absorbs light, there will be

differential absorption of EL and ER. The component with the larger index

of refraction or lesser velocity is preferentially absorbed and their

resultant vector e no longer oscillates along the circumference of a

circle but traces out an ellipse. This is called circular dichroism, a

differential absorption between circularly polarized beams. Diagrams

elucidating the waveforms involved along with more detailed elaborations

on the mathematics involvedin these transitions can be found in great

detail in texts by Crabbe' (1965), Djerassi (1960) and Urry (1970).

The circular dichroic signal which one measures on the spectropolari-

meter is reported as the observed ellipticity o in degrees. It can be

reported as the molar ellipticity -, where C is the molar concentration

of the optically active molecule and 1 is the path length in centimeters

required to obtain the molar ellipticity.

Spectra obtained from circular dichroic measurements are characterized

by so called "Cotton" effects of positive or negative maximums, minimums

and points of inflection. Since this work deals with the binding of drugs

to albumin, the factors which give rise to circular dichroic signals in

this system will be examined.

Macromolecules such as albumin have at least three types of dissym-

metry which may lead to optical activity. If the substituents on the a

carbon of an amino acid give rise to an asymmetric configuration,then the

primary structure of the protein is said to be inherently asymmetric and

may lead to optical activity. The secondary structure of albumin which

is helical in nature may give rise to optically active electron transitions.

Finally, the tertiary structure might be influential in that symmetrical

groups such as a drug molecule might be thrust into an asymmetric environ-

ment, as provided by the protein and give rise to a distorted electron

displacement which may lead to an induced optical activity. This case

usually gives a much weaker Cotton effect and occurs at wavelengths char-

acteristic of the drug molecule.

When analytically studying the effects of drugs binding to albumin

the researcher must usually work in the wavelength region above 300 nm

where there are negligible contributions from the albumin and measure the

induced ellipticity, if one arises. Difficulties arise when trying to

study binding at lower wavelengths due to the intrinsic ellipticity of

the albumin molecule. In the wavelength region between 250 nm and 300 nm

a strong CD signal arises from secondary structure, nonbackbone chromo-

phores. Aromatic amino acids, phenylalanine, tyrosine and tryptophan and

cysteine all give a strong signal. Tryptophan is especially influential

at 290 nm. In the region below 250 nm the backbone amino acids have a

very strong influence as well as the aromatic amino acids and histidine

and cysteine (Urry, 1970; Vallner, 1974).

Many small symmetrical molecules give rise to induced circular dichroic

activity when bound to albumin. Examples may be a variety of coumarins

and phenylpropionic acid derivatives (Perrin, 1973). Hydrophobic inter-

actions as well as van der Waal's forces, hydrogen and ionic bonding tend

to give rise to induced optical activity (Klotz, 1973). Structurally,

acidic drugs with large flat hydrophobic areas which are able to form a

rigid complex with the protein are also generally regarded as essential

criteria (Chignell, 1969; Rosen, 1970; Perrin, 1973).


Microcalorimetry has proven to be a useful analytical tool in the

study of drug-albumin binding. It has been used in the study of benzo-

diazepin-albumin interactions (Coassolo et al. 1978a,b), drug-cyclodextrin

interactions (Hardee et al. 1978) and drug-microorganism interactions

(Beezer, 1980).

Primarily, its use in this study arises from the fact that when drugs

bind to albumin the very small heat evolved or absorbed is proportional

to the amount bound. This allows one to calculate binding parameters

and also serves as a useful analytical tool for drug displacement studies.

It is advantageous in the binding studies of drugs with poor optical pro-

perties and is a non-invasive technique. Unlike the spectroscopic methods,

microcalorimetry does not alter the properties of the system under study

or introduce artifacts during measurements.

An LKB 2107-121 Flow Microcalorimeter (LKB, Bromma, Sweeden) will be

used in this study. It is fitted with two separate gold cells in which

heat reactions can take place. An albumin solution pumped into the calor-

imeter at about 14 ml/min reacts with a drug solution pumped through a

second pump and the binding reaction occurs in a gold mixing vessel. Heat

generated in this reaction flows across a thermocouple and into a heat

sink assembly. If heat is absorbed by the reaction, heat flows from the

heat sink to the mixing vessel.

Heat flowing across the thermocouple generates an electromotive force

proportional to the temperature difference. A second thermocouple acts

as a reference so that when the electromotive force in both thermocouples

is equal the voltage output is zero. A "Wheatstone bridge" circuit is

used to balance the thermocouples so that this zero obtained is actually

the experimental baseline which encompasses heats of dilution, flow effects,

etc. When an exothermic reaction occurs and is measured by the first

thermocouple, the heat in the second thermocouple is subtracted and an

output signal is amplified, fed into a recorder and read in microvolts.

The entire system is isothermal and kept in a Tronac Model 1005 water bath

with a PTC 40 temperature controller (Tronac, Orem, Utah) at 25 0.0020C.

A thorough description of this system has been given by Hardee (1980).

Disadvantages of this system include the large amount and concentra-

tions of reactants which must be used. To get a good measurable signal

in these experiments, albumin in the concentration range of 5.0 x 10-4 M

must be used. Drug concentrations of similar magnitude will also be used

and obviously solubility problems will limit this technique.

Fluorescence Spectroscopy
Fluorescence spectroscopy has proven to be one of the quickest and

most direct methods of studying drug-albumin interactions. Changes in

drug fluorescence on binding to proteins can be monitored and this tech-

nique has been used by many authors (Chen, 1967; Berde et al. 1979; Geddes

and White, 1979; Otagiri et al. 1979). Fluorescent markers have also

been used to study the displacement of drugs bound to albumin (Chignell,

1969; Sudlow et al. 1975). If a drug being studied does not fluoresce

or if the bound complex has poor fluorescence characteristics,then changes

in the native fluorescence of the albumin may be measured. This fluores-

cence quenching technique in which decreases in tryptophan fluorescence

are monitored has been studied and reviewed by Nieves (1980).

Measuring fluorescence enhancement of drugs upon binding to albumin

is the preferred technique for these studies due to the direct method in

which the binding parameters may be obtained and the low drug to protein

ratios which can be detected. A practical example of this technique will
be illustrated in the following experiment in which the binding of

phenprocoumon to albumin is investigated using fluorescence spectro-


Investigations Into the Binding of Phenprocoumon
to Albumin Using Fluorescence Spectroscopy

The high affinity of the coumarins for human serum albumin (HSA) has

received much attention, particularly since it was realized that their

anticoagulant action was frequently potentiated by the presence of other

drugs in the plasma. More recently, pharmacokinetic and pharmacodynamic

investigations of the enantiomers of warfarin have shown different meta-

bolic fates for the two forms, (Breckenridge et al. 1974) whereas the

enantiomers of phenprocoumon show differences in volumes of distribution

(Jahnchen et al. 1976). The more potent S(-) phenprocoumon is more

strongly bound to HSA than is the R(+) enantiomer. This observation has

been confirmed by a recent limited dialysis investigation (Brown et al.

1977). In the present investigation, the enhanced fluorescence of phen-

procoumon following interaction with HSA is used to investigate the

binding characteristics of the enantiomers to HSA at low drug to protein

ratios and to investigate the possibility of displacement of the drug

from its primary binding site by other commonly prescribed drugs.

Materials and Methods

HSA (lot No. 126C-8070) and BSA (lot No. 17C-8145), crystallized and

lyophilized, and essentially fatty acid free HSA (lot No. 76C-7480) were

obtained from Sigma Chemical Co., St. Louis, MO. Racemic phenprocoumon

(Oraganon, Inc., W. Orange, NJ), S(-) and R(+)-phenprocoumon (Hoffman-

LaRoche, Basel, Switzerland), phenylbutazone and acenocoumarin (Ciba-Geigy

Company, Summit, N J), sodium fenoprofen (Eli Lilly Company, Indiana-

polis, IN), and sodium 2(p-chlorophenoxy)-2-methylpropionate (CPIB) (ICI

LTD., Macclesfield Cheshire, U.K.) were used as supplied by the manufac-

turer. All other materials were reagent grade and all solutions were pre-

pared in deionized water.

All solutions were prepared in 0.1M phosphate buffer of pH 7.4 at

22 1C. HSA concentrations of 0.1 5.0 x 10-5M (M.W. 69,000) were

used. Fluorescence measurements were made using a Perkin-Elmer MPF-44A

fluorescence spectrophotometer (Perkin-Elmer, Norwalk, CT). The fluoro-

metric titrations were carried out as follows: 2.0 ml of the protein solu-

tion of an appropriate concentration in a 10 mm path length cell were

titrated by the successive additions of 2.0 pl volumes of phenprocoumon

solution to give a final drug concentration of 0.1 20 x 106M in the

cell. The fluorescence intensity was measured at 382 nm following exci-

tation at 340 nm. For the experiments in the presence of antagonists, the

fluorescence of phenprocoumon bound to albumin was measured before and

after the addition of the antagonist. The ratio of phenprocoumon to albu-

min was 0.1 and the ratio of competitor to protein ranged from 0.1 to 3.0.


The fluorescence intensity of phenprocoumon was enhanced when bound

to crystalline, essentially fatty acid free HSA, and to bovine serum albu-

min (BSA). The fluorescence peak was also shifted from 386 nm to 382 nm

following interaction with the albumin and excitation at 340 nm. Fluoro-

metric titrations were made by altering the drug concentration at a fixed

albumin concentration as shown in figure 2. For curve a, determined at

high protein to drug ratios, all the drug is bound and this is a measure

of the fluorescence of the drug-albumin complex. At lower albumin concen-

trations a curve is obtained as shown in figure 2, curve b. The fractions

of the drug bound, X, can be determined by using the equation:


10 15

Plots of relative fluorescence intensities as a
function of phenprocoumon concentration for the
phenprocoumon-HSA interaction.

Figure 2.

f f
X = p (12)
f fo
fb o

where fp and fo are the fluorescence intensities of a given concentration
of drug in a solution of low albumin concentration and in solution with-

out albumin. Fb is the fluorescence of the same concentration of fully

bound drug. For this treatment to be valid, the fluorescence intensity

of the bound drug must be a linear function of concentration. This is

the case only when the absorbance of the complex at the exciting wave-

length is low. A correction for this absorption can be made by the method

already described (Otagiri et al. 1979) and is made for all the data when

the absorbance at 340 nm is greater than 0.02.

This method of calculating the fraction bound gives the expected

binding constant for 1:1 complexes, and for higher complexes if the quan-

tum yields associated with the various binding sites are identical. Analy-

sis of the data of fig. 2, curve b, by an iterative least squares tech-

nique, assuming 1:1 complex formation were unsuccessful. Apparently the

binding of more than one ligand molecule to the albumin accounts for the

change in fluorescence. This observance is confirmed by the Job plot

shown in figure 3. Although the peak for the two HSA samples is near 0.5,

the value expected for 1:1 complex formation, the tangents at the origins

are not equal in magnitude and opposite in sign as is necessary for 1:1

complex formation (Job, 1928). The quantum yield associated with the lower

affinity sites appears to be much lower than that associated with the

first site. This means that equation (12) does not strictly apply to the

data obtained at high drug to albumin ratios, and the second binding con-

stant obtained by this method is only an estimate of the method. This

problem is shared by other spectroscopic techniques and is not peculiar

to the fluorescence technique (Perrin et al. 1975). It was found that the

0.2 0.4 0.6 0.8 1.0



Figure 3. A Job plot of relative fluorescence intensities
for the phenprocoumon-serum albumin interaction

data fitted a modified Bjerrum model for two independent sites (Naik

et al. 1975) using the following equation:

P + D Z PD (13)

S[PD] [PD2]
Ki [P] [D]' K2 [PD] [D] (14)

CD [D] KI[D] + 2KiK2[D]2
n = Cp 1 + Ki[D] + KK2[D]2 (15)

and (2 [2 (i 2- )[D] K + KK (16)

where P, D and PD are the concentrations of unbound albumin, unbound drug,
and the complex respectively. CD and Cp are the total concentrations of
drug and albumin and n is the moles of drug bound per mole of albumin.

A least squares analysis of the linear equation 16 gives the binding data

shown in table 1 for the phenprocoumon-HSA and BSA interactions, and in
table 2 for the enantiomers to HSA interactions. Table 1 also shows the

binding parameters calculated by the Scatchard method (Otagiri, M. et al.

1979) assuming two independent binding sites, enough material was not

available to obtain the data points at the high drug to albumin ratios

necessary for reliable estimates of the binding parameters of the enan-

tiomers by the Scatchard method.

The fluorescence intensity of the phenprocoumon-HSA and -BSA com-
plexes increases as the pH is increased from 6-9 (fig. 4) and is decreased
upon the addition of sodium chloride (fig. 5). The fluorescence intensity
of complexes is lowered by phenylbutazone and acenocoumarin, but not sig-
nificantly altered by clofibrate in fig. 6. The fluorescence is slightly

increased by ibuprofen and fenoprofen.

Table 1. Binding parameters for phenprocoumon-serum albumin complexes.
K1 and K2 are binding constants for two binding sites and r
is the correlation coefficient for data points.

Bjerrum pH 6.5 r pH 7.4 r pH 8.5 r

BSA K(n = 1) 0.9 x 106 0.997
K2(n2 = 1) 7.0 x 104

Ki(ni = 1) 1.0 x 106 0.994 1.1 x 106 0.999 1.3 x 106 0.997
K2(n2 = 1) 2.2 x 104 1.6 x 104 2.0 x 104

Ki(ni = 1) 1.0 x 106 0.998 1.0 x 106 0.998 1.4 x 106 0.998
K2(n2 = 1) 1.9 x 104 3.2 x 104 5.2 x 104


Ki(ni = 1) 8.0 x 10 -
K2(n2 1) 1.2 x 105

Ki(ni = 1) 1.0 x 106 9.5 x 105 1.6 x 106 -
K2(n2 = 1) 2.1 x 104 5.0 x 104 1.0 x 105

FFA K(n = 1) 9.8 x 105 1.9 x 106 1.9 x 106 -
K2FFA 1) 7.4 x 3.8 x 2.8 x 10
K2(n2 = 1) 7.4 x 104 3.8 x 104 2.8 x 104

Table 2. Binding parameters
to HSA at pH 7.4.
sites and r is the

of binding of phenprocoumon and its enantiomers
K1 and K2 are binding constants for two binding
correlation coefficient for data points.

Phenprocoumon K1 K2 r

S (-) 1.3 x 106 2.3 x 104 .998

R (+) 0.8 x 106 1.5 x 104 .998

RS (+) 1.1 x 106 1.6 x 104 .997

S--- x 10O
cU--- 0 -0- 0 v 0


Figure 4.



Effect of pH on the fluorescence intensities for
phenprocoumon-serum albumin interactions.
0: phenprocoumon alone 9 : phenprocoumon-
HSA system A : phenprocoumon-BSA system
















Figure 5.


Effect of sodium chloride on fluorescence
intensities for phenprocoumon-serum albumin
interactions at pH 7.4
* : HSA 0 : BSA


w F_



1.0 2.0
Figure 6. Effect of various drugs on
of phenprocoumon in the pr
0 : acenocoumarin
A: phenylbutazone

x 10 5 (M)
fluorescence intensity
esence of serum albumin
: CPIB /A: fenoprofen
|: ibuprofen

< W

z- L







The fluorescence of phenprocoumon is enhanced following the inter-

action with albumins, allowing a quantitative investigation of the binding

phenomena. Although the enhanced fluorescence is mainly the result of the

interaction at a single site, the data cannot be interpreted in terms of

a 1:1 reaction but gave a better fit with a two site model. A similar

situation has earlier been reported for the interaction of warfarin with

HSA, also investigated by this fluorescence technique (Otagiri et al. 1979).

The binding constants given in Table 1 show that the affinity of phenpro-

coumon for albumin increases as the pH is increased. This increased

binding is also reflected in the increased fluorescence of the complex as

the pH is increased. For the binding at the first site there is little

difference between crystalline and essentially fatty acid free HSA. How-

ever, the binding at the second site of crystalline HSA appears to be pH

independent, whereas with essentially fatty acid free HSA, the binding

constant increases with pH. The N B transition in HSA occurs over the

pH region 6-9 (Leonard et al. 1963; Harmsen et al. 1971) and apparently

the binding to the secondary site in the B form is enhanced following the

treatment to remove the fatty acids. It should be noted that the binding

at the first site is the phenomenon of clinical relevance and also that

the affinity of warfarin for HSA also increases as the pH is increased.

These changes as a function of pH must be due to changes in the protein

rather than the changes in the degree on ionization of the coumarins as

the pKa of phenprocoumon is 4.30 and of warfarin is 5.10 (Otagiri et al.

1978). Data of Table 2 show that the affinity for the S(-) enantiomer is

greater than the affinity for the R(+) enantiomer for both the first and

second site on crystalline HSA. The racemic form, the commercial form of

the drug, being of intermediate behavior. These observations are in

agreement with those reported earlier, however, the binding constants

reported here are significantly higher than those reported earlier (Brown

et al. 1977). In the current investigations much more data are available

at low drug to protein ratios, and so, greater reliability can be placed

on the computed binding constants than those of Brown et al. It is inter-

esting to note that although the binding sites seem to be nonspecific in

that a wide range of acidic drugs seem to share the same binding sites,

the sites do show some stereospecificity. Sodium chloride diminishes the

fluorescence of HSA and BSA complexes with phenprocoumon as shown in Fig.

5. Although chloride has a small effect on the N B transition at the

physiological pH (Harmsen et al. 1971), the effect of chloride appears to

be primarily one of displacement as was observed in the case of warfarin

(Wilting et al. 1979). Fenoprofen and ibuprofen slightly increased the

fluorescence of phenprocoumon-HSA complexes at pH 7.4; in an earlier

study ibuprofen had been shown to increase the fluorescence of warfarin-

HSA complexes (Otagiri et al. 1979). When the enhancement caused by these

nonsteroidal antiinflammatory agents is compared to that caused by pH

changes (fig. 4), it is possible to conclude that the two drugs cause an

increase in the B conformation of albumin. Sudlow, Birkett and Wade have

indicated that warfarin and ibuprofen do not share the same primary binding

site on HSA (Sudlow et al. 1976). Clofibrate did not significantly lower

the fluorescence of the phenprocoumon complexes, whereas acenocoumarin and

phenylbutazone did as shown in fig. 6. These observations are in agreement

with the classification of Sudlow et al. Binding constants of 8 x 105 M~1

for acenocoumarin and 1.2 x 105 M-1 for phenylbutazone can be estimated

from these data (Perrin et al. 1975); however, unpublished observations in

these laboratories suggest that phenylbutazone may have a small effect on

the N B transition.


Salicylates such as aspirin have long been used for their antiinflam-

matory effect in the treatment of rheumatoid arthritis. Large doses

required for therapy tend to cause side effects such as gastric distress,

occult bleeding and tinnitus. There is also a widely known interaction

with the coumarins which precludes their use with many geriatric patients.

Researchers have tried to develop drugs which have as good or better

therapeutic efficacy as the salicylates with a lesser degree of side

effects. Phenylpyrazolone derivatives such as phenylbutazone have been

tried but have a high incidence of side effects. Indole derivatives such

as indomethacin also have a high degree of side effects with chronic use.

A newer class of drugs with proven antiinflammatory activity and per-

haps less severe side effects are the phenylpropionic acid derivatives.

Ibuprofen, fenoprofen, naproxen and benoxaprofen are drugs of this class.

It has been hypothesized that the nonsteroidal antiinflammatory drugs

exert their therapeutic effect by inhibiting prostaglandin synthetase

(Vane, 1971; Vane, 1973). Recently, it has been found that other mecha-

nisms which are independent of prostaglandin formation might be respon-

sible for the efficacy of some of these drugs (Crook et al. 1976; Bonta

et al. 1977). Attallah and Lee (1980) have shown that indomethacin and

salicylates interfere with prostaglandin binding to plasma proteins in

vitro. They suggest that this will accelerate the clearance of prosta-

glandin from the blood.

Benoxaprofen appears to be novel in that its mode of action does not

have anything to do with inhibition of prostaglandin synthesis. Rather,

benoxaprofen interferes with the emigration of leucocytes into inflamma-

tory sites (Cashin et al. 1977; Hutchinson et al. 1977) and this alternate

mode of action may result in decreased ulcerogenic potential.

Pharmacokinetic studies with benoxaprofen have shown that after a

single oral dose of 100 mg, the half-life of elimination was 37 hrs and

the volumes of distribution in the central and peripheral compartments

were 6.8 and 3.2 liters respectively (Chatfield et al. 1977). Therapeutic

blood levels after a single dose reach 13 pg/ml while multiple dosing with

100 mg every 12 hrs allows equilibrium concentrations between 35 and 45

jg/ml (Smith et al. 1977). Plasma protein binding studies find benoxa-

profen to be extensively bound to plasma proteins (99.8%) and no displace-

ment of warfarin or salicylate has been noted (Chatfield et al. 1978).

No major metabolic transformation of benoxaprofen was observed, with the

compound excreted mainly as the ester glucuronide (Chatfield and Green,


It has been reported that patients with severe rheumatoid arthritis

have undergone abnormal serum protein alterations including a resultant

decrease in albumin concentration (Weimer et al. 1968; Clarke et al.

1970). McArthur (1979) reports that this disturbance of the plasma pro-

teins causes an increase in the binding of naturally occurring protective

molecules, with rheumatoid arthritis symptoms appearing when the active

free molecule concentration of these protectantss" decreases. These hypoth-

eses implicate the importance of further study of the protein binding

characteristics of the nonsteroidal antiinflammatory drugs.

Changes in the protein binding properties of a drug such as benoxa-

profen are especially interesting because it is so highly bound, with a

small volume of distribution and long half-life. Minor changes in the

bound fraction of the drug can cause large changes in the free fraction

which may have major physiological implications.

Albumin binding characteristics of benoxaprofen will be studied

using equilibrium dialysis, circular dichroism and flow microcalorimetry.

A high performance liquid chromatographic assay is developed and will aid

as an analytical technique in the equilibrium dialysis experiments. Drug

displacement interactions as well as pH induced conformational changes in

albumin which effect benoxaprofen binding will be examined. These in

vitro studies serve to provide a useful foundation for future clinical




Benoxaprofen (fig. 7), [2-(4-chlorophenyl)-a-methyl-5-benzoxazole-

acetic acid] has been shown to possess notable antiinflammatory activity,

being several times more potent than phenylbutazone (Dunwell et al. 1975;

Cashin et al. 1977). Two gas chromatographic (Ridolfo et al. 1979; Chat-

field and Woodage, 1978) methods have been used to determine benoxaprofen

in biological fluids, but they are time consuming, involving derivatiza-

tion procedures. The high pressure liquid chromatographic determination

described here is rapid in having only a single extraction step and a

retention time for benoxaprofen of 6.0 min. Sodium warfarin (fig. 7), has

been found to be an excellent internal standard for this procedure since

it has a retention time of 5.0 minutes. The use of sodium warfarin as an

internal standard greatly increases the precision and accuracy of the assay.

Sodium benoxaprofen (903.5 mg free acid per gram) and sodium warfarin,

were generously supplied by Eli Lilly (Indianapolis, IN) and Endo Labora-

tories, Inc. (Garden City, NY), respectively. A stock solution of 100 Pg/

ml benoxaprofen in 0.025 M Na2HP04 was prepared. The internal standard

stock solution contained 100 ug/ml warfarin in 0.025 M Na2HP04. Working

standards were prepared by appropriate dilutions with deionized water of

these stock solutions to give 10 ml samples of 2.5, 5.0, 15, 25, 35 and 50

ug/ml benoxaprofen, each containing 25 pg/ml of the internal standard.

Each plasma sample was spiked with 5 to 100 pl volumes of 100 ug/ml








Figure 7. Structures of Benoxaprofen and Warfarin

benoxaprofen stock solution to give final concentrations of 0.5, 1, 3, 5,

7 and 10 ig/ml.

Ether, phosphoric acid, hydrochloric acid and dibasic sodium phos-

phate were all analytical grade and supplied by Mallinckrodt Inc. (St.

Louis, MO). HPLC grade acetonitrile and methanol were obtained from Bur-

dick and Jackson (Muskegon, MI). All solvents including deionized water,

were filtered through 0.45 pm filters (Millipore Corp., Bedford, MA) prior

to use in the liquid chromatograph. Samples analyses were carried out on

a model 5000 liquid chromatograph equipped with a 10 pl manual loop injec-

tor and a Vari-Chrom variable wavelength UV detector (Varian Assoc., Walnut

Creek, CA). An alkylnitrile column, MicroPak CN-10 (Varian Assoc.) ofmedi-

um polarity was used. The mobile phase consisted of 25% acetonitrile in

water (pH 2.5 with H3P04). At ambient operating temperature, the flow rate

was 1.8 ml/min at a pressure of 160 atm. The column effluent was monitored

continuously at 309 nm. All UV spectra of stock solutions were taken on

a Cary model 219 UV spectrophotometer (Varian Assoc.).

Plasma samples of 1.0 ml, to which 5 pg of internal standard had been

added, are acidified with 400 ul of 1N HC1 and mixed thoroughly by hand

shaking. After the addition of 8 ml of ether, the mixture is vortexed for

30 sec. and then placed on a test tube rotator for 10 min. The sample is

centrifuged at 3600 rpm for 10 min. and then the ether layer is transferred

to another test tube and evaporated to dryness under a nitrogen stream.

The residue is reconstituted in 200 pl of methanol and a 10 pl aliquot

injected into the column.

Six point standard curves were obtained from the stock solutions.

Spiked plasma samples were compared to the stock solutions to obtain recovery

percentages. Data were obtained by plotting peak height ratios versus

benoxaprofen concentration and a linear least squares regression program

was utilized.

Figure 8 shows typical chromatograms for control plasma, plasma with

benoxaprofen and plasma with benoxaprofen and warfarin. The retention

times for warfarin and benoxaprofen were 5.0 and 6.0 min respectively.

No interfering peaks in the control plasma were noted.

Table 3 shows the peak height ratios of benoxaprofen to 5 ug warfarin

in the 0.5-10 pg range of benoxaprofen extracted from human plasma. Each

value represents the average of three samples. A linear regression curve

obtained from the data has the equation y=0.257X + 0.041 with r2=0.9996,

where r2 is the coefficient of determination.

Extraction recoveries were determined by injecting known concentrations

of benoxaprofen with internal standard and comparing them to the extracted

plasma samples (Table 4). The extraction recovery was 90.6 6.8% for 18


Stability tests were performed by adding 0.5, 1, 3, 5, 7 and 10 pg

of benoxaprofen to samples of plasma and freezing them. Analyses were

performed at 1, 4, 9 and 14 days, the results of which are listed in table

5. These findings show the samples are stable for this time period.

Two GC methods (Chatfield and Woodage, 1978; Ridolfo et al. 1979)

have been used to determine benoxaprofen levels but no liquid chromato-

graphic method has previously been reported. Radiolabelled [14C] benoxa-

profen has also been used in biological fluid assays (Chatfield and Woodage,

1978; Chatfield et al. 1978; Chatfield and Green, 1978). The extraction

03 6

10 3 6


Figure 8. Liquid chromatograms of a) control human plasma, b) human
plasma containing 5 ug benoxaprofen and c) human plasma
containing 5 pg warfarin and 5 pg benoxaprofen.

0113 6
03 6

Table 3. Peak height ratios for benoxaprofen plasma calibration curve.

Amount Added







Peak Height Ratio
(Mean SD)













Table 4. Benoxaprofen recovery from human plasma. (N=3)

Amount Added Amount Found Recovery, %
(pg) (pg) (Mean SD)

0.5 0.478 0.015 95.5 2.9

1.0 0.942 0.088 94.2 8.8

3.0 2.820 0.120 94.0 4.0

5.0 4.235 0.205 84.7 4.1

7.0 6.069 0.483 86.7 6.9

10.0 8.870 0.270 88.7 2.7

Ave. 90.6 6.8

Table 5. Effect of frozen storage on benoxaprofen stability in human

(pg) 0 1 4 9 14

recovered (pg)






































recovery obtained, 90.6 6.8%, is similar to the extraction efficiency

obtained in the radiolabelled assay (Chatfield et al. 1978).

Though not structurally similar to benoxaprofen, warfarin has proved

to be an excellent internal standard. Retention times were one minute

apart, namely 5.0 min for warfarin and 6.0 min for benoxaprofen. Peak

height ratios exhibited excellent linearity in the concentration range

studied (0.5-10 pg/ml). A variable wavelength UV detector was used to

monitor benoxaprofen levels at 309 nm, its absorbance maximum in the sol-

vent system used. Warfarin has an absorbance maximum of 308 nm in the

same system. The signal-to-noise ratios obtained were on the order of

2-3 times greater than when a 254 nm filter detector was used.

Stability studies have shown that benoxaprofen was stable for at

least two weeks in frozen plasma. It was found, however, that stock

solutions of benoxaprofen were not stable at room temperature during this

time interval. Preliminary studies in this laboratory have shown that

aqueous solutions of benoxaprofen undergo extensive photodegradation.

This is especially apparent when a sample is placed in an intense UV light

source, as in a fluorimeter. New stock solutions were prepared when neces-

sary and concentrations were confirmed by UV spectra.

The lowest level of benoxaprofen detected in this study was 0.5 pg.

Lower levels can be detected by decreasing the reconstituting volume or

using a higher detector sensitivity. However, the concentration range

studied was within the expected therapeutic range (Chatfield and Green,

1978; Ridolfo et al. 1979).



Equilibrium dialysis and microcalorimetry will be used as techniques

to study the binding parameters of benoxaprofen-albumin complexes. The

stoichiometry and strength of binding (n,K) as well as the thermodynamic

binding parameters (AH,AG,LS) will be determined.

Benoxaprofen has been found to be unstable in the spectrofluorimeter.

The intense light source causes photodegradation to occur, whereas in

aqueous solutions a white precipitate forms instantaneously. This pre-

cludes the use of fluorescence spectroscopy in studying the binding prop-

erties of benoxaprofen.

Materials and Methods

Human serum albumin, fraction V (lot No. 30F-02271), was obtained

from Sigma Chemical Co. (St. Louis, MO). Sodium benoxaprofen (903.5 mg

free acid per gram) was generously supplied by Eli Lilly and Co. (Indiana-

polis, IN). Monobasic, dibasic sodium phosphate and sodium chloride,

analytical reagent grade, were obtained from Mallinckrodt, Inc. (St.Louis,

MO). Dowex 50W-X8 cation exchange resin and 1-X8 anion exchange resin

were supplied by J.T. Baker Chemical Co. (Phillipsburg, NJ). Deionized

water was used in all experiments.

Prior to all experiments, human serum albumin (HSA) is deionized by

running a solution through a column containing cationic and anionic exchange

resins. The resins are prepared by adjusting the pH of the cationic and

anionic forms to 1 and 12, respectively. The pH is then adjusted to 4.5


for the cationic form and 7.5 for the anionic form by rinsing with de-

ionized water. Anionic and cationic resin are then added to a column

where they are again rinsed with water. An albumin solution is run through

the column and the final pH is adjusted to 7.4 in 0.1 M phosphate buffer.

Microcalorimetry is used to obtain a Job's plot for the determina-

tion of the stoichiometry of the benoxaprofen-HSA complex. HSA solutions

of 0-5.0 x 10-4 M are pumped into one half of the mixing cell while benoxa-

profen solutions of 5.0 x 10-4 -0 M are pumped into the other side. After

1:1 dilution in the mixing cell, the total final concentration for the

benoxaprofen-HSA complex is 2.5 x 10-4 M. The heats of binding in pV vs.

mole fraction benoxaprofen are then recorded.

A titration of HSA with benoxaprofen is done calorimetrically. HSA

at a constant concentration of 2.56 x 10-4 M is pumped into one side of

the mixing cell while benoxaprofen in concentrations of 0-5.12 x 10-4 M

is pumped into the other side. The binding affinity for the first site

can be obtained from this method.

Equilibrium dialysis is used to obtain a Scatchard plot from which n

and K can be determined. HSA at a concentration of 2.0 x 10-4 M is dia-

lyzed vs. benoxaprofen in concentrations of 2.0 x 10-5 8.0 x 10-4 M.

The system is rotated at 4 rpm in a water bath at 250C for 12 hr. Both

sides are analyzed for benoxaprofen using the previously described HPLC


Results and Discussion

Rather than use the charcoal treatment devised by Chen (1967) to de-

ionize albumin, a less harsh resin deionization is used. Perrin and

Vallner (1975) found that major differences in albumin preparations might

exist due to impurities arising from commercial manufacturing techniques.

Hardee et al. (1979) found that different heats of binding for the

salicylic acid-albumin interaction would be measured depending upon the

amount of ionic impurities present. It was later found by Chen and Koes-

ter (1980) that charcoal treatment causes major changes in the fluores-

cence characteristics of albumins and even after this treatment, major

changes in albumin samples may exist. In this experiment, albumin is

passively deionized to remove ionic impurities such as chloride, citrate,

lactate and various cations and fatty acids. Minimal perturbation is

made to the albumin. For microcalorimetric work, this has proven to be

an acceptable technique (Hardee et al. 1979).

A Job's plot (Job 1928) for the benoxaprofen-HSA interaction is shown

in fig. 9. An inflection point between 0.75 and 0.80 is a strong indica-

tion that at least three moles of benoxaprofen bind per mole of albumin.

Figure 10 illustrates a Scatchard plot (Scatchard, 1949) for the beno-
xaprofen-HSA interaction. Each data point represents the average of two

determinations from an equilibrium dialysis experiment. Both the bound
and free sides are analyzed since preliminary studies have shown that

5-6% of the drug is bound to the membrane. Computer fitting the data to

a three site model utilizes equation 17

nz KI[D] n2 K2[D] n3 K3[D]
1 + KI[D] 1 + K2T] 1 + K3[D] (17)

where r is the number of moles of benoxaprofen bound per mole of albumin,
[D] is the free benoxaprofen concentration and Ki and ni are the binding

constants and number of sites of the ith class. The binding constant

obtained for the first site (K1 = 6.02 x 106 L/mole) is high but not out

of the order for other drugs in its class as Ki for ibuprofen was reported
to be 2.73 x 106 L/mole (Whitham et al. 1979). A good graphical estimate

of n2 and n3 is not obtained since solubility problems with benoxaprofen

preclude getting higher values of r. Using lower HSA concentrations might

).4 Q6 0.8

Job's plot for the benoxaprofen-HSA interaction.
Final total concentrations = 2.50 x 104 M.

Figure 9.


25 K1 = 6.02 x 106 L/Mole

N1 = 1.0

K2 = 6.93 x 104
N2 = 2.0
K3 = 1.08 x 104

20 N3 = 3.0



0,5 1.0 1,5 2,0 2,5 3,0 3,5
Figure Scatchard plot of the benoxaprofen-HSA interaction.

Equilibrium dialysis experiment.

5 *


0.5 1.0 1.5 2.0 2.5 3.0 3.5

Figure 10. Scatchard plot of the benoxaprofen-HSA interaction.
Equilibrium dialysis experiment.

be a way of getting around this problem but a concentration of 2.0 x 10-4M

HSA is about the minimum necessary to achieve accuracy and precision with

equilibrium dialysis.

The association constant for the binding site of physiological signi-

ficance (Kj) obtained from the Scatchard plot is confirmed by a titration

of HSA with benoxaprofen done on the ricrocalorimeter. Figure 1 illus-

trates this titration curve where the heat flux generated is proportional

to the amount of benoxaprofen bound to albumin. More than one site con-

tributes to the heat flux, as is indicated by the linearity. If only one

site of high affinity was involved, the curve would be expected to plateau

soon after that site was saturated. Solubility problems with benoxaprofen

and the multiple number of binding sites limit attainment of a high enough

concentration to saturate the binding sites. The first binding constant

can therefore be calculated by mathematical manipulation of the data.

If it is assumed that at drug to protein ratios below 0.5 all of the

benoxaprofen is bound to the first site, the slope of a line drawn tan-

gent to these points and through the origin is represented by the dashed

line in fig. 11 and will give the value of the maximum heat produced by

the formation of one mole of bound complex. This heat is equivalent to

the plateau value if one could be obtained for a 1:1 complex and its units

are microvolts.mole-l.liter. A linear regression program is used to find

the value of 'Vmax and K1 is calculated from equation 18 where a and b are

the initial concentrations of reactants

Ki (18)
1 = (a-c)(b-c) (18)
and C is given by

S= (19)
representing the concentration of the 1:1 complex formed.


CR *


o C





n r-



U- i
0 i-


(An) Xnd IV3-

Using this method a value of 5.05 x 106 L/mole for Ki is obtained,

which is in excellent agreement with the value 6.02 x 106 L/mole obtained

for KI from the Scatchard plot. Equation 20 allows the heat of the reac-

tion in joules/mole to be calculated,

1 1
AH (joules/mole) = uvmax x .057 x total flow rate (20)

where 0.057 is the conversion factor for this microcalorimeter in uv/pw.

A value of -21,000 joules/mole is obtained for the heat of formation of

one mole of complex at 250C. From equations 21 and 22, AG and AS can

also be calculated and their values are -38.242 joules/mole and 57.9 joules/

mole K, respectively.

AG = -RT In K (21)

AS = AH AG (22)
Binding of Benoxaprofen to Hemodialyzed Plasma

Studies by Shoeman and Azarnoff(1972) and Boobis (1977) have suggested
that a change in the composition of albumin is a major factor in the

decrease of drugs binding to the plasma of uremic patients. Dirnhuber

and Shutz (1948) have found that in urea solutions above pH 6 cyanate is

in equilibrium with urea. A recent study by Erill et al. (1980) has

shown that cyanate can carbamylate plasma proteins and this carbamylation

results in decreased binding of acidic drugs.

Other factors such as hypoalbuminemia and increased levels of fatty
acids may also play a role in decreased acidic drug binding. It is there-

fore of interest to study the binding of benoxaprofen to uremic plasma
and to note the effects hemodialysis may have on the binding.

Two middle aged male uremic patients have 10 ml of whole blood drawn

at 0, 2 and 4 hrs of hemodialysis. The blood is immediately centrifuged

for 10 min and 4 ml of plasma is obtained. The plasma is spiked to give

a 40 pg/ml concentration of benoxaprofen. An equilibrium dialysis experi-

ment is devised so that 1.8 ml of plasma is dialyzed vs. 1.8 ml Sorenson

buffer (Schumacher, 1966) at pH 7.4 for 14 hr at 370C. Two samples from

each patient at each time are run. Normal human plasma is treated under

the same conditions for use as a blank. The previously described HPLC

procedure is used to assay the free concentration of benoxaprofen.

Results and Discussion

Table 6 gives the free concentrations of benoxaprofen after equilib-

riur has been reached. Due to the very high degree of binding of benoxa-

profen, the differences between the free concentrations in normal plasma

as compared to that in uremic plasma can at best be a result of inter-

individual differences.

Of significance is the decrease in free concentration of the drug in

uremic patients after 4 hrs of hemodialysis. There is not much difference

in the free concentrations between 0 and 2 hrs but after 4 hrs, major

decreases in the free concentrations are apparent. Again, interindividual

differences arising from the different types and levels of impurities in

each patient are probably responsible for different free benoxaprofen con-

centrations reported for the two patients. However, the trend is clear

in that the increase in the amount of bound benoxaprofen is a result of

the removal of competitive impurities from the plasma by hemodialysis.

Removal of urea might also lead to a decrease in the formulation of the

carbamylated products previously described. In any case, the effects of

hemodialysis upon patients receiving benoxaprofen should be carefully monitored.

Table 6. Effects of Hemodialysis on Benoxaprofen Binding to Human Plasma.

Hemodialysis Benoxaprofen
Patient Time (hr) Free Concentration (pg/ml) X

A 0 0.206 0.237

2 0.284 0.243

4 0.134 0.102
B 0 0.121 0.101
2 0.099 0.114
4 0.030 0.0204
Normal Plasma 0.130
0.118 0.125


Basic Structure

Proteins are high molecular weight copolymers of about twenty amino

acids linked together to form a polypeptide chain. Their length, amino

acid composition and sequence are genetically determined. Diversity is

introduced by residues attached to the a-carbon of each monomeric amino

acid unit. The amino acid residues are classified according to the pre-

sence of polar and/or apolar groups. There are six apolar aliphatic resi-

dues (glycine, alanine, valine, leucine, isoleucine and methionine); four

apolar heterocyclic and aromatic residues prolinee, phenylalanine, trypto-

phan and tyrosine); five polar aliphatic residues serinee, threonine, cys-

teine, asparagine and glutamine); and five ionizable residues (aspartic

acid, glutamic acid, lysine, arginine and histidine). As will be evidenced,

the last five ionizable amino acids (fig. 12) will be most relevant to the

conformational changes discussed later.

In an aqueous environment, hydrophobic residues entropically interact

with each other to reduce water contact while ionizable and polar residues

interact with water resulting in an enthalpy change that reduces AG. Thus

apolar residues tend to be on the inside of a protein molecule while charged

residues exist on the outer surfaces. The protein chain will assume the con-

formation which best satisfies the requirements of minimization of exposure

of hydrophobic residues to the aqueous environment, forming what is described

as a hydrophobic core. This leads to maximization of intramolecular inter-

actions such as hydrogen bonding, Van der Waals forces and ion pair forma-

tion. There is also a maximization of interactions between charged and


NH3- CH- CH2- CH2- CH2- C- oo


NH2- Cj- NH- CH2- CH2- CH2- q- C00


C- CH2- C- COO0




CHM.- C- CO0-





0- H

C- CH2- CP2- C- COo
0 NH3+


Figure 12. Ionizable Amino Acids.

polar residues with the solvent molecule described as "polar shell" forma-

tion. The resulting compact structure is characterized by the presence or

absence of chain segments in helical, B-sheet or "random coil" configura-

tions as well as B-turns.

Three-dimensional structure of a protein is a reflection of the bal-

ance of enthalpic and entropic forces of interaction between the amino

acid monomeric units (backbone peptide units and side chain residues) with

one another and with the solvent molecules. This results in a minimum AG.

The structure is stabilized by cooperative noncovalent interactions

(Brandts, 1969; Tanford, 1970). Small local changes in structure can be

readily transmitted by a "chain reaction" to the rest of the molecule

leading to the loss of native structure of the entire molecule.

The charge state of the protein is determined by the total number of

ionizable residues, accessibility to the solvent, pKa values, and solution

pH. pKa's are affected by inductive effects, temperature, chemical nature

of the solvent dielectricc constant), and ionic strength of the solvent.

Polymeric Forms
A major source of heterogeneity in serum albumin involves the presence

of dimer and even higher oligomeric forms. The presence of dimers was

first indicated by ultracentrifuge work and the more advanced work done by

Pedersen (Pedersen, 1962) with Sephadex chromatography clearly resolved the

monomer, dimer, trimer and tetramer forms.

Dimers and trimers are the result of the manufacturing process and

are not in the bloodstream (Andersson, 1966). Williams and Foster found

dimerization to be most probable at a pH of 3.3 and result from the forma-

tion of disulfide linkages through oxidative reactions (Williams and Fos-

ter, 1960). Cu+2 ion was also found to increase dimerization; and in fact

it is theorized that Cu+2 actually acts as a catalyst in the dimerization.
Thiol reagents are usually used to reduce the dimeric forms of albumin.

Oxidation with ferricyanide in 4 M guanidine hydrochloride at pH 8 is
used to artificially prepare the dimer.

Dimers usually have less affinity for drug binding than monomeric
albumin. A commercial albumin preparation has about 6% dimer content.

N-F Transition
Optical rotary dispersion studies have shown that there is a large

increase in the hydrodynamic volume of the albumin molecule at low pH.

This change occurs from coulombic repulsion incurred by the large positive

charge. These studies have also shown that the change occurring is a

helix-coil transition with a reduction of apparent helical structure from

51% to 35%. Thus, the pH expansion can be viewed as moving apart of the

disulfide loops due to electrostatic forces, and the accompanying break-
down of the helical structure (Sogami and Foster, 1968).

The two forms of albumin in equilibrium at low pH are N for native
and F for fast migrating. Physically, the N form is extremely soluble,

while the F form is insoluble in 3 M KC1 (Rachinsky and Foster, 1957).

This property can be attributed to the hydrophobic regions. This hypothe-
sis is supported by a study done by Wishnia and Pinder (Wishnia and Pinder,

1964), which showed that the N form binds substantial amounts of alkanes

such as pentane; this ability is lost in the F form.

Optical rotation has been a useful method of studying the N-F tran-
sition. At 233 nm, the albumin spectrum shows a deep trough in rotation

which is common to other proteins of high helical content. A smooth

decrease in intensity is displayed until a pH of 3.7 where a slight pla-

teau forms, then a decrease again until a plateau is reached at a pH of

2.9. These two steps are now commonly referred to as the N-F' and F-F-


The N-F transition can be described as the opening of the hydrophilic

interface, while the second step involves the opening of the hydrophobic

regions. Absorption spectroscopy has shown that there is a slight blue

shift in the spectrum at low pH (Williams and Foster, 1959). Perhaps this

is due to tyrosyl residues which are in a hydrophobic environment in the

N form, but exposed to an aqueous environment at the low pH's. This

results in an increase in polarizability, indicative of tryptophyl resi-

dues which have shown fluorescence intensity to diminish with decreasing

pH (Chen, 1966; Halfman and Nishida, 1971; Rudolph et al. 1975). A slight

blue shift is noted with the decrease in intensity. Sogami et al. have

concluded that this shift and intensity change accompany the N F tran-

sition. The decrease in fluorescence intensity can be due to quenching of

chromophores from protonated carboxyl groups (Sogami et al. 1973).

Another important aspect of the N-F transition concerns the hydrogen

binding properties of the protein. It has been hypothesized that up to

40% of the carboxylate groups are "masked" in the N state so that they

will not protonate (Stroupe and Foster, 1973). Though it is not likely

that charged carboxylate groups cannot be isolated from solvent molecules

without neutralization from positive charges, ion pairing may occur. The

partners may be protonated amino groups, imidazolium groups, or guanidin-

ium groups. The N-F transition involves the rupture of a number of these


N-6 Transition
Though much work has been done on the N-F transition, its relevance

to physiological conditions is doubtful. However, there is another tran-

sition which takes place much closer to the pH range encountered in blood.

Leonard and Foster (Leonard et al. 1963), using optical rotation studies

at 313 nm, first discovered a transition in the pH range of 7-9. This

wavelength was chosen because it monitors change in tertiary structure,

perhaps primarily alterations in aromatic chromophores. They noted that

the magnitude of this transition was smaller than that for the N-F transi-

tion and hypothesized that there was even less loss of helical structure

in the higher pH transition. This transition is now commonly referred to

as the N-B or neutral transition.

Histidine, whose imidazole residues have a pKa of 6.4 to 7.0, seems

to be involvedin this transition, and it is hypothesized than ten imida-

zolium residues are "hidden" in the N form and become available in the B

form (Harmsen et al. 1971). At physiological pH, albumin will exist in

two forms. Organs such as the liver and certain inflamed tissues have a

slightly lower pH; therefore, changes in protein conformation at these

sites resulting in differences in drug binding must be taken into account.

It is interesting to note that calcium ions in physiological concen-

trations favor the N-B transition. This has been confirmed by optical

rotation (Zurawski and Foster, 1974) and spectrophotometric studies (Harm-

sen et al. 1971; Wilting et al. 1979). With physiological levels of cal-

cium ion present there are nearly equal amounts of the two conformers since

the transition is pushed to the N form at physiological pH. Calcium ion

binding can be thought of as pH dependent (Katz and Klotz, 1953) and it

is possible that calcium ions compete with imidazolium residues for car-

boxylate binding sites (Pedersen, 1972a) supporting one reason for differ-

ences in binding constants for various drugs.

It has been found that calcium ions bind to albumin with increasing

affinity in the pH range 5-9 (Pedersen, 1972b). These studies have also

shown that calcium ions compete reversibly with twelve of the sixteen

imidazole groups on HSA with the calcium binding increasing over this pH

range. In a phosphate buffer at a pH of 6.9, the presence of calcium ions

results in 50% of the albumin existing in the B form. When no calcium

ions are present, 50% of the albumin is in the B form at a pH of 7.4.

Circular dichroism studies have shown that 80% of human albumin is in the

B form when calcium ions are present, but only 50% in the absence, at a

pH of 7.4.

Work done by Wilting et al. (Wilting et al. 1980a) has shown that high

concentrations of chloride ion (0.1 M) also alter the N-B transition.

He has shown that in warfarin binding experiments in the presence of cal-

cium and/or chloride ions, the N B transition is altered. Albumin is

"pushed" to the B form in the presence of these ions and in this form it

has a higher affinity for warfarin.

Physiologically the importance of this phenomenon is very important

as is the case with the anticoagulant drug which is mainly metabolized in

the liver. The pericellular pH in the liver is slightly lower than in the

blood, and the free fractions of warfarin available for metabolism and phar-

macological action are not accurately estimated from plasma sample deter-

minations. Slight changes in blood pH can be expected to alter free war-

farin concentrations dramatically. A shift in pH from 7.4 to 7.0 almost

doubles the free warfarin concentration in the presence of calcium ions.

These changes can be expected when a patient has diabetes mellitus or

severe renal failure.

The mechanics of the N-B transition are not as clearly classified as

with the N-F transition. The amount of helical structure involved in the

N-B transition is small and is almost unchanged by temperature (Wallevik,

1972). The apparent net loss of a-helix structure in the unfolding between


pH 7 and 9 is 2.5%, in the N-F transformation it is 8%. It could also be

concluded that the N-B transition causes a pK shift of imidazole groups

with protons released in the neutral regions. The highest pK was found

in the low pH conformation suggesting that several histidyl residues are

involved in salt bridges. In the presence of calcium ions, the affinity

of albumin for protons decreases, resulting in a shift to the neutral state.


Most of the studies of the pH induced N-B transitions in HSA have been

done with the coumarins. This class of drugs usually has good fluores-

cent properties and also gives a strong induced CD signal. Wilting et al.

(1979;1980a;1980b) and Otagiri et al. (1978) found that warfarin binds with

increasing affinity to HSA in the pH range from 6 to 9 and it was shown

that calcium and chloride effect this binding. Similar results were

obtained with phenprocoumon in Chapter I of this work.

Experiments in this chapter were designed to study the effects of pH,

calcium and chloride ions on the binding of benoxaprofen to HSA. Equilib-

rium dialysis and circular dichorism are the techniques used to perform

these experiments.

Materials and Methods
Human serum albumin, fraction V (lot No. 30F-02271) was obtained from

Sigma Chemical Co. (St. Louis, MO). Sodium benoxaprofen (903.5 mg free

acid per gram) was supplied by Eli Lilly (Indianapolis, IN). Monobasic,

dibasic sodium phosphate and sodium chloride, analytical reagent grade,

were obtained from Mallinckrodt, Inc. (St. Louis, MO). Calcium chloride

dihydrate, A.C.S. grade, was obtained from Fisher Scientific Co. (FairLawn,

NJ). Dowex 50W-X8 cation exchange resin and 1-X8 anion exchange resin

were supplied by J.T. Baker Chemical Co. (Phillipsburg, NJ).

Albumin was deionized as previously described and its molecular weight

was taken to be 66,500 g/mole. Concentrations were determined by UV


spectroscopy (e = 4.123 x 104 Z/mole-cm). All solutions were prepared
using deionized water.

Free concentrations of benoxaprofen were obtained by means of equilib-

rium dialysis using a Dianorm equilibrium dialyzer (Diachema, A.G.)

Rischlikon, Switzerland) with teflon cells of 10 ml total volume. Dialy-

sis membranes of hydrated cellulose were used with a molecular weight cut-

off of 10,000 (Diachema type 10.16). The samples were dialyzed at 4 rpm

for 12 hr at 25C. Benoxaprofen was analyzed using the previously described

HPLC procedure.

Circular dichroic (C.D.) measurements were made using a Jasco model

J-500 spectropolarimeter (Jasco International Co. LTD, Tokyo, Japan).

The extrinsic benoxaprofen-albumin signal was measured at a slit width

of 1 nm, wavelength expansion of 5 nm cm 1, chart speed 1 cm min-1, time

constant 32 sec and a sensitivity of 0.5 or 1.0 m cm-1. Pathlengths of

2, 5 and 10 mm were used. Observed ellipticities (eobs) are the actual

C.D. spectraof the benoxaprofen-albumin complex while the induced ellip-

ticity is the observed ellipticity of the complex minus the ellipticity

of albumin alone at the corresponding wavelength. The dynode voltage was

kept below 0.5 in all experiments.

Results and Discussion

Prior to beginning the study on the pH dependence of benoxaprofen

binding, it was necessary to determine if a constant ionic strength be

maintained when workingin the region of the N-B transition. Mixing dif-

ferent ratios of 0.1 M monobasic and dibasic phosphate buffer would be

used to adjust the pH but this would cause the ionic strength to vary.

Equilibrium dialysis was used to study the binding of a 1 to 1 ratio of

benoxaprofen to albumin in phosphate buffer concentrations of 0.05, 0.10,

0.20 and 0.30 M. From table 7 it can be shown that there would only be a

Table 7. Dialysis data. Effect of ionic strength on the binding of
benoxaprofen to HSA.

Peak Height Ratios (Benoxaprofen/Warfarin)

Phosphate Buffer Conc. (M) X S.D. (N=5)

0.05 1.50 0.09

0.10 1.34 0.11

0.20 1.31 0.03
0.30 1.26 0.07

[Benoxaprofen] = 2.5 x 10-4 M

[HSA] = 2.5 x 10-4 M

notable ionic strength contribution if the phosphate buffer concentration

went below 0.1 M. Therefore, adjusting the pH with 0.1 M phosphate buffer

should not effect benoxaprofen binding.

Benoxaprofen, when bound to HSA,gives rise to an extrinsic Cotton

effect with a positive maximum at 303 nm and a negative minimum at 333 nm.

These wavelengths can shift as is shown by the titration at pH 7.4 in fig.

13. The isosbestic point at 328 nm is maintained throughout the titration

at low drug to protein ratios, suggestive of a single binding site of high

affinity. When the drug to protein ratios exceed 1 to 1 the isosbestic

point is lost and at 3 to 1 the positive maximum shifts to shorter wave-

lengths and decreases in magnitude. This is easier to see when the same

data is plotted as induced ellipticities in fig. 14.

The sigmoidal character of this curve at low drug to protein ratios is

indicative of a secondary binding site of higherinducedellipticity or a

cooperative effect between the primary and secondary sites (Chignell, 1969).

Drug displacement studies done later in this work will tend to support the

first hypothesis. At a drug to protein ratio greater than 3 to 1 the ellip-

ticity decreases. This evidence of at least three binding sites correlates

with the datain the Scatchard and Job's plots (figs. 9 and 10) previously

described. Changes in the sign of the ellipticity upon increasing the drug

to protein ratio have also been observed with indomethacin (Ekman et al.

1980). Here, it was found that the first two binding sites give rise to a

negative ellipticity and the third site has a positive ellipticity.

The binding of various aromatic propionic derivatives has been studied

by Perrin (1973) and as with fenoprofen, it appears the extrinsic Cotton

effects of the benoxaprofen-albumin complex arise as a result of hydropho-

bic binding of the aromatic rings and hydrogen bonding of the carbonyl and


+14 -





8 +2-




2 300 310 320 330 30 350 3

Figure 13. Observed ellipticity of benoxaprofen-HSA complex
titration at pH 7.4. 0.5 cm. cell.









1.0 2,0

3.0 4.0 5.0

6.0 7.0

Figure 14.

Induced ellipticity of benoxaprofen-HSA complex
titration at pH 7.4 in 0.1 M phosphate buffer.
[HSA] = 5.0 x 10-5 M. 0.5 cm. cell at 300 nm.

ether oxygen to albumin. No Cotton effect is generated by benoxaprofen

itself. Studies done on a molecule similar to benoxaprofen, [2-(4-chloro-

phenyl)-4-oxazole]acetic acid, indicated that the molecule failed to

exhibit an extrinsic Cotton effect with albumin. It was postulated that

the absorption maximum of this compound was at too low a wavelength (281

nm) to distinguish the extrinsic ellipticity from the intrinsic ellipticity

of the albumin (Mitchell and Rosen, 1978). Benoxaprofen has one more

phenyl ring than this compound which gives it an absorption maximum of

309 nm. This, plus its added planar rigidity, probably gives rise to the

observable Cotton effect of benoxaprofen.

Albumin was titrated with benoxaprofen at pH 6.5 (fig. 15). It is
in the N form at the pH and the magnitude of the observed ellipticity is

greater than at pH 7.4. Since the pKa of benoxaprofen is between 3 and 4,

the changes in the nature of the binding are due to changes in the albumin

and not benoxaprofen. There also seems to be a loss in the distinction of

the isosbestic point in this conformation. Figure 16 shows the data at pH

6.5 plotted as induced ellipticities and there does seem to be a loss in

distinction between the primary and secondary sites as is noted by decreased

sigmoidal character of the curve at low drug to protein ratios. At ratios

higher than 3 to 1 the decrease in ellipticity is still observed.

At pH 9.2 the titration had to be done in a 0.2 cm cell as there was

too high a dynode voltage which was caused by the increased ionic strength

of the phosphate buffer (fig. 17). The induced ellipticities of these data

in fig. 18 exhibit the interesting observation that at drug to protein

ratios greater than 3 to 1 the ellipticity keeps increasing rather than

decreasing as was seen at pH 6.5 and 7.4. Much more sigmoidal character

is noted in this curve. It is clear from these observations that in the

B form, the character of albumin changes so that there is a greater


Figure 15.

Observed ellipticity of benoxaprofen-HSA complex
titration at pH 6.5. 0.5 cm. cell.

1.0 2.0 3.0 4,0 5,0 6.0

Figure 16. Induced ellipticity of benoxaprofen-HSA
pH 6.5 in 0.1 M phosphate buffer. [HSA]
0.5 cm. cell at 300 nm.

complex titration at
= 5.0 x 10 5 M.




? +1 -


290 300 310 320 330 340 350 360
Figure 17. Observed ellipticity of benoxaprofen-HSA complex
titration at pH 9.2. 0.2 cm. cell.

1.0 2,0 3,0 4.0

Figure 18.

5.0 6.0

Induced ellipticity of benoxaprofen-HSA complex titration
at pH 9.2 in 0.1 M phosphate buffer. [HSA] = 5.0 x 105 M.
0.2 cm. cell at 300 nm.

distinction between the primary and secondary sites for benoxaprofen.
The unfolding of the albumin in this conformation (Wallevik, 1972) and the

change in the ionization states of the imidazole groups (Harmsen et al.

1971) might favor a more rigid fit of the benoxaprofen at its third binding

site resulting in the change of sign to a positive ellipticity.

The effects of chloride and calcium ions on the N-B transition of

benoxaprofen-HSA complexes are studied with circular dichroism and equi-

librium dialysis. From fig. 19 it can be seen that the observed ellipti-

city of the benoxaprofen-HSA complex in 0.1 M phosphate buffer decreases

with increasing pH. This is contrary to the effects observed with warfarin

where the ellipticity increased with increasing pH. Under the conditions

in which this experiment was performed, there is no change in HSA with pH

so the observed signals arise strictly from the benoxaprofen-albumin complex.

The fact that the benoxaprofen-albumin and warfarin-albumin complexes have

ellipticities of opposite sign in the N-B transition is perhaps related

to the nature of their respective binding sites. Later, it will be proven

that they actually bind to two physically separate sites on HSA and the

different environments of these sites can be the reason for the opposite

ellipticities in the N-B transition.

Figure 20illustrates the effect sodium chloride has on decreasing the

ellipticity of the benoxaprofen-albumin complex. The mechanism involved

may be one of direct displacement of benoxaprofen, as the molar ratio of

chloride to benoxaprofen is 2000 to 1. It is also possible that chloride

"pushes" albumin to the B form, which when completed with benoxaprofen, has

a greater negative ellipticity. Calcium ion also has the same effect (fig.

21) but of a greater magnitude than with chloride ion.

The different effects of phosphate, chloride and calcium ions on the

N-B transition can be seen more clearly when they are plotted as induced


1o 3 3 0

Figure 19.

Observed ellipticity vs. pH in the presence of 0.1 M
phosphate buffer. [HSA] = 5.0 x 10 5 M = [benoxaprofen].
1.0 cm. cell.






290 30 30 30


I / / "" 9.65----

290 3C0 310 320 330

Figure 20.

3O0 350 360 370

Observed ellipticity vs. pH in the presence of 0.1 M
sodium chloride. [HSA] = 5.0 x 10 5 M = [benoxaprofen].
1.0 cm. cell (pH adjusted with 0.1 N sodium hydroxide).

Figure 21.

Observed ellipticity vs. pH in the presence of 2.50 x 10 3
calcium chloride dihydrate. [HSA] = 5.0 x 10-5 M = [Benoxa-
profen]. 1.0 cm. cell pH adjusted with 0.1 N sodium





(z. .-



0 )

-z4 *

4 .
2 r-


m *R

L (
O.- 0

4 -,


0 CO



(ow) e nDonNI








Figure 23. Dialysis data. 2.0 x 10-4 M HSA.
2.5 x 10-3 M CaC12-2H20 0.1
A 0.1 1M Phosphate buffer.

2.0 x 10~4 M Benoxaprofen.
M NaCl, M 0.1 M NaC1,

ellipticities in fig. 22. When albumin is in the N form, the effects of

chloride and calcium are emphasized, indicating that in this conformation

albumin is most sensitive to the effects of these ions. Table 8 lists

the parameters which are used to describe this type of curve. The pHso

is defined as the point at which the change from the neutral to basic

form is 50% completed. The range over which the change in the induced

ellipticity is from 10 to 90% completed is defined as the ApHo1,90o

HSA in 0.1 M phosphate buffer has a pHso at 7.4. The pH50 for the

benoxaprofen-albumin complex is 7.7, indicating that benoxaprofen itself

may "push" HSA to the basic conformation. Chloride ion does not have much

effect on the transition as the pH50 of 7.65 is almost identical to that

in phosphate buffer. Calcium ion has a major effect on the transition as

is seen by the pH50 of 6.90. As previously noted (Wilting et al. 1980),

this is a result of calcium ion inducing albumin into due B conformation.

Though the effect of chloride is mainly one of displacement, calcium seems

to both displace benoxaprofen and push the transition to the B conformation.

Calcium ions are reported to compete with carboxylate residues for imida-

zole binding sites (Pedersen,1972a,b)and this might be the displacement

mechanism involved.

Correlation of the C.D. data with the physical changes in the benoxa-

profen-HSA system has been done with equilibrium dialysis experiments as

shown in fig. 23. In phosphate buffer, the concentration of benoxaprofen

decreases from 0.200 to about 0.065 pg/ml as the pH is increased. When

chloride ions are present, the free concentration of benoxaprofen increases

as it is displaced by chloride throughout the N-B transition. At pH 7.4

the free benoxaprofen concentration is almost double in the presence of

0.1 M chloride than in phosphate buffer. When calcium ions are present

at a concentration of 2.50 x 10-3 M (with 0.1 M NaCl added to eliminate

Table 8. Parameters for benoxaprofen N-B curves.



0.1 M phosphate buffer 7.70 1.60

0.1 M chloride ion 7.65 2.30

2.5 x 10-3 M calcium ion 6.90 4.10

Table 9. Dialysis data. Variation of binding constants with pH.

[HSA] = 2.0 x 10-4 M [Benoxaprofen] = 2.0 x 10-4 M

0.1 M Phosphate Buffer
pH Ki(L/Mole)

6.00 1.38 x 106
6.50 1.46 x 106
7.00 1.70 x 106
7.50 2.66 x 106
8.00 3.82 x 106
8.50 3.88 x 106
9.00 3.34 x 106

0.1 M NaCl
pH K1(L/Mole)

6.35 6.75 x 105
6.85 6.29 x 105
7.30 6.19 x 105
7.80 8.81 x 105
8.40 1.07 x 105
9.10 1.33 x 105

0.1 M NaCI

& 2.5 x 10-3 M

pH KI(L/Mole)

6.30 3.72 x 105
6.59 4.17 x 105
6.81 4.98 x 105
7.35 6.08 x 105
7.74 6.16 x 105
8.12 6.07 x 105
9.13 5.61 x 105

CaC1 2H20

Donnan effects) there is an even greater displacement of benoxaprofen.

The flatness of the calcium curve at pH values greater than 7.0 confirms

that it does induce albumin into the B conformation. Solubility problems

encountered at low pH values made the plateau region for the N form inexact

but the general trend is still clear.

Table 9 lists the binding constants calculated from dialysis data

for 1:1 benoxaprofen-HSA complexes. In all cases, K1 increases as the

pH is increased. Displacement of benxoaprofen by chloride and calcium

ions is characterized by the smaller magnitude of the primary binding con-

stants in these cases as compared to phosphate buffer. As benoxaprofen

is very highly albumin bound (>99%), the bound fraction does not change

greatly but the free fraction can more than triple when pH, calcium or

chloride ion concentrations are changed. The free concentration of a drug

is responsible for its therapeutic or toxic effect.

Future protein binding studies with benoxaprofen should take careful

note of buffer composition and pH as this alone can significantly alter

its binding characteristics.


It is generally accepted that most drugs which are bound to albumin

are bound via only a few high affinity sites and a number of low affinity
binding sites (MUller and Wollert, 1979). Evidence has been increasing

which shows these high affinity sites possess a degree of specificity

for distinct classes of drugs such as coumarins and nonsteroidal anti-

inflammatory agents (Sudlow et al. 1975, 1976). Changes in the fluores-

cence intensity of probes which had separate and distinct binding sites

were monitored as competitors were added to the system. Using equilib-

rium dialysis as a confirming technique, they found that the relative

affinity of drugs for these binding sites was measured by their ability

to displace probes specific to these sites.

Two distinct binding sites of high affinity have been described and

drugs binding to these sites classified. Site II drugs tend to be aro-

matic carboxylic acids which are ionized at physiological pH. Examples

of drugs which bind to this site are ibuprofen, naproxen, ethacrynic acid,

clofibric acid and flufenamic acid. These drugs are characterized as having

an extended configuration with a negatively charged carboxylate group on

an aliphatic residue extended from a nonpolar aromatic region. Site I

drugs are usually aromatic acids which have a delocalized negative charge.

Examples of Site I drugs are warfarin, phenprocoumon, acenocoumarin, phenyl-

butazone, tolbutamide and sulfonamides. Tests performed with diluted

adult sera correlate with studies which used crystalline serum albumin.


Other authors have studied the specificity of the high affinity

sites on HSA and a wide range of drugs have been classified as to their

binding sites (Sjoholm et al. 1979). A third discrete site which binds

digitoxin has also been reported in these studies.

Much attention has been given the warfarin (site I) binding site as

displacement of the coumarins can have severe pharmacological consequen-

ces (McElnay and D'Arcy, 1980; Veronich et al. 1979; Wosilait and Ryan,

1979; Minn and Zinny, 1980). The nature of this site has been described

as involving the lone tryptophan residue on HSA (Fehske et al. 1979).

Modification of this site with different reagents has led to decreased

coumarin binding.

Less information is known about the nature of the diazepam site

(site II) but studies in this laboratory found that some nonsteroidal

antiinflammatory drugs binding to this site may have a tendency to alter

the conformation of HSA as well as cause displacement interactions. Evi-

dence for anionic molecules inducing changes in albumin conformation has

been given in the binding of fatty acids (Birkett et al. 1977; Ashbrook

et al. 1974; Muller, 1976).

The nature of benoxaprofen's binding site will be described in this

study and the conformational adaptability of this site will be investi-


Materials and Methods
Human serum albumin, fraction V (lot No. 30F-02271) was obtained from

Sigma Chemical Co. (St. Louis, MO). Monobasic and dibasic sodium phos-

phate, analytical reagent grade, were obtained from Mallinckrodt, Inc.

(St. Louis, MO). The following drugs were used as supplied by the manu-

facturers: sodium benoxaprofen (903.5 mg free acid per gram), Eli Lilly

and Co. (Indianapolis, IN); fenbufen, Lederle Laboratories (Pearl River,

NY); flufenamic acid, Aldrich Chemical Co. (Milwaukee, WI); sodium
clofibrate, Imperial Chemical Industries, LTD. (Great Britain); oxphenyl-

butazone and phenylbutazone, Ciba Pharmaceutical Co. (Summit, NJ); ibu-

profen, Upjohn Co. (Kalamazoo, MI); sodium warfarin, Endo Labs, Inc.

(Garden City, NY), phenprocoumon, Organon, Inc. (W. Orange, NJ).

Albumin was deionized as previously described and its concentration

was determined by UV spectroscopy (M.W. = 66,500; e = 3.946 x 104 /

All solutions were prepared in 0.1 M phosphate buffer at pH 7.4. Free

concentrations of benoxaprofen were obtained from equilibrium dialysis

experiments and detected by HPLC using techniques described previously.

Circular dichroic measurements were also done as described in Chapter VI.

Displacement studies using the microcalorimetric technique were per-

formed by pumping a 5.0 x 10-4 M albumin solution into one half of the

mixing cell and 5.0 x 10-4 M drug solutions into the other side. The

heats of reaction for drug 1-HSA, drug 2-HSA and then drugs 1,2-HSA were

obtained. At all times the drug to protein ratios were kept at 1:1.

Equilibrium dialysis experiments were done with 2.50 x 10-4 M HSA

solutions dialyzed vs. 1.25 x 10-4 M drug solutions. Benoxaprofen is

dialyzed vs. HSA and then benoxaprofen and competitor are dialyzed vs.

HSA. Drug to protein ratios were maintained at 0.5.

Circular dichroic displacement studies were performed by recording

the ellipticity of the benoxaprofen-HSA complex and then recording the

change in ellipticity as the concentration of competitor was increased.

Separate solutions were prepared for each run and all work was done at


Results and Discussion
Some experience was gained by studying the much more thoroughly
investigated binding of warfarin and this was used to ascertain whether

benoxaprofen was a site I or site II drug. As can be seen from table 10,

at a drug to protein ratio of 0.5 there is 1.00 Ig/ml free warfarin.

In the presence of phenylbutazone and oxyphenylbutazone which binds to

the same primary site, the free concentrations of warfarin increases to

1.77 and 1.66 ug/ml, respectively. Other nonsteroidal antiinflammatory

drugs, especially those with aliphatic carboxylate residues in an extended

linear configuration, do not bind to the same site (Sudlow et al. 1976;

Sjiholm et al. 1979; Otagiri et al. 1979) and evidence a smaller degree

of warfarin displacement. Benoxaprofen does not significantly displace

warfarin and appears to be a site II drug.

The drugs in table 10 are ranked in order of the ability to displace

warfarin by a significance test on the difference between two means (AX).

Mean values of free warfarin concentration are compared with and without

competitor and the 90% confidence limit of the difference between these

two means is calculated. If AX >C.L., the difference between the means

is due to drug displacement and not random scatter. If AX C.L.,the sam-

ples are not significantly different or there is an enhancement on binding

in the presence of a competitor. The magnitude of AX-C.L. is used to

rank the samples in terms of warfarin displacement. Ibuprofen gives a

negative value for AX-C.L. and this may signify an enhancement of warfarin

binding at this low drug to protein ratio. This observation has been made

by other authors (Sudlow et al. 1976; Otagiri et al. 1979) and previously

in Chapter I where the effects of ibuprofen on phenprocoumon binding were

noted. It has been hypothesized in the above studies that ibuprofen may

"push" albumin into the B conformation, which has a greater affinity for


Table 11 lists dialysis data for the interactions between benoxapro-

fen and competitors. It is displaced by flufenamic acid, clofibrate

Table 10. Dialysis data.
[HSA] = 2.5 x 10-4 M [Warfarin] =

Free Warfarin
(X S.D. ig/ml)





Flufenamic acid

Ethacrynic acid










1.18 +

1.13 +


1.01 +










1.25 x 104 M = [competitor]

C.L. (I = 0.05)*

















90% confidence limit for the difference between two means
**Largest value = greatest displacement

AX C.L.**

and oxyphenylbutazone. This data, plus the fact that it didn't displace

warfarin, confirms benoxaprofen as a site II drug. It is unusual that

ibuprofen does not displace benoxaprofen and oxyphenylbutazone causes a

slight displacement. These drugs may be altering the conformation of

albumin or causing some type of cooperative effect such that the distinc-

tion between site I and site II may not be as exact when benoxaprofen is

monitored. With phenylbutazone, this hypothesis has been advanced by

Maes et al. (1979) and Madsen and Tearne (1979).

A clearer explanation as to what type of mechanisms might be involved

in these site II displacements might be reached if this phenomenon is

examined with different experimental techniques. The heat flux of binding

for the mixture of a drug and competitor (AHAB) is compared to the sum of

the values obtained with each drug acting alone with HSA (AHA + AHg). If

AHAB is equal to AHA + AHB no interaction between drugs A and B has occurred.
If the value of AHAB is less than the sum of AHA + AHB, then a mutual dis-

placement is probable. If AHAB is greater than the sum, an interaction

causing enhancement of the binding has taken place (Hardee, 1980).

Table 12 lists the results for different displacement experiments

at pH 7.4. As expected, the heat flux generated for a mixture of two

site I drugs, warfarin and phenprocoumon, gave a heat which was less than

the additive heat when each was measured separately. This indication of

a displacement is in agreement with the dialysis data in table 10. Within

experimental error, there does not appear to be competition between benoxa-

profen and warfarin or phenprocoumon. There is a displacement of benoxa-

profen in the presence of flufenamic acid and clofibrate and this again

is confirmed by the dialysis data in table 10. In the presence of ibupro-

fen and fenbufen AHAB was greater than AHA and AHB indicating some type

Microcalorimetric data.

Binding site specificity and drug

Steady State
Heat Flux
(p j-oule sec-1)

Expected Value


r I. 1

Flufenamic acid
Benoxaprofen and
Benoxaprofen and
Benoxaprofen and
flufenamic acid
Benoxaprofen and
Benoxaprofen and
Benoxaprofen and
Warfarin and









(+) = greater than additive heat
(-) = less than additive heat
(o) = additive heat = observed heat

Table 12.











of cooperative phenomenon or conformational change resulting in the
enhanced binding of benoxaprofen may be taking place.

All of the drugs listed in table 12 reacted exothermally with HSA.

Phenylbutazone was to be included in this table but it was found that at

drug to protein ratio less than 1.0 an endothermic reaction occurred.

The value obtained for AH at this ratio was approximately +4.0 Kcal/mole

which is close to that seen by Maes et al. (1979). It was not clear

whether adding heats of different signs would be applicable to the model

described, so this problem was examined further by circular dichroism.

Induced ellipticities of benoxaprofen-HSA complexes in the absence

and presence of increasing amounts of phenylbutazone are shown in fig.

24. In the wavelength region above 315 nm the ellipticities overlap and

without the aid of a computer, are extremely difficult to interpret. For

clarity, the wavelength region of maximum change in ellipticity is pre-

sented and the scale has been expanded. The effects of flufenamic acid

and clofibrate have also been studied in this way but they have an induced

C.D. signal of their own in this region whereas phenylbutazone does not.

At drug to protein ratios greater than 0.5 of these competitors, the

ellipticity of benoxaprofen is "masked" so these spectra will not be pre-

sented. However, at 0.5 drug to protein ratios where the induced benoxa-

profen signal is not "masked" an increase in ellipticity of the benoxapro-

fen-HSA complex is observed. This is similar to the effect of phenylbuta-


Figure14 illustrates that as HSA is titrated with benoxaprofen, a

secondary binding site of higher ellipticity exists. Equilibrium dialysis

and microcalorimetry have shown that flufenamic acid and clofibrate both

displace benoxaprofen. The increase in induced ellipticity as these com-

petitors are added can best be explained by a displacement from its primary


Figure 24.

Induced C.D. curves for interaction of benoxaprofen-
HSA complex with phenylbutazone. [HSA] = 2.0 x 10-5
= [benoxaprofen] 0-3.0 D/P phenylbutazone

29 295 305 310 315 32

Figure 25.

Induced C.D. curves for
complex with ibuprofen.
0-4.0 D/P ibuprofen.

interaction of benoxaprofen-HSA
[HSA] = 2.0 x 10"5 M = [benoxaprofen




E+2- 3.0



I-- I V-- I -- I -
290 295 300 305 310 315 320

Figure 26. Induced C.D. curves for interaction of benoxaprofen-HSA
complex with fenbufen. [HSA] = 2.0 x 10-5 = [benoxaprofE
0-4.0 D/P fenbufen.

binding site to this secondary site of higher ellipticity. A similar

effect is seen with indomethacin, which also has a secondary binding site

of higher ellipticity (Ekman et al. 19801. Phenylbutazone does not bind

to the same primary site as benoxaprofen so the increase in the induced

ellipticity might be due to the phenylbutazone causing a conformational

change in the albumin which gives rise to the increased positive ellipti-

city. Figure 19 illustrates that as the pH is decreased and albumin is

induced into the N conformation, the ellipticity of the benoxaprofen-HSA

complex increases. As the ellipticity of the complex increases in the

presence of phenylbutazone, it too can possibly be inducing albumin into

the N conformation or it may be causing some other type of cooperative

effect. Studies in Chapter IX will confirm that phenylbutazone binding

to HSA involves a cooperative process rather than a B to N conformational


Figures 21 and 26 illustrate the induced CD signals of benoxaprofen

in the presence of ibuprofen and fenbufen. In both cases, the elliptici-

ties decrease as competitor is added. Microcalorimetric and dialysis data

(tables 12 and 11) have indicated that these drugs might enhance benoxa-

profen binding. Figure 19 shows that the ellipticity of the benoxaprofen-

HSA complex decreases as HSA is induced into the B conformation. It can

be hypothesized that both ibuprofen and fenbufen induce HSA into the B

conformation, resulting in the decreased ellipticities in figs. 25 and 26.

To confirm whether this is a correct hypothesis, the effects of these

competitors on site II binding must be studied at different pH values.

If they are involved in drug induced conformational changes as described

above, we would expect different competitive effects at pH 7.4 when HSA

is in both the N and B conformation as compared to pH 9 when HSA exists

solely in the B conformation. These experiments are presented in Chapter IX.


Fenbufen, 3-(4-biphenylcarbonyl)propionic acid (fig. 27, structure 1),

a nonsteroidal antiinflammatory drug, has been found to possess the same

spectrum of activity as aspirin, phenylbutazone and indomethacin. Its

two major serum metabolites, 3-(4-biphenylhydroxymethyl)propionic acid

(structure II) and 4-biphenylacetic acid (structure III) have a similar

degree of activity (DeSalcedo, 1975; Sunshine, 1975; Vergara et al. 1979;

Coutinho et al. 1976; Child et al. 1977).

Previous GC (Cuisinaud et al. 1978) and HPLC (Van Lear et al. 1978)

methods have been developed for the determination of fenbufen and its

metabolites in biological fluids. The GC method is very time consuming,

involving double extraction, derivatization and TLC separation before GC

injection. The established HPLC method has retention times of up to 22

minutes for the compounds of interest. A column heated to 450 and a 2 ml

plasma sample are also necessary.

In the new method the chromatographic procedure was improved to keep

the total retention time for all compounds under 9 minutes (table 13).

A plasma sample of only 1 ml is necessary and the column is operated at

ambient temperature.


Fenbufen, its metabolites and an internal standard, 4'-hydroxy-4-

biphenylacetic acid (structure IV), were kindly supplied by Lederle


/ / CH2CH2CH I





Figure 27. Structures of fenbufen (I) and major metabolites.

Table 13. Retention times for fenbufen and serum metabolites.

Compound Retention Time (min)

I 6.1

II 8.8

III 5.1

IV 2.9

Laboratories (Pearl River, NY). HPLC grade acetonitrile and isopropanol

were obtained from Burdick and Jackson (Muskegon, MI). Analytical grade

phosphoric acid, hydrochloric acid, sodium hydroxide and methanol were

supplied by Mallinckrodt, Inc. (St. Louis, MO). All chromatographic sol-

vents including deionized water, were filtered through 0.45micron filters

(Millipore Corp., Bedford, MA).

Sample analyses were carried out on a Varian model 5000 liquid

chromatograph equipped with a 10 pl manual loop injector and a column

heater (Varian Assoc., Walnut Creek, CA). An alkylnitrile column, Micro-

Pak CN-10 (Varian Assoc.) of medium polarity was used. Detection was made

on a Vari-Chrom variable wavelength UV detector.

During the extraction procedure, metabolite II forms a lactone (struc-

ture V) (Van Lear et al. 1978), as will be described later. The lactone

was prepared as follows: Met'abolite II was saturated in water at 400.

An excess of concentrated HC1 was added and the mixture was allowed to

stand for 5 minutes. The white precipitate was extracted into cyclohexane-

ether (7:3). The organic layer was evaporated under a nitrogen stream and

thelactone crystals were collected. Purity was determined chromatographi-

cally to be greater than 99%.

Chromatographic Conditions
The mobile phase consisted of water-isopropanol-acetonitrile-phosphoric

acid (84.5:10:5:0.5). The flow rate was 2.5 ml/min and the column was kept

at 25. Detection was made at 265 nm at 0.02 aufs. A 10 vl sample was

injected onto the column.

Analytical Procedure
The extraction procedure is similar to the one previously described

(Van Lear et al. 1978). Stock solution containing 200 ug/ml of fenbufen,

metabolites and internal standard are prepared in methanol. Accurate

volumes are pipetted into test tubes which will give final concentrations

of 0.5, 1.0, 3.0, 5.0, 10.0 and 25.0 ug/ml of plasma. The internal stan-

dard (2 pg) is then added. The methanol is evaporated in a stream of dry

nitrogen. Samples are reconstituted in 125 pl of 0.1 N NaOH and 1 ml of

plasma is added. Limited solubilities of fenbufen and its analogs neces-

sitate this procedure rather than direct spiking of plasma samples from an

aqueous stock solution.

After the contents of the tube are thoroughly mixed, 1 ml of concen-

trated HC1 is added and the samples are vortexed for 15 seconds. The sam-

ples are allowed to stand for 2 minutes and then 8 ml of cyclohexane-ether

(7:3 v/v) are added and the mixture is vortexed for 30 seconds. The sam-

ples are centrifuged at 3600 rpm for 12 minutes and the organic layer is

transferred to another tube and evaporated under a stream of dry nitrogen.

Samples were reconstituted in 100-200 pl of methanol and 10 il were injected

onto the column.

For absolute recovery experiments, spiked plasma samples were compared

to unextracted stock solutions. Peak height ratios were calculated and
amounts found were compared.

Precision determinations were performed by comparing the peak height

ratios of five extractions at each concentration.

Results and Discussion
Figure 28 shows a chromatogram of plasma containing fenbufen (I), two

metabolites (II) and (III) and internal standard (IV). The retention times

are listed in table 13.

Table 14 lists recovery data for compounds I, II and III. In the 0.5

to 25 Pg/ml range, the average recovery for I was 88.9 9.2%, II, 82.4

9.0% and III, 100.4 2.6%.