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University Microfilms International 300 N. Zeeb Road Ann Arbor, Ml 48106

8400151

Alexander, Michael Stephen

METABOLISM AND DISPOSITION OF ACEBUTOLOL

The Ohio State University Ph.D. 1983

University Microfilms International300 N. Zeeb Road, Ann Arbor, Ml 48106

METABOLISM AND DISPOSITION OP ACEBUTOLOL

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

Michael S. Alexander, B.S., M.S.

**********

The Ohio State University

1983

Reading Committee: Approved By

Brian D. Andresen, Ph. D.

Sarah Tjioe, Ph. D.

Howard Sprecher, Ph. D. Brian D. Andresen, PhD. Department of Pharmacology METABOLISM AND DISPOSITION OP ACEBUTOLOL

Michael S. Alexander, Ph. D.

The Ohio State University

Professor Brian D. Andresen, Advisor

Acebutolol disposition and metabolism have been studied in both rats and humans. Animal studies indicate acebutolol is metabolized in a two step process, with debutyration the rate-limiting step in the rat. Processes first examined in isolated, perfused rat livers were found to be reliable predictors of in vivo events. Specifically radiolabelled acebutolol was synthesized by an improved procedure and used as a probe to show the presence of several new metabol ites in the animal. In a large human study, the kinetics of acebutolol and both its known metabolites were simultaneous ly quantitated for the first time. It was shown that elimin ation of acebutolol following intravenous administration is triphasic, most probably reflecting filling and equilibra- ii tion of the enterohepatic subcompartment. About half the subjects showed elevated levels of acetolol, the metabolic intermediate in conversion of acebutolol to diacetolol, in their plasma following a 400 mg oral dose of acebutolol.

This suggests that a significant portion of the population will experience elevated levels of a circulating aniline derivative after taking oral acebutolol. ACKNOWLEDGEMENTS

Dr. Joseph Bianchine, for his acceptance and continuing personal and financial support.

Dr. Brian Andresen, for proposing this project, securing its funding, and giving both advice and the freedom of inquiry.

Dr. Matana Borrisud, for her material help and friendship.

Robert Kreitman, for living proof that unrestrained enthusiasm for science still exists.

The Central Ohio Heart Association for two years of needed grant support in my acebutolol study.

Dr. Sheila Auster, for reasons too long to list.

Okay, I'll try: love, patience, laughter, patience, support, patience, neat car, patience...

iii VITA

September 29> 1950...... Born - Cleveland, Ohio

1972...... B.S., University of Notre Dame, Notre Dame, Indiana

1972-1978...... Research Assistant, Dept of Chemistry, The Ohio State University, Columbus, Ohio

1978...... M.S., The Ohio State University, Columbus, Ohio

1978-1980...... Research Assistant, Dept, of Pharmacology, The Ohio State University, Columbus, Ohio

I98O-I983...... Research Associate, Dept, of Pharmacology, The Ohio State University, Columbus, Ohio

AWARDS

National Merit Scholarship, University of Notre Dame (1968 - 1972)

Winner, ICSABER Biomedical Research Competition, College of Medicine, The Ohio State University (1982)

PUBLICATIONS

Argentation thin-layer chromatography of arachidonic acid metabolites isolated from human platelets, J. Greenwald, M. Alexander, M. VanRollins, L. Wong and J. Bianchine, Prostaglandins, 21(1), 33 (1981).

iv Complete separation by high performance liquid chromatog raphy of metabolites of arachidonic acid from incubation with human and rabbit platelets, M. VanRollins, S. Ho, J. Greenwald, M. Alexander, N. Dorman, L. Wong, L. Horrocks, Prostaglandins, 2_0(3), 571 (1980).

Role of ferric iron in platelet lipoxygenase activity, J. Greenwald, M. Alexander, R. Fertel, C. Beach, L. Wong, J. Bianchine, Biochem. Biophys. Res. Comm., 96(2), 817 (1980).

"Prostaglandin alterations in pregnancy", J. Dicke, J. Greenwald, M. Alexander, J. Bianchine, F. Zuspan. In Pregnancy Hypertension, Sammour, Symonds, Zuspan and El-Tomi (eds.), Aim Shams University Press, Cairo, Egypt (1982).

ABSTRACTS

"HETE production in pregnancy", J. Dicke, J. Greenwald, M. Alexander, F. Zuspan, J. Bianchine, L. Wong, Society for Gynecologic Investigation, Denver, Colorado, 1980.

"Acebutolol metabolism in the isolated, perfused rat liver", M. Alexander and B. Andresen, American Society for Clinical Pharmacology and Theraputics, Lake Buena Vista, Florida, 1982.

"Salicylate determination in Reye’s syndrome by HPLC", M. Alexander, B. Andresen, J. Bianchine, American Society for Pharmacology and Experimental Theraputics/Society of Toxicology Joint Meeting, Louisville, Kentucky, 1982.

FIELDS OF STUDY

Major Fields: Pharmacology and Chemistry

Studies in Natural Product Synthesis and Characterization (Derek Horton, Ph.D.)

Studies in Prostaglandin Metabolism (Joseph Bianchine, M.D., Ph.D.)

Studies in Salicylate Kinetics (Brian Andresen, Ph.D.)

Studies in Acebutolol Dynamics (Brian Andresen, Ph.D.)

v TABLE OF CONTENTS

Page

DEDICATION...... ii

ACKNOWLEDGEMENTS ...... iii

VITA...... iv

LIST OF TABLES...... vii

LIST OF FIGURES...... iX

CHAPTER I Introduction...... 1 Statement of the Problem...... 16

CHAPTER II Experimental Chemical Syntheses...... 19 Isolated Liver Perfusion...... 31 In Vivo Animal Studies...... 35 Human Studies...... 35

CHAPTER III Results and Discussion Syntheses...... 55 Animal Experiments...... 62 Human Studies...... 98

CHAPTER IV Summary...... 129

BIBLIOGRAPHY...... 131 LIST OP TABLES

Table Page

1 Schedule for Collection of Blood Specimens Following AOOmg Acebutolol Oral Dose...... 37

2 Schedule for Collection of Blood Specimens Following lOOmg Acebutolol Intravenous Dose...... 38

3 Standard Statistical Table for Subject Weight Inclusion Criterion...... 39

A Subject Dose Randomization Code...... A3

5 Laboratory Evaluation Tests...... A6

6 Acebutolol Half-Lives for Patient # 1...... 53

7 Values for AUC, Clearance and Clearance/ Kilogram for Subjects Recieving AOOmg Acebutolol Oral Dose...... 106

8 Values for AUC, Clearance and Clearance/ , Kilogram for Subjects Recieving lOOmg Acebutolol Intravenous Dose...... 107

9 Terminal Half-Life, Time to Maximum Concentration and Maximum Plasma Concentration for Acebutolol in Subjects Recieving AOOmg Acebutolol Oral Dose...... 109

10 Terminal Half-Life, Time to Maximum Concentration and Maximum Plasma Concentration for Diacetolol in Subjects Recieving AOOmg Acebutolol Oral Dose...... 110

11 Terminal Half-life, Time to Maximum Concentration and Maximum Plasma Concentration for Acetolol in Subjects Recieving AOOmg Acebutolol Oral Dose...... Ill

12 Terminal Half-Life, Time to Maximum Concentration and Maximum Plasma

vii Concentration for Acebutolol in Subjects Recieving lOOmg Acebutolol Intravenous Dose...... 1 1 2

13 Half-Life Values for Plasma Concentration of Acebutolol In Subjects Recieving lOOmg Intravenous Dose Obtained by Curve Stripping...... 116

viii LIST OP FIGURES

Figure Page

1 Structures of Propranolol, Practolol, and Acebutolol...... 2

2 Effect of Oral Acebutolol on Isoprenaline Induced Tachycardia...... 5

3 Effect of Acebutolol on Isoprenaline Induced Tachycardia and Diastolic Hypertension, Showing Cardiac Selectivity...... 6

4 Quantitation of Plasma Levels of Acebutolol and Diacetolol by Gas Chromatography...... 9

5 Quantitation of Plasma Levels of Acebutolol and Diacetlol by High Performance Liquid Chromatography...... 12

6 Thin Layer Radiochromatogram of Crude Freidel-Crafts Reaction Mixture...... 23

7 Thin Layer Radiochromatogram of 2-Acetyl -4-Butyramidophenol...... 24

8 Thin Layer Radiochromatogram of Reaction Mixture of 2-Acetyl-4-Butyramidophenol with Epibromohydrin...... 26

9 Thin Layer Radiochromatograms of the Reaction of the Epoxide (III) with Isopropylamine...... 28

10 Thin Layer Radiochromatogram of Purified Acebutolol...... 29

11 Schematic Diagram of the Isolated Liver Perfusion Apparatus...... 32

•±x 12 Human Study Informed Consent Form 41

13 Schematic Diagram of the High Performance Liquid Chromatographic System...... 48

14 Graphical Example of Curve Stripping...... 54

15 Flow Chart of Acebutolol Literature Synthesis...... 56

16 Flow Chart of Improved Acebutolol Synthesis...... 58

17 Ultraviolet and Radiochromatograms of Purified Radiolabelled Acebutolol...... 60

18 Optimization of Scintillation Counter...... 61

19 Concentrations of Acebutolol and Diacetolol in Perfusate Following Liver Perfusion with 20mg Acebutolol...... 63

20 Concentration of Diacetolol in Perfusate Following Liver perfusion with 20mg Diacetolol...... 65

21 Concentration of Acetolol and Diacetolol in Perfusate Following Liver Perfusion with 20mg Acetolol...... 67

22 Chromatograms of Rat Urine Before and After Oral Administration of 20mg Acebutolol...... 68

23 Chromatograms of Rat Urine Before and After Intraperitoneal Administration of 20mg Acetolol...... 69

24 Chromatograms of Rat Urine Showing Lack of Effects on Acebutolol Metabolism Following Intestinal Sterilization by Antibiotics...... 72

25 Chromatogram of Basic Fecal Extract from Rat Given 25mg Oral Acebutolol...... 73

26 Ultraviolet Spectra of Peaks in Fig. 19***» 74

27 Bile Flow Rate During Liver Perfusion...... 76

x 28 Total Radioactivity in Bile During Perfusion with Labelled Acebutolol as a Function of Time...... 78

29 Total Radioactivity in Perfusate as a Function of Time for 20mg Acebutolol...... 79

30 Total Radioactivity in Perfusate as a Function of Time for 4mg Acebutolol...... 81

31 Ultraviolet Chromatogram of Perfusate 300rain after Dosing with 4mg Acebutolol.... 82

32 Blank Rat Bile Ultraviolet Chromatogram.... 84

33 Ultraviolet Chromatogram of Rat Bile 60min After Dosing with 20mg Acebutolol.... 85

34 Superimposition of Figure 33 on Its Reconstructed Radiochromatogram...... 86

35 Sequential Radiochromatograms of Bile During 3hr Perfusion...... 87

36 Continuation of Fig. 35...... 88

37 Superimposed UV and Radiochromatograms of Bile from Perfusion, Using Phenyl HPLC Column...... 91

38 Radiochromatograms of Bile Following Acid/ Base Fractionation...... 92

39 Radiochromatograms of Perfusate Following Acid/Base Fractionation...... 93

40 Concentration of Acebutolol and Diacetolol in the Rat Small Intestine 30min After 20mg Oral Acebutolol...... 95

41 Radiochromatograms of Urine and Feces from a Rat Given 20mg Oral Acebutolol...... 97

42 HPLC Retention Curve Families...... 101

43 Sample Chromatogram From Human Study...... 104

44 Typical Acebutolol and Diacetolol Plasma Concentration Curves for Human Subject Recieving lOOmg IV Acebutolol...... 113

’ x i 45 Plot of Maximum Acebutolol Plasma Concentration vs. Clearance for Oral and IV Acebutolol in Humans...... 118

46 Typical Plasma Concentration Curves in Human Subjects Recieving 400mg Oral Acebutolol...... 120

47 Plot of Maximum Plasma Concentrations of Acetolol vs. Diacetolol in Human Oral Subjects...... 122

xii CHAPTER I

INTRODUCTION

Acebutolol (DL-l-(2-acetyl-4-butyramidophenoxy)-2-hydroxy-3-isopropylaminopropane hydrochloride) is a beta - 1 specific adrenergic blocking agent currently manufactured by

May & Baker, Ltd, Dagenham, Sussex, England, which is under going final clinical trials prior to release for general use in the United States in 1984. The drug has been in use in

England and Europe for almost a decade, and has been tested 1-10 and found efficacious in control of cardiac arrythmias , 12-16 17-23 angina pectoris and hypertension . It was patented by Wollridge and Basil with rights assigned to May & Baker 24 in 1969 •

Acebutolol is a member of the beta-adrenergic receptor blocking group of drugs, which includes such compounds as propranolol, metoprolol and.timolol. Its chemical structure is very similar to the now-discontinued drug practolol, differing only in the addition of an acetyl group at the 2- position of the aromatic ring (Figure 1).

Its pharmacological effects on the myocardium are simi lar to propranolol, with which it has often been co-studied.

Propranolol often served as a reference compound for compar- 12,25-27 ison of physiological beta-blocker effects . In con-

1 2

PRACTOLOL

PROPRANOLOL

ACEBUTOLOL

Figure 1. Structural formulas of practolol, propranolol and acebutolol. 3 trast to propranolol, however, it shows a high degree of selectivity for the myocardial beta-receptors, a response similar to practolol. In practice, it shows little effect on the beta -receptors and thus is an attractive alternative 2 drug to the generally used propranolol where a beta-blocking drug is indicated in patients sensitive to nonspecific beta- blockade, such as asthmatics or diabetics.

The development of the current views of the metabolism and disposition of acebutolol has been a case of clinical knowlege co-evolving with analytical technology; .this has

been evident from the earliest literature studies and con-

ctinues to the present time. Indeed, even the currently

accepted values for the various pharmacological parameters

of the drug are by no means near a consensus. This has been

due in part to inadequate analytical techniques, to certain

biases of the investigators and in large part to the in

creasingly complex view of thie disposition of the drug

itself. 28 In 1971, Cuthbert and Owusu-Ankomah published the

first results of human studies with the investigational drug

M & B 17803A (acebutolol). They asessed the general toler

ance of volunteer subjects given oral doses up to 300 mg.

The beta-receptor blocking activity was assessed (by measur

ing reflex tachycardia levels, both in response to passive

tilting and intravenous isoprenaline; the latter to estab

lish the myocardial selectivity, of acebutolol) and the time J] course of such activity. In general, the drug was well- tolerated by the volunteers. There were no significant chan ges from control values in static supine blood pressure caused by acebutolol, and basic blood chemistry was unaffec ted. The drug appeared to have little intrinsic sympathomi metic activity compared to propranolol. At the 300 mg dose level,acebutolol produced significant changes in tilt-in duced reflex tachycardia at all times from 30-360 min post dose, with a peak effect at 90-120 min (decrease of 15-40 bpm from control values).

In a second series of experiments, the competitive blocking effect of acebutolol on isoprenaline-Induced tachy cardia was asessed. The response-time curve shown in Fig ure 2 gives the results from one such subject. The peak effect occured at 2-4 hrs, and the half-life of decline in the 10-24 hr region was about ten hours. This early study showed that acebutolol in a single dose showed significant physiological effects for an extended period of time and presaged the continuing debate over parent drug and metabol ite half-life that continues to the present. In 1973 Basil, 25 et al. determined the cardioselectivity of acebutolol by measuring the dose-related antagonism of acebutolol to isoprenaline-induced tachycardia and diastolic hypotension in several species. Results for the anesthetized cat are shown in Figure 3. The displacement of the response-log dose line clearly shows the cardioselectivity of acebutolol. The ratio 5

0 o in v) (D 0) M & B 17803A (300 mg) c *- 1 0 Propranolol (40 mg)

is a> cc

10 24 Time (h)

Figure 2. Degree and duration of beta-adrenoceptor bolckade produced by a single 300 mg oral dose of M & B 17803A (acebutolol) compared to 40 mg oral propranolol (from Cuthbert and Owusu-Ankomah, Br. J. Pharmac., 43> 639 (1971)* 6

M & B 17,803A Practolol

I I o o c MS> m o ■oO co c 05 M cft c« o. o. o •*o

10* IO-i 10* io> 10 • 10* 10° Intravenous dose of antagonist (mg/kg) Intravenous dose of antagonist (mg/kg)

Propranolol

I o

«* •oo cw S 0-5 fc. a. oM 02

|0» JO* |0-i 10* Intravenous dose of antagonist (mg/kg)

Figure 3* Regression lines computed for M & B 17803A (acebutolol), practolol and propranolol in antagonizing isoprenaline-induced tachycardia (______) and diastolic hypotension (----- ) in the anesthetized cat (from Basil, et al., Br. J. Pharmac., 48, 198 (1973)* 7 of this selectivity is in the range of 20-50:1, depending somewhat on the species and the exact method used. These 26 results were supported by Levy who also reported that the beta-receptors of the bronchioles were apparently unaffected by acebutolol at dose levels up to 3 mg/kg in the dog, a value 2-3 times the usual intravenous dose used in man

The first significant systematic study of acebutolol metabolism and disposition in humans was carried out by Kaye 29 et al. (1976). In this study six male volunteers recieved either an intravenous (100 mg infusion over 10 min) or oral

(100 mg capsule) dose of acebutolol. Plasma and urine sam ples were collected at regular interals. A spectrophoto- 30 metric method was used to measure plasma and urine levels of the drug.The technique did not distinguish between acebu tolol and its metabolites. Pharmacokinetic parameters were calculated for both oral and intravenous dosage regimens.

For both the IV and oral subjects, a terminal elimination half-life of 3.2 hrs was found. The IV distribution half- life was 0.60 hrs, and plasma clearance 818 ml/min. The mean cumulative urinary recovery of all compounds was 60% for the IV dose and 35% for the oral dose. This suggested to

the authors that about half the dose reached the systemic

circulation. It was an early demonstration of a significant

amount of nonrenal elimination of acebutolol. The existence

of a major acebutolol metabolite, diacetolol (the 4-acetami-

do congener of acebtuolol), was reported by the authors, although no evidence of its existence or of blood level measurements was provided. Since diacetolol was stated by the authors to possess cardiac activity similar to acebu tolol (although this was not substantiated), they argued that the nonspecific total plasma concentration curve should be taken to represent plasma levels of total pharmacologic ally active material. 31 In that same year Meffin, et al. , presented results from hospital clinical trials of both oral and IV adiminis- tered acebutolol in a variety of subjects. They were the first group to confirm the identity of the major metabolite, diacetolol, by combined gas chromatography-mass spectrosco py. They also clearly demonstrated the strong route-depen- dence of metabolite formation. Diacetolol was the predomin ant circulating compound after oral administration, but only a minor component following IV infusion. They were the first to use a specific assay (in this case, gas chromatography) to separately quantitate both acebutolol and its metabolite

(Figure 4).

They assumed for the analysis of the plasma a two- compartment open model with elimination from the central compartment only. They obtained for acebutolol a distribu tion half-life of 0.17 hrs and an elimination half life of

3.6 hrs.' The plasma clearance for acebutolol was 320 ml/min.

For the oral doses, 15-3% of the total dose was recovered as acebutolol and 27*6% as diacetolol in the urine. Based on 9

HOURS A f TER OOSE

5.000

< 1,000

100

0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 MINUTES FROM START OF INFUSION

Figure 4. Upper: plasma concentrations of acebutolol (I) and diacetolol (II) following 300 mg oral acebutolol. Lower: plasma concentrations of acebutolol (I) and diacetolol (II) following 1 mg/kg intravenous acebutolol, as determined by gas chromatography (from Meffin, et al, Res. Comm. Chem. Path. Pharmacol., 15(1), 31 (1976). 10

these numbers, which were obtained from a single individual, it was felt that the bioavailability was significantly lower than previously reported. This was because the assay in the earlier study included the metabolite as well as the parent

drug. 32 In 1976, Collins and George administered carbon-lA

labelled acebutolol to both humans and dogs. Pour hyperten

sive male patients were included in the study. Two recieved

200 mg (34 uCi) orally, and all four were administered 20 mg

(36 uCi) of acebutolol by IV infusion. Urine and feces were

collected for 2-5 days after the dose. In their graphical

data, 26-53% of acebutolol and its metabolites (% total

radioactivity) appeared in the urine, but up to 63% of the

dose was recovered in the feces. In anesthetized dogs, small

loops of small intestine were isolated _in vivo and either

filled with 0.9% saline solution for a one hour period or o continuously perfused with saline at 37 C; after which ra

dioactive acebutolol was administered IV in a bolus/constant

infusion regimen over the period of study. They found that

total biliary clearance of radioactivity over the study

period was 9-2% and that the radioactivity accumulated in

the intestinal lumen of the isolated loops. In the filled

loop, the concentration was 4-9 times that in the arterial

plasma at 1 hr. The data from the perfusion experiment led

these researchers to calculate that up to 8% of the total

body clearance might result from a direct elimination of 11 acebutolol across the membrane of the small intestine. These data are unfortunately fragmentary in many respects. Only total radioactivity was determined, not acebutolol' and dia cetolol separately. The total duration of the studies was not given unambiguously in all cases, so the clearance value obtained (12.6 ml/min/kg) must be interpreted cautiously.

The pH of the intestinal medium used was not measured or controlled. The membrane transfer processes for these weakly basic compounds are very sensitive to pH. Thus, while show ing direct transluminal elimination of acebutolol after IV dosing was possible, they did not show that it normally occured significantly in vivo. If the duration of the study was 1 hr, however, the finding that almost 10% of the total radioactivity was eliminated in the bile in this period indicates what a large contribution biliary excretion can make to the overall pharmacokinetic profile of the drug.

Finally, this was the first study to directly examine fecal elimination.

In three subsequent papers in 1977 and 1978, Meffin et 33-35 al. introduced an improved high performance liquid chromatographic (HPLC) technique for the simultaneous deter mination of acebutolol and diacetolol in biological fluids

(Fig. 5)* With this new analytical approach they reanalyzed the disposition of acebutolol following both intravenous and oral administration. For IV doses in 9 healthy volunteers, the blood clearance was found to be 550 ml/min. This value 0 120 240 360 480 600 720 TIME FROM START OF INFUSION (min)

Figure 5- Blood acebutolol concentrations during and after a 15 min 1 mg/kg constant rate intravenous infusion, with inset plot of weighted residuals, as determined by HPLC (from Meffin, et al., Clin. Pharmacol. Ther., 22(5), 557 (1977). 13 was found to be dose-independent over a range of levels from 0.25 to 1.0 mg/kg, indicating a linear relationship in the dose/AUC ratio over this concentration interval. The blood concentration data were analyzed by computer, using a two-compartment open model with elimination from the central compartment only. This yielded fast and slow half-lives of

6.1 and 157 min, respectively. The fraction of the dose excreted in the urine as the parent drug was M0.5%> Urinary excretion of diacetolol represented 13% of the total dose.

In the oral studies, the terminal half life was also deter mined to be 157 min. It was also observed that the normal ized AUC values increased in a nonlinear fashion with in creasing oral dose size, or in the final dose of a multiple oral dose regimen (patient at steady state). In the latter study, the diacetolol plasma concentration averaged 2.5 times higher than that found after a single oral dose of the same size. An important advance in these studies was the introduction of a relatively simple, rapid and sensitive assay technique to replace the earlier, more cumbersome spectrophotometirc or GC methods. 36 In 1979, Andresen and Davis synthesized a hexadeutero analog of acebutolol and used it with combined gas chromato graphy-mass spectroscopy techniques as a probe for new ace butolol metabolites. They reported the discovery of a new compound, the M-amino congener of acebutolol (acetolol), and 14 proposed that it was the metabolic intermediate in acebu- rats fed acebutolol, and later detected in the urine of human patients undergoing therapy with the drug. 37 In 1981, Gulaid et al. reported their results in a study of eight volunteers who recieved either a single 400 mg oral doses of acebutolol or repeated oral doses (400 mg b.i.d.). Their analyses utilized an HPLC-fluorescence meth- 38 od . For the single dose studies, a value for the slow half-life of 3«1 hrs was found for the post-dose period 4-12 hrs, similar to that previously reported. Investigation of the data showed, however, that there was another, shower half-life apparent at later times. The average value for this half-life was 11 hrs, which reduced the previously reported "terminal" half life to 2.6 hrs. The C values max for acebutolol and diacetolol were found to have mean values of 649 and 821 ng/ml, respectively. An examination by thin layer chromatography (details not given) of the urine sam ples from the subjects showed that four of the eight showed traces of acetolol (determined semiquantitatively by compar ison TLC). It is interesting to note that this 11 hr final half-life for the time region 12-36 hrs post-dose is almost identical to that found in the isoprenaline response/time curve described by Cuthbert in 1971* The two curves are remarkably similar, both in t and biexponential half max life. 15 39 In 1980, Munn,et al. examined acebutolol and diacetlol elimination patterns in patients with differing degrees of renal impairment following a 200 mg oral dose. They found that the relative importance of renal elimination for diace tolol is greater than for acebutolol. 40 Gabriel, et al. reported in 1981 on the first quan titative assay of total bioavailability in a human volunteer for both acebutolol and diacetolol. They discovered that after a 400 mg oral dose, 53$ was converted to diacetolol, and after a lOOmg IV dose, only 36$ was so converted to the major metabolite (considering both total urinary and fecal output). They also showed that the minimum bioavailability of acebutolol in terms of luminal absorption, is 89$. The proportion of acebutolol to diacetolol in the fecal samples was almost exactly 1:1 in both oral and IV doses, even though the total fecal recovery was 56 and 33$, respective ly. Analysis of the values reported indicated that in terms of diacetolol, the magnitude of the metabolic first-pass effect was at most 50$, and could be even lower. 16

STATEMENT OF THE PROBLEM

A comprehensive review of the literature shows that understanding of acebutolol dynamics has increased in a co- evolutionary manner along with the development of improved analytical instrumentation capabilities. It has become in creasingly clear that the disposition of the drug is com plex 3 depending on many variables. Even such basic values as half-life and bioavailability have been changed several times as the sophistication of the investigative tools has increased.

The clear variation in acebtuolol metabolism and dispo sition between IV and oral routes of administration has been

conclusively shown, and the importance of hepatic elimina tion has been stressed. The use of techniques of increasing ly higher sensitivity and selectivity has shown the pres ence of at least two new metabolites and hinted at the actual metabolic processes involved.

It seemed at the time of the initiation of this study

that an extension of the newer analytical techniques availa ble could be used to find answers to some of the outstanding questions and controversies surrounding acebutolol metabol

ism and disposition. It was decided that a combined approach 17 using both jLn vitro and in vivo techniques would be most useful.

Both preliminary distribution and metabolic studies would be carried out by the administration of acebutolol and its metabolites to isolated, extracorporeally perfused rat livers. This would allow a clear examination of the metabol ic pathway of acebutolol (under relatively controlled condi tions) and provide simultaneous samples of both perfusing medium and uncontaminated bile. This would allow a diagnos tic approach to such areas as hepatic recirculation and nonrenal excretion.

In vivo animal experiments were also to be performed to determine if the results from the isolated liver perfusion experiments could be extrapolated to a whole-animal system.

In order to obtain the sensitivity and selectivity of detec tion and analysis needed, and especially to search for any new metabolites, a radiolabelled acebutolol had to be syn thesized.

The techniques of HPLC previously used to quantitate acebutolol were to be further extended, using such methods as gradient elution to better separate the compounds. In order to identify minor metabolites in complex biological samples, a combined HPLC-radiotracer method had to be devel oped.These methods were to be developed in order to deter mine the metabolic pathway followed by acebutolol in the 18 liver, as well as to look for any evidence for extrahepatic metabolism.

Finally, the results from the animal work were to be applied to human studies, in order to better understand the distrubition and fate of acebutolol in man. CHAPTER II

EXPERIMENTAL

CHEMICAL SYNTHESES

All solvents and chemicals used were of reagent grade. 14 C-acetyl chloride (1.0 mCi, 1.4 mg) was obtained from New

England Nuclear, Boston, Mass. Radiochemical reactions were carried out in an efficient fume hood. Radiochemical reac tions were monitored by thin layer chromatography (LK6-D silica gel 5 x 20cm prescored plates, Whatman) and developed 41 in a Davidow solvent system. Plates were scanned for radioactivity using a Packard 2100 radiochromatogram scan ner.

N-(p-Methoxyphenyl)butanamide (I): p-Anisidine (20g) was dissolved in water by the slow addition of concentrated hydrochloric acid with stirring. The solution was clarified with activated charcoal. A solution of sodium acetate (100 g) was prepared in water (100 ml) with crushed ice (100 g).

The £-anisidine solution was placed in a large beaker with a magnetic stirring bar and 40ml of butyric anhydride was added, followed immediately by the cold sodium acetate solu-

19 20 tion. With vigorous stirring product (I) appeared almost immediately as an off-white, flocculent solid. Filtration, followed by repeated washing gave a light gray granular solid, which upon repeated crystallization from isopropan ol/water yielded a clear, crystalline product. The yield was

14 g. No attempt was made to optimize the recovery from this step.

2-Acetyl-4-butyramidophenol: A small portion of (I) (100 mg) was placed in a 4ml ReactiVial with a 1/4” teflon-coated 14 magnetic stirring bar. The 1- C acetyl chloride (1.4 mg,

1.0 mCi, New England Nuclear, Boston, Mass.) was purchased in a breakseal tube. The tube was scored for breaking, then the bottom allowed to rest on a cake of dry ice for 1 hr to condense the material. Nonradioactive acetyl chloride (45 ul) was dissolved in 500 ul of freshly distilled nitroben zene and placed in a 500 ul gastight Hamilton syringe with a teflon-faced plunger. In the hood, the cold tube of radioac tive acetyl chloride was opened by breaking the tube tube along the score line. A gentle stream of dry nitrogen was used during this proceedure to minimize hydrolysis of the acid chloride by atmospheric water. The acetyl chloride solution in the syringe was rapidly added to the tube. The tube was sealed with a rubber stopper, and vortexed to dissolve the materials completely. The nitrobenzene solution was then transferred to the reactivial containing (I) via 21 syringe. Both tube and transfer syringe were serially rinsed with portions of nitrobenzene which were added to the vial

(total volume 2 ml). a After the amide (I) had dissolved,

500 mg of anhydrous aluminum chloride (A1C1 ) was added to 3 the vial, which was quickly capped with a teflon-faced septum. The mixture was stirred magnetically at room temper- o ature (24 C) for 15 min. The vial was next placed in an oil o bath at room temperature and slowly heated to 65 C over 15 min. During this time the A1C1 dissolved completely and the 3 mixture took on a dark, orange-brown color. After 30 min, a

25gauge hypodermic needle was inserted through the septum to relieve internal pressure (HC1 gas was released). The solu- o tion was next heated to 110 C over 20 min (gas evolved) and held at this temperature to effect cleavage of the methyl ether. Eventually, the color changed to a brown-green with a small amount of solid flocculent material. The total

reaction time was 65 min.

The reaction mixture was removed from the oil bath, allowed to cool briefly, then pipetted into a 16 x 125 mm

test tube containing 5 g of crushed ice. The vial and pipet were rinsed with some of the melted water, and the material vortexed. A two-phase system resulted, with the aqueous layer a brilliant opaque yellow color. Concentrated HC1 (1.5 ml) was added to the tube and the material vortexed until

the color was an opaque white. This was centrifuged (2 min ffi

1000 x g) to give a clear aqueous layer and a light brown 22 nitrobenzene lower layer. The nitrobenzene was removed with a syringe and the aqueous layer extracted with dichloromethane (2 x 2 ml). The organic extracts were combined in another test tube, 2 ml of water was added, and concentrated

(2M) sodium hydroxide solution added dropwise with vortexing until the aluminum salts had dissolved and the aqueous layer was an intense yellow color. The aqueous layer was removed after centrifugation. The nitrobenzene was extracted with water and NaOH (2 x 2 ml) and the aqueous extracts combined.

The bright yellow aqueous sloution was acidified dropwise with concentrated HC1 until the yellow color was discharged and a white precipitate had formed. The aqueous material was extracted with dichloromethane (3 x 2 ml), and the organic extract dried by passing it through a 9” Pasteur pipet filled with anhydrous granular sodium sulfate held in place with a cotton plug. The pipet was washed with 2 ml of di chloromethane, and the combined organic solutions evaporated o under nitrogen in a sand bath at 40 C. The product dried on the tube walls as a light yellow powder. Figures 6 and 7 show radiochromatograms of both the crude reaction product and the extracted phenol (II). This material was not weighed or purified further, but used immediately in the next step of the reaction sequence.

1-0-(2,3-Bpoxipropyl)-2-acetyl-4-butyramidophenol (III): To the test tube containing the crude reaction product (II) PHENOL

~ r 5 CM

Figure 6. Radiochromatogram of crude reaction product from the Freidel-Crafts reaction of (I) with labelled acetyl chloride. PHENOL

10 15 CM

Figure 7. Radiochromatogram of (II) following extraction from the crude reaction mixture. 25 from the Freidel-Crafts reaction was added a 1/2" teflon-

coated stirring bar, 2 ml of dimethylsulfoxide and 1 ml of epibromohydrin. About lg of anhydrous -sodium carbonate was

added, and the mixture placed in an oil bath at room temper- o ature, which was brought to 70 C over 15 min. The mixture was stirred for 2 hrs, then poured into a centrifuge tube

(50 ml) containing 30 ml water and 15 ml dichloromethane

(CH Cl ). The tube was capped, shaken vigorously, centri- 2 2 fuged, and the dichloromethane removed with a syringe. The

process was repeated with 15 ml of fresh CH Cl , the organic 2 2 extracts combined, dried over sodium sulfate, and quantita

tively transferred to a 100 ml round bottom flask. Solvents

were removed on a rotary evaporator under reduced pressure

(water aspirator), followed by use of a high vacuum pump with liquid nitrogen cold trap to remove DMS0. The product

appeared as a yellow viscous oil which solidified after

several minutes under high vacuum. Figure 8 shows a radio

chromatogram of the crude epoxide reaction product (III).

This material was not purified, but used immediately in the

next reaction step.

1-(2-Acety1-M-butyramidophenoxy)-2-hydroxy-3-isopropylamino-

propane (acebutolol) (IV): The crude epoxide from the pre

vious reaction was dissolved in 4 ml of Isopropylamine and

transferred quantitatively to a capped ReactiVial with a

1/4" teflon-coated magnetic stirring bar. The mixutre was EPOXIDE

PHENOL

5 10 15 CM

Figure 8. Radiochromatogram of the reaction mixture of (II) with epibromohydrin. 27 stirred for 40 hrs at room temperature, at which time radio chromatography of the mixture (Figure 9) showed the starting material had almost completely disappeared and that decompo sition byproducts were significantly increasingr The isopro pylamine was evaporated under nitrogen, the resultant yellow oil dissolved in CH Cl , and extracted with 0.1M aqueous HC1 2 2 (2 x 3 ml). The aqueous layer was separated and made basic by dropwise addition of 2M NaOH, at which time a yellow oil came out of solution. This was extracted into CH Cl (3x2 2 2 ml), the extract reduced in volume under nitrogen, and applied to a thin layer chromatographic plate (20 x 20 cm silica gel 60 F-254, 0.25mm thickness, E. Merck, Darmstadt, 20 Germany) and developed in the Davidow system. Development was halted when the solvent front reached the top of the plate (about 2 hrs). Acebutolol (IV) was visualized under long wave ultraviolet light as a broad band of R = 0.3 f (blue fluorescence). This area was scraped off the plate, extracted with methanol, and dried to a light yellow oil under nitrogen. The oil was taken up in CH Cl and dried by 2 2 passing through anhydrous Na SO . The very pale yellow 2 4 solution was evaporated to an off white powder, which was o stored under dry nitrogen in a -70 C freezer. The yield of acebutol was 63 mg, or 38% in terms of the starting material

(I) (Figure 10). 28

ACEBUTOLOL

EPOXIDE A) 16 HRS

B) 24 HRS

C) 4 0 HRS

Figure 9. Sequential radiochromatograms of the reaction mixture of (III) with isopropylamine taken at A) 16 hours, B) 24 hours and C) 40 hours. 1 — T" 10 15 CM

Figure 10. Radiochromatogram of purified C- acebutolol.

N5 VO 30

2-Propionyl-4-butyraroidophenol (V): A solution.of (I) (4 g)

in nitrobenzene (20 ml) was placed in a screw-capped vial

with a teflon-coated magnetic stirring bar. Propionic anhyd

ride (3 g) and anhydrous A1C1 (8 g, large excess) were 3 added to the vial, which was loosely capped. The reaction

was performed as for (II). The product (V) was used directly

in the next step without purification.

1-0-(2,3-Epoxipropyl)-2-propionyl-4-butyramidophenol (VI):

The product (V) was dissolved in 20 ml of DMSO, 5 ml of

epibromohydrin and 5 g of anhydrous Na CO was added. The 2 3 proceedure followed was then identical to the preparation of

(III). The product (VI) was used in the next step without further purification.

l-(2-Propionyl-4-butyramldophenoxy)-2-hydroxy-3-isopropylaminopropane (VII): The product (VI) was dissolved in 40 ml of isopropylamine and stirred at room temperature for three days. The isopropylamine was then evaporated to give a light yellow solid. This material (VII) was then repeatedly re

crystallized from isopropanol/water to give fine white crys tals showing a single absorbance peak on HPLC. Of the mater ials synthesized as internal standards, (VII) was chosen as the working material, because of its favorable HPLC reten tion characteristics. 31

ISOLATED LIVER PERFUSIONS

Apparatus: The livers used in these experiments were main tained on an extracorporeal organ perfusion apparatus (Ambec

Two/Ten Perfuser, MX International, Inc., Aurora, Co.); the apparatus is described In Fig. 11. 42 Liver perfusion experiments utilized white, male

Sprague-Dawley rats. The rat was placed under full ether anesthesia. The abdomen was opened at the midline from pubis to sternum, and the bile duct and portal vein freed from other tissue by blunt dissection. The bile duct was ligated approximately 2 cm distal to the liver and carefully pierced with a fine needle. A fine polyethylene catheter (Intramedic

#50) with a sharpened end was then inserted 1-2 cm into the duct toward the liver and tied firmly in place, after which the distal portion of the duct was severed. The catheter was observed for a short time to confirm continued bile flow; occasionally, application of a slight suction was be neces sary to initiate the process.

The abdominal aorta was next exposed and 100 units of heparin were infused into the animal. After allowing the heparin to circulate, the animal was exsanguinated and the heparinized blood added to the reservoir of the perfusion apparatus.

The portal vein was then rapidly catheterized in the same manner as was used for the bile duct. A flow of oxygen- 12 ated complete Krebs bicarbonate buffer perfused the liver CHAMBER

BILE CATHETER

LIVER

HUMIDIFIER HEATED STAGE

SAMPLE PORT

PERISTALTIC OXYGENATOR PUMP

FILTER

PERFUSATE RESERVOIR

Figure 11. Schematic diagram of the isolated liver perfusion apparatus. w 33 through the catheter as it was being removed from the car cass to minimize tissue hypoxia. The liver was rapidly cut away from all supporting tissue, transferred to the heated stage of the perfusion apparatus, and the portal vein cathe ter connected to the perfusate pump. The pump circulated a flow of the oxygenated perfusing medium (100 ml of Krebs buffer with added rat blood) through the liver from a reser- o voir maintained at 37 C. The liver was allowed to equilib rate 15-20 min to assure that tissue oxygenation (as deter mined by color and overall appearance) and bile flow were adequate. After obtaining blank samples of bile and perfus ate (time zero), the drug was added as a solution to the reservoir, and timed aliquots of perfusate and bile were collected. The collected samples were quickly frozen until analyses could be performed.

Analytical methods

High performance liquid chromatography (HPLC) was the major method used for analysis of perfusate and bile sam ples. For kinetic studies an isocratic solvent system was used (60% acetonitrile:40# water, adjusted to pH 2.10 by addition of 85% orthophosphoric acid). For metabolic assays, samples were examined using gradient elution (acetonit rile rwater, linear gradient, 0-50% acetonitrile over 50min).

Solvent flow rates in all studies were 1.0 ml/min, and the columns used were Beckman Ultrasphere C-18 reverse-phase 34

(4.6 x 150 or 250mm) and Beckman C-8 reverse-phase (4.6 x

250mm). Detection of compounds was accomplished using either an Altex Model 153 ultraviolet detector (254nm, fixed wave length) or a Beckman Model 165 variable wavelength detector.

Sample preparation

For perfusate, a 100 ul aliquot was mixed with 5 ul of

85% H PO and 150 ul of acetonltrile (containing internal 3 4 standard) to precipitate proteins. The sample was centri fuged, and 200 ul of the clear supernatant was injected onto the column using a fixed loop injector. For bile, the same method was used for isocratic elution. In gradient runs, the high acetonltrile content of the sample lead to peak broade ning. For these samples 25 ul of bile were mixed with 5ul of

85% H PO and centrifuged in a capillary tube. A 20 ul 3 4 sample of this supernatant injected on the column.

Distribution of acebutolol and dlacetolol in the perfused rat liver

Perfusate samples (0.5 ml) were taken at 0, 1, 2, 5,

10, 15, 20, 30, 45, 60, 90, 120 and 180 min. Bile samples were collected in capillary tubes for quantitation, flame sealed, and frozen until analyses could be performed at a later time. 35

In vivo animal studies

In whole animal studies, the rat was placed in a metab olic cage overnight to obtain baseline urine and fecal samples. Food was restircted for 12 hrs prior to dosing; water was available ad lib. The drug was administered as an aqueous solution orally using a gavage needle, or intraperl- toneally. The rats were then returned to their cages; urine and feces were collected in chilled recievers, and then frozen.

For the analysis by HPLC, urine was worked up by the same proceedure as used for bile. Fecal material was homo genized in water (1:4). A uniform 250 ul aliquot was treated with 20 ul of 85% H PO , centrifuged, and 200 ul of the 3 4 supernatant injected onto the column.

HUMAN STUDIES

The purpose of this study was to assess the kinetic and metabolic profiles of acebutolol. It was designed as a single-dose, open-label, two-way crossover study of oral

(400 mg capsule) vs. intravenous (100 mg) administration of acebutolol in eighteen healthy male human volunteers. The subjects were randomly assigned to one of two groups for treatment as follows:

Treatment A: one acebutolol capsule (400 mg)

Treatment B: 100 mg acebutolol IV solution

Each dose was followed by a one week washout period. 36

For each group, samples of blood, urine and feces were collected prior to and at predetermined intervals after dosing (Tables 1 and 2). The following values were to be determined: area under plasma concentration vs. time curve

(AUC), intrinsic plasma clearance (Cl and Cl ), peak oral IV plasma concentration (C ), time to peak plasma concentra- max tion (T ) and elimination half-life (t ) max 1/2 Medical and social histories of all subjects were ob tained prior to the study. A complete physical examination with vital signs (temperature, radial pulse, respiration and standing and supine blood pressure), clinical laboratory safety tests (blood chemistry, hematology and urinalysis), including antinuclear antibody titer (ANA) and ECG were evaluated both before and within M8 hrs after the final drug administration. Side effects were assessed at each dosing.

Subject selection

Eighteen healthy male volunteers completed the study.

All were of legal age and not more than 40yrs old with weights within 15% (plus or minus) for height and frame as specified by the Metropolitan Life Insurance Standard Table

(Table 3)* None of the subjects chosen showed any signifi cant abnormal findings at the pretreatment physical examina tion as evaluated by the primary clinical investigator. All subjects presented with a normal ECG at the pretreatment evaluation, had no laboratory values outside the accepted normal ranges, and all eighteen completed the study. All 37

TABLE 1

SCHEDULE FOR COLLECTION OF BLOOD SPECIMENS

400 MG ORAL ACEBUTOLOL

SAMPLE NUMBER SAMPLE TIME

1 0 min

2 20 min

3 40 min

4 1.0 hr

5 1.5 hr

6 2.0 hr

7 3.0 hr

8 4.0 hr

9 6.0 hr

10 8.0 hr

11 10.0 hr

12 12.0 hr

13 24.0 hr

14 48.0 hr 38

TABLE 2

SCHEDULE FOR COLLECTION OF BLOOD SPECIMENS

100 MG INTRAVENOUS ACEBUTOLOL

NUMBER SAMPLE TIME

1 0 rain

2 5 min

3 10 min

4 15 min

5 20 min

6 25 min

7 30 min

8 40 min

9 1.0 hr

10 1.5 hr

11 2.0 hr

12 3.0 hr

13 4.0 hr

14 6.0 hr

15 8.0 hr

16 10.0 hr

17 12.0 hr

18 24.0 hr

19 48.0 hr 39 TABLE 3

MID-POINT OP WEIGHT.RANGE BY HEIGHT AND FRAME (POUNDS)*

HEIGHT DESIRABLE WEIGHT 15% UNDERWEIGHT 15% OVERWEIGHT

IN SMALL MED LARGE SMALL MED LARGE SMALL MED LARG] ICHES FRAME FRAME FRAME FRAME FRAME FRAME FRAME FRAME FRAM]

61 114 121 132 97 102 112 131 140 151

62 117 125 134 99 106 114 135 144 155

63 120 128 138 102 109 117 138 147 159

64 123 • 131 142 105 111 121 141 151 163

65 126 134 144 108 114 124 144 154 166

66 130 138 148 111 117 126 149 158 171

67 134 . 142 154 114 121 131 154 163 177

68 138 146 158 118 124 134 158 168 181

69 142 150 162 121 128 138 163 173 186

70 145 154 165 124 131 140 167 177 190

71 149 158 170 126 134 145 171 182 196

72 153 162 174 131 138 148 176 187 201

73 158 167 180 135 142 153 181 192 206

74 162 172 184 138 146 156 186 198 212

75 166 177 189 141 150 161 190 204 217

*Tables modified from Metropolitan Life Insurance Co., Statistical Bulletin 40, 1, 1959 and 47, 1, 1966. subjects participated in the study voluntarily, and signed a witnessed informed consent form before participation, with the right to withdraw from the study at any time without penalty explicitly stated (Figure 12).

Exclusion criteria for the study included:

1) Any medication (including over-the-counter drugs)

within fourteen days prior to Day 1 of the study.

2) Reception of any other investigational new

drug within four weeks prior to entry into the

study.

3) Reception within the previous three months of

any drug known to have a well-defined potential

for toxicity or enzyme-inducing potential (i.e.,

barbiturates) to a major organ system.

4) Any surgical or medical condition which could

interfere with the absorption, metabolism, distribu

tion or excretion of the test drug.

5) A history of chronic alcoholism, drug abuse or con- 4 sumption of more than eight cups of coffee or tea or,

for cigarrette smokers, of more than one pack per

day. No subject was allowed to consume caffeine con

taining or alcoholic beverages, or to smoke for at

least 24 hrs prior to and 36 hrs after dosing with

the test medication. TUB OHIO 3TATB UNIVERSITY Protocol Ho. H-'IO

4. Possible benefits for nubjecta/aoclety? Ho benflt to the volunteer but accumulation CONSENT TO SPECIAL TREATMENT OR PROCEDURE of data for the clinical evaluation of thla antlarrhythmlc drug say be a benefit to _____ society^ This drug may be of henefi1 to aathmatic patients.______

, hereby authorize or ilirect Joseph R . Blenchlne.H. D., Ph. P. or associates or assistants of hia or bur choosing, to perform tne following tre/itn^nJ ir procedure and such additional aerwlcea aa thay may deem reasonably necessary (n its per 5* Anticipated duration of subject's participation: Two Weeks. formance (describe In general teraa)To participate in a clinical research program. The study w U l evaluate the drug level In your blood etreem of an antlarrhylholc drug, Sectral*_faoehut'olon^ Poans wni^be^ administered aa one jntfa,?f .1^2 US- nectral* facebutoiolT or orally aa a 400 tag capaule of Sectral* Taccbutololl. TLlj___ 1 hereby acknowledge that Joseph R. Blanchlna,H.D.,Ri.D. haa provided information about Know whT(^~do3 ^you~^iTr~be~raceTv'ing prior to a dnlnlatratlon. _ Tc>u will be confined to _ the procedure described above, about ay rights a a a subject, and that he/ahe answered theClTnJcalPhernacolo^y Center for two nights and one day ger trealnent. Your_blood__ all queattons to ay satisfaction. I understand that I aay Contact him/her should 1 have will be drawn on 77 occasions over a two week period. You will alao collect urine^ and additional questions. He/ahe has explained the risks described above end I understand atoola_Jor~^dAy3-8fTer~dlacharRe oil an outpatient baala per treataent period . ______them; he/ahe has alao offered to explain all possible risks or complications. Upon MYSE L F ______• (myself or name of aubJectT I understand that the information obtained from me, or from the person I aa authorised to represent, will remain confidential unleas I specifically agree otherwise by placing The experimental portion of tha treataent or procedure latTou, along w ith 17 o ther _ __ __ my initials here ______. I understand that, where appropriate, the U.S. Food and persona will recelve either one Intravenous Injection of 100 mg Sectral* (acebutolol T or £ Drug Administration say inspect records of this research project. 400 mg oral capsule separated by one week Intervals. Tou will be observed Jor the. oafcty and^ tolerance of thts entTaj rhythmic compound. This wfll not be the _f i rs£ In hum ana I understand that I aa free to withdraw my conaent mod participation in this project at for these"foraula 11onsT Sectral»~TacebutoloQ la currently marketed In Canady U M ted _ any time after notifying the project direotor without prejudicing future care. Ho K) ngdom and Europe .______guarantee has been given to me concerning this treataent or procedure.

In the unlikely event of physical injury resulting from participation in this study, This is done as part of an Investigation entitled; An Open label, S lngle-Poaa, Abaoluts I understand that immediate medical treatment lo available at University Hospital of Bluaval labiHty Stud^r of Acebutolol (Sectral*) - 400 HU Capsule Versus'iOO H? Intravenous The Ohio State University. Questions about this ohould be directed to the parson named Solution in Hewlthy Adult Male Volunteers.______above. I alao understand that the coats of such treataent will be at ^ y ‘expanse and that financial compensation la not available.

1. Purpose of the procedure or treatment: To atudjr the amount of oral versus I have read and fully understand the conaant form. I sign It freely and voluntarily. lntravenoue drug in your blood atream.______A copy hae been given to me.

AH 2. Poaalblo appropriate alternative methods of treatment: Wot to participate. Itate:___ Time:______R4 Signed:

Wltnoaa: ______7. Discomforts and rieka reasonably to be expactad: Pain and discomfort may occur at (Person Authorized to Conaant for the intravenous site. Thera may be a rash and irrltatlon at the intravenous si te. Fain Subject — if Required) and discomfort end bt-ulalng may occur at the bl£od__drawinjg alto In your erm._ There may Wi tness:______also he tissue discoloration at the blood drawing ajte In jrour arm. Possible si'Da uffccts of Seetralb (acebutoloil £ro: headache, tlredneas and lightheaded ness. ft la also t certify that I have personally completed ell blanka In thia form and explained thorn to possible to have a drop in your H o o d presaure. This (a an__eip_erlmental drug, lhercfnrut the subject or his/her representative before requesting the subject or his/her represen there may also'be unknown side effects and risks._ Tou_mlll have your blood drawn tative to sign it. approximately 37 times -- the amount being 770 (|1 ot.L 1.5 cups) over a 7 wnuk period. Alao, there la a posslbl I ty jif faIntWir whi 1^ having your blood drawn. Tou also un«Jersfand"that wlthfn the two week period prior to the study and throughout tho duration Signed; ______of the study, no other drug therapy, Including^ alcohol, marljuenat over-the-cnunlor (Signature of tho Project Director or Authorized RepreeantatJveT modi cations, vitamins and investigational drugs will be allowed. You also must not smoke tobacco. Form II3-02MA (Rev. l2/5»/«l) J;»:c|.

Figure 12. Human study consent form. 42

6) A history of adverse reaction or hypersensitivity to

drugs with a chemical structure similar to acebut

olol.

7) A history of cardiac, renal, hepatic, gastrointes

tinal, hematological, neurological or psychiatric

disease.

8) Any other chronic illness not covered by the above.

Screening

Within 14 days prior to study entry, each subject underwent a screening evaluation consisting of the follow ing :

1) A brief interview during which a full explanation of

the nature and purpose of the study was provided.

Medical and social histories were obtained as dis

cussed above. Subjects were questioned with respect

to the inclusion/exclusion criteria discussed above.

2) A complete physical examination as discussed above.

3) A laboratory evaluation as discussed above.

4) A standard 12-lead electrocardiogram (ECG).

Drug administration and trial management

Eighteen normal subjects completed the three weeks of the study. After acceptance, each subject was randomly as signed to one of two treatment groups composed of nine subjects each (see Table 4 for randomization code). TABLE 4

RANDOMIZATION CODE FOR STUDY SUBJECTS

SEQUENCE 1

1 3 5 8 9 12 13 16 18

SEQUENCE 2

2 4 6 7 10 11 14 15 17

WHERE: SEQUENCE 1 = A B SEQUENCE 2 = B A

WHERE: A = 400 MG ACEBUTOLOL ORAL B = 100 MG ACEBUTOLOL INTRAVENOUS 44

The subjects were admitted to the treatment unit (Clin ical Pharmacology Unit, Means Hall, The Ohio State Universi ty Hospitals, Columbus, Ohio) the evening prior to dosing, where they began an overnight fast which continued at least four hours after administration of their assigned dose of medication. Each study drug (oral and IV) was taken with 250 ml of water; additional fluids were allowed ad lib one hour post-dosing. The oral test drug, administered as a gelatin capsule, was swalloed intact and a mouth check made to confirm ingestion. The IV drug dose was administered as follows: The subject, in a supine position, was adminis tered a slow continuous infusion of physiological saline and

5% dextrose through an arm or hand vein, maintained at a constant drip rate. Using sterile technique, 10 ml of the acebutolol solution (10 mg/ml) was taken up in a twenty milliliter disposable syringe fitted with a butterfly need le. The syringe was fitted to a Harvard constant-rate infu sion pump, and the butterfly was piggybacked to the dex trose/saline IV line. The drug was administered at a con stant rate over a fifteen minute interval, and the IV drip line was left in place for at least 5 min after dosing was completed. Blood samples were collected from the antecubital vein of the arm not used for the infusion IV.

A standardized, low-fat lunch was provided to all sub jects (oral and IV) after the four hour venipuncture. No strenuous activity was allowed during the treatment day. 45

Subjects remained under close medical supervision in the

treatment unit for at least 24 hrs following each drug administration.

Continung subject evaluation

Vital signs (temperature, pulse and respiratory rates,

supine and erect blood pressure) were taken at the one and

two hour post-dose intervals. During the 15 min period of

intravenous dosing, supine blood pressure and pulse were monitored at two-minute intervals, with the criteria for

discontinuation of infusion being a heart rate falling to

less than 50bpm or a diastolic blood pressure fall of

"20mmHg. Discontinuation was not required for any subject in

the study. After IV dosing was complete, supine blood pres

sure and pulse were monitored every five minutes for the

first fifteen minutes and every ten minutes for the remain

ing first hour post-dose.

Laboratory evaluation

All subjects underwent the clinical laboratory safety

tests listed in Table 5 at their initial screening and

within 96 hrs of the final drug administration. All subjects

were fasted (excepting water ac3 lib) eight hours prior to

all laboratory tests. 46

TABLE 5

SUMMARY OP LABORATORY EVALUATION TESTS

HEMOGRAM

Hemoglobin Hematocrit Leucocyte Count (Total and Differential) RBC .'Platelet Estimate (if abnormal, then total count)

URINALYSIS

Color Specific Gravity . pH Protein, Sugar, Ketones, Bile, Blood Microscopic Examination of Sediment

BLOOD CHEMISTRY

Creatinine Urea Nitrogen SGOT Alkaline Phosphatase Bilirubin (Total) Calcium Inorganic Phosphorus Cholesterol (Total) Protein (Total) Albumin Uric Acid Glucose Sodiun, Potassium, Chloride, Bicarbonate ANA Titer 47

Collection of plasma samples

On dosing days, blood samples were collected immediate ly prior to and after dosing at predetermined intervals.

Samples (10 ml) of venous blood were collected by aseptic technique in heparinized Vacutainer tubes and centrifuged at

1200 rpm for 15 minutes to obtain plasma. The plasma samples were transferred into labelled glass vials and frozen up- o right in a -20 C freezer until assays could be performed.

Assay methodology

A sensitive and specific high performance liquid chrom atography (HPLC) method was devised to assay all samples for acebutolol, diacetolol, acetolol and any other metabolites which might be detected.

The HPLC system used (Figure 13) consisted of an Altex

MP 322 dual pump gradient liquid chromatograph, a Beckman

Model 165 dual-wavelength scanning ultraviolet detector, a

SpectraPhysics System 1 computing integrator and a Kipp &

Zonen dual pen recorder. The column was a 4.6 x 250 mm 5- micron reverse-phase phenyl (Chromanetics, Inc.). The system was operated in isocratic mode. The solvent system was 25% of a 2:1 mixture of methanol:acetonitrile and 75% water

(made acidic to a pH of 2.05 by addition of 85% orthophosphoric acid). The solvents used were of HPLC-grade. The flow rate was 2.0 ml/min. Detection was performed at a wavelength of 230 nm. This value was a compromise; the lambda of max il del ecj^ peak

HPIC MASTER CONTROL PROCESS TIMER UV SCANS activate INTEGRATOR REMOTE RECORDER 2 KEYPAO

RECOROER 3 control How + RES gradient RES.

PUMP A PUMP B FRACTION collect1 COLLECTOR peak

MIXER

COLUMN INJECTOR

Figure 13. Schematic diagram of the high performance liquid chromatographic system. 49 acebutolol, diacetolol and acetolol were 236, 236 and 210 nm, respectively, using this system. Very poor detection limits for the acetolol metabolite were obtained at 236 nm;

230 nm was found to be an acceptable value for detection of all three compounds simultaneously. At this wavelength, the detector sensitivity was approximately four times lower for this compound than the other two. The detector sensitivity was 0.025 AUPS. The parameters for the integrator were set to a peak width of 20 sec and a slope sensitivity of 500 microvolts/sec.

Sample preparation

A solution of the internal standard (the propionyl homolog of acebutolol) in methanol (500 ul of a 2 mg/1 solution) was pipetted into a 16 x 125 mm borosilicate glass serum vial equipped with a teflon-lined screw cap and evap orated to dryness under nitrogen at room temperature. A 2.0 ml aliquot of serum was added to the tube, followed by 0.5 ml of 2M aqueous sodium hydroxide, with approximately 0.5 g of sodium chloride, and 10.0ml of diethyl ether. The tubes were tightly capped, then gently mixed for 30 min (Thermo- lyne Speci-Mix aliquot mixer, Sybron Corp., Dubuque, Iowa).

The tubes were then centrifuged at 3000 rpm for 10 min, the upper ethereal layer transferred to a 16 x 125mm test tube, and the ether evaporated at room temperature under a stream of dry nitrogen (Evaporation for extended periods under 50 elevated temperatures, as for example in a heating bath, can lead to significant sublimation and loss of the extracted compounds). To each tube was then added 0.3 ml of 2% orthophosphoric acid and 0.8 ml of dichloromethane. The tubes were vortexed, then centrifuged (3000 rpm for 10 min), and the upper, aqueous layer withdrawn by a 500 microliter

Hamilton syringe for injection onto the chromatograph col umn. The size of the injected sample was 200 ul, using a fixed-loop injection valve (Rheodyne 7120).

Calibration

Drug and metabolite concentrations were determined by the internal standard method. Standard curves were construc ted as follows: To serum tubes containing 500 ul of the internal standard solution were added methanolic solutions of acebutolol, diacetolol and acetolol, which were then evaporated to dryness under nitrogen. A 2.0 ml aliquot of plasma from a drug-free volunteer was then added to each tube, resulting in a final concentration of each compound at

4000, 2000, 1000, 500, 200,100, 50, 20 and 0 ng/ml. The samples were then worked up as previously described and analyzed. The resulting calibration curves of the three compounds were plotted as area compound/area standard vs. concentration. The best-fit linear regression line through the points was determined, and the equation of the line used to convert peak area to concentration for the samples. 51

Reduction of data

The area under the plasma concentration vs. time curve

(AUC) for both oral and IV doses was determined by. the trapezoidal rule for the interval t=0 to t=n. If the concen tration had not fallen to zero within the sampling interval, the area corresponding to t=n to t=infinity was estimated.

The slope of the concentration curve for the last three points (t=n-2 to t=n) on a semilogarithmic plot of the data was determined by linear regression, and the x-intercept of this line (t ) was taken as the value corresponding infinity to C=0. Assuming a first order decrease in this terminal phase, the estimated area is then C /2 x (t - t ). n n infinity Terminal half-lives were estimated by determining the best fit linear regression line through the points in the terminal phase of the semilogarithmic time vs. concentration curve. Data points included were determined by visual in spection of the graph with the criteria that no value prior to t=l80 min was used for the IV values, no value prior to t=240min for oral values and no concentration of less than

20ng/ml was included. In cases where the decision for in clusion/exclusion was unclear, an iterative linear regres sion of the values was performed in the direction of dec reasing time until the correlation coefficient of the best fit line decreased for two consecutive data points; these two points were then discarded and the remainder used to estimate t . In all cases, at least four data points were 1/2 52 used to find the terminal half-life.

For the determination of multiple half-lives in the IV data, a model-independent method was used. From the best-fit line for the terminal half-life calculations, concentrations for the compound of interest at the data collection times were computed. These were then subtracted from the original values 1 and the resulting concentrations plotted semilogar- ithmically as a graph of first residual, or stripped, val ues. The time at which the concentration difference between the actual value and the value from the best-fit line reached zero (i.e. where the experimental and calculated lines intersect) was taken as the final data point to be used in calculating the secondary half-life. This stripped graph was then analyzed by linear regressive methods as described earlier, although the point inclusion/exclusion criteria were somewhat different, because fewer data points were available. If the stripped graph still showed a definite inflection (nonrandom deviation from a straight line), the stripping process was repeated a second time, using the same method as described earlier, to obtain a teritary half-life (see Table 6 for a numerical example of this process, and Figure 14 for a graphical interpretation). 53

TABLE 6

ACEBUTOLOL HALP-LIVES FOR PATIENT #1: INTRAVENOUS

CONC BEST-FIT 1ST LOG BEST-FIT 2ND LOG (NG/ TERMINAL RESIDUAL 1ST RESIDUAL ML) LINE LINE RESIDUAL LINE SAMPLE TIME (NG/ML) (LOG (LOG (LOG NO (MIN) NG/ML) NG/ML) NG/ML)

47523 0 0 0 12832 5 1203 360 2.926 2.775 2.394 23473 10 1634 354 3.107 2.721 2.877 08516 15 2100 349 3.243 2.646 3.116 53203 20 1145 343 2.904 2.582 2.623 21986 25 833 337 2.695 2.518 2.220 07035 30 652 331 2.507 2.453 1.575 61412 40 545 320 2.352 79389 60 384 300 1.924 11953 90 315 271 1.643 03902 120 273 245 1.447 01167 180 203 200 0.477 92179 240 166 163 0.477 67597 360 109 109 68064 480 73 22261 600 44 51156 720 35 16114 1440 10 23850 2880 0

HALF-LIFE ESTIMATES FROM STRIPPED VALUES

TIME INTERVAL 180-720 30-180 15-30 LINE CORRELATION VAL. -0.996 -0.990 -0.995 Y-INTERCEPT 2.564 2.839 4.643 SLOPE -0.00146 -0.01286 -0.10052 HALF-LIFE 206 min 23*4 min 2.99 min 54

t,/2^f°r biexponential model = 2 .78hrs(2.61) t J" " " «7.8mln(6.l) 1/2*

2 N o z o

i/2, = 38min

=4.2min i / 2 of

2 4 6 8 10 12 HOURS

Figure 14. Graphical example of curve stripping for the various half-lives of acebutolol, using a typical log con centration/time curve. The plotting symbols are dots=acebutolol plasma concentration, circles=plot of first residual values, stars=plot of second residual values. The three half-lives thus determined are listed next to the best-fit regression lines for each segment. The upper two half-life values are those obtained using a biexponential model, with the accepted literature values in parentheses. >

CHAPTER III

RESULTS AND DISCUSSION

Syntheses

There were two synthetic goals In this work: production of a specifically labelled radioactive acebutolol, and det ermination of the best internal standard for chromatographic work. For the latter, several acebutolol congeners were synthesized.

The synthesis of acebutolol with a radioactive label at an unambiguous position in the molecule was seen as essen

tial for animal metabolic studies. However, this presented

significant practical difficulties if the proceedures recor ded in the chemical literature were used, especially If a microscale ('100 mg) synthesis was to be performed.

The published synthesis of acebutolol Is shown in Fig ure 15* The salient features are the last three steps in the

synthesis. The literature yield (the net throughput on these

steps) is 5-6% • The aluminum chloride-catalyzed Fries rear

rangement in step 4, with an average yield of 15%> is the major stumbling block in this synthesis. Working on the micro scale, . the yield would in all probability be even

lower. Thus, there were three choices for the synthesis:

incorporate the label at a step prior to the Fries rear-

55 © ^7) 1 PY 93%

AICI3 150°C 10- 20%

synthesis. Figure 15. Flow chart of acebutolol literature 57 rangement and accept a low yield of product, Incorporate the label after the rearrangement, or devise an Improved synthe sis. The first was rejected because it would be too costly in terms of the amount of labelled compounds needed. The second was also rejected due to the unavailability and/or cost of labelled intermediates ( in both steps the reagents used, epichlorohydrin and isopropylamine, are used in large excess). Accordingly, a new synthetic scheme was devised, 14 starting from p-anisidine and the readily available 1- C acetyl chloride as the labelled intermediate (Figure 16).

14 Radiochemical purity by HPLC: Approximately 200 ug of C- acebutolol was dissolved in 5 ml of water with a few drops of 85% orthophosphoric acid. A 20 ul aliquot was injected onto the HPLC (C-18 Ultrasphere ODS reverse-phase column,

Beckman Instruments, Inc, Berkeley, Cal., 4.6 x 150 mm; C-18

Apex, Jones Chromatography, London, Eng., precolumn, 4.6 x 40mm, chromatographic mobile phase 60% acetonitrile/40% water, pH 2.10, flow rate 1.0 ml/min, detection by UV at 236 nm). Fractions of the eluent from the column (0.4 ml) were colected serially in 7 ml plastic scintillation vials (Medi cal Frontier Enterprises, Beliefontaine, Ohio). To each vial was added 6 ml of scintillation fluid (NEN-963, New England

Nuclear, Boston, Mass.). The vials were vortexed and coun ted in a well-type scintillation counter (Packard Tri-Carb,

Packard Instruments, Inc., Downers Grove, 111.) for 2 min. 58

och3 u OCH3 * CH3COCI AICU(excess) o 0 . ^ NaOAc nitrobenzene heat HN

U > ^ B r i

DMSO NogCOj HN

Figure 16. Flow chart of the reaction pathway used to syn thesize specifically radiolabelled acebutolol in this study. The label site is indicated by an asterisk (*). This synthe sis is an improvement over the previously used Fries rear rangement because the final yield is about three times higher. It is more amenable to microscale techniques, and gives a cleaner product suitable for metabolic studies. 59

The ultraviolet chromatogram (Figure 17) shows a slight impurity as a shoulder on acebutolol, but this is not seen in the reconstructed radiochromatogram. Radiologically, the labelled material was greater than 99*9% pure.

14 Determination of C-acebutolol specific activity: A solu tion of acebutolol hydrochloride was prepared in water at a concentration of 1 mg/ml. This was diluted to concentrations of 1,3,5 and 7 mg/10ml. A 20 ul aliquot of each was injected on the HPLC, and a calibration curve of peak area vs. con centration was constructed.

The labelled acebutolol was dissolved in acetonitrile.

An aliquot of this material was diluted 1:9 and 10 ul of the diluted solution was counted for radioactivity. This estab- 4 lished a value of 4.8 x 10 cpm/ul of the stock material. A

20 ul aliquot of the diluted stock solution was then injec ted into the HPLC and its concentration determined using the standard curve previously constructed. The stock solution of labelled material was found to have a concentration of 5»3 mg/ml. The specific activity of the labelled acebutolol was 6 then calculated and found to be 9*21 x 10 cpm/mg, calculated as the hydrochloride salt.

Optimization of gain in scintillation counter 14 The gain was optimized by counting a sample of C- acebutolol in the same scintillation fluid used in all 60

10 20 30 40 30

FRACTION NUMBER

14 Figure 11. Chromatograms of the purified C- acebutolol from HPLC. The lower trace Is the ultra violet chromatogram; the upper trace Is a radio chromatogram constructed by scintillation counting of sequentially collected 0.4 ml fractions of the HPLC eluate. 10 15 20 25 30 35 % GAIN

Figure 18. Graph of counts per minute vs. percent gain for sample of radioactive acebutolol in the scintillation coun ter. The gain was set at 20% for the studies. 62

subsequent experiments at various gain values from 0 to 32%

(Figure 18). Maximum sensitivity was found at a value of

20$, andthis setting was used in all subsequent measure

ments .

Animal experiments (Metabolic Studies)

In order to examine the basic dynamics of acebutolol

distribution and metabolism in the perfusion experiments, a

series of studies were carried out in which rat livers were

administered acebutolol, diacetolol and acetolol.

To insure that acebutolol was not subject to nonmeta-

bolic degradation under the study conditions, a sham experi

ment was performed by running a solution of Krebs buffer,

whole rat blood and 20 mg of acebutolol through the perfu

sion apparatus for three hours with no liver attached. At

the end of that time, a perfusate sample was examined by

HPLC and found-to contain only acebutolol by ultraviolet

detection. This showed that the compound was stable in this

environment.lt also showed that blood enzymes had no signif-

' icant effect on acebutolol.

Figure 19 is a plot of the perfusate time-concentration

curve for a liver given a 20 mg dose of acebutolol. The

acebutolol concentration drops rapidly during the distribu

tive phase, then begins a slow, constant decline. After a

short induction period, diacetolol begins to appear in the

perfusate, and also rises in a slow constant manner. 63

12.5t

io.o -

-J 7.5- 2 O o

C5 z 5.0-

ACEBUTOLOL

DIACETOLOL

0 50100 150 MIN

Figure 19* Graph of acebutolol and diacetolol concentration in the perfusate of a liver perfusion experiment with a dose of 20 mg acebutolol. 64

Several deductions can be made from this experiment.

First, both elimination and metabolic pathways are satura

ted, resulting in zero-order kinetics at this acebutolol

concentration. Second, the tissue affinity of acebutolol is high; extrapolating the concentration back to time zero

gives an equilibrium acebutolol concentration in the perfus

ate of 4.9 mg/lOOml. With the liver weight of 13 g, this

results in a distribution coefficient liver:perfusate of

24:1 (this value will be returned to in the discussion of

human kinetics). Third, diacetolol is returned directly to

the central compartment from the liver.

A series of liver perfusions with acebutolol were per

formed at concentrations (initial) of 50, 20, 10, 5 and 2

mg/lOOml. In all cases, the distribution coefficient was in

the range 22-24:1. This argues against a specific, saturable

binding site in the liver for acebutolol and also indicates

the tissue is not saturated, even at very high doses (rough

ly estimating, the dose-equivalent for the 50 mg perfusion

is on the order of 40-50 mg/kg; by contrast, an average IV

infusion in man is in the region of 1-2 mg/kg, and the

average oral dose is about 400 mg).

Figure 20 shows the results of a perfusion with 20 mg

of diacetolol. It is evident that this metabolite has a much

lower tissue affinity than acebutolol; the equilibrium dis

tribution coefficient is 2.5-'l> or ten times lower. This is

not surprising, as one would expect an acetamide to be less 65

25 n

20-

DIACETOLOL 5 O o o

0 50 100 150 MIN

Figure 20. Graph of diacetolol concentration in the perfus ate of a liver perfusion experiment with a dose of 20 mg diacetolol. 66

lipophillic than a butyramide, other things being equal.

Both compounds posess the same functional groups. The nega

tive slope of the latter part of the curve confrims that

diacetolol is actively secreted by the liver in the perfu

sion model.

Figure 21 shows the results of a perfusion with 20 mg

of acetolol. The most interesting finding from this experi ment was the rapidity with which acetolol is converted to

diacetolol. Under the same conditions, conversion of acebut

olol to diacetolol is more than ten times slower. This would

seem to indicate that debutyration of the parent compound,

rather than acetylatiuon of the metablic intermediate, is

the rate determining step in acebutolol metabolism, at least

in the rat.

An extrapolation of the decay curve for acetolol back

to zero time gives an equilibrium concentration of 15 mg/100

ml, essentially identical to that of diacetolol. Thus acet

olol, like diacetolol, also has an approximately tenfold

lower tissue affinity than the parent drug. This will also

be returned to in the discussion of human data, especially

in relation to the relative half-lives of acetolol and

diacetolol in the human system.

To determine whether the same metabolic patterns are

found in the intact animal, two additional experiments were

performed. In the first, a rat recieved 20 mg of acebutolol

by oral gavage, and the 0-8 hr urine was collected. Gradient 67

20 ■

_ l Z o o DIACETOLOL s o ACETOLOL Z

0 50 100 150 MIN

Figure 21. Graph of acetolol and diacetolol concentration in the perfusate of a liver perfusion experiment with a dose of 20 mg acetolol. RAT URINE-ACEBUTOLOL R 0. DUAL WAVELENGTH SCAN 4701

Figure 22. Ultraviolet chromatograms of rat urine prior to ,and after administration of 5U mg oral acebutolol: acebut olol (®), diacetolol (•)• 69

CONTROL URINE

0 - 4 HRS URINE ACETOLOL DIACETOLOL

T r T — I— 4 0 7 0 M I N

Figure 23. Ultraviolet chromatograms from gradient HPLC of urine from a rat given 20 mg acetolol intraperitoneally. Upper trace=control, lower trace=0—4hrs pooled urine. 70

HPLC of the urine (Figure 22) showed most of the dose was

recovered in the urine as acebutolol. In another experiment, a rat recieved 20 mg of acetolol intraperitoneally, and the

0-4 hr post-dose urine was collected and analyzed by HPLC

(Figure 23)* The results showed that in this case the total dose excreted in the urine was ”90% diacetolol. Thus, the

isolated liver system seemed to accurately predict the meta bolic trends in the whole animal.

A question that arose early in this study was whether

or not there was significant extrahepatic metabolism of

acebutolol. Specifically, the possible role of the intestin

al flora or. the possibility of prehepatic metabolism in the

intestinal mucosa was examined.

A fresh rat fecal pellet was homogenized in 20 ml of

complete Krebs bicarbonate buffer at a pH of 7*4, 5 mg of

acebutolol was added to the suspension, and the mixture o incubated anerobically for 36 hrs at 39 C. At the end of

this time, a buildup of gas pressure and froth in the sample

indicated that active fermentation had taken place. HPLC

analysis of an aliquot, however, showed only acebutolol pre

sent in the solution at that time, with no measurable metab

olites .

To test in vitro the possibility of intestinal mucosal metabolism, a section (5 cm) of fresh rat small intestine

was removed and everted to expose the inner lumen. This was o incubated at 37 C for 12 hrs -in a solution of 25 ml of 71 oxygenated Krebs buffer containing 5 mg acebutolol at pH

7.4. At the end of this time, only acebutolol could be found by HPLC. These results indicate there is no significant intestinal metabolism of acebutolol.

To test this result in vivo, one rat was placed on an oral antibiotic regimen (tetracycline 100 mg, bacitracin 200 mg, neomycin 200 mg) for 48 hrs prior to recieving an oral acebutolol dose of 20 mg; the second rat recieved no antibi otics, but was given the same dose. The urine from both was collected and analyzed. Figure 24 shows the HPLC results from these experiments. There is no significant difference in metabolism between the two animals, ruling out any significant contribution to acebutolol metabolism by the gut flora in vivo.

In view of the documented nonrenal elimination of ace butolol, an examination of fecal contants in the rat was performed by HPLC. A 2 g sample of fecal material from a rat which had recieved 25 mg acebutolol orally was homogenized in water, made basic with sodium hydroxide solution, and extracted with ethyl acetate. The extract was evaporated to dryness, reconstituted in water, and injected onto the HPLC for gradient elution. Figure 25 shows the resulting UV chromatogram. In addition to the presence of both acebutolol and diacetolol, two things were apparent. One was the abscence of detectable acetolol; the second was the appearance of a small peak which eluted between diacetolol and w VJ V. Q 8-24hro 24-48hra 48 *72 hrs V. J \J V u J VAw 0 "8 h rs 8 - 2 4 hre 24"48hr* 4B~72brs

CONTROL RAT ANTIBIOTIC-TREATED RAT URINE URINE

Figure 24. Ultraviolet chromatograms of rat urine collected following an oral dose of 20mg acebutolol. Left series, control rat; right series, rat given antibiotic regimen for 48 hrs prior to acebutolol administration. - 4 ro Figure 25- Ultraviolet gradient liquid chromatogram of basic fecal extract from a rat fed 25 mg acebutolol orally. The peaks at 3637 and 4710 sec are diacetolol and acebutolol, respectively. The peak at 4386 sec does not appear in a sample of blank fecal extract. The thin vertical lines represent the portion of the peak scanned for the ultravio let spectra of the compounds. DIACETOLOL ACEBUTOLOL

UNKNOWN METABOLITE 2 3 6

325

325 325

nm nm nm

Figure 26. Ultraviolet spectra of the three compounds scanned in Figure 25. The unknown compound has the same absorbance maxima as diacetolol and acebutolol. The differ ing absolute intensities are due to concentration differ ences. 75 acebutolol. An ultraviolet spectrum of this peak was ob tained by scanning during elution and compared to spectra obtained of diacetolol and acebutolol during the same run

(Figure 26). All three spectra show absorption maxima at 236 and 325 nm, and the ratios of the intensities were-esential- ly equal. This was the first strong evidence of any new metabolites of acebutolol.

Labelled perfusion results

The combination of isolated liver perfusion with

HPLC/scintillation counting produced a large quantity of data useful in comparison with later in vivo studies.

One basic question always present in perfusion work is the condition of the organ itself: all metabolic activity is related to the health of the organ, which will be continual ly degrading over the course of the experiment. Empirically, monitoring of bile flow offers an estimate of basic organ activity.

Figure 27 shows a plot of bile flow in microliters/min ute for two experiments. In both cases, there is a gradual fall in flow rate, with the flow decreasing by about half to two-thirds over three hours. In all perfusion experiments where bile flow was measured, there was no evidence for either a choleretic or anticholeretic effect of any signifi cance. iue 7 Gahsoigbl lwrt i lmnfr two Graphshowingbilerate inflow ul/min for 27. Figure separate isolated perfusion liverpreparations.

BILE FLOW (ul/min) 10 * MIN 76

Examination of the bile was also essential in the investigation of enterohepatic recirculation of acebutolol. 32 Early studies had shown that acebutolol was secreted Into the intestine of the dog following intravenous administra tion. It has also been shown that acebutolol is secreted in 29 human bile , and that a significant portion of an intra- MO venous dose is lost in the stool in humans

In a perfusion experiment, labelled acebutolol was mixed with unlabelled acebutolol to give a total dose of 20 mg. Both timed perfusate and timed bile samples were collec ted .

The radioactivity of each bile fraction was determined and calculated. It was found that the concentration rapidly rose to a constant average value within 30 minutes of admin istration (Figure 28).

The same proceedure was applied to the perfusate sam ples to construct time vs. total perfusate activity plots.

Figure 29 shows such a plot for the liver given a total dose of 20 mg acebutolol. Since the perfusion apparatus was operated in recycle mode, any decrease of radioactivity must either be due to increased sequestration by the organ or loss through the bile. Total biliary radioactivity loss over 6 the course of the experiment was 5*1 x 10 cpm. The decrease in total perfusate activity over the bile collection period 6 was however only 2.5 x 10 cpm, leaving the question of why the two values were not in closer agreement. Figure 28. Total radioactivity in bile during perfusion with labelled with time. perfusion of during function bile a in as acebutolol radioactivity Total 28. Figure CPM-MO HRS 78

CPM/IOO iue 9 Gah f oa rdociiy nte perfusate total of radioactivity in the Graph 29* Figure fromaperfusion proceedure employing 20 acebutolol. mg 20 40 OA PRUAE ACTIVITY PERFUSATE TOTAL 20 MG20 ACEBUTOLOL 10 ALIQUOTS UL 60 80 MI N

100

120 4 160 140

180 VO —5 80

Another perfusion with labelled acebutolol was per formed, but the total dose was reduced by a factor of ap proximately five (4.3 mg). The graph of total serum radioac

tivity is quite different. It reaches a minimum after

approximately 30 minutes, then begins a slow, constant in

crease (Figure 30). The ultraviolet chromatogram of the perfusate at the end of the experiment indicates the reason

for this (Figure 31). At this time, the concentrations of

acebutolol and diacetolol are essentially equal. It will be

recalled from early experiments with large doses of acebut

olol that the ability of the liver to convert the parent drug to the major metabolite is saturated above dose levels

in the 4-5 mg range, and that also the affinity of diacet

olol for liver tissue is much smaller than that of acebut

olol. The change in the shape of the curve for total circu

lating labelled material is due to the combination of smal

ler initial dose and lower tissue binding for diacetolol.

An increasing proportion of the total activity is present in

this form as the total amount of parent drug decreases.

Metabolic profiles

In humans, acebutolol is known to have one major and

one minor metabolite (with the minor metabolite, acetolol, ✓ being the reaction intermediate between the two). These

three materials account for essentially all of the total

dose. However, the presence of minor metabolites cannot be from a perfusion experiment employing 4 mg acebutolol. 4 mg employing experiment perfusion a from iue 0 Gah f oa rdociiy n h perfusate the in radioactivity total of Graph 30. Figure

CPM/IOOO 20 10 • • 50 T O T A L P E R F U S A T E ACTIVITY ACTIVITY E T A S U F R E P L A T O T L O L O T U B E C A 4 O M U A S T O U Q I AL 0 5 UL 100 MIN 150 200 0 5 2 0 0 3 81 82

r r T T i 0 5 10 15 20 MIN

Figure 31. Ultraviolet chromatogram of perfusate 300 min after dosing with 4 mg acebutolol. 83

dismissed. The bile represents a highly favorable hunting

ground for these compounds.

A severe drawback to analysis of the bile by HPLC is

illustrated in Figures 32 and 33. Figure 32 shows a UV

chromatogram of a blank bile sample at 236 nm, the absorb

ance maximum of acebutolol and diacetolol. Figure 33 is a

chromatogram of bile from the same experiment, taken at 60 minutes after administration of 20 mg acebutolol. It is

obvious that identification of minor metabolites by UV in

such a matrix would be difficult.

The HPLC eluent from the UV detector was collected at

0.5 min intervals and radioactivity measured in a scintilla

tion counter. Such a technique offers greatly enhanced sens

itivity (the samples can be counted for as long as neces

sary) with relatively little loss in resolution. Figure 3^

is a superposition of Figure 33 and its accompanying radio

chromatogram , showing the correlation of UV and radioactiv

ity in the sample.

An examination of the radiochromatograms (Figures 35

and 36) obtained from sequential bile samples collected

during a perfusion shows a consistent pattern. Acebutolol

appears in the bile Immediately and maintains an essentially

constant concentration throughout the experiment. Diacetolol

begins to appear in quantity after an induction period of

about 30 minutes; this is about the same amount of time

required for measurable traces to appear in the early i— --1— — i— 0 10 20 ~ 30~ 40 MIN

Figure 32. Blank rat bile ultraviolet chromatogram. IV

T —I— 30 MIN

Figure 33. Ultraviolet chromatogram of rat bile 60 min after dosing with 20 mg acebutolol. oo ui 20 30 40 MIN

Figure 34. Superimposition of Figure 33 on its reconstructed radiochromatogram. oo CT\ 87 16

BILE RAOIOCHROMATOGRAM

20-i

7.5— 15.5 MIN

CPM/IOOO

20 25'

30 40 50 60 70 HPLC FRACTION

Figure 35* Radiochromatograms of bile from a liver perfusion experiment using 4 mg labelled acebutolol. Top, 0-7*5 min fraction; middle, 7*5-15 min fraction; bottom, 25-35 min fraction. 88

2 4 1

5 5 6 5 M I N 20

CPM/1000 2 4

1 0 5 121 M I N

3 0 4 0 5 0 6 0 7 0 HPLC FRACTION

Figure 36. Continuation of Figure 35, showing bile radio chromatograms from: top, 55-65 min; bottom, 105-121 min. 89 perfusion experiments with cold acebutolol. The concentra tion of diacetolol increases steadily throughout the course of the perfusion in the bile, eventually becoming slightly higher than the parent drug. Along with the increasing diacetolol concentration, several new peaks of radioactivity appear, grouped around the major metabolite and equalling

10-15% of the total label output (None of these newly dis covered metabolites is acetolol, which appears much earlier under these chromatographic conditions; this compound was never detected in any perfusion experiment). There are at least six of these new metabolites seen in the bile.

That so many new compounds could be discovered is not surprising. In the first place, the samples used are perhaps the most favorable for such a discovery. Second, the use of a label allows one to greatly enhance the sensitivity and selectivity of the assay over conventional ultraviolet or fluorescence methods (in these, sensitivity is a function of both the molecular chromophore, the incident radiation wave length, the absorption background and the residence time in the detector). The selective concentration of acebutolol and its metabolites by the liver in the bile is impressive; the ratio of total activity between bile and perfusate averages over 100:1. It should be realized, however, that the total amount of these new metabolites is still very small, relative to the total dose, even in the highly favor able conditions of the perfusion experiment. 90

In later perfusion experiments, a reverse-phase phenyl column was used for HPLC gradient separations. The substi tution of a reverse-phase phenyl column offered an enhanced separation capability for these samples. Figure 37 shows superimposed ultraviolet and radiochromatograms for a bile sample subjected to a linear gradient with this column. The greater hydrophobicity of the phenyl column packing relative to the C-18 material tends to result in earlier elution of more water-soluble components; in addition, there may be enhanced interaction of the stationary phase with compounds posessing an aromatic center.

Samples of bile and perfusate from another perfusion were subjected to acid/base fractionation. Figure 38 shows

HPLC/radiochromatograms for such a fractionation performed on a bile sample. It can be seen that there are several acid-extractable components in the bile. Figure 39 shows the results from a similar proceedure performed on perfusate, with similar results. The relative concentration of these components, however, is greater in the perfusate than in the bile, indicating that, like diacetolol, they are more polar

(a fact reinforced by their early elution). This is the first evidence that Phase I metabolism (other than debutyration) occurs in acebutolol. 91

18,0 0 0

16,0 0 0

14.0 0 0

12.0 0 0

. 10,0 0 0 co 5 u_ Z3 Ql < 6 8,000 o o

6,000

4.0 0 0

2.0 0 0

6 0 MINUTES

Figure 37* Superimposed ultraviolet (236 nm) and radiochrom atograms of rat bile from an animal given an oral dose of labelled acebutolol. The bile was analyzed by gradient elu tion on a reverse-phase phenyl column. Acebutolol is marked by one dot and diacetolol by two dots. The use of a radio label permits identification of minor metabolites in a com plex biological matrix. 92

BILE RADIOCHROMATOGRAMS

B) BASE EXTRACT

O 6

2 A) ACID EXTRACT

• •

0 10 20 30 40 50 60 70 80 90 100 FRACTION NUMBER

Figure 38. Radiochromatograms of bile obtained from isolated rat liver which was perfused with labelled acebutolol and then subjected to acid/base fractionation and gradient HPLC. A)acid extractible compounds, B)base extractable compounds. The peaks marked with a dot are acebutolol, with two dots, diacetolol, and with stars, previously undetected metabol ites. A C-8 reverse-phase column was used. 93

PERFUSATE RADIO CHROMATOGRAMS

B) BASE EXTRACT

o _

______CL A)ACID EXTRACT

FRACTION NUMBER

Figure 391 Radiochromatograms of perfusate obtained perfu sion of isolated rat liver with labelled acebutolol after acid/base fractionation and gradient HPLC. A)acid extractable compounds, B)base extractable compounds. The peaks marked with a dot are acebutolol, with two dots, diacetolol, and with stars, previously undetected metabolites. A C-8 reverse-phase column was used. 94

In vivo animal experiments

The success of the isolated liver perfusion technique in studying acebutolol distribution and metabolism is sig nificant only if the findings can be substantiated in the living animal. Here, the conditions are less favorable and the experiment less easy to control.

The in vitro perfusion experiments showed that acebut olol and diacetolol appeared rapidly in the bile following administration of the drug. This conclusion was tested in directly in vivo in the following experiment.

A rat which had been fasted overnight was given a dose of 20 mg of acebutolol by oral gavage. After thirty minutes, the animal was sacrificed. The small intestine was removed and sectioned into 2 cm pieces, which were split and washed with dilute phosphoric acid. The washings were then chrom atographed. The results are shown in Figure 40. It can be seen that even at the farthest end of the intestine, corres ponding to the earliest time, diacetolol is present in the luminal contents, indicating that metabolism and biliary excretion begin as soon as acebutolol reaches the intestinal lumen. As one looks farther back up the intestine, one sees that the concentration of diacetolol is steadily rising, becoming very high near the opening of the bile duct itself.

This is the same pattern seen in the perfused organ discussed earlier.lt has already been shown that extrahepatic metabolism of acebutolol is minor. In this experiment one UG/ML 4 0 iue 0 Cnetainv. lengthofacebutolol anddia Concentrationvs. Figure 40. eoo i telmnlcnet fart ml intestine small theluminal30contents aftermin administrationofa rat of 20 oral acebutolol.mg in cetolol ACEBUTOLOL • o DIACETOLOL

M INTESTINE CM 20 0 3

0 4 vO VJI 96

can also see that direct elimination into the gut across the

mucosal lining must also be very minor. Such an elimination

would have the effect of raising the entire curve off the

baseline. The results show minimal amounts of either com

pound in the troughs between large, peaks which must repre

sent sequential stomach emptyings.

Figure 4l shows HPLC/radiochromatograms obtained from

urine and feces of a rat administered 20 mg of labelled

acebutolol orally. As can be seen, new metabolites appear in

both urine and fecal material, indicating these compounds

are formed in vivo as well as in vitro. The pattern shows more of the polar metabolites in the urine and more of the

less polar metabolites in the feces, repeating the results

seen in the liver perfusion experiments. 97

9 8 A) FECES 7 6

3 2 o o o +

URINE

• •

FRACTION NUMBER

Figure 41. Radiochromatograms of A) feces and B) urine from an intact rat following oral administration of labelled acebutolol. The urine was acidified and injected onto the HPLC column. The fecal material was extracted with acetonitrile, evaporated, reconstituted with water, and then injec ted. A C-18 reverse-phase column was used with gradient elution. 98

HUMAN STUDIES

The ultimate goal of most drug investigations is eluci dation of the effect of the compound on humans. The value in a study of a drug in human volunteers lies in the determina

tion of the drug's kinetic parameters, both of the parent

compound and any significant metabolites. In addition, the physical effects or side effects of these compounds must be assessed. The determination of such values as average half

life, clearance, peak concentration and others is essential for rational prescribing and use. If in addition the drug

can be administered by more than one route, the metabolic and kinetic profiles of each route must be determined and

compared, ideally by administering the drug by both routes

to the same individual. Also importantly, the spread of values in different individuals must be assesed. The "aver

age" is often most misleading in any given individual. This

is especially important when oral administration is contem plated. This is often the most variable of the routes used.

HPLC methodology

For the large number of samples to be examined in this

study, a new HPLC separation procedure was developed,

centered on the use of a reverse-phase phenyl column. This

type of column offered certain advantages over the

traditional C-18 reverse-phase column. Considerations

Instrumental in the choice of a phenyl reverse-phase column 99 were:

1) The use of methanol as a co-solvent. It is more capable of hydrogen bonding than acetonitrile. Methanol also en hances the solubility of compounds of the type being anal yzed on the phenyl column, leading to shorter retention times. In contrast, the use of methanol with the C-18 column lead to such a decrease in retention time that the compounds overlapped unless the organic fraction of the mobile phase was decreased to a very small value. This led to a severe broadening and tailing of the compounds, especially acetolol, and made area quantitation less reliable.

2) Unique retention properties of the phenyl column. The phenyl column was initially used during a series of deter minations of aspirin and salicylic acid in serum samples.

The ortho-configuration of the hydroxyl and carboxylic acid groups in salicylic acid give it an anomalously low pK value compared to the other positional isomers of this compound.

As a result, the use of a simple charge-suppression mobile phase with a C-18 column is Ineffective in producing a well- defined chromatographic peak for this compound. The pK is close to the lower operational pH limit of the silica-based column. This resulted in severe tailing of the salicylate peak. In the past, this problem has been overcome by use of buffers utilizing ion-pairing. It was desired, however, to keep the system as simple as possible. It was found that use of simple ion-suppression in conjunction with the phenyl 100 column gave sharp, well-defined peaks for both salicylate and aspirin with reasonable retention times. Since a similar problem was occuring with the separation of acebutolol and its metabolites (the aniline nitrogen of acetolol was only partially protonated under charge-suppression techniques, again leading to peak tailing), it was decided to use the phenyl column to solve this problem, which it did in a very satisfactory manner.

3) Simplicity of the solvent system. The phenyl column allowed use of a very simple solvent system, not requiring precise buffers or ion-pairing agents. This offered substan tial savings in time and money. Since use of ion-pairing agents is known to shorten the life of a bonded-phase col umn, the savings could be substantial. In practice, a single phenyl column was used for over 1000 injections during a five month period before any significant deterioration in performance was noted. Some of this may have been attribu table to lack of a guard column, however.

To aid in the selection of optimum separation parame ters, a family of retention curves was generated for the four compounds of interest (Figure 42). The data were plot ted as percent organic mobile phase vs. retention time for three different organic mobile phase compositions: methanol/acetonitrile 1:4, 1:1 and 2:1 (vol:vol). As can be seen in the figure, all the components with the exception of acetolol show an inverse exponential relationship of 101

50-1

48- 46-

44- 42-

4 0 -

38-

36-

34-

30"

2 6 - \ V

2 2 - X

20- V v

200 300 400 500 600 700 800 900 RETENTION TIME (SEC)

Figure 42. Families of retention time curves for acebutolol, diacetolol, acetolol and internal standard, both as a func tion percent organic solvent (%B) and organic solvent compo sition. (®)=acebutolol, (<§))=diacetolol, ( O) =acetolol, (®) =internal standard. Dotted lines=l:4 methanol/acetonitrile, solid lines=l:l methanol/acetonitrile and broken lines=2:l methanol/acetonitrile. 102

retention time to percent organic solvent, with the curves displaced toward increasing retention times as the relative

ratio of acetonitrile is increased. Acetolol on the other hand shows a logarithmic relationship under the same condi

tions, the overall result was that the regions of maximum

retention time variability were at different portions of the graph for the aniline vs. the amide compounds. A compromise

solution was reached by balancing several simultaneous fac

tors as follows:

1) System pressure vs. retention time: Increasing the meth anol concentration decreased retention time for all com pounds and allowed a faster sample throughput, but at the

cost of high column back pressure (due to the increasing viscosity of the solvent mixture). Excessively high column

back pressure leads to rapid column deterioration and shor

tened effective life. The maximum methanol concentration

consistent with a system total back pressure of '4000 psig was chosen.

2) Resolution from background impurities: Increasing the methanol/acetonitrile ratio resulted in earlier elution of

interfering background peaks. The limiting factor here was

the desire to have all the UV-active impurities eluted and

the detector output back to baseline prior to elution of the

earliest peak of interest (acetolol).

3) Maximum separation of the four peaks of interest: This is

a function of both relative retention time and peak 103 sharpness and symmetry. Resolution was most easily con trolled by choosing a range of % organic values where the acetolol retention time was essentially constant. In prac tice this was in the region 22-28%.

4)Sensitivity to pH: A minor drawback to this system is sensitivity to minor changes in the acidity of the mobile phase; a change of 0.05 pH units in the sensitive region can result in a change in retention time of 20-30%. Such a variation can occur between changing bo.ttles of solvent. By adjusting the range between 22-28% in the organic phase, however, minor day-to-day variations were quickly compen sated while retaining good separation and retention times.

In final practice, the system was stabilized initially at 25% organic phase, and calibrated for retention time and optimum separation by injecting a standard mixture of all four compounds prior to the start of a sample series. Any significant deviation from the previous series could then be compensated for by a small change in the organic phase. Such changes were never greater than 2% for the duration of the study (Figure 43)•

Accuracy of the assay

Periodic calibration curves were constructed throughout the assay period of the study. At a minimum, a new curve was constructed for each new batch of mobile phase prepared, since minor batch-to-batch variations could affect peak 104

® o <0 « o ® 0 SO

• — 1 fi° e o EACH PEAK o 200ng/ml STANDARD 500 ng/ml

1 HPLC OF STANDARDS

Figure 43* Ultraviolet chromatogram of acebutolol, diacet olol, acetolol and internal standard, under the conditions used for the human study assay. The concentration of stan dard was fixed at 500 ng/ml for all samples. The peaks represent 200 ng/ml of each of the other compounds. The time values are for retention in seconds. The chromatogram is taken from a standard curve, where the compounds were ex tracted from 2 ml of blank plasma. 105 shape significantly. Also, the primary focussing mirror of the detector needed to be cleaned of tarnish periodically

(this is easily accomplished by rubbing the surface of the mirror with a fingertip and a slurry of ordinary chalk dust and water). Since reflectivity in the ultraviolet is very sensitive to the condition of the mirror surface, the total beam intensity passing through the sample cell could vary significantly, with concomitant effects on detector perfor mance. The coefficient of variation between slope values is less than 3% for each group, and that the correlation coef ficient for each individual line is greater than 0.999*

Clearance values

The areas under the time-concentration plasma curves

(AUC) for the oral and intravenous doses are listed in

Tables 7 and 8, respectively. The average value for the AUC in the oral doses was 357+110 mg/L/min and for the IV dose

199+^6 mg/L/min. The clearance was calculated by dividing the dose by the AUC value, and the clearance per kilogram of body weight by dividing by the clearance value by the sub ject's body weight as measured at the start of the regimen.

The individual values are also listed in Tables 7 and 8. The mean+the standard deviation for all subjects were: oral,

1193+279 ml/min and 16.1+3-8 ml/min/kg; IV, 505+115 ml/min and 7.2+1.5 ml/min/kg. 106

TABLE 7 VALUES FOR AUC, CLEARANCE AND CLEARANCE/KILOGRAM FOR 400MG ACEBUTOLOL ORAL DOSE

AUC(MG/ML/MIN) CLEARANCE(ML/MIN) CLEARANCE(ML/MIN/KG)

315 1268 17.4 542 738 10.2 230 1735 20.3 309 1292 15-0 305 1310 17.2 682 586 9-2 350 1142 15.3 335 1192 17-5 331 1207 15-5 279 1432 23.4 253 1575 21.4 373 1072 14.8 313 1276 18.3 378 1056 12.8 380 1052 13.0 347 1149 16.1

357+110 1193+279 16.1+3.8 107

TABLE 8 VALUES FOR AUC, CLEARANCE AND CLEARANCE/KILOGRAM FOR 100MG INTRAVENOUS ACEBUTOLOL

AUC(MG/ML/MIN) CLEARANCE(ML/MIN) CLEARANCE(ML/MIN/KG)

159 630 . 9.0 157 635 8.8 207 483 5-6 258 387 5-1 228 437 6.9 143 699 9-3 229 437 6.4 164 610 7.8 300 333 5-4 156 642 8.7 173 578 8.0 170 588 8.4 219 457 5*5 175 571 7.1 245 407 5-7

199+46 508+115 7. 2+1. 5 108

Table 9 shows the values for terminal half-life, time to maximum concentration, and maximum plasma concentration for the oral doses. The mean+standard deviation for oral t is 202+36 min, and for C 1023+304 ng/ml for 1/2 terminal max acebutolol. Table 10 shows the same values for oral diacet olol, with mean t =459+127 min and mean 1/2 terminal C =974+379 ng/ml. Table 11 gives these values for oral max acetolol, with mean t =202+74 min and 1/2 terminal C =265+147 ng/ml. Table 12 gives these values for the IV max doses, with mean acebutolol t =201+20 min and 1/2 terminal mean acebutolol C =2553+563 ng/ml. max Figure 44 shows a typical set of plasma acebutolol and diacetolol concentrations during, and following an infusion of 100 mg acebutolol at a constant rate over 15 min. There is a very rapid fall off of acebutolol concentration after the infusion ceases, then a more gradual decline. Diacetolol concentration also increases rapidly, but levels off quickly after the end of the infusion at a relatively low level, generally in the range of 100-150 ng/ml, and thereafter remains essentially constant for many hours. The level of acetolol present at any time in the IV doses was usually near the detection limit of the chromatograph and was never significant. The mean clearance value calculated for acebu tolol of 508 ml/min, or 7.2 ml/min/kg, was in close agreement 34 with those found by Meffin et.al of 555 ml/min, or 6.6 ml/min/kg in nine subjects. Areas under the curve for both 109

TABLE 9 TERMINAL HALF-LIFE, TIME TO MAXIMUM CONCENTRATION AND MAXIMUM CONCENTRATION OF ACEBUTOLOL FOR 400MG ORAL DOSE

(MIN) t (MIN) C (NG/ML) max max 210 40 1385 293 180 687 196 180 570 142 180 1207 170 40 924 190 180 1517 254 90 631 179 120 1134 227 240 988 193 180 918 183 40 679 227 180 1143 171 180 904 207 120 901 202 120 1538 205 120 1245

202+36 1023+304 110

TABLE 10 TERMINAL HALF-LIFE, TIME TO MAXIMUM CONCENTRATION AND MAXIMUM CONCENTRATION OF DIACETOLOL FOR 400MG ORAL DOSE t (MIN) t (MIN) C (NG/ML) 1/2 max max

435 180 813 547 180 613 304 240 871 395 240 946 314 120 1304 551 240 731 398 120 755 403 240 637 452 240 775 547 180 1658 308 180 1209 379 240 734 457 240 679 792 120 1098 469 120 1934 604 180 835

459+127 974+379 Ill

TABLE 11 TERMINAL HALF-LIFE, TIME TO MAXIMUM CONCENTRATION AND MAXIMUM CONCENTRATION OF ACETOLOL FOR 400MG ORAL DOSE t (MIN) t (MIN) C (NG/ML) 1/2 max max

144 60 308 146 180 502 179 180 173 151 180 397 140 90 143 334 240 372 235 90 377 151 180 534 221 240 89 146 180 160 156 240 99 197 180 384 182 240 266 165 240 108 344 120 131 347 180 194

202+7A 243+15A 112

TABLE 12 TERMINAL HALF LIFE, TIME TO MAXIMUM CONCENTRATION AND MAXIMUM CONCENTRATION OF ACEBUTOLOL FOR 100MG IV DOSE

fc (MIN) t (MIN) C (NG/ML) 1/2 max max 206 15 2100 192 10 2663 236 15 2023 183 15 3225 192 15 3385 215 15 3094 181 15 2037 .199 15 2478 177 20 2070 211 15 3173 237 10 2004 191 15 2253 191 15 1786 212 15 2708 168 15 2622 213 15 3343

201+20 2553+563 LOG (NG/ML) iue 4 Graphofacebutolol(®) anddiacetolol(o) concen Figure 44. subject recieving100 intravenous mg acebutolol. rto v. ie ntepam farpeettv human time intheplasma ofrepresentative a vs. tration 2 2.5- 3.0 3.5- . 0 - 120 0 6 4 360 240 ACEBUTOLOL • DIACETOLOL ° MINUTES 600 720 1440

113 114 those data and the present values were calculated by the trapezoidal rule.

Perhaps the major discrepancy in the reported data when compared to the literature values are the half-life esti mates. The generally accepted values are those determined by 34 Meffin et al. which give a fast half-life of 6.1 min and a slow half-life of 157 min, whereas the value determined in this study is 202 min, a 45 min discrepancy.

The answer to this difference seems not to lie in the data themselves, but in the method of analysis. Meffin assumed a biexponential model of elimination and used a recursive best-fit computer routine to determine his kinetic parameters. For this study, the analysis was model-indepen dent, using curve-stripping routines described earlier.

Figure 14 illustrates the effect of these differing proceedures. This is a graph of the values of acebutolol plasma concentration for a typical subject. Fast and slow half-lives were calculated from this data, first assuming a biexponential best-fit, then using curve stripping. The slow half-life from a biexponential fit was l66min, in excellent agreement with the 157 min literature value; the fast half- life was found to be 7-8 min, again in good agreement with the literature’s 6.1 min. When the curve was succesively stripped, however, a different picture appeared. The termin al half life(’’slow") was found to be 192 min, in good agree- 29 ment with the earlier value proposed by Kaye of 192 min 115

also. The plot of the first residual curve, however, showed

a definite inflection, indicating the existence of another

exponent. A second stripping gave a line with a half-life of

37.6 min, and the fast half life was reduced to 4.2 min.

In his analysis of Kaye's previous work, Meffin pointed

out that in his analysis Kaye calculated his curve fit

assuming a bolus dose instead of the constant rate infusion

actually performed, resulting in an underestimation of the

area in the eraly portion of the plasma concentration curve.

Purely fortuitously, the nonspecific spectrophotometric method of analysis used by Kaye measured diacetolol as well

as acebutolol, so that areas inm the later portion of the

curve were overestimated, the slope was decreased, and the

half-life found to be larger, as well as giving a clearance

value in close agreement with Meffin's data. In fact, It

appears that both were correct in part, and that ignoring

the actual shape of the curve lead both to different ans

wers. An examination of the data published by Meffin in fact

seems to show that his curves are doubly inflected, a fact

reinforced by his plot of weighted .residuals from the fitted

curve. The values for the three half lives for the nine

subjects in this study who showed this trend are shown in

Table 13. The point of this discussion is not to argue over

which half-life is right. It seems more productive to

speculate on the origin of the intermediate value instead. 116

TABLE 13 HALF-LIFE VALUES FOR ACEBUTOLOL PLASMA CONCENTRATION IN MINUTES t t t alpha 1/2 beta 1/2 gamma 206 23.4 3.0 236 27.4 4.1 192 26.2 3.0 215 26.8 2.0 181 23.5 3.2 199 20.5 4.4 237 15-1 2.7 191 23.6 1.9 213 27-7 2.4

206+19 23.8+4.0 3.0+0.8 117 40 In a preliminary communication by Gabriel , the total bioavailability of acebutolol both orally and intravenously was determined by analysis of 0-96 hr fecal and urine sam ples from a single volunteer. It was found that after a 100 mg intravenous dose, 36% of the material had been metabol

ized to acebutolol, 33% of the total drug plus metabolites was eliminated in the feces, and the fecal component was

50.5% diacetolol. For a corresponding 400 mg oral dose, 53% of the drug had been converted to the acetyl metabolite.

Thus, while the C and AUC values of diacetolol are 5-7 max times lower in the intravenous subjects than the oral ones, most of this due to the fourfold lower IV dose. The magni

tude of the"first-pass effect", which looks very large in

deed upon casual examination of the curves, is at most an

increase in metabolite production of 50% in terms of total

excreted material; this is certainly not in the league with

drugs such as the orgainc nitrate antianginals, where the

same effect is often several thousand percent. The first

pass effect seems therefore more important in terms of

distribution (t , C of acebutolol, for example) than max vmax■ in terms of metabolism. This point will be returned to in

the discussion of the results of the oral doses.

In Figure 45 a plot of the clearance per kilogram of

body weight vs. the maximum plasma acebutolol concentration

is shown. The values form a narrow band, and the slope of

the best-fit line indicates that a 50% decrease in C max 118

3600-1

o 3200 - 8

2800'

iv=o

2400- ORAL = • O

O ~ 2000 E S.O) c

o,E 1600

1200-

• • 800-

4 0 0 -

\ I I i i "I 4 8 12 16 20 24 28 CLEARANCE (ml/min/kg)

Figure 45* Plot of maximum plasma acebutolol concentration vs. calculated clearance for subjects recieving 100 mg IV (O) and 400 mg oral (•) acebutolol. 119 corresponds almost exactly to a similar increase in clear ance per kilo. It is difficult to see how a purely random error, such as could be produced by insufficient time res olution of t at the early portion of the curve, could max produce such a systematic spread. Furthermore, most of the values are bunched at the extremes of the range, giuving a pronounced bimodal distribution to the data, such that the mean values correspond to the most lightly populated area of the graph. There is clearly a significant distribution in the population studied with regard to the IV clearance.

Oral

Figure 46 shows typical sets of plasma acebutolol, diacetolol and acetolol concentrations vs. time following ingestion of a 400 mg capsule of acebutolol. There is a slower, sometimes stepped rise in the concentration of all components when compared to the IV data; acebutolol and acetolol peak ^earlier than diacetolol. In contrast to the IV doses, diacetolol is usually the predominant plasma species after 3hrs. Also in contrast to the IV subjects, acetolol was a significant species in all persons assayed, although the amounts varied by a factor of five or more. The mean clearance value calculated for acebutolol was 1193+239 ml/min (16.1+3*8 ml/min/kg). This compares to values derived from data by Kaye of 1556 ml/min and by Meffin of 1288 ml/min (17*9 ml/min/kg). The mean terminal half-life found 120

3.0

.J S

o o

3.0

24 48 HOURS

Figure 46. Typical concentration curves for acebutolol (•), diacetolol (★) and acetolol (□) in human subjects recieving 400mg oral acebutolol. 121 for oral acebutolol was 201+36 min. In a small patient sample (6), Meffin found a half-life of 2.6hrsJ using vary ing oral doses, Kaye in a study of eight volunteers found a terminal half life of 3.1hrs, with mean peak acebutolol concentrations of 649ng/ml and peak diacetolol concentra tions of 821 ng/ml. The present study found values of 974 and 1023 ng/ml, respectively.

In Figure 45 the lower portion of the graph shows a plot of clearance vs. C for the oral doses. The spread in max clearance is broader than the IV data. The mean clearance is about twice that of the IV subjects, showing the effect of the first pass. The clearance shows a unimodal distribu tion, with more than half the values clustering around the

15-18 ml/min/kg range, different than the bimodal distri bution seen in the IV data.

When the C values for diacetolol and acetolol are max plotted against each other for the oral group an interesting pattern emerges (Figure 47). For those subjects with a diacetolol value below 800ng/ml, the acetolol value begins rising rapidly. There is a subpop.ulation in the group who either acetylate the acetolol more slowly or debutyrate the acebutolol more rapidly.

In his study of oral acebutolol kinetics, using the 37 same dose as used in the present study, Kaye examined the urine of his subjects for the presence of acetolol qualita tively using thin layer chromatography, and found it present a E ro ACETOLOL 400 - 400 600 0 - 500 200 0 - 300 100 -i - - aiu pam ictll ocnrto o ua subjects plasmadiacetolol concentration for human maximum Figure 47. Plotof maximum plasma acetolol concentration vs. recieving 400mg oralacebutolol. 0 400 200 0 80 00 20 40 60 1800 1600 1400 1200 1000 800 600 i i ----- Cmax DIACETOLOL 1 ----- ~ " ' I I ' "H r~

2000 122 123

in four of eight volunteers. The present study is the first

reported to quantitate acetolol in human plasma, and the

pattern is similar. There is a subpopulation in the oral

group who show.much higher plasma acetolol levels. Further more, these show a correlation with lower plasma diacetolol

values. In his study, Kaye determined the acetylator status

of his subjects for sulfadimidine, and found no correlation

between this value and diacetolol formation. If the acetyla

tor status of the subjects in the present study is similarly

invariant, then the higher acetolol levels seen in some

subjects would be due to more rapid debutyration.

The perfusion experiments showed that the tissue affin

ity of acetolol , like diacetolol, is tenfold lower than

acebutolol. In these subjects, the faster rate of acetolol

production leads to loss from tissues (essentially the liv

er) and movement into the central compartment.

Another explanation for the observed higher acetolol

levels in some subjects could be related, not to intrinsicly

faster debutyration, but rather to faster absorption from

the intestinal lumen. Metabolism could still be proceeding

under first order conditions, but the total output would be

higher, due to the higher levels of acebtuolol in the liver

which would result from the faster absorption. This would

then lead to saturation of acetylation and accumulation of

acetolol. There is, however, no correlation between acebut

olol t and acetolol production, so this seems unlikely, max 124

The terminal half-life of acetolol Is essentially Iden tical to acebtuolol. With its much lower tissue affinity, one might expect the concentration profile to parallel that of diacetolol, but this is not the case. This may be due to decreased tubular reabsorption by the kidney, or greater tubular secretion. In view of the low levels of acetolol seen in both urinary and fecal samples in other studies, however, it seems more likely that its clearance and half- life are primarily due to metabolic conversion to acebut olol. In any event, further studies are necessary to answer this question unequivocably.

When one examines the pattern of diacetolol curves in the IV data, one is struck by their esential similarity, with little deviation from a plateau value of 100-200ng/ml, in sharp contrast with the concentration swings of the two metabolites seen in the oral doses. It seems possible that an intravenous dose distributes rapidly into the peripheral tissues, while an oral dose is initially seen entirely by the liver (Kaye has shown the bioavalability of acebutolol 40 is at least 90% ; the lower values seen in the literature reflect the highly nonrenal mode of elimination for this drug). It is reasonable to assume that the larger amount of acebutolol seen by the liver in the oral dose may saturate the acetylation capability of some persons, leading to a buildup of acetolol in the plasma. This could also explain the fact that in several earlier studies, oral acebutolol 125 has sometimes resulted In appearance of acetolol In the urine, sometimes not; It would be Instructive to determine the acetylation capability of those persons who show high levels of acetolol following oral acebutolol.

The rat studies showed that significant amounts of diacetolol and acebutolol are excreted in the bile, and that diacetolol has a much lower tissue affinity than the parent drug. Thus, diacetolol blood levels may be controlled by a) biliary excretion rate, b) acetylation activity lev els,c) reabsorption of excreted material from the intestinal lumen and c)excretion by the kidney. One would expect intes tinal reabsorption to be lower than the parent drug, due to the decreased solubility of diacetolol in tissue .

Meffin has shown that diacetolol blood levels are 2.5 times higher in a person on a multiple oral dosage regimen at 400 mg t.i.d. than for a person recieving a single 400 mg oral dose, and that in a series of rising doses given to the same individual there is a disproportinately large increase in the AUC. This accumulation may be a result of acetyla tion proceeding faster than biliary excretion, which has become saturated, thus shifting the entire elimination curve upward. It will be recalled in the rat studies with isolated livers that very large doses of acebutolol resulted in zero- order kinetics for drug elimination and metabolite forma tion. If this explanation for the increased diacetolol con centration is correct, it implies that elimination is 126 saturated at concentrations of acebutolol lower than those needed for saturation of acetylation. The disproportionate growth of the AUC of acebutolol noted by Meffin and others with increasing dosage was explained as being due to a) in creased bioavailability or b) decreased clearance. Meffin mentioned that a decreased clearance could be due to de creased hepatic or renal uptake, and speculated that the metabolite diacetolol could cause these increases by inhibi ting hepatic or renal processes, but felt it most likely that the abscense of increasing t values with increasing 1/2 dose size made an incresae in systemic availability more likely. Such an availability could be the result of satura tion of the biliary excretion mechanism for acebutolol as well as diacetolol. One implication of the above discussion is that the doses of acebutolol currently used in therapy may be close to the upper limit of first-order processes for the average population, and that higher dose levels may show the appearance of mixed-order-type kinetics. The nonlineari ty of the acebutolol dose/AUC curve at higher dose levels may be a manefestation of this effect.

In this study, many of the IV concentration graphs show a small peak in the diacetolol curve (and acetolol curve as well; usually, acetolol is seen in the IV doses only in the early samples) around 30-60min post-dose. This could be due to the transiently higher acebutolol concentration in the plasma before primary distribution is complete, with a 127 corresponding briefly higher acebutolol level In the liver.

This would be reflected In a temporary Increase In diacet olol and acetolol production. Both In this and earlier studies the diacetolol curve settles Into a long flat region extending out 10-12hrs. Now, the diacetolol concentration can remain constant only if it is being replaced as fast as it is being lost, that is, if the relative rates of metabol ism and renal and biliary elimination are constant. This could be the case if all three are first order concentra tion-dependent processes; it is possible that at the low levels of acebutolol present in the liver during elimination of an IV dose this is what occurs. This also explains the abscence of acetolol in the IV doses; the acebutolol levels never get high enough in the liver to cause saturation of the acetylating enzymes. This may be a function of both the route of adimiistration and the fact that the dose was four times lower.

The source of the intermediate half-life seen in the IV data seems most likely to be a result of distribution rather than an elimination process, although the definition here is rather arbitrary. In all likelyhood, this section of the concentration curve represents the filling and equilibration of the enterohepatic compartment with the rest of the sys tem. The variability of this system was reflected in a small minority of the subjects who did not show a clearly defined set of three half-life values. When the concentration curves were stripped, the plot of the first residuals did not show a cleanly inflected line, but neither was it smoothly linear (which would indicate a two-compartment system). Instead, it showed a smooth declining curve.

This may indeed be a reflection of deviation from first- order kinetics in the biliary excretion/luminal reabsorption of acebutolol in a portion of the population. CHAPTER 4

SUMMARY

This has been a study of the disposition and metabolism of acebutolol in rats and humans. The animal experiments, which included oral and intraperitoneal dosing studies in whole rats, as well as administration to perfused livers, indicate that acebutolol is metabolized by a two-step process. The rate-limiting step is debutyration. The behavior observed in the perfused liver was an excellent indicator of the behavior of the drug in the whole animal.

A number of new metabolites were discovered in the course of the animal studies via the use of specifically radiolabelled acebutolol as a probe. An improved synthesis of acebutolol was developed and used to prepare the labelled compound.

The human study, which was the largest kinetic study reported to date, included the first simultaneous quantitation of the kinetics of acebutolo and both of its known metabolites. Intravelous doses were shown to result in triphasic elimination of acebutolol. It is felt that this represents the filling and subsequent equilibration of the enterohepatic subcompartment. Elevated levels of acetolol, the intermediate in the conversion of acebutolol to

129 130 diacetolol, were found in the plasma of approximately half of the subjects after' a 400 mg oral dose was administered.

Thus, It would seem that a large portion of the population will have elevated levels of a circulating aniline derivative following oral doses of acebutolol in the normal theraputic range. This is of concern due to the direct physiological effects of activated aniline derivatives, and also for the potential of reaction with other materials in the gut, such as nitrites, which could -result in the formation of potenitally carcinogenic nitrosoamines. BIBLIOGRAPHY

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