THE INFLUENCE OF H-2 RECEPTOR ANTAGONISTS, CIMETIDINE AND RANITIDINE, ON THE PHARMACOKINETICS OF FENTANYL IN DOGS

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Nenad, Robert Eugene, Jr.

THE INFLUENCE OF H-2 RECEPTOR ANTAGONISTS, CIMETIDINE AND RANITIDINE, ON THE PHARMACOKINETICS OF FENTANYL IN DOGS

The University of Arizona M.S. 1986

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University Microfilms International

THE INFLUENCE OF H-2 RECEPTOR ANTAGONISTS,

CIMETIDINE AND RANITIDINE, ON THE

PHARMACOKINETICS OF FENTANYL IN DOGS

by

Robert Eugene Nenad, Jr.

A Thesis Submitted to the Faculty of the

DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY

In Partial Fulfillment of the Requirements For the Degree of

MASTER OF SCIENCE WITH A MAJOR IN TOXICOLOGY

In the Graduate College

THE UNIVERSITY OF ARIZONA

19 86 STATEMENT BY AUTHOR

This thesis has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.

Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgement of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department of the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be ob­ tained from the author.

SIGNED

APPROVAL BY THESIS DIRECTOR

This thesis has been approved on the date shown below:

afcr-. /2- , /•?££, Sifpes^/ Date and Head, Pharmacology and Toxicology Anesthesiology, Pharmacology ACKNOWLEDGEMENTS

This project could not have been completed without the assistance and support of numerous individuals. The author would like to acknowledge Drs. I. G. Sipes, A. J. Gandolfl and David Earnest for guidance, moral support and encouragement. In addition, special thanks to Dr. James Borel, Dr. John Bentley and Terry Gillespie for their supervision, assistance and expertise.

The author is grateful to the University of Arizona College of

Medicine for providing the USPHS research grant and extremely apprecia­ tive of Dr. Alan du Bois for his unselfish generosity during the pursuit of my education.

Finally, to my father, Robert Nenad, Sr., my brothers Mark and

David, fellow graduate students and friends for their support and friendship throughout this project.

iii TABLE OF CONTENTS

Page

LIST OF ILLUSTRATIONS vi

LIST OF TABLES vii

ABSTRACT viii

CHAPTER

1. INTRODUCTION 1

Fentanyl 1 Pharmacokinetics 3 Metabolism 4 Toxicity—Adverse Effects 6 Ciraetidine 9 General Information 9 Pharmacokinetics 12 Metabolism 12 Toxicity—Adverse Effects 13 Drug-Drug Interactions . 13 Ranitidine 15 Pharmacokinetics and Metabolism 15 Toxicity—Adverse Effects 16 Drug-Drug Interactions 16 Drug Absorption, Distribution, Metabolism and Excretion 17 Statement of Problem 20

2. METHODS ..... 22 Supplies 22 General 22 Dog Anesthesia 23 Sample Preparation 26 Chromatography 28 Pharmacokinetic Analysis 29

3. RESULTS 32 Gas Chromatography 32 Standard Curve 32

iv V

TABLE OF CONTENTS—Continued

Page

Individual Dog Results 38 Dog 322 38 Dog 510 48 Dog 603 48 Dog 655 49 Summary 49

4. DISCUSSION ..... 51 Model 51 Choice of Animal 51 Choice of Drugs 52 Extraction and Chromatography ... 54 Pharmacokinetics 56 Discussion of Pharmacokinetic Results 58 Renal Excretion 58 Enterohepatic Recirculation 59 Interference with Metabolism 59 Hepatic Blood Flow ^2 Clearance and Volume of Distribution ^5

5. CONCLUSION 66

APPENDIX I. SERUM CONCENTRATIONS OF IV FENTANYL (100 pg/kg) WITH NO PRETREATMENT BY H-2 ANTAGONISTS 67

APPENDIX II. SERUM CONCENTRATIONS OF IV FENTANYL (100 jig/kg) AFTER PRETREATMENT WITH CIMETIDINE 68

APPENDIX III. SERUM CONCENTRATIONS OF IV FENTANYL (100 yg/kg) AFTER PRETREATMENT WITH RANITIDINE 69

LIST OF REFERENCES 70 LIST OF ILLUSTRATIONS

Figure Page

1. Chemical Structures of Fentanyl, Cimetidine and Ranitidine 2

2. Theoretical Pathways of Fentanyl Biotransformation .... 5

3. GC Chromatogram of Fentanyl (Peak 1) 90 Minute Sample 1:32 Attenuation and Internal Standard Alfentanil (Peak 2) 33

4. Standard Curve Fentanyl/Internal Standard vs. Concentration ..... 35

5. Standard Curve Fentanyl/Internal Standard vs. Concentration 36

6. Diagrammatic Illustration of Pharmacokinetic Parameters Applied to Fentanyl 37

7. Serum Fentanyl Levels after Intravenous Administration of Fentanyl (100 jjg/kg) in Dogs 322, 510, 603, 655 ... 43

8. Plasma Elimination of IV Fentanyl (100 yg/kg) from Dog 322 with No Pretreatment, with IM Cimetidine Pretreatment, and with IM Ranitidine Pretreatment ... 44

9. Plasma Elimination of IV Fentanyl (100 yg/kg) from Dog 510 with No Pretreatment with IM Cimetidine Pretreatment and with IM Ranitidine Pretreatment .... 45

10. Plasma Elimination of IV Fentanyl (100 yg/kg) from Dog 603 with No Pretreatment, with IM Cimetidine Pretreatment, and with IM Ranitidine Pretreatment ... 46

11. Plasma Elimination of IV Fentanyl (100 yg/kg) from Dog 655 with No Pretreatment, with IM Cimetidine Pretreatment, and with IM Ranitidine Pretreatment ... 47

vi LIST OF TABLES

Table Page

1. Pharmacokinetic Parameters of IV Fentanyl (100 yg/kg) following Pretreatment with IM Normal Saline (Control) 39

2. Pharmacokinetic Parameters of IV Fentanyl (100 yg/kg) following Pretreatment with IM Cimetidine 38

3. Pharmacokinetic Parameters of IV Fentanyl (100 yg/kg) following Pretreatment with IM Ranitidine 41

4. Summary of Pharmacokinetic Parameters of IV Fentanyl (100 yg/kg) 42

vii ABSTRACT

The influence of pretreatment with H-2 antagonists on the pharmacokinetics of intravenous fentanyl was studied in dogs. Three groups of dogs were studied. The first received, only fentanyl with no pretreatment. The second group received pretreatment with IM cimetidlne and the third group was pretreated with IM ranitidine.

Serial blood samples were drawn at selected time points and following extraction fentanyl levels were determined on a gas chro- matograph. Pharmacokinetics were calculated by the method of Benet and Galezzi.

Cimetidine pretreatment statistically prolonged the terminal elimination half life of fentanyl. Cimetidine also caused a trend toward decreased clearance of fentanyl but statistical significance was not achieved. Fentanyl's volume of distribution was not altered by cimetidine. Pharmacokinetic parameters of fentanyl were not sig­ nificantly altered by ranitidine.

It was concluded that cimetidine's effect on fentanyl pharma­ cokinetics was most likely due to inhibition of cytochrome P-450 enzymes.

viii CHAPTER 1

INTRODUCTION

Fentanyl

Fehtanyl [N-(l-phenethyl-4-piperidyl) propionalide] citrate,

Subliraaze, has the molecular formula C22H28H2°2C6H8°7 and a molecu^ar weight of 528.6 (see Figure 1). It is a synthetic narcotic analgesic derived from phenylpiperidine and functions as an opiate receptor agonist. Fentanyl's analgesic potency is approximately 100 times that of morphine.

Fentanyl is a very lipid-soluble drug. It has been shown to have a heptane/water coefficient 6 orders of magnitude greater than morphine at pH 7.4 (Kitahata and Collins 1982). Fentanyl has a pKa of

7.7, and at pH 7.4, 67% of it.is unionized. This unionized form is responsible for the drug's relatively high binding to plasma proteins

(60-70%), and many of its pharmacokinetic properties are the result of its high lipid solubility. This was shown by Hug and Murphy (1979), who demonstrated a direct relationship between the CNS concentration of fentanyl related to lipid solubility and its associated respiratory depression.

Fentanyl's primary use is as an intravenous anesthetic agent.

It is often used with ^0 for short-term general anesthesia or com­ bined with droperidol, a neuroleptic agent.

1 2

Fentanyl Citrate

CH2CH2—N y-NCOCR2CH3 6 H3C6H5O7

Cimetidine

CH3N—•PH2SCH2CH2NHCNHCH3

N^N N-C=N

Ranitidine

(CH3)2NCH2^^^CH2SCH2CH2NHyNHCH3*HCI

chno2

Figure 1. Chemical Structures of Fentanyl, Cimetidine and Ranitidine. 3

Pharmacokinetics

Fentanyl's rapid onset and short duration of action are due to its rapid CNS penetration followed by redistribution to tissues. Hug and Murphy (1981) studied radio-labeled 3H-fentanyl distribution in rats. They found that fentanyl concentrations of highly perfused organs, such as brain, heart and lung, equilibrated with plasma within

1.5 minutes and rapidly declined. Redistribution was demonstrated by the delayed uptake of fentanyl by fat and muscle, with equilibrium being reached in 120 minutes. At 5 minutes, muscle contained 56% of an 3 IV dose of H-fentanyl and fat accounted for only 6% of the dose. By

30 minutes, the fat content had increased to account for 17% of the dose. The actual concentration of fentanyl was 2-4 times greater in muscle and as much as 35 times greater in fat than in plasma. Thus, fentanyl is initially distributed according to blood flow but later accumulates in fat soluble tissues.

The terminal elimination phase starts following redistribution of fentanyl from storage sites. The affinity of fentanyl to these sites may be the rate limiting factor in its elimination. Computer fitting of plasma concentration versus time curves generates a three- compartment model (Murphy, Olson, Hug 1979). Fentanyl equilibration occurs so rapidly between vessel-rich tissues (i.e., brain, heart, lung) that they cannot be distinguished kinetically, and thus they all constitute the central compartment (Hug and Murphy 1981).

Initially, fentanyl is rapidly removed from the blood. In the dog anesthetized with enflurane, 98.6% of an IV dose of fentanyl was 4

eliminated from the plasma within 5 minutes (Murphy, Olson and Hug

1979). In man, McClain and Hug (1980) showed 98.6% of an IV dose was

eliminated in 60 minutes. In 72 hours, 85% of the dose was recovered in urine and feces, with only 8% appearing as unchanged fentanyl.

Therefore, fentanyl is quickly redistributed out of plasma and ex­ tensively metabolized before elimination.

Metabolism

Because of the low concentrations of fentanyl required to pro­ duce anesthesia, direct measurement and identification of fentanyl metabolites has been difficult. Determination of fentanyl metabolites 3 has been estimated by subtracting recovered H-fentanyl from the ini- 3 tial H-fentanyl administered. Hug and Murphy (1981) found that metabolites represented 25% of the dose at 15 minutes and 80% at 4 hours.

Efforts have been made to identify the metabolites. Hug and

Murphy (1981) have identified despropionyl fentanyl in extracts of rat 3 liver and kidney in vivo following IV H-fentanyl administration.

Lehmann, Moeseler and Daub (1981) incubated mouse liver homogenates 3 with H-fentanyl and identified phenylacetic acid and norfentanyl as well as four other metabolites (see Figure 2).

Excretion of parent fentanyl by the kidney is doubtful. Be­ cause fentanyl is so lipophilic, it must be metabolized to more polar products prior to excretion by the kidney. The bulk of parent drug presenting to the proximal renal tubule would likely be passively parahydroxylalion / \ II amide CH2-CH2-N VN-C-CHj-CHJ. | (ling oaidalion) hydrolysis ^ / \ « Fen'a"V' |\ H0ALrcHj-CH2-N Y—c—cHz— 3 | CH2-CH2-o NH • hydroiylallon • conjugation

Despropionyl fentanyl

N-dealKylation OH conjugation I N-dealkytation N-dealkylatlon /—Vf VN-C "-CH2 -CH3

amide 'N ^-NH- -+ hytiroxytation —» conjugation -oCH2-CH2- a N-C-CHjCHi 1hydrolysis Nor-fenlanyl

X s glucuronlda (ether or esler) or ethereal sulfite OX Despropionyl nor-tentanyl

Figure 2. Theoretical Pathways of Fentanyl Biotransformation (Kitachata 1982).

Ln 6 reabsorbed. This corresponds to the observed 4-8% parent drug recovered in the urine.

Several factors have been shown to alter the parmacokinetics of fentanyl. Bentley, Borel and Nenad (1982a, 1982b) showed that the elderly have a prolonged terminal elimination phase and an increased terminal elimination half-life (t^^B). Despite its lipophilicity, there appears to be no alteration in fentanyl serum concentration be­ tween obese and non-obese (Bentley et al. 1981). The terminal elimina­ tion phase of fentanyl from plasma is prolonged in patients with liver failure (Kitahata and Collins 1982).

Although the liver is the main site of biotransformation of fentanyl, it may be metabolized by other tissues. Hug et al. (1981) studied acutely hepatectomized dogs versus sham-operated animals. The excretion of fentanyl was reduced but not eliminated by hepatectomy.

They concluded that fentanyl was therefore metabolized by extrahepatic tissues, although the sites of metabolism were not determined. Lehmann et al. (1981) showed mouse kidney and adrenal homogenates were able to metabolize fentanyl, with oxidative dealkylation of only minor impor­ tance.

Toxicity—Adverse Effects

The acute toxicity associated with fentanyl is similar to that of other narcotics, i.e., respiratory depression, miosis and, ulti­ mately, coma. Fentanyl produces analgesia, sedation, respiratory de­ pression and finally apnea as the dosage is increased. Side effects 7 commonly observed with toxicity from other narcotics may not be seen with fentanyl because its higher potency allows for the use of smaller doses. Fentanyl's toxic and therapeutic effects can both be reversed by opiate antagonists such as naloxone.

Fentanyl has minimal effects on the cardiovascular system.

Mild bradycardia occasionally occurs and is rarely significant. It is usually relieved by atropine (Collins 1976). In addition to respira­ tory depression, fentanyl has been associated with increased tone of respiratory muscles (stiff chest), laryngeal and bronchiolar spasm.

This usually occurs after rapid IV administration and is thought to be caused by stimulation of spinal reflexes. Neuromuscular blocking drugs may diminish but do not eliminate the tonic muscular state, and nar­ cotic antagonists will counteract the effect (Collins 1976, Dundee and

Wyant 1974). Unlike morphine (Philbin et al. 1981), fentanyl is rare­ ly associated with histamine release (Oddis 1981). The chronic use of fentanyl like other narcotics may result in tolerance and addiction

(Gilman, Goodman and Gilman 1980).

There have been instances of delayed respiratory depression following apparent recovery from fentanyl anesthesia.. This is a rela­ tively rare event, but it is potentially dangerous. Several explana­ tions have been proposed. One explanation follows the observation that pain and other types of stimulation antagonize fentanyl's effects.

Therefore, when a patient is moved from a recovery room to a quiet hospital room, he may lose this stimulation, fall asleep and experience respiratory depression. 8

Several other mechanisms have been proposed to account for delayed respiratory depression seen with fentanyl. Stoeckel,

Hengstmann and Schuetter (1979) proposed enterohepatic recirculation of fentanyl, such that fentanyl excreted into gastric juice is then reabsorbed from the alkaline medium of the small intestine. However, even with continuous nasogastric suctioning, secondary plasma peaks are noted (Bentley et al. 1982b). Another proposed mechanism was the sequestration of fentanyl in muscle with subsequent release upon re­ sumption of activity (Bentley et al. 1982b). This theory was discarded when the development of plasma peaks were noted during anesthesia while the patients were still paralized. Bentley et al. (1982b) then pro­ posed that fentanyl may be trapped in the lungs because of altered ventilation-perfusion relationships during surgery and anesthesia. As these relationships return toward normal, fentanyl is washed out of the lungs.

It has been suggested that the secondary peaks actually repre­ sent fentanyl metabolites which are too similar to fentanyl to escape detection by current methods (Gandolfi personal communication 1984).

As fentanyl is metabolized, metabolites begin to accumulate in plasma.

The appearance of metabolites would then coincide with the appearance on increased respiratory depression. There appears to be no univer­ sally accepted mechanism for the appearance of delayed respiratory depression, and the presence of secondary fentanyl peaks may represent multiple causes. 9

Since fentanyl mediates its effects via an opiate receptor,

any additional opiate analgesics will act in an additive manner with

pre-existing fentanyl. The analgesic effects of fentanyl terminate

prior to the elimination of fentanyl from the body, it is therefore

likely that additional opiate analgesics would be potentiated by

residual fentanyl. The amount of fentanyl remaining in the body may

not be realized prior to this interaction and could be potentially

hazardous.

Cimetidine

Cimetldine (N^-cyano-N-methyl N"^ (2-[5-metylimidazole-4-yl)]

formula (C^ff^H^S) and molecular weight of 252.4. At 25® C in 0.1M

HC1, it has a pKa of 7.08, with protonation occurring in the imidazole

ring (Brimblecombe et al. 1978)". It is available parenterally or as a

300 mg oral tablet (see Figure 1).

General Information

Cimetidine, Tagamet®, is a histamine-2 (H-2) receptor antago­

nist. Development of cimetidine followed the observation that hista­

mine-! (H-l) receptor antagonists failed to block histamine stimulated

gastric acid secretion and vasodilation (Brimblecombe et al. 1978).

Early antihistamines discovered in the 1940's, such as mepy- ramine, were classified as H-l receptor antagonists. A second hista­ mine receptor, initially classified non-H-1 (and now H-2), was

identified in the mid 1960's (Brimblecombe et al. 1978). By modifying

histamine chemically, over 200 compounds were tested for receptor 10 antagonistic actions. The first true H-2 antagonist was burimamide.

This was followed by metiamide. Metiamide proved to be of therapeutic value in duodenal ulcer disease but saw limited use because of its association with reversible granulocytopenia (Brimblecombe et al. 1978).

Cimetidine was the next compound in the stepwise modificiation of histamine. The imidazole ring of histamine was found to be important in H-2 receptor binding and was retained in the structure of cimetidine.

Cimetidine was approved for clinical use in the U.S. In 1977, with indications for the treatment of acute peptic ulcer disease. Clini­ cally, it suppresses the release of gastric acid, thereby increasing gastric pH and allowing the ulcerated tissue to heal.

There are three compounds which stimulate acid secretion by the stomach: 1) gastrin, 2) acetycholine, and 3) histamine. Gastrins are peptides secreted predominantly by G-cells of antral muscosa. Their release is stimulated by the effects of products of protein digestion on antral mucosal G-cells, presumably by direct action on microvilli.

The effects of gastrin can be partially inhibited by atropine, suggest­ ing a cholinergic inhibitory system. Stimulation of the vagus nerve also promotes gastrin release. The transmitter for vagal release of gastrin has not been identified (Wyngaarden and Smith 1982).

Acetycholine is released by post-gangllonic cells of the vagal nerve which anastamose with short reflex arcs within the walls of the stomach. Both cephalic influences and gastric distension enhance acetylcholine release. The vagus nerve facilitates gastric acid secre­ tion by: 1) direct cholinergic stimulation of parietal cells, 11

2) release of gastrin, and 3) sensitization of the parietal cell to

stimulation by gastrin (Wyngaarden and Smith 1982).

Histamine is released near parietal cells by cells in the lamina

propria. It enhances the activity of gastrin and acetylcholine on

parietal cells (Wyngaarden and Smith 1982). Cimetidine and other H-2 antagonists function by blocking histamine's actions on the parietal cell and therefore inhibiting the activity of gastrin and acetylcholine as well (Guyton 1981).

Although cimetidine was initially intended only for acute treatment of duodenal ulcers, it was soon discovered that removal of

the drug after the recommended 8 weeks of therapy was often (50-60%) followed by recurrence of peptic ulcer disease. Thus, some patients are maintained on cimetidine for extended periods.

It has been proposed that cimetidine be used during anesthesia to reduce the secretion of gastric acid and thereby reduce the risk of pneumonitis, also called Mendelson's Syndrome (Husemeyer, Davenport and

Rajasekaren 1978, and Weber and Hirshman 1979). This is a potentially lethal complication of anesthesia in which acidic gastric contents are inhaled into the lungs. A pH value of less than 2.5 of an aspirate is generally considered the critical level for inducing pulmonary damage

(Roberts and Shirley 1974). Toung and Cameron (1980) showed in 28 patients given three preoperative doses of cimetidine that 93% had gastric pH greater than 2.5. Similar results were also noted by Weber and Hirshman (1979) and Stoelting (1978). Since cimetidine lowers the 12 volume of gastric secretion volume and increases its pH, it Is often used preoperatlvely to reduce the risk of aspiration pneumonitis.

Pharmacokinetic's

The bio-availability of cimetidine in man was studied by Walk- enstein et al. (1978). A 300 mg dose given either intravenously or intramuscularly resulted in similar blood levels. A first pass effect following oral administration has been noted, and approximately 60% of the oral dose reaches the circulation. A blood concentration of 0.5 ng/ml suppresses basal gastric secretion by 80%, and gastrin or food stimulated secretion by 50% in most people (Pounder et al. 1976).

Walkenstein et al. (1978) found a blood concentration of 0.5 ng/ml is usually reached within 30-60 minutes following IV, IM or oral doses of 300 mg cimetidine. Levels remain above 0.5 ng/ml for approxi­ mately 4 hours. The mean half-life of cimetidine is 1.7-2.1 hours

(Richards 1983), and approximately 20% of • cimetidine present in the blood will be bound to protein.

Metabolism

Following oral administration to dogs, cimetidine is rapidly absorbed and excreted in the urine. A small first pass effect occurs in man, but not in the rat.or in the dog (Taylor, Cresswell and

Bartlett 1978). No difference in metabolites of cimetidine was noted between oral and parenteral administration in rats or dogs (Taylor et al. 1978). In both man and dog, the principal metabolite is formed by oxidation of the side chain sulfur to a sulfoxide. In dogs, 84% 13 3 of an IM dose of H-cimetidine is excreted in the urine, and of this,

75% is unaltered parent drug (Taylor et al. 1978). Despite similari­ ties of the Imidazole ring to histamine, cimeditlne does not undergo

N-methylation of the ring structure (Taylor et al. 1978). The mechanism of sulfoxidation and enzymes responsible for it, while presumably cyto­ chrome P-450 mediated, have not been identified.

Toxicity—Adverse Effects

Prior to its clinical release, toxicity tests of cimetidine toxicity were performed for up to 24 months in the rat and 12 months in the dog. At daily doses of 30-100 times those recommended for humans, dogs were observed to have underdeveloped prostates, occasional liver and kidney damage and episodes of transient tachycardia (Brimble- combe et al. 1978).

Following release for clinical, use, side effects in man includ­ ing mental confusion (particularly in the elderly), occasional eleva­ tions of serum transaminase (SGOT), alkaline phosphatase activities, and elevations of creatinine have been observed without other evidence of hepatic or renal dysfunction (Bennett 1983). Gynecomastia and im­ potence have developed, but are generally associated with very high doses (Gilman et al. 1980).

Drug-Drug Interactions

The most common adverse effect of cimetidine was the observa­ tion that it interfered with the metabolism of several drugs. This inhibition resulted in reduced clearance of warfarin (Serlin et al. 14

1979), Increased elimination half-life of theophylline (Jackson et al.

1981), chlordiazepoxide (Desmond et al. 1980), and antipyrine (Serlin

et al. 1979), among others. Inhibition of metabolism occurs at the

level of the mixed-function oxidase system. It does not affect the

glucuronldation of oxazepam and lorazepam (Klotz and Relmann 1980a,

Patwardhan et al. 1980). It has been shown that cimetidine binds to

cytochrome P-450 in vitro. This binding results in a type-II spectral

shift that is attributed to the Interaction of the nitrogen groups of

its imidazole ring with the hememoiety of P-450 (Pelkonen and Puurunen

1980). The P-450 binding is noncompetitive.

An alternative explanation for the influence of cimetidine on

the clearance of other drugs could derive from the reported effect of cimetidine decreasing blood flow (Feely, Wilkinson and Wood 1981).

Feely reported a positive correlation between cimetidine steady-state

blood concentration and a reduction in propranolol oral clearance fol­ lowing oral administration. An unexplained phenomenon was a lack of change in propranolol half-life despite reduced systemic clearance and unaltered volume of distribution. Jackson (1981) suggested cimetidine interfered with the transport, storage or metabolism of indolyamine dye used to measure the hepatic blood flow.

Since cimetidine is secreted by the renal organic cation trans­ port mechanism (Dubb et al. 1978), it might inhibit the secretion of actively transported compounds. This potential interaction with drugs

has not been investigated. 15

The principal mechanism by which cimetidine influences the pharmacokinetics of other drugs appears to be through Interactions with the mixed-function oxidase systems. Further investigation is necessary to determine if other determinants of clearance also contribute.

Ranitidine

Ranitidine hydrochloridet H[2[[[5— £(dimethylamino)methyl]-2- furanyl]methyl]thio]ethylJ-N'-methyl^-nitro-ljl-ethenediamine, hydro­ chloride, is a newly introduced H-2 receptor antagonist (Gaginella and

Bauman 1983) (see Figure 1). Unlike cimetidine, which is a substituted imidazole, ranitidine is a substituted alkylfuran. Like cimetidine, it inhibits gastric acid secretion stimulated not only by histamine but by gastrin and vagal releast of acetylcholine. Ranitidine is five to ten times more potent than cimetidine (on a weight or molar basis) in inhibiting stimulated acid secretion in patients with duodenal ulcer

(Walt et al 1981). It is therefore given in lower dosage.

Pharmacokinetics and Metabolism

Ranitidine is rapidly absorbed following oral administration with peak plasm concentrations within 1-3 hours. Oral bio-availability compared to IV administration ranged from 41-57% (Woodings et al 1980).

Like cimetidine, ranitidine undergoes significant "first pass" metabo­ lism with approximately 30-50% of an oral dose recovered as unchanged drug in the urine (compared to 60% of cimetidine) (Berner et al 1982).

The elimination half-life after a single oral dose is 2-3 hours, and its volume of distribution is 75L (1.2-1.9L/kg)(Brogden et al. 1982). 16

Renal clearance was observed to be higher than creatinine clearance, suggesting active tubular transport of the unchanged drug (Berner et al. 1982). There are at least three metabolites of ranitidine: desmethylranltldine, the N-oxide, and the S-oxide (Louis et al. 1981, and Carey, Mantin and Owen 1982).

Toxicity—Adverse Effects

The incidence of adverse effects caused by ranitidine has been low. It has not been associated with gynecomastia, impotence or nephrotoxicity. It has been implicated in some cases of mental confu­ sion (Hegarty 1984) and hepatotoxicity (Black, Scott and Kanter 1984, and Souza 1984).

Drug-Drug Interactions

Ranitidine does not appear to interact significantly with the cytochrome P-450 system. Knodell et al. (1982) noted high binding affinity of cimetidine for cytochrome P-450, but found no evidence of ranitidine binding. He utilized rat and human liver microsomes.

Cimetidine inhibited meperidine and pentobarbital metabolism, whereas ranitidine did not. Conjugation of morphine was unaffected by either drug. Rendic, Kolbak and Kajfez (1982) found that ranitidine would bind to P-450 from phenobarbital-treated rats. At lower concentrations the oxygen ligand formed, and at higher concentrations the nitrogenous or thioether ligand of ranitidine interacted. It is a different type of binding than seen with cimetidine and has an affinity 10 times lower. Ranitidine does not alter metabolism of warfarin (Serlin, 17 Kolbach and Breckenridge 1981), pentobarbital (Serlin et al. 1981),

diazepam (Klotz, Reimann and Ohnhaus, 1983), lldocaine (Jackson et al.

1983) or theophylline(Breen et al. 1982). This suggests it has little clinical effect on hepatic metabolism by cytochrome P-450.

Ranitidine has been reported to impair the oxidative pathway

involved in acetaminophen metabolism in vitro (Mitchell et al. 1981).

Lee et al. (1982) found ranitidine inhibited fentanyl metabolism in a rat microsomal system.

Drug Absorption, Distribution, Metabolism and Excretion

The metabolism of most substances intended as pharmacological agents follow a similar pattern upon administration: i.e., absorption, distribution, metabolism, and excretion. These factors make up the

pharmacokinetic parameters of a particular drug. Many factors influ­

ence a drug's pharmacokinetics. Host factors include pathologic, physi­ ologic, genetic, anatomic, metabolis, age, and environmental differ­ ences. Drug factors include mode of administration, vehicle, composi­

tion, ionic characteristics, and many others. A major concern in this age of increasing polypharmacy is the potential for drug-drug interactions.

Because this thesis involves the interaction of one drug with another, a brief discussion of possible drug-drug interactions will follow.

The effect of one drug on another is often very difficult to predict and usually involves observations from clinical experiences.

This difficulty can be attributed partly to the wide spectrum of po­

tential drug interactions involving absorption, distribution, metabo­ lism, and excretion. 18

There are several mechanisms by which one drug can alter the

bio-availability of another orally administered drug. Bio-availability

is the rate or extent of absorption. Fhysiochemical interactions may

result in complexing, chelation or absorption. The gastrointestinal

state may be altered, resulting in changes in gut motility, blood flow

or pH. Parenteral absorption is influenced by blood flow, site of in­

jection, ionic character, and vehicle composition.

Once administered, distribution is highly influenced by

regional blood flow. Highly perfused organs such as the liver, kidney

and brain receive initial exposure. Changes in blood flow may be

locally mediated, due to vasodilation or vasoconstriction, in a particu­

lar region. Blood flow changes may also be indirectly mediated through

changes in cardiac output, causing systemic alterations. Other factors

influencing distribution are the drug's partition coefficient, degree

of protein binding, and availability of transport mechanisms (i.e.,

saturation of active transport mechanisms). As the portal circulation

delivers an orally administered drug to the liver, metabolism by the

liver enzyme may occur, reducing the amount of drug that is subsequent­

ly available to the general circulation. This is termed the "first

pass" effect, and this will result in decreased bio-availability

(Gibaldi and Perrier 1975).

The action of drugs which have their effects mediated through

specific receptors may be influenced by other drugs specific for the

same receptor. This is the basis for the reversal of opiates by naloxone (Gilman et al. 1980). In addition, receptor number may be 19 altered in response to chronic application of certain drug types, i.e. steroids (Gilman et al. 1980). There may also be indirect effects on receptors such as depletion of cholinesterase by neostigmine.

Many drugs undergo metabolism to more polar compounds prior to excretion. This does not necessarily imply inactivation, and in fact may produce the pharmacologically and/or toxicologically active agent (Levine 1978). Drugs may alter metabolism of other drugs by com­ peting for specific enzymes, by inhibiting metabolizing enzymes, or by inducing formation of metabolizing enzymes. The majority of drug metabolizing enzymes are found in the liver, however other sites such as lung, kidney, intestinal mucosa, and gut bacteria may contribute.

The cytochrome P-450 dependent, mixed function oxygenase (MFO) enzymes are the principal hepatic enzymes involved in drug metabolism. Induc­ tion of these enzymes by phenobarbital will Increase the capacity of the liver to metabolize many MFO substrates. On the other hand, a substance which interferes with the access of compounds to the MFO's will decrease the liver's ability to metabolize drugs (Powell and Donn

1983).

Two types of inhibition occur. The first is when the inhibitor acts as a substrate for the enzyme. In this instance, the inhibition is competitive, and the concentration of the inhibitor receptor at the site must be greater than the substrate (Powell and Donn 1983).

The second form involves an inhibitor irreversible binding to and in­ activating a specific component of the MFO. This type of inhibition is 20 non-competitive. Other forms of inhibition can be competition for enzyme cofactors, destruction or decreased biosynthesis of an enzyme.

Some inhibitors slow cytochrome P-450 activity in either a competitive or non-competitive manner, depending on the substrate and inhibitor concentration (Powell and Donn 1983). Changes in cytochrome

P-450 characteristics can have marked influences on the bio-availability of orally administered drugs undergoing first pass effects.

Another area of potential drug-drug interaction occurs in the kidney, which is the final elimination site of most drugs. Agents may affect renal blood flow either directly or systemically, resulting in changes in the glomerular filtration rate. If the drugs share a tubu­ lar transport mechanism involving secretion and/or reabsorption, they can have pronounced effects on another drug's pharmacokinetics. An example of this is the use of probenecid to decrease the renal excretion of penicillin (Gilman et al. 1980). Drugs undergoing passive diffusion from the proximal renal tubule may be greatly influenced by changes in urinary pH.

Drug-drug interactions most likely involved in this study would include alterations in liver enzyme metabolism, influences on liver blood flow, and changes in enterohepatlc recirculation.

Statement of Problem

The histamine-2 receptor antagonist, cimetidine, decreases the volume and increases the pH of gastric secretions. It has become quite popular in the chronic treatment of patients with peptic ulcer disease 21

and is now seeing increased use as a pre-operative prophylaxis for

pulmonary acid aspiration syndrome. For these reasons, the administra­

tion of anesthesia to patients on either acute or chronic cimetidine

therapy is steadily increasing. Yet, cimetidine has been shown to bind

in vitro to cytochrome P-450 and is reported to reduce liver blood flow

in vivo. Cimetidine has been shown to alter the metabolism of a wide

variety of compounds, including antipyrine, warfarin, lidocaine, pro­

pranolol, and diazepam. Therefore it may alter the metabolism of anesthetic agents as well.

Ranitidine is another selective H-2 receptor blocker which was recently released for clinical use. It is five to ten times more po­ tent than cimetidine and binds very weakly to microsomal cytochrome

P-450. Preliminary studies have shown that ranitidine does not inhibit the metabolism of aminopyrine, antipyrine, and hexabarbital. Suggest­ ing minimal interference with cytochrome mediated metabolism. This drug is seeing widespread clinical use in this country.

The effect of cimetidine and/or ranitidine on the metabolism and elimination of intravenous anesthetic adjuvants has not been re­ ported. Therefore, we Investigated the influence of both these drugs on fentanyl pharmacokinetics using dogs as models. CHAPTER 2

METHODS

Supplies

The following materials and supplies were utilized in this study. Ethanol 95% was obtained from the University of Arizona, and

USP absolute pure ethyl alcohol, reagent quality, was obtained from

U.S. Industrial Chemical Company, New York, NY. Sodium hydroxide beads, S686 Reagent A.C.S. pellets* were acquired from Ashland Chemi­ cal Co., Columbus, OH. Hexane (UV), glass distilled, was obtained from

Burdick Jackson Labs, Muskegon, MI. Janssen Pharmaceutica, of Beerse,

Belgium provided both fentanyl citrate and alfentanll hydrochloride.

Dimethyldichlorosilane, Pierce #83410, was obtained from the Pierce

Chemical Company, Rockford, IL. Certified A.C.S. tolulene was acquired from Fisher Scientific, Fairlawn, NJ. Smith, Kline and French Lab Co. of Carolina, PR supplied cimetidine hydrochloride. Glaxo, Inc. of Ft.

Lauderdale, FL kindly provided ranitidine hydrochloride. The gas chromatograph utilized was a N-P-510 gas chromatograph, model 5710A, manufactured by Hewlett Packard Co. of Corvalis, OR.

General

Four healthy adult mongrel dogs weighing 21.6 to 28.6 kg were purchased by, and maintained at the Division of Animal Resources,

University of Arizona, and approved for research by the University

22 23

Laboratory Animal Committee. Each dog was utilized three times:

1) fentanyl without either cimetidlne or ranitidine; 2) fentanyl and' cimetidlne; and 3) fentanyl and ranitidine. Dogs were rested a minimum of 2 wks between studies. Animals were fasted 24 hours prior to fenta­ nyl administration.

All dogs received fentanyl (100 yg/kg) XV bolus over 30 sec­ onds. The cimetidlne group received 10 mg/kg cimetidlne IM 12 hrs before and 5 mg/kg IM 90 minutes prior to fentanyl injection. • This corresponds with the recommended human dosage/kg. Ranitidine dogs fol­ lowed the same protocol but were given 2 mg/kg IM 12 hrs before and 1 mg/kg IM 90 minutes before fentanyl injection. This reflects the in­ creased potency of ranitidine (5 times) over cimetidlne in inhibiting gastric secretions. Control dogs were pretreated with 2cc normal saline.

Dog Anesthesia

Dogs were initially given IV pancuronium bromide (Pavulon® .15-

.2 mg/kg) while breathing 50% nitrous oxide by mask. They were placed supine on an animal surgical table and restrained. After topical lidocaine (1-2 mg/kg) administration to the trachea, a cuffed oral endotracheal tube was inserted.

Ventilation was mechanically controlled to maintain at

40+5 torr and PaO^ at greater than 100 torr as confirmed by periodic arterial blood gas analysis. Pancuronium bromide given, IV, facilitated controlled mechanical ventilation. 24

Halothane anesthesia (0.25-0.5%) was provided only during

placement of a catheter in the forelimb cephalic vein and groin cut-

down for femoral artery catheter placement. The cephalic vein catheter

was connected through a three-way stopcock to 5% dextrose in Lactated

Ringer's solution at a rate of 3-5 ml/hr. This catheter was also used

to replace blood loss (due to sample withdrawal) with equal volumes of

5% albumin and for injection of all intravenous medication. Following

each injection the catheter was flushed thoroughly with lactated

Ringer's solution.

The femoral artery catheter was utilized for blood pressure

monitoring by a pressure transducer for sample collection. The cathe­

ter line was cleared prior to sample collection by withdrawing 10 cc

of blood. A 10 ml volume of blood was then immediately withdrawn,

which served as the sample. It was then transferred to a Vacutainer® red-top tube and allowed to clot. The first 10 ml blood sample was

reinjected into the animal, followed by a thorough flush of IV dextrose in lactated Ringers.

The blood pressure transducer was attached to an oscilloscope for continual monitoring. EKG leads were attached to the appropriate

limbs and monitored on the oscilloscope.

Since the effect of potent volatile anesthetic agents on the

pharmacokinetics of short-acting narcotics was not known, halothane

administration did not exceed 30 minutes and was discontinued at least

30 minutes prior to the injection of a narcotic. Halothane exposure 25 was also limited to simulate pharmacological conditions under which

these narcotics are used clinically.

Animal temperature was monitored by placement of a rectal

temperature probe. Body temperature was maintained at 30° C by plac­

ing heating pads under the animals and draping the animals with large

plastic bags.

. Fentanyl (100 yg/kg) was injected as an intravenous bolus given

over a 30-second period. Arterial blood samples (10 ml) were collected

at 1, 2, 5, 10, 15, 30, 60, 90, 120, 180, 210, 240, 270, 300, 330, 360,

390, and 420 minutes, transferred to empty Vacutainer® red-top test

tubes, allowed to clot, and then refrigerated. Albumin (5%) was ad­

ministered to the dog to replace lost blood volume.

The cimetidine and ranitidine groups differed from controls

only by the prior injection of IM cimetidine or ranitidine, respec­

tively. All other parameters were constant.

After the last blood sample was withdrawn and refrigerated,

the catheters were removed and the incisions closed. EKG monitors

were also removed. Naloxone was used to reverse the effects of fenta­

nyl, and physostigmine was used to reverse the effects of pancuronium.

Animals were observed until recovery was assured and then returned to

the post-operative cages.

Samples were then centrifuged at 3000 rpm for 15 minutes, fol­

lowed by transfer of the serum, using disposable pipettes, to fresh

Vacutainer® red-top glass test tubes. Samples were then stored at

4° C until analyzed. 2.6

Sample Preparation

The method used to analyze the serum samples was that of

Gillespie et al. (1981). This procedure employs a gas chromatograph with a nitrogen phosphorus detector.

New glassware, with the exception of disposable glass pipettes, were silanized with 5% dimethyldichlorosilane in toluene. All glass­ ware and Teflon caps were washed by hand in phosphate-free reagent- quality detergent, rinsed twice with distilled water and twice with 95% ethanol to ensure removal of potentially interfering impurities. New disposable glass pipettes were used without alteration.

Reagents were prepared as follows using reagent-grade chemicals throughout. Stock solutions of 2.0 N NaOH and 0.1 N HC1 were prepared in one-liter Nalgene and glass bottles, respectively. A 19:1 solution of glass-distilled hexane and 100% ethyl alcohol was prepared in a one-liter glass bottle. This was then capped tightly to prevent evapo­ ration of solvent.

An internal standard was prepared from stock solution of al- fentanil hydrochloride by dilution to a concentration (670 ng/ml) which elicited a peak height approximately equal to 4 ng of fentanyl in an extracted standard when 50 pi. internal standard was used.

A standard curve was prepared from a stock solution of 1.57 mg/ml fentanyl citrate (1 mg/ml free base). This primary stock solu­ tion was used throughout the experiment. A working stock solution of

1 yg/ral was prepared in 100% ethanol. A 64- ng/ml standard solution was prepared by removing 0.64 ml of working stock solution and combining 27 with 9.4 ml of human plasma. From this serial dilutions with plasma were made to create concentrations of 32, 16, 8, 4, 2, 1 ng/ml. Addi­ tionally, both water and plasma blanks were run.

Aliquots (2 ml) of fentanyl serum samples and fentanyl stan­ dards were pipetted into 12 ml glass culture tubes containing 0.5 ml 2

N NaOH, 5 ml of extraction solvent (19:1, haxanesethanol) and 50 ]il of internal standard. Tubes were capped with PTFE-lined caps and placed in a mechanical shaker for 10 minutes.

Samples were then removed and centrifuged at 3000 rpm for 15 minutes. The upper hexane phase (containing fentanyl) was then trans­ ferred to a second set of extraction tubes containing 5.0 ml of 0.1 N

HC1. These were again shaken 15 minutes and centrifuged at 3000 rpm for 15 minutes.

The hexane phase was l-emoved and discarded and the aqueous phase was adjusted to pH 13 with 0.5 ml 2 N NaOH. Fresh hexane extrac­ tion solvent was added and samples were again shaken and centrifuged.

Following centrifugation, the hexane layer was transferred to 12 ml conical tubes, being cautious not to include aqueous phase. Samples were then evaporated to dryness in a 40° C water bath under a stream of nitrogen.

The sides of the conical tubes were then rinsed with 1 ml eth- anol, vortexed briefly and again evaporated to dryness. The samples were then reconstituted in 30 lil 100% ethanol and vortexed immediately prior to their injection onto the gas chromatograph. Because of the 28 inclusion of internal standard, variations in sample injection volume were permitted to obtain maximal detector response.

Chromatography

A Hewlett Packard 5710A gas chromatograph equipped with a nitrogen/phosphorus detector was used for analysis. A 2 mm x 2 mm id

Silanized glass column packed with 3% OV-17 on Gas Chrom Q, 80/100 mesh was used for separation. A voltage of 15 V was applied to the ceramic alkali bead in the detector. The injector port and detector were operated at 300° C and the column at 280° C. Helium carrier gas flow was 30 ml/min, air 50 ml/min, and hydrogen 4 ml/min. Electrometer range and attenuation were optimized for maximum sensitivity.

Standards were injected using a glass microsyringe from highest to lowest concentration. Peak height ratios were utilized for deter­ minations because of the large height-to-width ratios of the peaks.

Peak heights were determined by measuring maximum peak height from a tangent to the baseline. Corrections for detector attenuation were made and a standard curve and correlation coefficient were obtained by linear regression. When linearity was assured, fentanyl samples were injected onto the column from anticipated highest to lowest concentra­ tion. Peak height ratios of fentanyl to internal standard were de­ termined as above, and fentanyl concentrations were obtained from the standard curve.

When sample concentrations fell below approximately 5 ng/ml, a second standard curve was used. It consisted of concentrations 0, 1, 29

2, 4, 8, ng/ml. This more accurately reflects the concentration range of samples collected in the terminal elimination phase of fentanyl. It is essential that these concentrations be accurate, since pharmaco­ kinetic elimination parameters would be calculated from them.

Pharmacokinetic Analysis

The model indepentent method of pharmacokinetic analysis used by L. A. Benet and R. L. Galeazzi (1979) was chosen. This method re­ quires two assumptions: 1) the system must respond linearly, and 2) the exit of tracer or drug from the body must be directly from the mea­ sured site, i.e., plasma or central compartment. The model independent method eliminates errors of model assignment. For these reasons, the method of Benet and Galeazzi is gaining popularity and I chose to utilize it since my data satisfied the two assumptions.

Log plasma concentration versus time curves were constructed and slope, point fit and y-intercept were calculated using a pre­ programmed Hewlett Packard 34C (HP34C) calculator. Terminal elimina­ tion (3) constant was defined as the product of slope and -2.303.

0 = 2.303 (slope).

Terminal elimination half-life 3 was defined as:

Tl/2e = °-693/B'

The volume of distribution at steady state equals: 30

tCpdt iAUM(W Vd = dose = -dose ss AU( 2 ' Cpdt E W 0 where t is time, Cp is plasma concentration, AUMC is area under the moment curve (defined below) and AUC is area under the curve.

rtplast AUMCQ = tCpdt + tCpdt tplast

where, tCpdt . tplast

tplast 00 AUC = Cpdt + Cpdt 0 tplast

where, fr Cpdt = tUsfPlaBt + . J tlastfcl ast "" RB"

Integrals were approximated using the linear trapezoidal rule.

To eliminate potential errors, linear regression on a Hewlett Packard

34C (HP34C) calculator was used to determine CP last, corresponding to the Y-intercept at final collection time.

Dose The steady state clearance is: cl ss AUC^

Both clearance and volume of distribution were adjusted for kg body weight. 31

For ease of calculation and elimination of mathematical errors,

ftCpdt L program was designed for the HP34C calculator. Cp, tcp , and J0

•t t Cpdt were determined with this program. 0 CHAPTER 3

RESULTS

Gas Chromatography

A typical chromatogram of dog serum containing fentanyl (peak

1) and internal standard alfentanil (peak 2) is shown in Figure 3. The chromatogram shows good resolution of both compounds. Fentanyl eluted following a solvent front of unknown endogenous substances.

Because of the large peak height in proportion to peak width, peak height was used as the determinant of concentration. Attenuation adjustments were used to maximize peak height. Corrected peak heights were then compared to a standard curve for conversion into serum con­ centration.

Standard Curve

Serial dilutions of fentanyl in spiked human plasma were used to generate the standard curve. A constant amount of internal standard was added to each sample. Standard samples were extracted simultane­ ously with dog serum samples. Once linearity of the standard curve was assured, dog serum samples were serially injected into the chroma- tograph.

Because of the rapid distribution of fentanyl into tissues fol­ lowing IV injection, a broad range of serum concentrations were found.

One minute serum concentrations of fentanyl were in the range of

32 33

Peak 1

Peak 2

c o o

Time

Figure 3. GC Chromatogram of Fentanyl (Peak 1) 90 Minute Sample 1:32 Attenuation and Internal Standard Alfentanil (Peak 2). — Conditions: 3% OV-17 on gas chrom Q, 80/100 mess detector 15V. Injector and Detector temp 300oC, column temp 280"C. Helium 30 ml/min, air 50 ml/min; hydrogen 4 ml/min. N-P detector. 3:4

200-300 ng/ml. However, as fentanyl was rapidly distributed, these concentrations decreased rapidly, then stabilized, and by 120-150 minutes they were in the range of 1 ng/ml.

To improve the accuracy, a standard curve consisting of values

0, 1, 2, 4, 8, 16, 32, 64 ng/ml was utilized for dog serum concentra­

tions greater than approximately 5 ng/ml. A second standard curve, excluding the 8, 16, 32, 64 ng/ml values was utilized for dog serum concentrations below 5 ng/ml. The two curves tended to converge be­ tween 4-8 ng/ml. The goal was to alleviate the tendency for the higher standard concentrations to overwhelm the contributions of the values 0,

1, 4, 8 ng/ml in determining the slope. By using the lower concentra­ tions only, Improved precision in the critical serum range of 0-5 ng/ml was achieved. Since pharmacokinetic parameters of Tan^ 3 are cal­ culated within this range, the increased accuracy is essential.

A standard curve using peak height ratios of fentanyl/internal standard vs. concentration was constructed. Linear regression analysis was performed on a HP34C calculator. Figure 4 shows a standard curve using all values 0-64 ng/ml. Figure 5 shows the standard curve after exclusion of values 8, 16, 32, 64 ng/ml.

As discussed earlier, dog fentanyl levels were measured follow­ ing XM cimetidine pretreatment, IM ranitidine pretreatment or as con­ trols using IM normal saline pretreatment. Pharmacokinetic parameters are illustrated in Figure 6. 35

2(H

15- Peak Height Ratio 10-

5-

-i 1— -r- -~r~ —T— 0 8 16 32 48 64 80 Plasma Cone, (ng/ml)

Figure 4. Standard Curve Fentanyl/Internal Standard vs. Concentration. --Range0-64ng/ml. 36

Peak Height Ratio

Plasma Cone, (ng/ml)

Figure 5. Standard Curve Fentanyl/Internal Standard vs. Concentration. — Range 0-8 ng/ml (omitting concentra­ tions 16, 32, 64 ng/ml). 37

50- Dog 803

»- Fentanyl

10-

3.0- O) c o 3.0 • c

« E (QCO 5L t.0-

0.S-

0,3-

I I -I 1 1 1 1 1 1 1 1 1 1- 30 60 90 120 190 100 210 240 270 300 330 360 390 Time (min.)

Figure 6. Diagrammatic Illustration of Pharmacokinetic Parameters Applied to Fentanyl. 38

Individual Dog Results

Please refer to Tables 1, 2, 3, and 4 for pharmacokinetic parameters and Figures 7-11. Each dog will be discussed individually.

Dog 322

The plasma elimination of fentanyl from the serum of dog 322

(Figure 3) was altered by pretreatment with cimetidine, but it was not altered by pretreatment with ranitidine. The terminal elimination rate constants were .00533 and .00461 for nonpretreatment (control) and ran­ itidine pretreatment respectively. However, the serum elimination rate constant for fentanyl was decreased when pretreated with cimetidine.

The elimination rate constant was .00299 for cimetidine pretreatment, which correspondingly resulted in a longer terminal than the con­ trol group. The control fentanyl was 130 minutes, while the cimetidine pretreated was 231 minutes.

The terminal elimination phase of fentanyl when pretreated with cimetidine appeared later than either control or ranitidine pre­ treatment. The cimetidine pretreatment group also displayed higher serum concentrations during the redistribution phase than the other groups.

When compared to both ranitidine and non-pretreatment groups, cimetidine pretreatment decreased both clearance and volume of dis­ tribution. 39

Table 1. Pharmacokinetic Parameters of IV Fentanyl (100 ug/kg) following Pretreatment with IM Normal Saline (Control)

Dog 322 510 603 655 kg wt 19.8 24.4 17.0 29.4 yg dose 1950. 2440. 1700. 2950. min"1 0 .00533 .00588 .00698 .00704 min & 130. 124. 99. 98.

L Vdss 143. 143. 64. 79.

L/kg Vdss/kg 7.2 3.9 3.8 2.7 ml/min CI 1796. 958. 1215. 920. ml/min/kg Cl/kg 90.7 39.3 71.9 31.3 Table 2. Pharmacokinetic Parameters of IV Fentanyl (100 yg/kg) following Pretreatment with IM Cimetidine

Dog // : 322 510 603 655 kg wt 20.8 24.6 22.6 28.6

Pg dose 2050. 2450. 2250. 2850. min ^ 0 .00299 .00316 .00241 .00222 min T1/2 213. 219. 288. 312.

L Vdss 98. 109. 185. 163.

L/kg Vdss/kg 5.7 8.2 4.4 4.7 rnl/min CI 904. 437. 1169. 638. ml/min/kg Cl/kg 43.5 17.8 51.7 22.33 41 Table 3. Pharmacokinetic Parameters of IV Fentanyl (100 yg/kg) following Pretreatraent with IM Ranitidine

Dog # : 322 510 603 655 kg wt 21.6 27.4 24.0 28.6 yg dose 2150. 2750. 2400. 2850. min ^ 3 .00461 .00726 .01009 .00369 min 150. 95. 69. 188.

L Vdss 178. 219. 88. 178.

1/kg Vdss/kg 8.2 8.0 3.64 5.07 ml/min CI 1830. 1829. 1584. 923. ml/min/kg Cl/kg 84.7 66.7 66.0 32.3 42 Table 4, Summary of Pharmacokinetic Parameters of IV Fentanyl (100 jjg/kg)

A B C Control Cimetidine Ranitidine

Wt (kg) 22.7 ± 3.5 24.2 ± 3.5 25.4 + 3.2

Dose (jig) 2260 ± 553 2400 ± 342 2538 ± 322

Strain"1) .00623 ± .00090 .00270 ± .00045*o .00641 ± .00288

T1/2 3(rain) 113 ± .17 263 ± 45Ag 126 ± 54

Vdss(L) 107 ± 42 139 ± 42 157 ± 55

Cl(ml/min) 1222 ± 404 787 ± 318 1542 ± 428

* p < .05 from Co o p < .05 from R A p < .005 from Co § p < .005 from II

Column A shows the pharmacokinetic profile of fentanyl in control dogs Column B shows the pharmacokinetic profile of fentanyl in dogs pre- treated with IM ciraetidine (5 rag/kg) Column C shows the pharmacokinetic profile of fentanvl in dogs pre- treated with IM ranitidine (1 rag/kg) (ANOVA with Student-Newman-Kewls) 43

t

so- Fentanyl

30- • Dog 322 • Dog 510 * Dog 603 • Dog 655

10-

E 5.0-

O 3.0-

1.0-

0.5-

0.3-

-I- —r~ I ~l 1 1 1 1 1 1 1 1 1 T— 30 60 90 120 1GO 180 210 240 270 300 330 360 3S0 420 Time (min.)

Figure 7. Serum Fentanyl Levels after Intravenous Administration of Fentanyl (100 yg/kg) in Dogs 322, 510, 603, 655. 44

Dog 322

Fentanyl Fentanyl & Clmetldlne Fentanyl & Ranitidine

10-

g 5.0- O) c. cj 3.0 c 8 a E Mrt D- 1.0

Time (mln.)

Figure 8. Plasma Elimination of IV Fentanyl (100 |ig/kg) from Dog 322 with No Pretreatmenta with IM Clmetldlne Pretreatment, and with IM Ranitidine Pretreatment. 45

Fentanyl Fentanyl & Cimetldine Fentanyl & Ranitidine

_r_ ~l 1 1 1 1 1 1 1 1 1 1 1— 30 60 90 120 150 180 210 240 270 300 330 300 390 420

Time (min.)

Figure 9. Plasma Elimination of IV Fentanyl (100 yg/kg) from Dog 510 with No Pretreatment, with IM Cimetidine Pretreatment, and with IM Ranitidine Pretreatment. 46

Fentanyl Fentanyl & Clmetldine Fentanyl & Ranitidine

T—i 1—i—i—i i —i—i—i—i—i—I I" 30 00 00 120 ISO 180 210 240 270 300 330 380 300 420 Time (mln.)

Figure 10. Plasma Elimination of IV Fentanyl (100 yg/kg) from Dog 603 with No Pretreatment, with IM Cimetidine Pretreatment, and with IM Ranitidine Pretreatment. 47

SO- Dog 655

SO- • Fentanyl • Fentanyl & Cimetldine • Fentanyl & Ranitidine

10-

.E 5.0- CD O 10- c (D E a0> a. 1.0-

oa-

0.3-

"T I 1 1 1 1 1 1 1 1 | 1 •(••••!— 30 60 00 120 150 180 210 240 370 300 330 300 390 420

Time (min.)

Figure 11. Plasma Elimination of IV Fentanyl (100 Ug/kg) from Dog 655 with No Pretreatment, with IM Cimetidine Pretreatraent, arid with IM Ranitidine Pretreatraent. 48

Dog 510

The plasma elimination of fentanyl from the serum of dog 510

(Figure 4) was again similar in both nonpretreatment (control) and rani- tidine-pretreated groups. The terminal elimination rate constants were

.00588 (for control) and .00726 for ranitidine pretreatment. Again, cimetidine pretreatment lowered the terminal elimination rate constant for fentanyl (to .00316). This resulted in increasing the of fentanyl from a control time of 124 minutes to a cimetidine pretreated time of 219 minutes. As seen in Figure 4, the presence of cimetidine shifted the curve upwards, decreasing the slope and increasing the half- life of fentanyl.

When compared to control, the volumes of distribution and clearance were increased by ranitidine pretreatment but decreased by cimetidine pretreatment.

Dog 603

The plasma elimination of fentanyl from the serum of dog 603

(Figure 5) differed slightly from that of the other dogs. The terminal elimination rate constant for nonpretreatment (control) dogs was .00698.

Cimetidine pretreatment. lowered the terminal elimination rate constant to .00241, which was consistent with that of the other dogs. The terminal elimination half-life of fentanyl was prolonged by cimetidine from a control time of 99 minutes to 288 minutes. Cimetidine had neg­ ligible effects on clearance of control fentanyl but did increase volume of distribution. 49

This dog was unusual in that ranitidine caused a decrease in

the terminal °f fentanyl to 69 minutes. The elimination rate

constant was increased in the presence of ranitidine to .01009. Raniti­

dine caused mild increases in both volume of distribution and clear­

ance when compared to control.

Dog 655

The plasma elimination of fentanyl from the serum of dog 655

(Figure 6) showed a lowering of the terminal elimination rate constant

of fentanyl by both ranitidine and cimetldine. Cimetidine showed the

more pronounced effect. Terminal elimination rate constants were

.00704, .00369, and .00222 for nonpretreatment (control), ranitidine

pretreatment, and cimetidine pretreatment respectively. These corres­

ponded to an increase in the terminal half-life of fentanyl from 98

minutes control to 188 minutes, in the presence of ranitidine, and 312

minutes in the presence of cimetidine.

The volume of distribution of fentanyl was increased by both ranitidine and cimetidine pretreatment. Cimetidine caused a decrease

in clearance of fentanyl, whereas ranitidine was without effect.

Summary

Cimetidine caused a decrease in the terminal elimination rate

constant and an increase in the terminal half-life of fentanyl in all individual dogs. In each case, cimetidine caused a decrease in fentanyl clearance. Cimetidine's effect on the volume of distribution was in­ consistent. 50

Ranitidine caused minimal alterations in the terminal elimination rate constant or the half-life of fentanyl in the individual dogs. Its effect was inconsistent. In some cases it increased the terminal elimination rate constant, such as in dog 603, and in other cases it decreased it, as in dog 655. In all instances, ranitidine in­ creased both the volume of distribution and clearance of fShtanyl.

Cimetidine pretreatment groups showed statistically significant lowering of the fentanyl terminal elimination rate constant compared to both non-pretreated (p < .05) and ranitidine-pretreated dogs (p < .05).

Also, the fentanyl half-life was significantly increased from non- pretreated (p < .005) and ranitidine pretreated dogs (p < .005). There were no significant changes In either volume of distribution or clear­ ance between control, cimetidine, or ranitidine. The ranitidine- pretreated dogs did not differ significantly in half-life, volume of distribution or clearance from non-pretreated dogs. CHAPTER 4

DISCUSSION

Model

Choice o£ Animal

Dogs were chosen as the experimental model in this study for

several reasons. First, the pharmacokinetics of fentanyl (100 yg/kg IV)

had been previously studied in the dog (Murphy et al. 1979), as were the

initial studies on the metabolism and excretion of cimetidine (Taylor

et al. 1978). Taylor et al. (1978) found that the metabolism of cimeti­

dine in the dog differs slightly from that found in man but in most re­

spects was similar. In both man and dog, cimetidine is excreted

predominantly unchanged. Cimetidine's microsomal enzyme inhibition was

shown by Felkonen and Fuurunen (1980) to be non-competitive." Taylor et

al. (1978) noted an increase in polar metabolites in man when cimetidine

was administered orally, but he was unable to characterize the metabo­

lites. This observation was not seen in either the rat or the dog.

Parenteral administration did not exhibit this difference. He concluded

that a small first pass effect occurred in man but not in the rat or

dog. We used both male and female dogs in this study and utilized

parenteral administration of cimetidine.

When this study was initiated, ranitidine was not yet approved

by the FDA for human use. Thus, the potential interaction of fentanyl

51 52

and ranitidine could not be determined in humans. Therefore, dogs

served as an inexpensive and readily available substitute for humans.

It was important to use a large animal in order to obtain sufficient

blood for the time points needed in a pharmacokinetic study. Addition­

ally, the dogs could be re-utilized in each study group, following a

rest period, and thus serve as their own controls.

The disadvantage in using dogs occurs when one tries to ex­

trapolate the results obtained in dogs to human patients. This criti­

cism cannot be overcome short of repeating the study in humans. A major disadvantage in this study was the small number of animals I had available for study. The small number of dogs made it difficult to achieve statistical significance unless results were dramatic. If I were to repeat the study, I would increase the number of dogs studied in an effort to achieve greater certainty and significance in the results.

Choice of Drugs

Fentanyl was chosen as the reference drug. The analytical pro­ cedure for the measurement of fentanyl in serum was developed and available in the Department of Anesthesia at the University of Arizona.

Fentanyl is a widely used anesthetic agent and is likely to be used in combination with a Histamine-2 receptor antagonist. Fentanyl is highly metabolized by microsomal enzymes, and its metabolism is hepatic blood flow limited (Hug et al. 1981). Thus, agents that alter hepatic 53 metabolism and/or hepatic blood flow could alter the clearance of this potent intravenous anesthetic.

Histamine antagonists, cimetidine and ranitidine, were chosen because of the liklihood that they would be co-prescribed with fentanyl in the prevention of aspiration pneumonitis during anesthesia. Cimeti­ dine has been identified as interfering with the hepatic mixed function oxidase enzymes in a non-competitive manner (Pelkonen and Puurunen

1980).

It is also reported that cimetidine may reduce hepatic blood flow in man by acting on H-2 vascular receptors (Feely et al 1981).

Therefore, two pharmacological actions of cimetidine may result in re­ duced clearance of fentanyl by the liver, Feeley and Guy also reported

(1982) that ranitidine reduced liver blood flow in man.

Ranitidine is approximately five times more potent than cime­ tidine in blocking mesenteric gastric acid secretion. For this reason, the administered dose chosen was one-fifth that of cimetidine.

To ensure absorption, I administered cimetidine and ranitidine

IM. Walkensteln et al. (1978) found similar cimetidine blood level curves after intravenous and intramuscular administration in dogs, in­ dicating these routes may be used interchangeably. The bioavailability of ranitidine is unchanged whether given intramuscularly or intravenous­ ly (personal communication, J. Bauman, Glaxo, Inc. 1984). Therefore, I elected to pretreat dogs with intramuscular ranitidine.

It was hoped that the use of cimetidine and ranitidine might clarify whether prolongation of the terminal phase of elimination of 54 drugs results from microsomal enzyme inhibition or from reductions in vascular flow. If both drugs affect clearance, the mechanism is prob­ ably mediated by vascular response. If only cimetidine alters fentanyl levels, it could be argued that the prolongation of fentanyl levels is the result of enzyme inhibition. Because of the structural dissimilar­ ity, it could also be postulated that there is a subdivision of Hg receptors which cimetidine influences but ranitidine does not. This has not been addressed to my knowledge. To prove the theory, one would have to show the presence of vascular receptors which would bind radio­ labeled cimetidine but would not bind radiolabeled ranitidine.

Extraction and Chromatography

The extraction and gas chromatographic analytical technique of

Gillespie et al. (1981) was used in this study. The extraction tech­ nique is rather tedious, and precautions to avoid contamination are crucial. Fentanyl concentrations at the later time collections are in the range of 0.5 to 1.5 ng/ml. Avoidance of nitrogen-phosphorus con­ taminants on glassware and in reagents helped eliminate potential interferences.

Prior sillzation of glassware helped to increase recovery of fentanyl. The back extraction technique proved adequate for the removal of parent fentanyl from serum samples. It is doubtful that metabolites were extracted by this technique, because of the specificity of back extraction techniques and the absence of metabolite peaks on the chromatogram. The internal standard, alfentanil, was chosen because of 55 its chemical similarity to fentanyl and desirable chromatographic retention time and resolution.

Good resolution of fentanyl from the internal standard was achieved. An unknown solvent front preceded fentanyl. It is doubtful that this affected peak height determinations, since it represented minimal concentrations at the retention time of fentanyl. Attempts to identify the component of the solvent front were unsuccessful. Samples were injected from highest concentration (earlier collection points) to lower concentrations (later collection points). This served two pur­ poses. Firstj it minimized the effects of residual fentanyl on the column (if any), and second, with experience, one could predict the next optimal attenuation and thereby maximize peak height. Since the initial collections of serum had very high fentanyl concentrations com­ pared to the amount of internal standard present, attenuation changes were made between the elutions of fentanyl and internal standards. As fentanyl concentrations approached that of the internal standard, no attenuation changes were needed. The time to elute both fentanyl and alfentanil was approximately 20 minutes. Attempts to decrease the elution time tended to decrease the resolution between fentanyl and the solvent peak and were subsequently abandoned.

No attempt was made to measure fentanyl metabolies. There are several postulated metabolites (Hug and Murphy 1981, Lehman et al. 1981) of fentanyl (see Figure 2). To identify metabolites, I would use 56

radiolabeled fentanyl and try to isolate metabolites from the extraction

solutions not containing parent fentanyl.

No attempt was made to measure serum concentrations of either

cimetidine or ranitidine. Cimetidine has shown consistency in plasma

levels following IM administration equal to IV administration. Raniti­

dine has also shown consistent bio-availability whether given IV or IM

(J. Bauman, Glaxo, Inc., personal communication).

Pharmacokinetics

Fentanyl pharmacokinetics have been reported in the literature

as conforming to a three-compartment mamillary model. In this model, final elimination occurs from the central compartment, which is the vascular compartment. Model assignment is dependent on estimates of

tangents drawn to the concentration vs. time plots of fentanyl. Varia­

tions in fentanyl serum values may cause estimates to be inaccurate and

thus the resulting computer fit and model assignment would be in error.

To avoid the problems of model assignment, the method of Benet and

Galeazzl (1979) was chosen. The Benet and Galeazzi method is model- independent; however, certain assumptions must be made: 1) the system must follow first-order kinetics, and 2) the exit of the drug must be from the measured site, i.e., the plasma or central compartment. Both of these assumptions were satisfied by fentanyl.

Pharmacokinetic parameters chosen for the characterization of fentanyl were the terminal elimination rate constant, which represents the combined effects of biotransformation in the liver, and renal 57 excretion by the kidneys (Hug 1978). The terminal half-time, is defined as the proportion or fraction of the drug eliminated per unit time. For drugs obeying first-order kinetics such as fentanyl, °

0.6931/3.

Any inhibition of fentanyl metabolism or excretion by either ranitidine or cimetidine will result in a lengthened half-life and de­ creased 3. Inhibition may occur as a result of metabolism interfer­ ences, changes in hepatic blood flow (hepatic clearance), enterohepatic recirculation, bile excretion, and renal excretion.

Total body clearance at steady state, CI , is a product of the S3 rate constant and the volume of distribution at steady state, Vd . It ss is the total drug fraction irreversibly removed from the body as a func­ tion of time multiplied by the volume of distribution on which the rate constant is operating (Hug 1978). Total body clearance can be calcu­ lated by dividing the dose by the area under the plasma concentration time curve from time zero to infinity. It is a model-independent terra.

Clearance is altered by changes in the elimination rate constant due to changes in drug metabolizing enzymes or excreting mechanisms and/or by altered distribution of the drug (Hug 1978).

Volume of distribution at steady state is hypothetically de­ fined as the volume of body water which would be required to contain the amount of drug in the body if it were uniformly present in the same concentration in which it is in the blood at steady state (Notari 1980).

It is influenced by plasma protein, blood cell, and tissue binding of the drug. 58

Discussion of Pharmacokinetic Results

Compared to control, cimetldlne pretreatment resulted in a

statistically significant decrease in the terminal elimination rate

constant and thus an increased serum half-life for fentanyl. These

changes were not observed following administration of ranitidine.

There are several mechanisms through which cimetldlne could accomplish

this. Because {3 is an integral part of I will discuss the effect

of cimetldlne pretreatment on the terminal elimination half-life of

fentanyl.

Half-life ^ depends on Interchanges between compartments,

as well as upon elimination from the central compartment (Notari 1980).

The most likely causes for the effect of cimetldlne on fentanyl are

interferences with renal excretion, enterohepatic recirculation, micro­ somal enzymes, and hepatic blood flow.

Renal Excretion

The effect of cimetldlne on renal excretion of drugs has not

been well studied. Dubb et al. (1978) showed cimetldlne can inhibit the active secretion of creatinine into the renal tubule. Cimetldlne Is

cleared by the kidneys in young healthy adults at a rate of over 600 ml/rain. Hence, tubular secretion plays a significant role in the drug's renal elimination. McNeil et al. (1981) reported mean renal clearance of ranitidine to be 489 to 512 tul/min after IV administration, suggest­ ing it too is actively secreted. I would expect both drugs to give a similar response to fentanyl at the level of the renal tubule. This was 59

not the case. Therefore it seems unlikely that cimetidine is affecting

renal transport of fentanyl. However, it must be remembered that five

times aa much cimetidine on a milligram basis was administered due to

its lower potency relative to that of ranitidine. In addition, it is

unlikely that fentanyl is actively secreted. Very little unchanged -

fentanyl is excreted in the urine because its lipophilic properties

allow it to be readily reabsorbed from the proximal renal tubule

(Kitahata and Collins 1982).

Enterohepatic Recirculation

Enterohepatic recirculation of fentanyl has been postulated

(Stoeckel et al. 1979) and has been used to explain some of the delayed

effects attributed to fentanyl, such as delayed respiratory depression.

In a study by Bower and Hull (1982), it was found that the clearance of

fentanyl approximated hepatic blood flow, and thus the hepatic extrac­

tion ratio approached unity. On this basis, fentanyl secreted into the

stomach and removed via the portal vein should be completely extracted

by the liver and metabolized. Morphine also undergoes enterohepatic

recirculation, but a study by Mojaverian et al. (1982) found no influ­

ence of oral cimetidine on morphine distribution in man. It seems un­ likely, in light of the high hepatic extraction ratio of fentanyl, that

enterohepatic recirculation is a significant factor.

Interference with Metabolism

Both cimetidine and ranitidine have been studied with regard to cytochrome P-450 binding in vitro. Rendic et al. (1979) using liver 60 microsomes from rats pretreated with various inducers found cimetidine

underwent a ligand type (Type 11) binding to cytochrome'P-450. Cimeti­

dine could be competitively displaced from the active site of cyto­

chrome P-450 by diethylphenylphosphine, known to form a ligand complex

with P-450.

Pelkonen (1980), using benzo(a)pyrene, also studied the effect

of cimetidine on rat liver microsomes. He found in vitro Type II bind­

ings to cytochrome P-450. Cimetidine non-competitively interfered with

N-demethylatlon of aminopyrine and hydroxylation of benzo(a)pyrene.

This was attributed to the binding of the imidazole ring of cimetidine

to the heme moiety of cytochrome P-450. This pattern of binding, char­

acterized by a Type II spectral shift, has been observed with other

nitrogen-containing compounds (Rogerson, Wilkinson and Heturski et al.

1977). Knodell (1982) found that cimetidine inhibited both meperidine

and pentobarbital metabolism in rat and human microsomes. It had no

effect on conjugation of morphine. Thus, cimetidine alters the metabo­

lism of many cytochrome P-450 metabolized compounds due to its Type II

binding to cytochrome P-450. It inhibits microsomal metabolism in

either a competitive or non-competitive manner, depending on the sub­ strate.

In vivo-, cimetidine prolonged the half-life of antipyrine

12-37% in man (Puurunen 1980). Antipyrine is highly dependent on the activity of the cytochrome P-450 system for clearance. Other studies have shown that cimetidine inhibits the metabolism of warfarin (Serlin 61 et al. 1979), theophylline (Roberts et al. 1981), and diazepam (Klotz and Reimann 1980a). All of these are metabolized by cytochrome P-450.

• Drugs which are conjugated directly and therefore bypass the cytochrome P-450 system were not affected by cimetidine. These include oxazepam and lorazepam (Patwardham et al. 1980) and morphine (Mojaverian et al. 1982). These results suggest that cimetidine alters drug bio­ transformation due to an effect on the hepatic cytochrome P-450 system rather than by reducing blood flow. One would expect a reduction in hepatic blood flow to affect the rate of conjugation as well as P-450 mediated events.

Ranitidine is structurally dissimilar to cimetidine, in that it has a furan, as opposed to an imidazole ring, and a different side chain. Investigators have shown that ranitidine does bind to cytochrome

P-450 of liver microsomes but has a tenfold lower binding affinity than that of cimetidine (Rendic et al. 1979). No effect of ranitidine on meperidine or pentobarbital metabolisra in rat or human microsomes was noted by Rnodell et al. (1982). Conjugation of morphine was unaffected as well. One study measured the effect of ranitidine on fentanyl metabolism in vitro and found that while cimetidine showed a non­ competitive effect, ranitidine exhibited a mixed effect (Lee et al.

1982). The study measured disappearance of fentanyl from the microsomal system and the disappearance of NADPH. Concentrations of cimetidine and ranitidine were estimates of therapeutic concentrations. Whether these are actual concentrations present in the liver remains to be determined. 62

Ranitidine in vivo does not decrease the half-life of antipyrine (Henry et al. 1980). Ranitidine did not interfere with me­ tabolism of theophylline in man (Powell and Donn 1984). It did not affect prothrombin time or plasma warfarin concentrations in man, nor influence pentobarbitone sleeping time in rats (Serlin et al. 1981).

Ranitidine did not affect diazepam or lidocaine levels in man (Klotz et al. 1983, Jackson et al. 1983). There is no evidence for inter­ ference with Phase II conjugation reactions by ranitidine (Powell et al. 1983).

In summary, it appears certain that cimetidine can mediate some of its drug interactions by interfering with Phase I metabolism of drugs. It does this by participating in Type II binding to cytochrome

P-450. While there is some evidence to support the binding of raniti­ dine to cytochrome P-450, the clinical experience and in vivo experi­ ments make this an unlikely consequence of ranitidine use. It appears that the effects of cimetidine and ranitidine on bio-transformation of drugs are based on structural differences between the two antagonists and not on H-2 antagonist properties per se.

Hepatic Blood Flow

Feely et al. (1981) reported cimetidine caused a decrease in liver blood flow. Indocyanine green (ICG) is employed as a measure of hepatic blood flow. ICG is metabolized nearly completely (97%) by the liver and is highly bound to albumin (Caesar et al. 1961). Feely et al. (1981) found a single 600 rag oral dose of cimetidine decreased ICG 63 clearance by 25 percent. This was Interpreted as a lowering of liver blood flow. Propranolol was also used as a marker of liver blood flow.

The effect of seven days pretreatment of cimetidine on oral and IV propranolol administration was studied. Cimetidine caused a 25% de­ crease in both oral and IV propranolol clearance as well as a decrease in calculated liver blood flow. Since both oral and IV Clearances of propranolol were affected equally, they argued there was no change in bio-availability. Therefore, since extraction is equal to 1 minus bio­ availability, they conclude that cimetidine does not alter extraction of propranolol. Similar, although less dramatic effects, were seen when they repeated the experiment using ranitidine (Feeley and Guy

1982). They concluded that the effect of cimetidine and ranitide was attributable to vascular H-2 receptors which caused decreased liver blood flow.

The conclusions of Feely et al. 1981 and Feeley and Guy 1982 have been criticized. Jackson (1981) suggested cimetidine may alter

ICG transport, storage or metabolism in the liver. He challenged the assumption that propranolol is wholly metabolized by the liver with no metabolism taking p.lace in the lungs or gut. The authors themselves pointed out that evidence exists for a regulatory role of atrial H-2 receptors, and bradycardia has been reported after the use of cimeti­ dine. They therefore could not rule out the possibility of a syner­ gistic effect by propranolol or cimetidine on the sino-atrial node.

Morphine is eliminated by conjugation, whereas lidocaine and propranolol are primarily oxidized. Mojaverian et al. (1982) showed 64

that cimetidine pretreatment did not influence morphine clearance.

Therefore, this seems a more reliable indicator of a lack of effect of

cimetidine on liver blood flow. SKF 525A, another inhibitor of cyto­

chrome P-450, decreases lidocaine clearance in cats by 60%, a result

totally due to decrease in hepatic extraction and not due to liver

blood flow (Lautt and Skelton 1977).

Daneshmend et al. (1984) found chronic cimetidine treatment did

not reduce liver blood flow, as measured by ICG, but did reduce anti-

pyrine clearance in man.

In my study, cimetidine decreased fentanyl clearance, but

ranitidine did not. Fentanyl, like morphine and propranolol, is highly

dependent on liver blood flow for elimination. If histamine antagonists

altered vascular receptors, both cimetidine and ranitidine should show

similar effects, which they do not.

Because of the decreased metabolic activity In the gastric

mucosa and the resultant reduction in hydrogen ion secretion caused by

histamine antagonists, one would expect decreased metabolic demands and

perhaps decreased gastric blood flow. Whether this causes decreased

hepatic flow or whether it just shunts the blood to other portal areas is unknown.

Clearly, there may be an effect of cimetidine on hepatic blood flow. However, our evidence, the evidence of Mojaverian et al. (1982) and others tend to contradict the conclusion made by Feely. Our results suggest that cimetidine increases the half-life of most agents by de­ creasing their bio-transformatlon by the cytochrome P-450 system. 65

Clearance and Volume of Distribution

Clearance of fentanyl was decreased In the presence of

clmetldlne and Increased In the presence of ranitidine. Statistical

significance could not be shown In either case. The decreased clear­

ance with clmetldlne Is consistent with other studies on theophylline

(Jackson et al. 1981), warfarin (Serlln et al. 1979) and several others.

The decreased clearance of fentanyl 1s consistent with the prolonged

half-life seen with cimetidine. The Increased clearance seen with

ranitidine is more difficult to explain and may be an artifact due to

the small number of subjects studied.

Volumes of distribution were inconsistently changed by either cimetidine or ranitidine. In seven studies investigating the influ­ ence of cimetidine on antipyrine pharmacokinetics, no consistent al­ teration was seen in the volume of distribution (Somogyi and Gugler

1982). Cimetidine did not affect the volume of distribution of diazepam (Klotz and Reiman 1980b). I saw no significant trend of

either cimetidine or renitidine on volume of distribution, which was consistent with the results of other investigators. CHAPTER 5

CONCLUSION

The effect of pretreatment with IM clmetidlne or IM ranitidine

on fentanyl pharmacokinetics in dogs was studied. There was a statis­

tically significant prolongation of the terminal elimination half-life

and decreased terminal elimination rate constant of fentanyl by cimeti- dine but not of ranitidine.

Cimetidine caused a trend toward decreased clearance of fenta­ nyl, but statistical significance was not achieved. There was no consistent alteration of volume of distribution by cimetidine or ranitidine.

The effect of cimetidine on fentanyl levels is probably related to its imidazole structure and mediated by Type 11 binding to cyto­ chrome P-450 enzymes, thus inhibiting the metabolism of fentanyl. Ran­ itidine showed no effect, probably due to its structural dissimilarity to cimetidine and its lack of cytochrome P-450 influences. It is un­ likely that changes in liver blood flow, enterohepatic recirculation, or renal clearance contributed to the effect of cimetidine on fentanyl pharmacokinetics.

66 Appendix I. Serum Concentrations of IV Fentanyl (100 yg/kg) with No Pretreatment by H-2 Antagonists

Time Dog Dog Dog Dog Dog (mln) 322 510 603 655 X =

0 61.58 76.84 292.5 297.25 182.04 + 130.5

2 48.33 38.85 92.4 123.5 75.77 + 39.46

5 30.00 24.32 33.0 90.74 44.52 ± 31.03

10 16.1 21.78 14.2 37.57 22.43 + 10.59

15 10.54 18.65 9.95 25.89 16.25 + 7.55

30 7.09 13.70 4.48 13.78 9.76 + 4.71

60 4.43 9.61 3.02 10.30 6.84 + 3.65

90 2.30 8.11 1.70 7.49 4.90 + 3.37

120 1.47 5.14 1.37 5.36 3.34 + 2.21

150 1.02 4.49 1.27 3.83 2.65 ± 1.76 t - O « o C r

180 0.77* - 1.02 3.61 + 1.57

210 0.69 3.28 0.77 2.74 1.87 + 1.34

240 0.47 3.19 0.58 2.50 1.69 + 1.37

270 0.50 2.88 - 1.97 1.78 ± 1.20

300 - 2.30 0.52 1.85 1.56 + 0.93

330 0.33 - - 1.32 0.33 + 0.70

360 0.30 1.60 0.30 1.38 0.90 ± 0.69

390 - - - 0.97 0.97

420 0.21 0.27 1.13 0.54 + 0.51

67 Appendix II. Serum Concentrations of IV Fentanyl (100 yg/kg) After Pretreatment with Citnetidine

Time Dog Dog Dog Dog Dog (min) 322 510 603 655 X =

0 155.83 275.21 242.66 318.49 248.05 + 68.88

2 83.68 140.33 ,105.86 174.17 126.01 39.67

• 5 51.66 59.86 53.14 100.14 66.20 22.91

10 27.20 36.46 29.94 55.39 37.25 + 12.79

15 18.77 30.46 11.00 25.75 21.50 + 8.49

30 13.05 17.36 7.60 5.93 10.99 ± 5.23

+ 60 9.13 11.55 4.27 - 8.32 3.71

90 5.53 10.24 1.99 4.88 5.66 + 3.42

120 4.73 9.14 1.35 4.00 4.81 + 3.23

150 3.13 7.66 1.31 3.60 3.93 + 2.68

180 1.89 8.76 1.09 3.36 3.78 + 3.45

210 1.38 7.88 0.94 3.01 3.30 + 3.18

240 0.87 6.27 0.85 3.34 2.83 + 2.57

270 0.69 6.47 0.90 2.97 2.76 + 2.68

300 0.64 4.79 0.73 . 3.00 2.29 + 1.99

330 - - . - - -

360 0.53 4.67 0.70 2.53 2.11 + 1.93

390 - - - - -

420 0.44 3.50 0.70 2.38 1.76 + 1.45

68 Appendix III. Serum Concentrations of IV Fentanyl (100 Ug/kg) After Pretreatment with Ranitidine

Time Dog Dog Dog Dog Dog (min) 322 510 603 655 X -

0 61.49 41.27 172.54 . 240.00 158.01 ± 90.14

2 46.27 18.50 58.97 134.00 79.75 + 47.41

5 26.25 18.50 . 41.70 69.8 39.06 £ 22.65

10 16.75 13.44 15.92 36.8 20.73 + 10.81

15 13.29 9.47 13.72 24.8 15.32 + 6.60

30 8.20 8.68 11.12 12.7 10.18 + 2.11

60 3.56 5.25 4.84 5.47 4.78 + 0.85

90 213 4.81 3.16 3.95 3.51 ± 1.14

120 1.70 4.52 2.17 4.14 2.61 + 1.24

150 1.36 3.61 1.73 3.75 2.61 + 1.24

180 0.988 2.44 . 1.21 3.08 1.93 + 1.00

210 0.785 2.42 1.05 3.15 1.85 + 1.12

240 0.715 1.58 0.65 2.45 1.35 + 0.85

270 0.662 1.03 0.57 2.59 1.21 + 0.94

+ 300 0.472 0.98 - 2.19 1.21 0.88

+ 330 0.472 - - 1.81 1.14 0.95

+ 360 0.441 ' - - 1.64 1.04 0.85

390 0.419 - - 1.57 0.99 ± 0.81

420 0.282 0.489 1.36 0.71 + 0.57

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