The effect of experimental diabetes on drug metabolism

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Title:
The effect of experimental diabetes on drug metabolism
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ix, 85 leaves : ill. ; 29 cm.
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Ackerman, Dennis M., 1946-
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Subjects / Keywords:
Diabetes Mellitus, Experimental   ( mesh )
Pharmaceutical Preparations -- metabolism   ( mesh )
Pharmacology thesis Ph.D   ( mesh )
Dissertations, Academic -- Pharmacology -- UF   ( mesh )
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bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1975.
Bibliography:
Includes bibliographical references (leaves 81-84).
Statement of Responsibility:
by Dennis M. Ackerman.
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Typescript.
General Note:
Vita.

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University of Florida
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notis - AEK7324
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TH:E _-FCT OF EXPERIMENTAL DIABETES ON DRUG METABOLISM











By


DENNIS M. ACKERMAN


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL CF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIRE2MENTTS FOR THE DEGREE
OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA















ACKNOWLEDGMENT S


The author wishes foremost to thank his supervisory

corrmittee chairman, Dr. Kenneth C. Leibman, for his sug-

gestions, assistance, and most of all his patience

throughout the course of this study. Recognition is also

extended to the other members of his committee, Dr. Owen

M. Rennert, Dr. David M. Travis, and Dr. Betty P. Vogh.

Recognition is also extended to Dr. Ira Weinstein for his

assistance.

The author also wishes to acknowledge Mr. George

WJynns for his assistance in surgical procedures and is

grateful for the aid of Dr. Robert J. Cohen in assisting

with cyclic AMP assays.

The author would like to acknowledge and thank

Kathleen Ackerman for her encouragement and support pro-

vialed during a large part of his tenure as a graduate

st-Udent. Sincere appreciation is also extended to the

author's parents for their everlasting support.

A special thanks is extended to Mrs. Elsa Couto for

her assistance, companionship, and warmth.

The author would also like to express his appreci-

L; ion for the friendship and aid of Mrs. Theresa Fulford

-s well as thanking her for typing this ma-uscript.















TALE OF CONTENTS

Page

Acknowledgments .......................................ii

List of Tables.. ..................................... iv

List of Figures .......................................vi

Abstract...... ....................................... vii

Historical Review ............................ ........... 1

Goals .... ....................... ........................19

Materials a,'1d Methods ............... -....24

Results............................................... 34

Discussion......... ... ................................ 67

SurMLmary. ...............................................78

References......... .....................................8

Biographical Sketch.................................... .
























ii.
















LIST OF TABLES


Table


1. Summary of Literature.............................14

2. Hexobarbital Sleeping Times after Adminis-
tration of Diabetogenic Agents .................42

3. Effect of Streptozotocin on Kinetic Constants
for Hexobarbital and Aniline Hydroxylations....43


4, Effect of 6-Arinonicotinamide on Kinetic Con-
stants for Hexobarbital and Aniline......
Hydroxylations

5. Effect of N-Methylacetamide on Kinetic Con-
stants for Hexobarbital and Aniline
Hydroxylations..........................

6. Hexobarbital and Aniline Hydroxylations One
Week after the Administration of
Strephozotocin.................... ........

7. Effect of Alloxan on Hexobarbital and Aniline
Hydroxylations..........................

S. Effect cf Streptozotocin on Hexobarbital and
Aniline Hydroxylations with Isolated
Microsomes as Enzyme Source..............

). Effect of 6-Aminonicotinamide on Hexobarbital
and Aniline Iydroxylations with Isolated
Microsomes as Enzyme Source ..............


......45




... ..46



...... 47


...... 49



......50



......51


10. Effect of Glucose Infusion on Hexobarbital
Metabolism..................................

11. Effect of Diabetogenic Agents Added in Vitro
on Hexobarbital Metabolism................

12, Effect of Diabetogenic Agents Added In Vitrn
on Aniline Metabolism........................


Page


r-,








Table


13. Effect of Insulin In Vitro on Hexobarbital and
Aniline Metabolism Following Diabetogenic
Agents..................... .................... 57

14. Effect of Diabetogenic Agents on Hepatic Micro-
somal Protein Concentration .............. .....58

15. Effect of Diabetogenic Agents on Cytochrome
P-450 Concentration in Liver Microsomes........60

16. Effect of Diabetogenic Agents on Cytochrome
P-450 Binding to Hexobarbital and
Aniline....... .................................61

17. Localization of Inhibitor of Hexobarbital
Metabolism Present in 100,000g Super-
natant Fraction of Rat Liver after
Pretreatment with Streptozotocin and
6-Aminonicotinamide............................63

18. Localization of Inhibitor of Aniline Meta-
bolism Present in 100,000g Supernatant
Fraction of Rat Liver after Pretreatment
with Streptozotocin and 6-Aminonicotin-
amide............................................64

19. Effect of Diabetogenic Agents on Hepatic c-AMP
Concentrations................. .. ....... ..66


Page
















LIST OE FIGURES


Figure Page

1. Structures of Diabetogenic Agents..................25

2. Relationship between Serum Glucose and Dose
of Streptozotocin..............................36

3. Relationship between Serum Glucose and Dose of
6-Aminonicotinamide............................38

4. Relationship between Serum Glucose and Dose
of N-Methylacetamide ......... ................40

5. Metabolic Pathway of Hexobarbital..................69

6. Metabolic Pathway of Aniline...................... 70









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



THE EFFECT OF EXPERIMENTAL DIABETES ON DRUG METABOLISM

By

Dennis M. Ackerman

December, 1975

Chairman: Dr. Kenneth C. Leibman
Department: Pharmacology and Therapeutics

The purpose of this investigation was to examine the

effect of experimental diabetes on the hepatic microsomal

drug-metabolizing system of the liver in male Holtzman rats

and to elucidate a mechanism for these effects. Three dia-

betogenic agents were used: streptozotocin, 6-aminonicotin-

amide, and N-imethylacetamide. Streptozotocin and 6-amino-

nicotinamide produce an insulin-deficient animal whereas

N-mechyiacetamide does not affect insulin production or

release but causes a condition in which insulin is incap-

able of lowering blood glucose.

Both in v;vo and in vitro experiments were performed

to investigate the effects of the diabetic state induced

by these three agents on hepatic drug metabolism. The

results of the in vivo experiments correlated with the

results of those in o -tro in that inhibition of certain

pathways of drug metabolism was seen in animals pretreated

with streptozoiocin and 6-a.minonicotinamide but not in ani-

rmals retreated with N-mn:ehy iacetamide even though ali








treated animals were hyperglycemic.

The rate of hydroxylation of hexobarbital was not

significantly different in preparations from rats that had

been infused with glucose and those from saline-infused

control animals. This demonstrates conclusively that the

inhibition caused by diabetogenic agents was not due to

hyperglycemia.

Neither insulin nor any of the diabetogenic agents had

any direct effect on drug metabolism in vitro. Further-

more, hepatic microsomal protein and cytochrome P-450

contents were not significantly different in any of the

diabetic animals than those of the control animals.

The binding of both hexobarbital and aniline to he-

patic cytochrome P-450 was inhibited in animals pretreated

with streptozotocin but only that of hexobarbital was in-

hibited in 6-aminonicotinamide-pretreated animals. The

fact that the affinity of aniline to cytochrome P-450 was

increased in microsomes from animals pretreated with N-

methylacetamide somewhat obscures the interpretation of

these results.

It had been shown previously that treatments that in-

creased the endogenous levels of cyclic 3',5'-adenosine

monophosphate (c-AMP) in the liver were inhibitory to cer-

tain drug metabolic pathways and that this was due to the

release of an inhibitor of drug metabolism into the liver

cytosol. The presence of an inhibitor of hexobarbital and


viii










aniline hydroxylation was shown to occur in the liver

cytosol of animals pretreated with streptozotocin and an

inhibitor only of hexobarbital hydroxylation in animals

pretreated with 6-aminonicotinamide. This correlated with

elevated levels of hepatic c-AMP observed in animals pre-

treated with streptozotocin and to a lesser degree in

animals pretreated with 6-aminonicotinamide. The levels

of c-AMP were not elevated in animals pretreated with N-

methylacetamide, as compared to control animals.

In conclusion, it appears that the inhibition of drug

metabolism in animals made diabetic with certain drugs is

not caused by hyperglycemia, but is rather the consequence

of insulin lack. It is proposed that this lack of insulin

causes an elevation of hepatic c-AMP which, in turn, causes

the release by liver microsomes of an inhibitor of the

metabolism of xenobiotics.















HISTORICAL REVIEW


Many of the metabolic alterations due to diabetes are

well understood but the effect of the disease on hepatic

drug metabolism is not clear, even though this has been

studied for over 15 years. No definitive conclusions can

be drawn from the data accumulated because of the consid-

erable amount of conflicting results that have appeared

in the literature.

In 1958 (1), the half-life of tolbutamide was deter-

mined in both diabetic and nondiabetic humans of both

sexes. The half-life was found to be similar in both

groups (7.8 hours in the diabetic group and 8.0 hours in

the nondiabetic group). Prolonged use (2.5 5 months)

of tolbutamide in the diabetic patients was shown to

decrease the half-life of the drug in these patients

about 17% (7.3 hours to 6.5 hours). This decrease is

probably due to the fact that tolbutamide is an inducer

of the liver microsomal oxidase system that metabolizes

foreign compounds (2) and thus it stimulates its own

metabolism. Large deviations among diabetic and non-

diabetic subjects as well as the small sample size pre-

vented statistical analysis of these data.








It was shown in 1961 by Dixon et al. (3) that the

activities of drug-metabolizing enzymes of liver micro-

somes were decreased by short-term alloxan diabetes (the

duration of the diabetic state was never longer than three

days). In this study, microsomal liver preparations of

male Holtzman rats that had been pretreated with alloxan

had a diminished ability to metabolize hexobarbital (side

chain cxidation), chlorpromazine (oxidation of ring sulfur),

and codeine (O-demethylation). In vivo, hexobarbital

sleeping time was prolonged after alloxan pretreatment,

which correlated well with the reduced microsomal enzyme

activity toward hexobarbital. Both the in vitrc and in

vivo effects could be reversed by treating the diabetic

animal with insulin. Neither alloxan nor insulin had any

effect when added to the incubation mixtures in vitro.

The authors concluded that the diabetic state had an

inhibitory effect on the ability of these animals to

metabolize drugs.

A follow-up study (4) in 1963 was done to see the

effect of long-term (1 3 month duration) alloxan dia-

betes on certain pathways of the hepatic microsomal

mixed-function oxidase drug-metabolizing system. In this

study, male rats of the Long-Evans strain were used as

well as the Hoitzman strain because the former appeared

to be more resistant to the toxic effect of alloxan

and the following diabetic condition. A decreased rate








of metabolism in vitro and a prolonging effect on hexo-

barbital action in vivo was again seen. Also, a decreased

rate of in vitro metabolism of aminopyrine (which is

demethylated) was observed, whereas an increased rate of

in vitro metabolism of aniline (which is hydroxylated)

was seen. Insulin treatment returned the rate of hexo-

barbital metabolism back to control levels but had no

effect on aminopyrine metabolism. The rate of aniline

metabolism was decreased below the control rate after

insulin treatment. From this study, the authors concluded

that the diabetic state had varying effects on drug meta-

bolism which were substrate-dependent.

In the same year, it was reported by Gillette et al.

(5) that the administration of alloxan to immature female

rats did not alter zoxazolamine hydroxylation or mono-

methyl-4-aminopyrine demthylation. On the other hand,

the O-dealkylation of phenacetin was stimulated by the

alloxan-diabetic state.

In 1965, Kato and Gillette (6) again demonstrated

that alloxan diabetes of three days' duration decreased

the activity of the hepatic microsomal enzyme system of

male Sprague-Dawley rats toward aminopyrine and hexobar-

bital but did not change the activity of the system

toward zoxazolamine. An enhancement of aniline hydrox-

ylation was again seen. The effect of the alloxan dia-

betic state in female Sprague-Dawley rats, though, was

much different. In these animals, the metabolism of








aminopyrine, aniline, and zoxazolamine was increased by

the diabetic state, but the activity of the hexobarbital-

metabolizing enzyme system was not significantly affected.

When female rats were castrated and given miethyltestos-

terone, the results were different from those observed

with intact female rats, but were similar to those seen

with male rats. Also, ailoxan diabetes did not produce a

further reduction in hexobarbital hydroxylation or amino-

pyrine demethylationin male rats that had been castrated.

Castration of male diabetic rats, however, did not pre-

vent the increased activity of the microsomal system

toward aniline that was also seen in the non-castrated

male diabetic rats. These results showed that certain

hepatic microsomal pathways in diabetic rats, such as

that of aminopyrine, were sex-dependent; whereas other

pathways, such as that for aniline, were not.

A cytochrome termed "P-450," found in the hepatic

microsomal fraction, is considered to be the final oxygen-

activating enzyme in many NADPH-dependent drug-metabolic

pathways. It catalyzes the reduction of an oxygen mole-

cule with placement of one oxygen atom into water and

simultaneous insertion of the other oxygen atom into a

foreign substrate. This cy-tochrome can be measured quan-

titatively by the difference spectrum observed when it is

reduced (usually with dithionite) and corn-Lr.ed with carbon

monoxide.





5


Drugs combine with cytochrome P-450 (7) to produce

difference spectra of two general types, known as types I

and II. Type I compounds, exemplified by hexobarbital and

aminopyrine, give difference spectra with max in the range
max
of 385 390 nm and A min in the range of 418 427 nm.

Type II compounds, exemplified by aniline, give A
max
between 425 435 nm and Xin between 390-405 (8). These
min
spectral changes resulting from substrate interaction with

cytochrome P-450 have been presumed to represent the pri-

mary binding of substrate for enzyme action and a change

in the degree of binding may be indicative of a change in

the amount of substrate being metabolized. The strength

of binding can be determined spectrophotometrically by

plotting the reciprocals of the spectra change vs. those

of substrate concentration in a Lineweaver-Burk type of

plot to give by extrapolation the spectral dissociation

constant (K ).

In 1970, Kato et al. (9) studied the effect of alloxan

diabetes on Wistar rats and mice of the dd strain. In this

study, the effect of allcxan diabetes on the in vitro meta-

bolism of several substrates and on the content of cyto-

chrome P-450 and NADPH-cytochrome c reductase (the first

enzyme in the electron-transport chain involved in drug

metabolism in the liver) were examined. In male rats, a

decrease was again seen in the rates of both aminopyrine

N-dei.ethylation and hexobarbital hydroxylation but in









female rats and male and female mice, an increase was

observed in the rates of metabolism of both of these sub-

strates. An increase in the rate of aniline hydroxylation

was seen in both sexes of rats and mice.

The activity of NADPH-cytochrome c reductase was not

altered by alloxan diabetes in either the male or female

rats but was increased by alloxan diabetes as compared to

controls in both sexes of mice. The cytochrome P-450 con-

tent in liver microsomes from the diabetic animals was

slightly increased over that of controls in the female

rats but was not affected by the diabetes in male rats or

male or female mice. The authors concluded that the oxi-

dative activities of the liver microsomes of the male rats

toward certain substrates such as hexobarDital and amino-

pyrlne are enhanced by androgen and that this enhancement

is blocked by alloxan diabetes. In mice, this androgen-

dependent enhancement is not present. Aniline hyaroxyl-

ation is presumed not to be androgen-dependent and there-

fore alloxan diabetes does not increase its rate of

hydroxylation. Why the rate of its hydroxylation is

increased, however, was not explained.

The same authors (10) studied the effect of alloxan

diabetes on cytochrome P-450 content, substrate inter-

action with hepatic cytochrome P-450, and kinetics of

several drug-metabolic pathways in male and female Wistar

rats. Alloxan diabetes did not alter the amount of cyto-

chroe-. P-450 in the livers of male rats but slightly









increased the cytochrome P-450 content in the livers of

the female rats of this species. The affinity of the

cytochrome P-450 for hexobarbital and aminopyrine was

lower in male diabetic rats than it was in normal male

rats. Also, a decrease in the rates of both hexobarbital

and aminopyrine oxidation was observed. Aniline, however,

was h-droxylated at a greater rate in the diabetic male

rats than in controls although there was no significant

increase in the binding of the cytochrome P-450 to aniline

from the diabetic rats as compared to controls. An

increase in the hydroxylatjon of zoxazolamine as well as

an increased affinity for cytochrome P-450 was also seen

in the diabetic male rats as compared to control animals.

In female diabetic rats, increases in the degree of binding

of hepatic microsomal cytochrome P-450 to both hexobarbital

and aniline, as compared to those of control rats were

seen.

Inasmuch as hexobarbital and aminopyrine, which are

both type I compounds, show both decreased binding to

cytochrome P-450 as compared to controls as well as

decreased rates of oxidation, whereas aniline, a type II

compound, shows no change in affinity to cytochrome P-450

compared to control but an increase over control animals

in the rate of its metabolism, it was concluded that the

effect of alloxan diabetes on hepatic drug-metabolic

enzymes may be related to the type of spectral change seen

when the drug binds to cytochrome P-450. The results seen








with zoxazolamine, a type I compound, in the male rats

were not explainable. Also, the effect of alloxan diabetes

does not appear to be related to the type of spectral

change a drug gives with cytochrome P-450 in female rats.

In 1972, Dajani and Kayyali (11) studied the meta-

bolism of phenacetin in male New Zealand White rabbits

which had been made diabetic by the administration of

alloxan. They found that the urinary and plasma concen-

trations of the unchanged drug were higher in the diabetic

animals than in the controls, and that the concentrations

of the major metabolite, acetaminophen, were lower. This

was suggestive of a decreased metabolism of this drug in

the diabetic animal. A decreased rate of in vitro metabo-

lism of phenacetin in liver microsomes from alloxan-

diabetic rabbits proved that the metabolism of phenacetin

was inhibited in these animals. Both the in vivo and in

vitro inhibition of phenacetin metabolism were reversible

by the administration of insulin to the animals. To

attempt to explain this inhibition of metabolism of phen-

acetin in alloxan-diabetic rabbits, the contents of

hepatic microsomal protein and hepatic NADPH, a necessary

cofactor for the hepatic microsomal drug-metabolizing sys-

tem, were measured, and both were found to be lower in the

diabetic animals than in control animals. They concluded,

therefore, that the inhibition that occurred in the

alloxan-diabetic state in the male rabbit was probably due









Lo a deficiency of micrcsomal drug-metabolizing protein

and/or a deficiency in hepatic NADPH.

In a follow-up study the following year (12), the

metabolic transformation of phenylbutazone (which is

hydroxylated) was examined in the alloxan-diabetic Wistar

rat. As in the previous study, the rates of both the in

vivo and in vi-to metabolism of the drug were decreased

in alloxan-diabotic animals as compared to control animals

and treatment of the diabetic animals with insulin

returned the in vivo and in vitro metabolic rate to near

control levels. Again, this inhibition of metabolism in

the alloxan-diabetic animal was stated to probably be due

to a decreased hepatic microsomal protein and/or hepatic

NADPH content, which they showed to occur in the diabetic

rats.

In 1974, this same group (13) examined the metabolism

of phenacetin and acetaminophen by insulin-requiring dia-

betic men and women and nondiabetic men and women. Aceta-

minophen was used in this study to see if a diabetic state

in humans affects the conjugating enzyme system of the

liver. Acetaminophen, unlike phenacetin, already pos-

sesses a center for conjugation and thus does not require

an initial biotransformaticn to form this center. The

diabetic group was deprived of insulin for 48 hours pre-

ceding the administration of the drugs and during the 12

hours following the administration. With phenacetin, it








was shown that the diabetic group excreted six to ten times

the amount of nonmetabolized drug as did the nondiabetic

group. Also, the excretion of the principal metabolite,

acetaminophen, was from two to seven times greater in the

control than in the diabetic group. Treatment of the

diabetic group with insulin returned the excretion rate of

nonmetabolized phenacetin and acetaminophen nearly to con-

trol values. It was also shown that there was a sub-

stantial individual variation in the rate of excretion of

both nonmetabolized phenacetin and acetaminophen in the

diabetic group, which consisted of only five subjects, and

that this variation appeared to correlate with the serious-

ness of the diabetic state as measured by blood glucose

]evel.

When acetaminophen was administered, twice as much

unchanged drug and 40% as much conjugated derivatives of

acetaminophen were found in the urine of the diabetic

group compared with that of the control group. From these

results, the investigators concluded that the inhibition

of metabolism of phenacetin and acetaminophen was due to

the diabetic state of the patients. They suggested that

the abnormality in these diabetic patients responsible for

this inhibition may be a reduction in hepatic NADPH.

The fundamental defects to which most of the wide-

spread biochemical abnormalities seen in diabetes can be

traced are a reduced entry of glucose into various peri-

pheral tissues and an increased release of glucose into









the circulation by the liver. The immediate result of

these defects is hyperglycemia. Several investigators

have studied the effect of a hyperglycemic state on drug

metabolism.

In 1951, Lamson et at. (14) showed that more than 25%

of dogs anesthetized with pentobarbital returned to sleep

when given an intravenous injection of glucose shortly

after awakening. This could not be correlated with blood

sugar leves and no explanation could be given why some dogs

did not respond at all. Other species were also investi-

gated and the hamster, rabbit, pigeon, chicken, and guinea

pig showed this effect nearly 100% of the time whereas the

mouse, rat, goldfish, and tadpole did not show this effect.

They also found that certain metabolites of glucose, i.e.,

lactate, pyruvate, levulose, and galactose, elicited a

similar response in guinea pigs. Moreover, they found

that lactate and pyruvate increased the rate of entrance

of barbital into the brain of guinea pigs whereas glucose

did not. These results indicated that glucose and some

of its metabolites potentiated the anesthetic action of

barbiturates in certain species and it was at least

partially due to an increased penetration of the barbitu-

rate into the brain.

In 1971, Strother st al. (15) studied the influence

of high sugar consumption by Swiss-Webster mice on the

duration of action of barbiturates and the in vitro meta-








bolism of barbiturates, aniline, and p-nitroanisole.

They found that mice kept for two days on a diet of ground

chow ad liibtum and a 35% glucose solution as their sole

source of fluid had an increased sleeping time after

administration of hexobarbital, pentobarbital, secobarbi-

tal, amobarbital, or phenobarbital, compared to control

animals allowed ground chow ad Zibitum and water as their

sole source of fluid. The treated animals tended to com-

pensate for the high level of glucose intake in that their

sleeping times returned to normal after three to four days

on this diet and then they apparently overcompensated

after five to eight days on this diet and actually slept

for a shorter length of time than did the control animals.

The mechanism of these changes in sleeping time is not

apparent because possible changes in the brain concen-

trations of the barbiturates can not be ruled out, inas-

much as they were not measured. The in vitro metabolism

of aniline was not affected under these conditions. None

of these effects could be correlated with blood glucose

level, as the treated animals never exhibited a hypergly-

cemia and actually had a 20% lower mean blood sugar level

than did the control group.

There are a number of similar disturbances seen in

both starvation and diabetes, such as a negative nitrogen

balance and ketosis. Another apparent disturbance is an

alteration in hepatic drug metabolism. 'i`e effects of

starvation on drug metabolism, however, are also confusing









because of conflicting results reported by many researchers.

In 1970, Kato et aZ. were able to obtain results that

closely resembled those of Dixon et a1. (16) and Kato and

Gillette (17). These results showed that starvation for

a 36- to 48-hour period increased the activity of the

hepatic drug-metabolizing enzymes in both sexes of mice and

female rats for nearly all substrates tested, including

aniline, whereas it decreased the activity of the system

in male rats toward hexobarbital and aminopyrine. These

results are similar to those seen in the alloxan-diabetic

state in that an inhibition in the drug-metabolizing sys-

tem of male rats toward type I substrates occurs, whereas

either no inhibition or stimulation is seen with female

rats and male and female mice for types I and II

substrates.

Table I saimarizes the work described so far in

animals in vitro.

From the previous discussion, it is apparent that

there is a high degree of uncertainty concerning the

effects of a diabetic state on the hepatic drug-metabo-

lizing system and until recently, no investigation has

added any insight as to how diabetes may be affecting this

system.

The secretion of insulin from the pancreas is stimu-

lated under conditions that raise intracellular concen-

trations of cyclic 3',5'-adenosine monophosphate (c-AMP)























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in the pancreas (18). Diabetes is one of the conditions

that raise the intracellular level of c-AMP in the pan-

creas (19,20) but because the islets of Langerhans are not

functional (in classical diabetes), no insulin release

occurs. Diabetes also has been shown to increase the

levels of c-AMP in other tissues, e.g. liver (21).

Weiner et al. (22) showed in 1972 that certain treat-

ments that raised the endogenous levels of c-AMP, such

as the administration of glucagon and theophylline, were

inhibitory to the in vivo metabolism of hexobarbital

(as measured by sleeping time) and the in vitro metabolism

of both hexobarbital and p-chloro-N-methylaniline in male

Sprague-Dawley rats. Direct support for the involvement

of c-AMP in the metabolism of these substrates was pro-

vided by the demonstration that the administration of

dibutyryl cyclic AMP increased hexobarbital sleeping time

and, in addition, inhibited the in vitro metabolism of

hexobarbital and p-chloro-N-methylaniline. The dibutyryl

derivative of c-AMP was used because it has been shown to

produce parallel effects caused by c-AMP at lower concen-

trations, due to greater penetration through cellular

membranes and lower susceptibility to destruction by the

enzyme phosphodiesterase (23, 24). Weiner et al. concluded

that increasing endogenous c-AMP concentrations or admin-

istering a c-AMP analogue is inhibitory to certain pathways

of drug metabolism and that this inhibition is mediated in

the liver.









Further studies by Weiner at aV. (25, 26) into the

mechanism of inhibition of drug metabolism by cyclic

adenine nucleotides have shown that an inhibitor of the

in vitro metabolism of hexobarbital and of F-chloro-N-

methylanilina is released into the liver cytosol in

response to treatment with dibutyryl c-AMP. This inhi-

bition was observed as early as ten minutes and as late as

24 hours after the administration of dibutyrl c-AMP. Pre-

liminary characterization of this inhibitor has shown that

it has a molecular weight of less than 5,000 and that it

is probably not a protein.

Ross et al. (27) confirmed in 1973 the finding that

cyclic nucleotides are inhibitory to certain pathways of

the hepatic mixed-function oxidase system. They found

that male and female Holtzman rats pretreated with c-AMP

showed a decreased in vitro metabolism of aniline, whereas

in vitro aminopyrine demethylase activity was depressed in

the male but not in the female. Microsomal content of

hepatic cytochrome P-450 fell only in the male after pre-

treatment with c-AMP. When dibutyryl cyclic AMP was used

at an equivalent dose, inhibition was seen in both the in

vitro metabolism of aniline and aminopyrine. Cytochrome

P-450 content was decreased in both sexes after pretreat-

ment with dibutyryl cyclic AMP. Also, pretreatment with

dibutyryl cyclic AMP increased the spectral dissociation

constant (K ) of hexobarbital and cytochrome P-450 but had
s





18


no change in the K of aniline and cytochrome P-450.

They concluded that cyclic nucleotides exert a sex-

dependent inhibitory effect on the hepatic mixed-function

oxidase system and that this effect is at least partially

due to qualitative and quantitative changes in hepatic

cvtochrome P-450.














GOALS


The primary purpose of this investigation was to study

the effect of experimental diabetes on the microsomal drug-

metabolizing system of the liver and to elucidate a mecha-

nism for this effect. Until now, the only work done in

studying the effect of diabetes on drug metabolism has

involved induction of the diabetic state with alloxan, a

chemical which destroys the insulin-producing beta-cells of

the islets of Langerhans in the pancreas. Alloxan, however,

has a number of unfavorable actions. First of all, it is

a highly toxic chemical and a mortality rate in Sprague-

Dawley rats as high as 40% has been observed with a normal

diabetogenic dose (28). Also, the nephrotoxicity of

alloxan is well established (29, 30). Finally, there is a

recovery of insulin production in alloxan-diabetic animals

approximately three months after they receive the chemical

(31). A remission of the diabetes, consisting of an

increased body weight, decreased mean blood glucose level,

and a measurable rise in plasma insulin level as a response

to the administration of corticotropin, occurs concomi-

tantly with this renewed production of insulin.








Because of these disadvantages, I have chosen three

other chemicals, streptozotocin, 6-aminonicotinamide, and

N-methylacetamide, to induce experimental diabetes.

Streptozotocin, an N-nitroso derivative of glucosamine (32)

produces diabetes by a mechanism similar to that of alloxan

(33, 34) but has some distinct advantages over alloxan.

The mortality rate in rats is as much as ten times lower

with streptozotocin than with alloxan (28, 35). Also,

streptozotocin has a higher specificity as a beta-cytotoxic

agent in the rat than does alloxan (34, 36). Another

advantage is that it is much easier to achieve a graded

diabetogenic response with streptozotocin than with alloxan

(37). With alloxan, a graded diabetic response is nearly

impossible either the animal does not become diabetic or

it becomes severely diabetic and ketoacidotic. With strep-

tozotocin, on the other hand, it is fairly easy to get a

graded diabetic response and the animal only becomes keto-

acidotic at high doses. Finally, animals made diabetic by

streptozotocin do not recover from the diabetes (31).

The second agent used, 6-aminonicotinamide, an anti-

metabolite of NADP synthesis, produces diabetes by blocking

insulin release (38). It is hypothesized that NADPH, which

is formed in the pentose-phosphate shunt during the cata-

bolism of glucose, is needed for the release of insulin,

and that 6-aminonicotinamide inhibits the synthesis of

NADPH by blocking the formation of its precursor, NADP.

The blocking of the in vitro release of insulin from an








individual islet by 6-aminonicotinamide can be reversed by

the addition of either NADP or 'iADPh. The effects of 6-

aminonicotinamide are not irreversible and wear off with

time.

The third diabetogeric agent, N-methylacetamide, pro-

duces diabetes by making the animal insulin-resistant (39).

Neither insulin synthesis nor release are affected, but

the insulin that is released is ineffective in lowering

the blood glucose level, even though the plasma insulin

level rises concomitantly with that of glucose. The

effects of this drug are irreversible and its toxicity is

such that a rat treated with it will usually be dead or

too moribund for further experimentation 48 hours after its

administration.

The administration of insulin to animals pretreated

with alloxan, streptozotocin, or 6-aminonicotinamide

reverses the diabetic effects caused by these agents but

has no effect on those in animals pretreated with N-methyl-

acetamide.

To investigate the effect that the diabetes induced

by these three agents had on hepatic drug metabolism in

male Holtzman rats, both in vivo and in vitio experiments

were performed. The in vivo experiments involved measuring

the length of hypnosis after the administration of hexo-

barbital sodium in animals which were made diabetic. The

ir vitro experiments were of several types. After the

diabetes had been induced with the three agents, the








kinetic constants for the metabolism of hexobarbital and

aniline were measured at several time periods. Also, the

metabolism of hexobarbital and aniline were measured one

week after the administration of streptozotocin to deter-

mine if the effects seen on drug metabolism existed for

more than several days. To examine the effect of hyper-

glycemia on drug metabolism, animals were infused intra-

venously with glucose, after which the in vitro metabolism

of hexobarbital was measured. To rule out any direct

effect that the diabetogenic agents may have had, they

were added to in vitro incubation mixtures from control

animals in which hexobarbital and aniline hydroxylations

were measured. To rule out any direct effect that insulin

may have had, it was added to similar in vitrj incubation

mixtures from control animals and those pretreated with

the diabetogenic agents. Hepatic microsomal protein and

cytochrome P-450 content were also measured. In addition,

the characteristics of binding of both hexobarbital and

aniline to hepatic cytochrome P-450 from the diabetic

animals were studied.

As was mentioned in the historical review, increasing

the endogenous levels of c-AMP or administering c-AMP de-

rivatives to animals is inhibitory to certain hepatic drug-

metabolic pathways. This increase in c-AMP content has

also been shown to be responsible for the release of an in-

hibitor of drug metabolism into the liver cytosol. To

attempt to elucidate a mechanism for the effects seen





23



with experimental diabetes on drug metabolism, hepatic

c-AMP content under the different diabetic conditions

was determined and these were correlated with the appear-

ance of an inhibitor of drug metabolism in the liver

cytosol.















MATERIALS AND METHODS


Materials

6-Aminonicotinamide and N-methylacetamide were pur-

chased from Aldrich Chemical Company, Inc., Milwaukee,

Wisconsin. The streptozotocin was a kind gift from Dr. W.

E. Dulin of The Upjohn Company, Kalamazoo, Michigan. The

structures of these three compounds are shown in Figure 1.

Alloxan was purchased from Eastman Kodak Company, Rochester,

New York.

Hexobarbital sodium was purchased from Winthrop

Laboratories, New York, New York and aniline hydrochloride

from Eastman Kodak Company, Rochester, New York. Both the

crystalline zinc insulin and the protamine zinc insulin

were purchased from Eli Lilly, Inc., Indianapolis, Indiana.

NADP, NADPH, glucose 6-phosphate, glucose 6-phosphate

dehydrogenase and Tris buffer were all purchased from

Sigma Chemical Company, St. Louis, Missouri.

The c-AMP radioimmunoassay kit was purchased from

Schwarz/Mann of Orangeburg, New York.

All other chemicals used were of reagent grade.

Animals

Adult male rats weighing 100-250 g were obtained

from Holtzman Laboratories, Madison, Wisconsin. All animals







CH20H
O

HO OH
NH-C-N-CH3
1i I
0 NO

STREPTOZOTOCIN


NH9




C-lNH2

-- Arn- I .U I IrOT I N!'! PE



0
li
CH3-C-NH-CH3

E fYLACETAM T IDE
N:-: E F-ii,.Y .. I ., ,

FIGURE 1
DTRUCTURFS OF DIPABETOGENTC AGENTS








were maintained on standard laboratory chow and water ad

Zibitum for a minimum of 48 hours in the Department of

Pharmacology and Therapeutics before experimentation. All

animals, except for those that were used in the glucose

infusion experiments were fasted overnight for 17 hours

preceding the experiment but were allowed water ad libitum.

Streptozotocin and 6-aminonicotinamide were dissolved

immediately before use, the former in a citrate buffer, pH

4.5 (26.5 ml of 0.1 M citric acid + 23 ml of 0.1 M sodium

citrate) and the latter in Krebs-Ringer bicarbonate solu-

tion. These drugs were both administered intraperitoneally.

Control animals used with streptozotocin-treated animals

were injected with the citrate buffer and those with 6-

aminonicotinamide were injected with the Krebs-Ringer

bicarbonate solution. N-Methylacetamide is a liquid and

was administered intragastrically. Control animals used

with N-methylacetamide animals were given normal saline

intragastrically. Alloxan was dissolved in normal saline

and injected subcutaneously at a dose of 115 mg/kg and

control animals used with alloxan-treated animals received

normal saline. Animals were considered diabetic only if

their blood glucose exceeded 200 mg/100 ml.

Enzyme Preparation

Animals were killed by a blow on the head followed by

decapitation. The livers were immediately removed, weighed,

and homogenized in four volumes of ice-cold 0.1 M Tris

buffer, pH 7.5. The homogenate was centrifuged at 9000,








for 15 minutes in a Servall refrigerated centrifuge at

0 C. The supernatant fraction from this homogenate was

used for the enzyme assays. When microsomal protein deter-

mination was necessary, part of this supernatant fraction

was centrifuged at 100,000g in aBeckman Model L ultracen-

trifuge for one hour at 40 C. The microsomal pellet was

then resuspended in 0.15 M KC1 and recentrifuged at

100,000g for another hour. This microsomal pellet was

then resuspended and a protein determination was performed

(40).

Isolated liver microsomes were also used in some

experiments. For these experiments, the liver was homog-

enized in four volumes of ice-cold 0.25 M sucrose solution

containing 1 mM EDTA. The supernatant fraction obtained

from centrifugation at 9000g for 15 minutes at 00 C in a

Servall refrigerated centrifuge was then centrifuged at

100,000g in a Beckman Model L ultracentrifuge at 4 for one

hour. The microsomal pellet was then suspended in 0.15 M

KCl and recentrifuged at 100,000g for one hour. This

microsomal pellet was then resuspended in 0.15 M KC1 con-

taining 0.05 M Tris buffer, pH 7.5.

Incubation Conditions

The in vitro metabolism of hexobarbital was determined

by assay for unchanged hexobarbital by the method of Cooper

and Brodie (41). The incubation system used was as follows:

2 ml of 9000g supernatant fraction equivalent to about 500

mg of liver, 1 micromole of hexobarbital, 30 micromoles of









nicotinamide, 25 micromoles of MgClz, and 0.26 micromole of

NADP. Tris buffer (0.1 M, pH 7.5) was added to bring the

volume to 4 ml in a 120-ml glass-stoppered bottle. The

incubation system used in microsomal experiments was the

same as above except that microsomes equivalent to 500 mg

of liver were used, and 1.8 micromoles of NADPH were sub-

stituted for the NADP. Also, the following were added:

25 micromoles of glucose 6-phosphate and 2 units of glucose

6-phosphate dehydrogenase. When either the 9000g fraction

or the microsomes were used as the enzyme source, the

mixtures were incubated with shaking at 370 C for 20 min-

utes and at the end of the incubation period, all bottles

were packed in ice for five minutes to stop the reaction.

Then, in succession, 1.3 ml of phosphate buffer, pH 5.5

(96.5 ml of 0.2 N NaH2P04 + 3.5 ml of 0.2 N Na2HPO4), 1.5

g NaC1, and 60 ml of heptane were added. The bottles were

then placed in a Burrel wrist-action shaker for 45 minutes,

after which the mixtures were centrifuged for 15 minutes

at room temperature in an International Centrifuge, Model

CS, at 40,000g. Then 40 ml of each organic solvent phase

was transferred to a 60-ml glass stoppered bottle containing

4 ml of phosphate buffer, pH 11 (8.5 ml 10 M NaOH + 200 ml

0.8 M Na2HOP4). This was then shaken for three minutes

after which 3 ml of the aqueous phase was used to determine

the spectrophotometric absorbance at 245 nm in a Gilford

spectrophotometer, Model 2400.








The in vitro metabolism of aniline was determined by

assay for p-aminophenol, the major metabolite formed in

rats, by the method of Brodie and Gillette (42) and modi-

fied by Kato and Gillette (43). The incubation system

used was as follows: 2 ml of 9000g supernatant fraction

equivalent to 500 mg of liver, 4 micromoles of aniline,

0.15 micromole of NADP, 15 micromoles of glucose 6-

phosphate, 15 micromoles MgSO4, and 0.5 ml of 0.3 M Tris

buffer, pH 7.5. Distilled water was used to bring the

final volume to 4 ml in a 120-ml glass-stoppered bottle.

The difference in conditions when microsomal experiments

were performed were the same as with the hexobarbital

experiments. In experiments with both 9000g supernatant

and microsomal fractions, the mixtures were incubated for

15 minutes with shaking at 370 C and then placed in an

ice-bath for five minutes. Then 2.5 g of NaCl and 50 ml

of ether were added, after which the bottles were shaken

in a Burrell wrist-action shaker for 15 minutes. Then 40

ml of each ether extract was removed and shaken with 3 ml

of a 0.1 M NaOH solution containing 1% phenol for three

minutes. This was allowed to stand for 30 minutes, after

which the ether was aspirated off and the absorbance of

the aqueous layer was determined at 620 nm in a Coleman

Junior spectrophotometer, Model 6A.









Determination of Kinetic Constants

For these experiments, the 9000g supernatant fraction

was used as the enzyme source and the incubation conditions

were as previously described except that the concentrations

of hexobarbital and aniline were different. Hexobarbital

was added to the incubation mixtures so that the final con-

centrations of hexobarbital were 0.5, 0.8, 1, 1.6, and 2 mM.

Aniline was added to the incubation mixtures so that the

final concentrations of aniline were 0.125, 0.167, 0.25,

0.5 and 1 mM. The kinetic constants were obtained by plot-

ting the reciprocals of the concentrations vs. the recipro-

cals of the initial velocities of the reaction.

Glucose Infusion

In these experiments, rats weighing between 200-250 g

were used and allowed laboratory chow throughout the exper-

iment. Their right jugular veins were cannulated under light

ether anesthesia, after which the animals were allowed to

recover for 24-36 hours. They were then placed in plastic

restraining cages and were infused with 40% glucose solu-

tion or normal saline solution for 24 hours with a Harvard

infusion/withdrawal pump at a rate of 0.074 ml/hour for a

total of 1.78 ml, equivalent to 710 mg of glucose.

Cytochrome P-450 Content

Cytochrome P-450 content was measured by the carbon

monoxide-induced difference spectrum of the reduced hemo-

protein according to the method of Omura and Sato (44).

Three ml of a microsomal suspension containing 2 mg/ml









protein were placed in quartz cuvettes and dithionite was

added to reduce the cytochrome P-450. Carbon monoxide was

then gently bubbled into the cuvette for one minute and the

change in absorbance at 450 nm minus that at 489 nm was

measured in an Aminco-Chance recording spectrophotometer

operated in the split-beam mode. Cytochrome P-450 content

was measured according to Beer's Law by the following

formula:

AA x 1000
c ----
x d x (p)

where c is the concentration of cytochrome P-450

expressed as nanomoles per mg of protein

E is the molar absorption coefficient

(91. mM- cm-l)

d is the optical path length (1 cm), and

(P) is the concentration of protein, expressed

as mg/ml

Drug Binding of Cytochrome P-450

The binding of drugs to cytochrome P-450 was measured

by the method of Remmer et aL. (7). Three ml of a micro-

somal suspension containing 2 mg of protein per ml were

placed in quartz cuvettes and either hexobarbital or

aniline was added. Hexobarbital was added in increments

which gave final concentrations of 0.125, 0.15, 0.187,

0.25, 0.375, and 0.75 mM. Aniline was added in increments

which gave final concentrations of 0.0625, 0.075, 0.094,









0.125, 0.188, and 0.375 mIM. The change in absorbance after

each addition was measured and the reciprocal of this

change was plotted us. the reciprocal of the drug concen-

tration; the apparent spectral dissociation constant (Ks)

was determined by extrapolation to the x-axis.

Determination of the Presence of an Inhibitor of Drug
Metabolism in Hepatic 100,000g Supernatant Fraction

Microsomes were prepared as described previously

except that after the first one-hour centrifugation at

100,000g, the microsomal and supernatant fractions of

control and streptozotocin- and 6-aminonicotinamide-

pretreated rats were separated and various combinations

were prepared. The soluble fractions were added to the

microsomes immediately and were rehomogenized and then

kept on ice for 15 minutes. This homogenate was then

recentrifuged and the resultant microsomal pellet was

resuspended as previously described for the preparation

of the final microsomal suspension used for the enzyme

assays.

Miscellaneous

C-AMP levels were measured by the method of Steiner

et aZ. (45). The Schwarz/Mann c-AMP radioimmunoassay

kit was used for these determinations.

Blood samples for determination of blood glucose

were taken from the tail vein and were analyzed by the

o-toluidine reagent method (46).

Hexobarbital sleeping time was measured as the time










between the loss and recovery of the righting reflex after

intraperitoneal injection of 100 mg of hexobarbital sodium

per kg.

Statistics

The results are expressed as the mean standard

error of the mean (S.E.). The p-values were calculated

by applying the Student t-test, which is a measure of the

probability that the differences observed are due to

chance (47).














RESULTS


Hyperglycemic Response to Diabetogenic Agents

Since the criterion chosen for a diabetic state was a

blood glucose concentration in excess of 200 mg/100 ml,

doses of the diabetogenic agents that would yield such a

blood glucose level had to be determined. The response to

streptozotocin at the dosages of 55 mg/kg and 65 mg/kg are

shown in Figure 2. The lower of the two doses was chosen

for further studies because there was little difference in

the hyperglycemic response and the higher dose was associ-

ated with a greater toxicity (40% of those animals died

within 24 hours compared with no mortality with the lower

dose). Figure 3 shows the response to 6-aminonicotinamide

at dosages of 25 mg/kg and 35 mg/kg. No animals died

within 24 hours after each dose. The higher dose was

chosen here because the lower dose did not produce a blood

glucose level that met the above criterion. Figure 4 shows

the response to N-methylacetamide at dosages of 6.75 ml/kg

and 10.0 ml/kg. The lower dose was chosen here because

the higher dose proved too toxic (60% died within 24 hours

vs. 0% at the lower dose).

























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Hexobarbital Hypnosis

To ascertain if the diabetes induced by the diabeto-

genic agents affected the in vivo sleeping time induced by

a hypnotic, 100 mg/kg of hexobarbital sodium was injected

intraperitoneally into rats. Table 2 shows the results

of these experiments; both streptozotocin and 6-aminonico-

tinamide increased the sleeping time, whereas N-methylace-

tamide had no effect.

Effect of Diabetogenic Agents on Kinetic Constants for
Hexobarbital and Aniline Hydroxylations

To determine if the diabetes induced by the three

diabetogenic agents affected the in vitro metabolism of

hexobarbital and aniline, the rates of hydroxylation of

these substrates were measured. Table 3 shows the kinetic

constants for both hexobarbital and aniline hydroxylation

in liver 9000g supernatant fractions 24, 48, and 96 hours

after the administration of streptozotocin. The table

also shows the effect on these constants after the daily

subcutaneous injection of three units of protamine zinc

insulin for three days beginning 24 hours after the in-

jection of streptozotocin. The results indicate that the

K values for both hexobarbital and aniline hydroxylations

were increased and the values of V decreased at all time
max
periods tested after the animal had been treated with

streptozotocin. The changes in these parameters for

aniline metabolism, although statistically significant,

were quite small. Treatment with insulin in vivo was able




















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to reverse these effects and return the K and V for
m max
both hexobarbital and aniline hydroxylation to control

values.

Table 4 shows the kinetic constants for hexobarbital

and aniline hydroxylations 24 and 48 hours after the ad-

ministration of 6-aminonicotinamide. The K values for
m
hexobarbital hydroxylation were increased and those of

V were decreased at both 24 and 48 hours. The K of
max m
aniline hydroxylation was increased 24 hours after admin-

istration of the diabetogen, and the Vmax was decreased.
max
These changes, although statistically significant, were

quite small. At 48 hours, neither of these parameters

was significantly different from those in preparations

from control animals.

Table 5 shows that 24 hours after the administration

of N-methylacetamiae, there was no change in any of the

kinetic constants for either hexobarbital or aniline.

Effect of Streptozotocin Diabetes of One Week Duration on
Hexobarbital and Aniline Hydroxylations

To determine if the effects of streptozotocin diabe-

tes on drug metabolism lasted for longer than three days,

hexobarbital and aniline hydroxylations were measured in

vitro one week after the administration of streptozotocin.

Table 6 shows that the rates of both hexobarbital and ani-

line metabolism were still below control values one week

after administration of streptozotocin.















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Effect of Alloxan Diabetes on Hexobarbital and Aniline
Hydroxylations

As was noted in the historical review, a number of

researchers had reported that the rates of metabolism of a

large number of drugs, including hexobarbital, were

decreased in alloxan-diabetic animals but those of a few

drugs, such as aniline, were enhanced. These results were

reexamined, as shown in Table 7. The metabolism of hexo-

barbital in vitro was inhibited while that of aniline was

enhanced.

Effect of Diabetogenic Agents on Hexobarbital and Aniline
Hydroxylations Using Isolated Microsomes as Enzyme Source

To be sure that the inhibition observed in drug meta-

bolism in animal preparations from rats pretreated with

streptozotocin and 6-aminonicotinamide was due to changes

in the microsomal enzyme system rather than to an altera-

tion in the NADPH-generating system, isolated liver micro-

somes were used as the enzyme source with an added NADPH-

generating system, to measure the metabolism of hexobarbi-

tal and aniline after animals had been pretreated with

these diabetogens. N-methylacetamide was excluded in these

studies because no inhibition in drug metabolism was seen

when animals were retreated with this agent. Tables 8

and 9 show that an inhibition in hexobarbital and aniline

metabolism occurred when preparations of liver microsomes

from animals that had been treated 24 hours in advance with

streptozotocin and 6-aminonicotinamide were used as the

enzyme source for the assay.

































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Effect of Glucose Infusion on Hexobarbital Hydroxylation

To test if hyperglycemia alone could be responsible

for the inhibition seen in drug metabolism, rats were

infused intravenously for 24 hours with glucose; each ani-

mal received a total of 710 mg of glucose. All such ani-

mals had blood glucose concentrations that exceeded 200 mg/

100 ml by the end of the infusion. Control animals were

similarly infused with normal saline solution. The data

in Table 10 demonstrate that hyperglycemia produced by

glucose infusion had no effect on the in vitro metabolism

of hexobarbital.

Effect of Diabetogenic Agents Added in Vitro on Hexobarbi-
tal and Aniline Hydroxylations

To rule out any direct effect that the diabetogenic

agents may have had on the in vitro metabolism of hexobar-

bital and aniline, they were added at concentrations of

10-2, 103, and 10-4 M to incubation mixtures containing

preparations of livers from untreated animals. Tables 11

and 12 show that the in vitro addition of these agents had

no effect on either hexobarbital or aniline hydroxylation.

Effect of Insulin Added in Vitro on Hexobarbital and Aniline
Hydroxylations Following Treatment with Diabetogenic Agents

To determine whether addition of insulin in vitro

would reverse the effects of pretreatment with the diabetc-

genic agents, crystalline zinc insulin ranging in amount

from 0.001 to 1 unit was added to 4-ml incubation mixtures

containing preparations of livers from animals treated



























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with streptozotocin and 6-aminonicotinamide. Livers from

IJ-nm.thylacetamide-treated animals were not used because

inhibition of neither hexobarbital nor aniline was seen

when animals were treated with this diabetogenic agent.

Table 13 shows that insulin had no direct effect on the in

v-itro metabolism of hexobarbital or aniline after the ani-

mal had been treated with streptozotocin or 6-aminonico-

tinamide.

Effect of Diabetogenic Agents on Hepatic Microsomal Protein

To see if the alterations in hepatic drug metabolism

may be due to changes in hepatic microsomal protein con-

tent, the effect of the diabetogenic agents on hepatic

microsomal protein concentration was determined. Table 14

shows that protein concentration was not affected 24, 48,

or 96 hours after the administration of streptozotocin, 24

or 48 hours after the administration of 6-aminonicotinamide,

or 24 hours after N-methylacetamide. Also, the administra-

tion of three units of protamine zinc insulin for three

days beginning 24 hours after the animals had been treated

with streptozotocin had no effect on hepatic microsomal

protein concentration.

Effect of Diabetogenic Agents on Cytochrome P-450 in Liver
Microsomes

To examine the possibility that the changes seen in

drug metabolism may be due to alterations in cytochrome

P-450 content in liver microsomes, the effect of the

diabetogenic agents on the microsomal concentration of this







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cytochrome was determined. Table 15 shows that pretreat-

ment 24 hours previously with neither streptozotocin,

6-aminonicotinamide, nor N-methylacetamide had any effect

on the concentration of cytochrome P-450 in the liver

microsomes.

Effect of Diabetogenic Agents on Cytochrome P-450 Binding
to Hexobarbital and Aniline

To ascertain if the changes observed may be due to

changes in the strength of binding of hexobarbital and

aniline to cytochrome P-450, the spectral binding constants

(Ks) were determined in liver microsomes from control ani-

mals and those that had been treated with the diabetogenic

agents. Table 16 shows that the affinity of cytochrome

P-450 for hexobarbital was significantly lower in micro-

somes from streptozotocin- and 6-aminonicotinamide-diabetic

animals than in controls, whereas the binding to hexobarbi-

tal was not affected in microsomes from N-methylacetamide-

treated animals. In the case of aniline, a decrease in

affinity of cytochrome P-450 was seen only in preparations

from animals pretreated with 6-aminonicotinamide, and the

affinity of cytochrome P-450 for aniline was increased in

microsomes from animals treated with N-methylacetamide.

Effect of Hepatic Homogenate Fractions on Hexobarbital and
Aniline Hydroxylation

To determine if an inhibitor of hexobarbital or aniline

metabolism may have been present in the 100,000g supernatant

fraction of livers of rats pretreated with streptozotocin or

6-am.inonicotinamide, various combinations of microsomes and


















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supernatant fractions from control and treated animals

were mixed together as described in the Methods section.

Table 17 shows that in vitro inhibition of hexobarbital

hydroxylation was seen when enzyme preparations containing

microsomes from animals pretreated with either streptozo-

tocin or 6-aminonicotinamide were preincubated with either

their own or with control supernatant fraction. More

important, however, is that enzyme preparations containing

microsomes from control animals which had been incubated

with supernatant fractions from animals pretreated with

either of the diabetogenic agents also showed an inhibi-

tion of hexobarbital metabolism.

Table 18 shows the same experiment except that aniline

was the substrate. The in vitro inhibition was again seen

when enzyme preparations containing microsomes of animals

pretreated with both diabetogenic agents were added with

either their own supernatant fraction or with control

supernatant fraction. Microsomes from control animals

which were combined with supernatant fraction of animals

pretreated with streptozotocin did show an inhibition of

aniline metabolism but when microsomes from control animals

were added to supernatant fraction of animals pretreated

with 6-aminonicotinamide, no inhibition occurred.














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Effect of Diabetogenic Agents on Hepatic c-AMP Concen-
trations

C-AMP concentrations were measured in the liver

following treatment with the diabetogenic agents to

determine whether a correlation existed between the type

of diabetic state produced and the hepatic content of

c-AMP, since it had already been shown that elevation of

hepatic c-AMP is inhibitory to certain drug-metabolic

pathways. Table 19 shows that streptozotocin and, to a

lesser extent, 6-aminonicotinamide, elevated hepatic

c-AMP concentrations, whereas N-methylacetamide had no

effect as compared to control values.

































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DISCUSSION


It has been shown previously that alloxan diabetes was

inhibitory to certain pathways of drug metabolism in several

species (3 6, 9 12, 17). This study was undertaken to

investigate the effect of three newer diabetogenic agents

on drug metabolism. Streptozotocin and 6-aminonicotinamide

produce an insulin-deficient animal while the third agent,

N-methylacetamide does not produce an insulin-deficient

animal but instead produces a condition whereby insulin is

produced and released by the pancreas but is incapable of

lowering blood glucose. The results of the present experi-

ments with these three diabetogenic agents indicate that,

with male Holtzman rats, it was not hyperglycemia that was

responsible for the inhibition of certain drug-metabolic

pathways in diabetes but rather that the inhibition was a

result of the lack of insulin.

Hexobarbital and aniline were chosen for these studies

because they have both been used extensively as model sub-

strates in studies of the mixed-function oxidase system in

the liver, and because their metabolism in normal rats is

well understood. Also, as mentioned in the Historical

Review, they differ in their binding to cytochrome P-450

in that the difference spectra produced when they are added








to liver microsomes are qualitatively different.

Figure 5 shows the major metabolic pathway of hexobar-

bital. In the rat, the major metabolite is 3'-hydroxy-

hexobarbital; 3'-ketohexobarbital is a very minor metabo-

lite (48). As described in the Methods section, the meta-

bolism of hexobarbital was measured by the disappearance

of the substrate rather than by the appearance of a meta-

bolite.

Figure 6 shows the metabolic pathway for aniline. In

rats, there is six times more p-aminophenol formed than o-

aminophenol (49). The metabolism of aniline was measured

by the appearance of the metabolite, p-aminophenol.

Hexobarbital sleeping time has often been used to meas-

ure hepatic drug metabolism for several reasons. First,

the substance is apolar enough to be rapidly absorbed and

distributed in the body. Second, its hypnotic activity is

such that convenient doses can be injected intraperitoneally

to produce a rapid sleep of moderate duration so that an

alteration in the length of sleep can be measured with

acceptable precision. Lastly, and most important, hexobar-

bital is rapidly metabolized in animals so that there is

little time for its accumulation in body fat. Thus, the

duration of its hypnotic activity is a reflection of meta-

bolism rather than of tissue redistribution (50). Table 2

shows that rats pretreated with both streptozotocin and 6-

aminonicotinamide slept significantly longer than controls




69




cO
-r-

0 -'I F-
C)




0=-






4+ I
-r-
0=






LU
C Do




00 "

o 0






I I -

OD
O =I | =
0=0 0 -
I- O L-l- U3














I^<1 ^














ANILINE


NH2


NH2/
, 2-


OH


OH


P-AMINOPHENOL


o-AMINOPHENOL


FIGURE 6


METABOLIC PATHWAY OF ANILINE







while those animals pretreated with N-methylacetamide did

not sleep any longer than the controls.

To see if the in vivo results just described could be

correlated with the in vitro experiments, the metabolism

of hexobarbital and aniline were measured in in vitro

incubation mixtures using 9000g supernatant fractions from

liver homogenates as the enzyme source. The fact that the

Km values increased and the Vmax decreased for both hexo-

barbital and aniline 24, 48, and 96 hours after the

streptozotocin and 24 hours after 6-aminonicotinamide

indicated that the in vitro metabolism of these substrates

was inhibited after pretreatment with these diabetogenic

agents. The effects of 6-aminonicotinamide upon insulin

release are reversed with time as the agent is eliminated

from the body; this was reflected by the less intense inhi-

bition of hexobarbital metabolism and the lack of inhibition

of aniline metabolism 48 hours after the administration of

6-aminonicotinamide. The fact that the kinetic constants

for both hexobarbital and aniline were unchanged in animals

24 hours after they were treated with N-methylacetamide

again showed that this diabetogenic agent does not affect

the metabolism of either of these substrates. These results

and the fact that hyperglycemia produced by infusion of

glucose did not cause inhibition of in vitro hexobarbital

metabolism (Table 10), showed that the hyperglycemia of

diabetes was not responsible for the inhibition of drug

metabolism caused by streptozotocin and 6-aminonicotinamide.








Since the diabetogenic effect of streptozotocin was

irreversible but not lethal at the dosage used, it was

important to determine if the inhibitory effect of strepto-

zotocin-diabetes on hexobarbital and aniline hydroxylations

lasted for longer than three days. Table 6 showed that

the in vitro metabolism of hexobarbital and aniline were

still well below control values one week after the animals

had received streptozotocin.

As was mentioned in the Historical Review, previous

researchers had noted that the in vitro metabolism of

hexobarbital was inhibited in animals pretreated with

alloxan but that the in vitro metabolism of aniline was

enhanced (3, 4, 6, 9, 10). Since in the present studies

with streptozotocin, a diabetogenic agent similar in mecha-

nism of action to alloxan, no enhancement of aniline meta-

bolism was observed, it was important to reexamine the

results with alloxan reported previously. The results

shown in Table 7 are qualitatively similar to those

reported by other investigators, although the extent of

enhancement of aniline hydroxylation was not as great as

that previously reported. Nevertheless, it would appear

that the enhancing effect of alloxan on the rate of aniline

hydroxylation is not related to the diabetogenic activity

of this agent.








Since both hexobarbital and aniline are metabolized

by liver microsomes and not by any nonmicrosomal hepatic

cells, it was important to determine if the inhibition that

was seen with the in vitro incubation mixtures using hepatic

9000g supernatant fraction as the enzyme source could be

duplicated when isolated liver microsomes were used as

the enzyme source. This was necessary to show that the

inhibition was not due to an inability of the drugs to

reach the microsomes, or to a deficiency in the NADPH-

generating system. Tables 8 and 9 demonstrated that the

metabolism of both substrates after treatment with either

streptozotocin or 6-aminonicotinamide was inhibited in

isolated liver microsomes.

Addition of streptozotocin, 6-aminonicotinamide, and

N-methylacetamide to incubation mixtures were made to

determine the direct effect these agents may have had on

in vitro enzyme activity. These diabetogenic agents at

concentrations ranging from 10-2 to 10-4 M had no direct

effect on either hexobarbital or aniline hydroxylation.

The highestof the concentrations of the diabetogenic agents

used in these experiments was greater than any of the con-

centrations that could have occurred in vivo, assuming

complete absorption.

The direct effect of crystalline zinc insulin on hexo-

barbital and aniline hydroxylations was also examined.

Table 13 shows that insulin added to incubation mixtures

that contained preparations of livers from control animals








or those treated with streptozotocin or 6-aminonicotinamide

had no effect on the in vitro metabolism of either of these

substrates. The concentrations of insulin added in these

experiments both mimicked and exceeded the concentrations

of insulin that would occur in vivo.

The inhibitory action of streptozotocin and 6-aminonico-

tinamide on both hexobarbital and aniline metabolism could

not be attributed to an alteration in hepatic microsomal

protein (Table 13) or to an alteration in hepatic micro-

somal cytochrome P-450 (Table 14). Also, N--methylacetamide

did not alter hepatic microsomal protein or cytochrome P-450

content.

The binding of hepatic microsomal cytochrome P-450 with

a substrate has been assumed to be an initial step for the

oxidation of drugs, and with certain drugs there is a simi-

larity between the dissociation constant of this binding

and the Michaelis constant for the metabolism of the same

drugs (7, 51). Table 15 showed that the binding of hexo-

barbital, a type I substrate, to hepatic microsomal cyto-

chrome P-450 from animals pretreated with streptozotocin

and 6-aminonicotinamide was inhibited while the binding of

the hexobarbital to cytochrome P-450 from N-methylacetamide-

treated animals was not inhibited. With aniline, a type II

substrate, however, binding was only inhibited in microsomes

from 6-aminonicotinamide-treated animals and was not

inhibited in those from streptozotocin-treated animals.

With N-methylacetamide, the binding of aniline to hepatic









cytochrome P-450 was actually enhanced. These results

indicate that the inhibition of hexobarbital metabolism in

animals pretreated with streptozotocin and 6-aminonicotin-

amide may be partially due to an interference in binding

of hexobarbital to the hepatic microsomal cytochrome P-450

from these animals. This cannot be the case with aniline,

however, inasmuch as inhibition in binding was seen only

in animals that were pretreated with 6-aminonicotinamide

and not in those treated with streptozotocin, even though

animals pretreated with streptozotocin demonstrated an

inhibition in their in vitro metabolism of aniline. Also,

the enhancement in binding of aniline to cytochrome P-450

from animals pretreated with N-methylacetamide is not

explainable because no enhancement of the metabolism of

aniline had been noted in preparations from animals pre-

treated with this compound. It is apparent that the

strength of binding of substrate to hepatic cytochrome P-450

is not always an accurate measure of the degree of the

metabolism of the substrate. Indeed, it has become

increasingly obvious in recent years that the earlier

expectations that the spectral binding constants of drugs

with cytochrome P-450 are closely related to the kinetic

constants for microsomal metabolism have not been borne

out by further investigations (52).








As was mentioned in the Historical Review section,

intracellular levels of c-AMP in the liver are known to

be elevated in classical diabetes (21). Also, treatments

that increase endogenous levels of c-AMP in vivo are

inhibitory to certain pathways of drug metabolism; this is

thought to be due to the release of an inhibitor of drug

metabolism by liver microsomes into the cytosol (22, 25).

The presence of an inhibitor of hexobarbital hydroxylation

was shown in the present work to exist in preparations

from animals pretreated with streptozotocin and 6-amino-

nicotinamide, and an inhibitor only of hexobarbital

hydroxylation in those of animals pretreated with 6-amino-

nicotinamide. When hepatic c-AMP concentrations were

determined in animals pretreated with the diabetogenic

agents, it was found that the levels in streptozotocin-

pretreated animals were nearly twice those of control ani-

mals, while those in 6-aminonicotinamide-pretreated animals

were increased to a lesser extent. The levels of c-AMP in

liver preparations from N-methylacetamide-pretreated ani-

mals were not significantly different from those of control

animals. These results correlate with the fact that prep-

arations from streptozotocin-pretreated animals always

showed a greater inhibition of both hexobarbital and aniline

hydroxylations than did those from animals treated with

6-aminonicotinamide. The fact that no increase in hepatic

c-AMP content was observed in preparations from N-methyl-

acetamide-pretreated animals, which never showed an





77


inhibition of drug metabolism, further substantiates the

finding that levels of hepatic c-AMP higher than normal

are inhibitory to certain drug-metabolic pathways.















S lUr1jpRY


Until now, all the work done in studying the effects

of experimental diabetes on the microsomal drug-metabo-

lizing system of the liver has involved the induction of

diabetes with alloxan, a toxic chemical that destroys the

beta-cells of the pancreas. The purpose of this investi-

gation was to study the effect of experimental diabetes on

drug metabolism with newer, less toxic diabetogenic agents

and to elucidate a mechanism for these effects. Two of

the diabetogenic agents, streptozotocin and 6-aminonico-

tinamide, produce an insulin-deficient animal while the

third agent, N-methylacetamide, does not produce an insulin-

deficient animal but instead causes a condition whereby

insulin is produced and released but is incapable of

lowering blood glucose.

To investigate .the effect that the diabetes induced by

these agents had on hepatic drug metabolism, experiments on

male Holtzman rats were performed both in vivo and in vitro.

In the in vivo experiments, the duration of hypnosis was

measured after the administration of hexobarbital sodium

to animals that had been made diabetic. The results showed

that there was an inhibition, as compared to control ani-

mals, of the in vivo metabolism of hexobarbital only in

animals pretreated with streptozotocin and 6-aminonico-








tinamide and not in those pretreated with N-methylacetamide.

The in vitro experiments were of several types. After the

animals had been treated with these three agents, the

kinetic constants for hexobarbital and aniline hydroxyl-

ations were determined with 9000g supernatant fraction as

the enzyme source. Again, only preparations from animals

pretreated with streptozotocin and 6-aminonicotinamide

showed an inhibition as compared to control animals. Simi-

lar results were also obtained when isolated microsomes

were used as the enzyme source. Also, the in vitro meta-

bolism of hexobarbital and aniline was still inhibited as

compared to preparations from control animals one week after

the animals had been treated with streptozotocin. To

examine the effect of hyperglycemia on hepatic drug meta-

bolism, animals were infused intravenously with glucose,

after which the in vitro hydroxylation of hexobarbital was

measured and found not to be inhibited as compared to

saline-infused control animals.

Neither insulin nor any of the diabetogenic agents had

any direct effect on drug metabolism in vitro. Furthermore,

hepatic microsomal protein and cytochrome P-450 contents

were not significantly different in any of the diabetic

animals than those of the control animals.

The binding of both hexobarbital and aniline to cyto-

chrome P-450 was inhibited in animals pretreated with

streptozotocin but only the binding of hexobarbital was

inhibited in 6-aminonicotinamide-pretreated animals. The









fact that the affinity of aniline to cytochrome P-450

was increased in microsomes from animals pretreated with

N-methylacetamide somewhat obscures the interpretation of

these results.

It had been shown previously that treatments that

increased the endogenous levels of c-AMP in the liver

were inhibitory to certain drug-metabolic pathways and

that this was due to the release of an inhibitor of drug

metabolism into the liver cytosol. The presence of an

inhibitor of hexobarbital and aniline hydroxylations

was shown to occur in animals pretreated with streptozoto-

cin and an inhibitor only of hexobarbital hydroxylation in

animals pretreated with 6-aminonicotinamide. This cor-

related with elevated levels of c-AMP observed in livers

of animals pretreated with streptozotocin and to a lesser

degree in animals pretreated with 6-aminonicotinamide. C-

AMP levels were not elevated in animals pretreated with

N-methylacetamide as compared to control animals.

In conclusion, it appears that the inhibition of drug

metabolism in animals made diabetic with certain drugs is

not caused by hyperglycemia, but is rather the consequence

of insulin lack. It is proposed that this lack of insulin

causes an elevation of hepatic c-AMP which, in turn, causes

the release by liver microsomes of an inhibitor of the meta-

bolism of xenobiotics.















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84



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BIOGRAPHICAL SKETCH


Dennis M. Ackerman was born in Baltimore, Maryland,

March 30, 1946. He attended Baltimore City College High

School and was graduated from there in June, 1963. In

February, 1969, he received the Bachelor of Science Degree

in Business Administration from the University of Maryland;

and in June, 1970, he received the Bachelor of Science De-

gree in Pharmacy from the same university.

Mr. Ackerman began his graduate education at the

University of Florida in September, 1970. He was sup-

ported in his studies by a training grant from the Na-

tional Institutes of Health.








I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality
as a dissertation for the degree of Doctor of Philosophy.




Kenneth C. Leibman, Chairman
Professor of Pharmacology



I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality
as a dissertation for the degree of Doctor of Philosophy.




Owen M. Rennert
Professor of Pediatrics



I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality
as a dissertation for the degree of Doctor of Philosophy.




David M. Travis
Professor of Pharmacology



I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality
as a dissertation for the degree of Doctor of Philosophy.




Betty P. Vogh
Associate Professor of Pharmacology









This dissertation was submitted to the Graduate Faculty
of the College of Medicine and to the Graduate Council,
and was accepted as partial fulfillment of the require-
ments for the degree of Doctor of Philosophy.

December, 1975


Dean, Graduate School



































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