THE METABOLISM AND PLASMA CONCE~~RATIONS OF

L-ALPHA-ACETYLMETHADOL AND ITS METABOLITES IN MAN

by

Bryan Smith Finkle

A dissertation submitted to the faculty of the University of Utah in partial fulfillment of the requirements

for the degree of

Doctor of Philosophy

Department of Pharmacology

University of Utah

June 1977

THE UNIVERSITY OF UTAH GRADUATE SCHOOL

SUPERVISORY COMMITTEE APPROVAL

of a dissertation submitted by

BRYAN SMITH FINKLE

I have read this dissertation and have doctoral degree.

�-/q- 27 Dale

I have read this dissertation and have found it to be of satisfactory quality for a doct{;;i �gE� Z2

.�� (/ .� Date Salvatore :v. Fidone, Ph.D.

Member, Supervisory Committee

I have read this dissertation and have found it to be of satisfactory quality for a

doctoral degree.

<{- 77 Dale

Michael R. Franklin, Ph.D. Member, Supervisory Committee

I have read this dissertation and have found it to be of satisfactory quality for a

do�toral deere!;:.

�- 9� 77 C M.D., Ph.D.

Member. Supervisory Committee

I have read this dissertation and have found it to be of satisfactory quality for a doctor�e?": �

� ...J tZ�/ � ',r S, -i,..t... ... " ....

Date J Louis S. Goodman, M .• D., Ph.D. Member. Supervisory Committee

have read this dissertation and hay 1 for a dOC;7;%

a Dale /

I have read this dissertation and

dOCIoraizre% 5'��='7

Date '

have found it to be of satisfactory quality for a

Oi on M. Woodbury, P � . Member. Supervis0ry Committee

THE UNIVERSITY OF UTAH GRADUATE SCHOOL

FINAL READING APPROVAL

To the Graduate Council of The University of Utah:

I have read the dissertation of Brya n Smi th Fi n k 1 e

m lts

final form and have found that (l) its format, citations, and bibliographic style are

consistent and acceptable; (2) its illustrative materials including figures, tables, and

charts are in place; and (3) the final manuscript is satisfactory to the Supervisory

Committee and is ready for submission to the Graduate School.

May 18, 1977

Date

Approved for the Major Department

Chairman! Dean

Approved for the Graduate Council

ABSTRACT

The plasma disposition of L-a-acetylmethadol

(LAAM) and its two active metabolites, nor-LruLM and

dinor-LAAM, has been determined in 12 human subjects

as part of a controlled, clinical pharmacological

study. h~ was administered orally three times per

week for ten doses, ranging from 0.73 mg/kg to 1.51

mg/kg (80 mg doses), with five subjects receiving 1.0

mg/kg. Plasma concentrations were followed at short

intervals after the first three and last doses and for

a total of approximately 1000 hr, where possible. A

new quantitative, specific, and very sensitive analy­

tical method was developed for the study and is re­

ported in detail. The method involves organic solvent

extraction, amide-derivative formation of the metabo­

lites, and gas chromatography-chemical ionization­

multiple ion monitoring-quadrupole mass spectrometry,

with methane as carrier and ammonia as reagent gases.

Deuterated stable isotopes of LAAM and the two metabo­

lites are used as internal standards. The method has

quantitative sensitivity to 5 ng/ml for a 2.0 ml plasma

sample with 10-15% accuracy and precision. For the

range used in the study, the dose of LAAM is not a

critical variable with respect to ultimate plasma con­

centrations achieved and cannot be used to predict max­

imum concentrations or the possibility of drug or

metabolite accumulation. The mean time taken to reach

maximum plasma concentration after the first dose of

LAAM was 4.4 hri for nor-LAAM, 5.6 hri and for dinor­

LAAM, 6.6 hr. There was no change in these values

after the tenth and final dose. Maximum concentrations

after the first dose varied from 52 to 510 ng/ml for

LAAMi 65-175 ng/ml for nor-LAAMi and 11-92 ng/ml for

dinor-LAAM. Three subjects cont~nuously accumulated

L~1 and both metabolites; these three plus two others

accumulated LAAM, and the five plus one other accumu­

lated dinor-LAAM. The otherffix subjects reached plateau

concentrations after the anticipated 4-5 half-lives of

elimination. The mean half-lives in hours were LAAM

t~~ 2.4, t~S 37.5 (first dose), t~B 46.8 (last dose)i

nor-LAAM, t~B 38.2 (first dose), t~B 64.6 (last dose);

dinor-LAAM, t~B 168 (last dose). The maximum plasma

concentrations increased over the ten-dose period by

a factor of approximately 2.0 for LAAM, 2.0-4.0 for nor­

LAAM, and 4.0-10.0 for dinor-LAAM. The t~B for LAAM was

independent of dose throughout the study, and the plasma

concentration-time-course profiles are generally con­

sistent with a two-compartment, first-order kinetic

model.

vi

ACKNOWLEDGEMENTS

I wish to take this means of expressing my deep

appreciation to certain members of the College of Medi­

cine, Pharmacology Department, faculty and staff without

whose interest, advice, and constant support my educa­

tion and this research could not have been completed.

They are Drs. Louis S. Goodman, Ewart A. Swinyard, James

w. Freston, and James W. Gibb.

In addition, the skillful and dedicated techni­

cal assistance of Messrs. Thomas A. Jennison and Dennis

M. Chinn at the Center for Human Toxicology was an inte­

gral and indispensable part of the analytical work.

Clinical management of the human subjects was carried

out by Drs. Walter Ling, Elaine D. Holmes, and Stuart

SeIter at the Veteran's Administration Hospital,

Sepulveda, California. Their cooperation in executing

protocols involving drug administration and specimen

withdrawal in very exacting circumstances is acknowl­

ledged.

Finally, the research was financially supported

throughout by the National Institute on Drug Abuse

under contract numbers DA-01391 and 271-76-3323.

TABLE OF CONTENTS

ABSTRACT. . . . . . .

ACKNOWLEDGEMENTS.

INTRODUCTION. .

METHODS . . •

Analytical Methods.

Analytical Method Critique.

Extraction Procedure ••.

Gas Chromatography-Mass Spectrometry ..

GC-MS Data, Calculation, and Manipulation •

Analysis of Biological Test Samples •

Clinical Study. • . . •

RESULTS . . • • •

DISCUSSION.

REFERENCES ..

FIGURES .

APPENDIX:

VITA. . •

Tables .

Page

v

vii

1

12

12

18

18

22

27

30

31

36

45

53

60

224

266

INTRODUCTION

In 1965 Dole and Nyswander reported on the use

of methadone as a medical treatment for heroin addic­

tion, and the following year Dole et ale (1966) des­

cribed the rationale for methadone maintenance programs ..

They stated that, by inducing tolerance to methadone in

the heroin addict and subsequently maintaining the tol­

erant condition by daily oral doses of methadone, the

pharmacological action of heroin was blocked and the

addict's compulsion to use opiate narcotic drugs was

eliminated. Thus, methadone maintenance was found to

be an effective means of treating heroin dependence

in individuals who were apparently unresponsive to other

forms of rehabilitation.

Since 1969 there has been a flood of papers

published on almost every conceivable aspect of metha­

done maintenance programs. Those by Jaffe (1972),

Goldstein (1972), Kreek (1973), ~1artin et ale (1973),

Lipsitz and Blatman (1974), and Harms (1975) are repre­

sentative. Methadone has not proved to be a panacea

for all cases of narcotic drug dependence. Al though

some of the problems related to its clinical use can be

attributed to poor administration at some clinical

2

facilities, other problems are indirectly related to

the pharmacology of methadone. The drug has a duration

of action of approximately 24 hr and, therefore,

patients must visit the clinic every day to receive the

drug or they must be allowed to self-administer the

medication away from the clinic. Daily visits are a

major inconvenience to the patient and inhibit cooper­

ation; hence, providing the patient with take-home

weekend dosages has become a common practice. There

are serious problems associated with permitting metha­

done to be taken out of the clinic (Jaffe et al., 1970),

including the opportunity for nontolerant individuals,

especially children, to be accidentally poisoned and

the possible illegal diversion of the drug for sale or

illicit use. Both of these possibilities have been all

too frequently realized. Occurrence of these and other

problems would be curtailed if alternative modes of

successful therapy were available, or if patients could

be adequately maintained on a narcotic agonist with a

longer duration of action. Currently, the latter al­

ternative appears to be the best hope for the thousands

of heroin users seeking treatment for their addiction.

The acetylmethadols were synthesized as deriva­

tives of the parent compound, methadone, in an attempt

to find a drug with negligible addiction liability

(see review of Way and Adler, 1960). Figure 1 shows the

synthesis of l-a-acetylrnethadol (LAAM) from methadone.

The synthesis of this drug and preliminary preclinical

investigations of its pharmacology were first carried

out by Chen in 1948. LAAM has an analgesic effect

maintained up to 72 hr in animals (Keats and Beecher,

1952) and man (Fraser and Isbell, 1952). Jaffe and

coworkers (1970) supposed that other pharmacological

effects of LAAM might also persist beyond that known

for methadone, especially those effects which prevent

narcotic abstinence syndrome in physically dependent

individuals. If this proved to be correct, then LAAM

might be useful in lOllg-term maintenance treatment of

heroin addicts. Early work (Fraser and Isbell, 1952)

suggested this reasoning was valid, and more recently

it was concluded from a study in which LAAM and metha­

done were compared in the treatment of heroin users

(Jaffe et al., 1970) that LAAM may possess the sought­

after practical therapeutic advantages over methadone.

Later, it was reported (Jaffe and Senay, 1971) that

3

LAAM provided effective weekend treatment for narcotic

addicts, and it has been suggested that the frequency of

visits to the clinic by addicts could be reduced to

three times each week (Levine et al., 1973). Further­

more, because LAAM is apparently less active than some

of its metabolites (Smith, 1974; Nickander et al., 1974),

the onset of action is delayed until the metabolites

are bioavailable. One result of this is that LAAM is

not likely to reinforce drug taking, because drug

users generally desire drugs with a rapid onset of

action. In addicts, LAAM is more rapidly effective

orally than intravenously, the preferred route of

heroin addicts (Blackly, 1971; Fraser, 1952).

In June, 1971, the Special Action Office for

Drug Abuse Prevention (SAODAP) was established by

Executive Order of the President, and, because of the

potential advantages of LAAM over methadone, it man­

dated the expansion of research on long-lasting, non­

addictive blocking and antagonist drugs or other

pharmacological substances for the treatment of heroin

addiction. Dr. Jerome Jaffe, Director of SAODAP, in­

itiated in 1971 a comprehensive review of the status

of LAAM. The conclusion was drawn from the review

that LAAM was the most promising compound available at

the time but that its pharmacological development was

not proceeding. SAODAP created a governmental mech­

anism for developing LAAM, and in the Spring of 1972,

an Interagency Pharmacology Task Force was formed to

review, plan, and coordinate development work toward

a New Drug Application (NDA) to the Food and Drug Ad­

ministration (FDA).

When SAODAP was phased out of existence in the

Fall of 1974, the coordination and direction of the

L~1 Project, including the Pharmacology Task Force,

4

5

were transferred to the Division of Research, National

Institute on Drug Abuse. Today almost all the Phase I

new drug research is complete, much of Phase II is

complete or in progress, and LAAM is now on the verge

of undergoing large-scale Phase III clinical trials

with FDA approval. Despite this rapid and broad prog­

ress, the pharmacology and toxicology of LAAM are still

not thoroughly studied or well documented. This is

particularly true for the pharmacodynamics and metabo­

lism of the drug in man. There are several reasons

for these deficiencies, but perhaps most important was

the lack of an analytical method by which LAAM and its

principal metabolites could be accurately and simul­

taneously quantitated in small-volume plasma and urine

samples, and specifically identified. It was, there­

fore, the first and foremost objective of the research

described in this report to develop such a method. If

successful, the method would be applied in a controlled

clinical study to determine the plasma time-course dy­

namics of LAAM and its metabolites in human subjects.

The first metabolic study on LAAM was carried

out by Sung and Way in 1954 using rats and mice. The

drug was determined in tissues by a modified methyl

orange-complex technique, but unfortunately, the sen­

sitivity of the procedure was very poor and the re­

sulting quantitative data unreliable. However, it was

noted that uptake was rapid and that high tissue

levels resulted after parenteral administration. The

morphine-like effects did not become apparent until

4 to 6 hr after injection but persisted for at least

24 hr. Following oral administration, the tissue

6

levels were very low, and the onset of action was about

1 hr later. The authors concluded that LAAM was con-

verted to an active metabolite, a process which oc-

curred more rapidly after oral than after parenteral

administration. The effect of prior administration of

the microsomal enzyme inhibitor, SKF 525-A, was also

studied (Veatch et al., 1964) on the assumption that if

LAAM was converted to an active metabolite by liver

microsomal biotransformation, then the SKF 525-A would

inhibit the pharmacological effects, such as analgesia,

of the drug. The study revealed that SKF 525-A did de­

crease the analgesic response to LAfu~. In 1965 McMahon

et ale showed, by means of c14-radiolabelled drug in

rats, that nor-acetylmethadol (nor-LAAM) is a major

metabolite of LAAM. The second methyl group was also

removed to give the dinor compound (dinor-LAAM). It

appeared that the first demethylation occurred at ap­

proximately three times the rate of the second, so that

the primary metabolite, nor-LAAM, tended to accumulate

in the body. Subsequently, this work was confirmed

(Billings et al., 1973) in the rat: there are two active

metabolites of LAAM which are formed by successive

enzymatic N-demethylations, nor-LAAM and dinor-LAAM.

Billings et al. used a gas chromatographic method for

the analysis of nor-LAAM which involved forming the

trichloroacetamide derivative and electron capture de­

tection. The metabolite identities were established

7

by mass spectrometry. Although it is very sensitive,

this method is, unfortunately, complex and time­

consuming and does not lend itself to routine use.

Figure 2 shows the biotransformation of LAAM, includ­

ing a deacetylation pathway to methadols which have

been detected in animal urine. In 1976, Kochhar con­

firmed the identity of the methadols as metabolites of

LAAM after in vitro incubation of the drug with a

microsomal fraction obtained from rat liver homogenates

and, also, from the urine of rats which were given the

drug intraperitoneally.

In a further study, Billings et al. (1974) ad­

ministered 100 mg of LAAM three times a week to three

human subjects and measured the nor and dinor metabo­

lites in blood and urine, using their derivative gas

chromatography procedure. Analyses were made after the

first dose and after repeated doses of the drug. The

results showed that the total blood levels of the two

metabolites were variable between subjects, ranging from

250 to 370 ng/ml of plasma after the first dose, and 300

to 700 ng/ml after the twentieth dose. Kaiko and

Inturrisi also described two slightly different gas

chromatographic assays for LAAM and its metabolites

in 1973 and 1975. In one method, a benzomorphan de­

rivative was used as an internal standard and, in the

other, SKF 525-A was used. Flame ionization was the

method of detection in both procedures, but no account

was made of percent recovery from plasma for LAAM or

the metabolites at the solvent extraction stage in

either method. In consequence, sensitivity limits

8

were barely at the submicrogram level, and the accuracy

of the quantitative analyses is suspect. The methods

were used to determine plasma levels in eight out­

patients receiving 40-60 mg of LAAM three times a week

and to correlate the results with observed changes in

pupil diameter. Peak plasma levels of acetylmethadol

occurred at 4 hr post-administration and almost com­

pletely disappeared at 24 hr. Nor-LAAM plasma concen­

trations peaked in 48 hr and declined slowly over the

following 40 hr. Dinor-LAAM apparently remained con­

stant throughout the treatment dosing interval. The

reported half-lives for LAAM, nor-LAAM, and dinor-LAAM

were extremely variable at 2-12 hr, 13-78 hr, and in­

finitely long, respectively.

In a 1975 report, Henderson and Lau monitored

plasma concentrations of LAAM and metabolites in

9

patients receiving 60-85 mg of LAAM three times each

week and found that plasma levels of the parent drug

were only slightly higher after 90 days than in the

initial period, whereas the two metabolites were 5-10

times greater. These investigators used a gas chro­

matographic method involving halogenation of the drugs

by trichloro-acetic anhydride and electron capture

detection. The method is sensitive in the low nanogram

range but suffers from complex chemistry and interfer­

ences on the gas chromatogram caused by derivatized

biochemical artifacts which disturb the usable sensi­

tivity and quantitative accuracy of the procedure.

Goldstein, in an unpublished 1975 report, also

found by gas chromatography analysis very low plasma

concentrations of LAAM at 72 hr post-administration in

patients maintained on the drug, but dramatically in­

creased levels of nor-LAAM and dinor-LAAM were present

at 24-48 hr, with no decrease in concentrations through

72 hr.

Although several animal studies, including pri­

mates, have been carried out to evaluate the biodispo­

sition and metabolism of LAAM (Sung and Yay, 1954;

McMahon et al., 1965; Billings et al., 1973; Sullivan

et al., 1973; Welling, 1974; Lau and Henderson, 1975;

North-Root and Henderson, 1975; Musta et al., 1976;

Henderson, 1976), the only human studies reported are

10

those of Billings, Kaiko and Inturrisi, Henderson and

Lau, and Goldstein, discussed previously. In each

case, the data were incidental to monitoring heroin

addict patients undergoing treatment with methadone and

LAAM and were obtained by analytical methods with seri­

ous limitations in either sensitivity or quantitative

accuracy. The only other published analytical methods

require tritium or carbon-14 labels for LAAM followed

by thin-layer chromatography and scintillation counting

(Misra et al., 1975), enzyme immunoassay (McIntyre et

al., 1975), or thin-layer chromatography alone (Kuttab

et al., 1976). All these techniques are qualitatively

nonspecific and best suited to urine screening analysis

for the drug.

Nonetheless, it is evident from the animal

studies and limited human data that the active metabo­

lites of LAAM play an important role in the general

action of the drug. An appreciation of their signifi­

cance is critical to understanding the slow onset and

long duration of action, and the apparent cumulative

effects which have been observed. Large variations in

the rates of formation and elimination of LAAM and its

metabolites were noted in all the studies, as well as

considerable variation between individuals in plasma

concentrations of LAAM, nor-LAAM, and dinor-LAAM. If

confirmed, these individual variations, with respect

11

to the metabolism of LAAM, could possibly explain some

of the clinical response differences seen in patients

undergoing therapy with the drug, including adverse

side effects such as anxiety, confusion, and aggression

(Billings et al., 1974; Fraser and Isbell, 1952). The

questions of safety in chronic treatment and cumulative

toxicity still remain to be scientifically investigated.

It is against this background that the need for

a controlled human study to determine accurately the

plasma disposition of LAAM and its metabolites became

clear, with the essential prerequisite of a new analy­

tical method which would be qualitatively specific for

the drug and metabolites, sensitive to concentrations

below 10.0 ng/ml, and have accuracy and precision of

about 10-15% at this concentration. The subject of

this research report is the development of such a

method and its application to determine plasma concen­

trations of LAAM, nor-LAAM, and dinor-LAAM in humans

following oral administration of the drug in acute and

chronic therapeutic doses.

METHODS

Analytical Methods

The first objective of the research was to

develop a new analytical method suitable for routine

detection and simultaneous quantitation of LAAM and

its two principal metabolites, nor-LAAM and dinor-

LAAM, in small-volume blood and urine samples. The

complete procedure is new in all its essentials, in­

cluding sample preparation and extraction, derivati­

zation, and gas chromatography-chemical ionization-

mass sepctrometry-selected ion monitoring, with methane

as the carrier gas and ammonia as the reagent gas. The

d 3 , deuterated stable isotopes of LAAM and its metabo­

lites are used as internal standards. Computer programs

have been written for a desk-top instrument capable of

automatic calculation of plasma sample concentrations

from the raw analytical data, mathematical manipula­

tion, and graphic plotting of time-course concentration

curves. The procedure permits analysis of batches of

40 samples plus ten standards per week. Sensitivity to

less than 5 ng/ml and precision of 15% at 10 ng/ml is

easily maintained.

A schematic flow diagram of the solvent ex­

traction procedure and amide derivative formation

are shown in Figure 3. The following materials are

required:

Apparatus

1. 50 ml g.g. stoppered extraction tubes

2. 50 ml centrifuge tubes

3. Pasteur pipets

4. 2 ml Class A serological pipets

5. 0.5 ml Class A serological pipets

6. Mechanical shaker

7. Centrifuge

8. "Repipets" for solvent dispensing

9. vortex mixer

10. Water-bath with thermostat control

Reagents

1. De1ory-King (1945) carbonate-bicarbonate buffer

(pH 9.78)

13

0.1 M sodium carbonate and 0.1 M sodium bicar­

bonate in a volume ratio 4:6. 0.1 ml of buffer

added to 2.0 ml of plasma gives a pH 9.2

2. n-Butyl chloride. J.T. Baker Chemical Company

3. Chloroform. Ma1linckrodt nanograde

4. Methanol, anhydrous. Mallinckrodt AR

5. 0.2 N Hydrochloric acid

6. 5.0 N Sodium hydroxide

14

7. Drifilm sc-a7. 5% in toluene for siliconizing

evaporation tubes. Pierce Chemical Company.

Standards

Stock solutions in water: 10 ~g/ml of LAAM-HCl,

nor-LAAM-HCl, and dinor-LAAM-maleate. Equivalent to

9.06, 9.00, and 7.40 wg/ml of free base, respectively.

Working standards are prepared by dilution of the

stock solution with blank plasma, as required.

Internal Standards

D3 - LAAM

D3 - nor-LAAM

D3 - dinor-LAAM

(l-a-acetyl methadol, -2,2,3,2H3-HCl)

2 (l-a-acetyl- H3-normethadol·HCl)

(1-a-acetYl-2H3- N,N-dinormethadol.HCl)

Stock solutions of the stable isotope internal

standards are prepared in methanol to 1.0 mg/ml, and a

2.0 ml aliquot is diluted to 1.0 1 with water for a

stock solution. The stock solution is diluted 1:10 in

blank plasma for a mixed working internal standard solu-

tion of 200 ng/ml. The pure LAAM and its metabolites

and the deuterated stable isotopes used in this study

were obtained from Research Triangle Institute (Chemistry

and Life Sciences Division), Research Triangle Park,

North Carolina, U.S.A. through the courtesy of the

National Institute of Drug Abuse, Rockville, Maryland,

U.S.A.

15

Procedure

One ml of mixed internal standard solution and

1.0 ml of buffer are added to 2.0 ml plasma in a 50 ml

g.g. stoppered centrifuge tube. (= 200 ng into std/ml

of plasma.) Ten mls of n-butyl chloride is added and

the mixture is shaken mechanically for 10 min and then

centrifuged at 1500 rpm for 5 min to separate the

phases.

The organic phase is transferred to a second 50 ml

g.g. stoppered tube and extracted with 10.0 ml 0.2 N

hydrochloric acid. The mixture is centrifuged to sep­

arate the phases and the organic solvent then dis­

carded by aspiration.

One-half ml of 5.0 N sodium hydroxide is added to

the acid phase to bring the pH to 13 and the mixture is

then incubated at room temperature for 30 min and then

in a 70°C water-bath for 10 min. This step converts the

metabolites and metabolite-internal standards to their

amide derivatives.

Following incubation, the mixture is cooled and

extracted with 10 ml chloroform by vortex mixing. The

phases are again separated and the chloroform is trans­

ferred to a 50 ml silanized centrifuge tube and evapo­

rated to dryness under an airstream of not less than

200cc/min. The residue is reconstituted in approximately

25 ~l of methanol for GC-CI-MS analysis.

16

Quantitative Analysis by GC-CI-MS--Multiple Ion Monitoring

Instrument: FINNIGAN MODEL 3200 F GC-

MS, fitted with a chemical

ionization source and a six-

channel programmable multi-

pIe ion monitor with a six

pen Rikadenki strip chart

recorder.

Strip chart recorder for

total ion monitoring and

light beam oscillograph for

recording complete mass

spectra.

Gas Chromatographic Conditions

Column: 1.5 m x 2 rom id: glass U-

tube packed with 3.0% OV-17 on

Gaschrom Q. AW. DMCS. 80-

100 mesh.

Carrier Gas: Methane. Flow: approxi-

mately 12 cc/min through the

column, adjusted to achieve

a pressure of 500 ~ in the

MS source.

Temperatures: Injection port and oven:

17

Relative Retention Times: LA~, 0.59; dinor-L~l, 1.00;

nor-LAAM, 1.3.

Elapsed GC Time Per

Analysis: Approximately 3 min.

CI-Mass Spectrometer Conditions

Reagent Gas:

Diverter Valve:

Source Temperature:

Ionizing Voltage:

Filament Emission

Current:

Electron Multiplier:

Multiple Ion Monitor

LAAM

nor-LAAM

dinor-LAAM

Sampling Time:

Recorder:

Ammonia (Union Carbide-Linde.

Speciality Gas Products

Group) 200 ~ source pressure.

open 18 sec post-GC sample

injection.

100 EV.

0.70 milli-amps.

1700 V.

354 amu, D3-LAAM 357 amu

340 amu, D -nor-LAAM 3 343 amu

326 aniu, D3-dinor-LAAM 320 amu

100 milli-sec each channel.

0.5Hz filter.

Chart speed, 1 em/min. Full-

scale deflection adjusted be-

tween 50-100 mv for required

sensitivity.

Typical Analytical Recording

See Figure 9.

GC-MS Data, Calculation and Manipulation

S.P. 4000 Microprocessor with two analog-to-

digital converters (Spectraphysics, Santa Clara,

California) interfaced to the GC-MS multiple-ion mon­

itor and a Teletype-printer output.

18

This device is programmed to integrate, auto­

matically and on-line, the monitored ion peak areas,

calculate the peak area ratios drug:internal stand­

ard for each component, refer the ratio to a stored

standard concentration regression curve, and calculate

and print-out the final concentrations of LAAM, nor­

LAAM, and dinor-LAAM.

The analytical concentration data for LAAM

and the metabolites are entered into a mini-computer via

a keyboard, TEK 4051 (16 K) with Interactive Printer­

Plotter, TEK 4662 Tektronix. The data can then be

manipulated in a variety of ways for statistical anal­

ysis, tabulation, and graphic display.

Analytical Method Critique

Extraction Procedure

Although there are some similarities to the ex­

traction procedures published by Kaiko, Billings, and

19

Lau et al., there are significant differences which con­

tribute to better recovery, accuracy, and ultimate sen­

sitivity. In particular, these are the use of deuterated

LAAM, nor-LAAM, and dinor-LAAM as internal standards,

initial extraction of the sample at pH 9.2, and conver­

sion of the metabolites to their amide derivatives by

incubation at alkaline pH greater than 13, for not less

than 30 min and at least 70°C. No hexane wash or other

clean-up steps are necessary. The final solvent evapo­

ration is carried out in silanized glass tubes, which

also enhances the recovery. The recovery is in the range

of 65-70% for each of the drugs, but it only has meaning

relative to sensitivity limits of the method, because the

isotope standards internally compensate for this and all

other loss factors throughout the procedure. There are

a number of critical steps in this simple procedure to

which careful attention must be paid if best sensitivity

and accuracy are to be achieved. n-Butyl chloride and

pH 9.2 were chosen as the extraction conditions after

experiments clearly showed that extraction efficiency

was pH-dependent and that other solvents, notably chloro­

form at any pH value above neutral, gave very dirty gas

chromatograms and, therefore, demanded that clean-up

steps be introduced--with a subsequent loss in recovery.

The pH dependency of n-butyl chloride has previously

been noted by Walen (1968) in regard to propoxyphene and

20

methadone extractions from plasma. It is simply not

possible to extract nor-LAAM and dinor-LAAM at any pH

above neutral without some spontaneous formation of the

amide derivative. In consequence, after the decision

was made to convert the metabolites to their amides for

quantitative analysis, pH 9.2 assisted the transforma­

tion from the first step of the extraction procedure.

The use of n-butyl chloride does finally produce

very clean gas chromatograms, and the hexane wash pre­

viously reported by Kaiko (1975) is not necessary. How­

ever, following centrifugation to separate the plasma

and solvent phases, a thin, white interface invariably

occurs. It consists of lipid and must not be trans­

ferred with the solvent to the second extraction tube.

Very careful removal of about 8.0 ml of the solvent with

a pasteur pipet is required. Following extraction into

0.2 N hydrochloric acid, the solvent can be removed by

aspiration with a very clean interface. The alkaline

incubation stage was tested to determine the optimal

conditions of pH, time, and temperature for complete

conversion of the metabolites to the corresponding

amides, over a wide concentration range (25 ng -2 ~g in

approximately 5 ml of solution). All experiments were

conducted at pH 13, because it was known that strong

alkaline conditions were required, and this pH value was

easy to achieve by adding sodium hydroxide. It was

21

further determined that temperature was an important

consideration and must be maintained at about 70°C.

Excessive heat will degrade LAAM. Time can also be

critical, and it is essential that at least 30 min be

allowed, otherwise at higher concentrations amide for­

mation will not be complete. Table 1 shows the com­

parative efficiency of various incubation conditions.

The final extraction is with chloroform, be­

cause it is efficient and readily evaporated. The

evaporation tubes were siliconized by standing them for

10 min in 5% Drifilm in toluene, oven drying, and then

water washing, and again drying. Evaporation of the

chloroform extract, either in a 70°C water-bath with

approximately 200 cc/min air flow or at room temperature

(25°C) overnight, provides the best recovery. Supporting

experimental data are given in Table 2. The efficient

reconstitution and concentration of the extract residue

in approximately 25 ~l of methanol were greatly facili­

tated by the siliconized tubes which prevented any ad­

sorption on the glass walls and gave a smooth solvent

film evenly distributed over the tube walls.

If these points are carefully followed and con­

sidered, then the extraction scheme shown in Figure 3

is simple and efficient and lends itself to routine,

multiple-batch analysis for LAAM and its metabolites.

Gas Chromatography-Mass Spectrometry

It was originally thought that GC-electron

impact MS-selected ion monitoring of LAAM and its

22

metabolites would be possible. This notion was quickly

abandoned because, as described in the flAnalytical

Methods fl discussion, the extraction chemistry inevi-

tably converted the LAAM metabolites to their amide

forms and, as even a cursory inspection of the complete

EI spectra shows, the M + 1 protonated molecules were

useless for quantitative purposes, representing less

than 1.0% of the base peak, Figures 4A and 4B. The

only potentially useful ions in the spectrum occurred

below 100 amu, at M/e 85 and 83 but, unfortunately, were

associated with background interference, and could not

be used without an on-line computer or interactive data

system to subtract background. In any event, the in-

tensities of these ions were only approximately 20%.

Electron-impact spectra studies were, therefore,

stopped in favor of chemical ionization, selected ion

monitoring, with an emphasis on utilizing the parent

(M + 1) ions and methane as conventional GC carrier and

reagent gas. Although the CI-mass spectra of LAAM and

the amide-metabolites were clean, the M + 1 ions were

less than 2% of the base peak and gave M + 2 and M - 1

peaks of sufficient intensity to cause concern for

23

intolerable interference when D3-stable isotopes were

used as internal standards. Furthermore, other peaks

in the spectra were generally less than 10% of the base

peak (see Figures SA, B, and C). In consequence, both

isobutane and ammonia were tested as reagent gases. A

summary of the results is shown in Table 3. From a

practical standpoint, isobutane proved to be just as

inadequate as methane and, additionally, was a poor GC­

carrier gas--producing distorted peaks and inferior

resolution. Although the M + 1 ions of the amide­

metabolites increased to 18%, the protonated molecule

of LAAM remained at about 1%. This was important be­

cause, as expected, many of the plasma samples in the

clinical study contained concentrations of the parent

drug in the low nanogram range, particularly late

samples after a single dose.

For the ammonia studies, the gas was intro­

duced directly into the MS source and methane was again

used as the GC-carrier gas. Under these conditions, the

mass spectra showed intense M + 1 ions with virtually no

M - 1 or M + 2 contributions. The protonated molecule

for LAAM was also the base peak, and both amide­

metabolites gave M + 1 ions of 30% intensity, Figures

6A, B, and C. Various source pressures of ammonia were

tried, but no significant differences were observed be­

tween 150 and 250~. Two-hundred microns pressure was

24

selected for routine operation, because it was within

the reliable, useful range of the instrument control

valve. Figure 7 compares the molecular-ion monitoring

of LAAM and metabolites with methane alone or methane­

ammonia. The figure indicates a sensitivity increase

with the use of the gas mixture; 3-SX for LAAM, 7-7X for

nor-LAAM, and 26X for dinor-LAAM over methane alone_

There are two important instrument operating

conditions which were carefully evaluated for their

quantitative effects on the analysis. These were

the timing and use of the GC effluent diverter valve

following sample injection, and the source temperature

differential which occurs when the filament is off or

on. The filament on-off time following sample injection

into the GC was, therefore, important. It was found

that unless the source temperature was maintained in a

steady state (120°C with the filament on), the frag­

mentation patterns varied uncontrollably and seriously

affected quantitation. A compensating heater was de­

signed, built, and installed so that, when the filament

was off, the heater maintained the source temperature

at 120°C. This solved the problem. A similarly simple

solution to control the GC effluent during operation of

the diverter valve was achieved by constructing an auto­

matic, timed control for the valve which is located be­

tween the GC column exit and the MS source_ The valve

25

is open for exactly 18 sec during each analysis immed­

iately following the sample injection, so that all

components of the extract eluted from the GC column

prior to LP~, the metabolites, and internal standards

are diverted to a roughing vacuum pump and do not enter

the MS source. This device, plus stabilizing the MS

source temperature and the very short GC analysis

time, does much to maintain a clean source and low

background in the MID chromatogram. Although as little

as 50-100 pg of pure LAAM and metabolites can be easi­

ly detected following injection into the gas chromato­

graph, extraction, derivatization and reconstitution

from 2.0 ml plasma had previously indicated that the

method could maintain good precision to 15-20 ng/ml for

parent LAAM and 10-15 ng/ml fo.r the amide metabolites.

By modifying the analytical extraction procedure and

conditions for amide derivative formation, as described

earlier, sensitivity limits were greatly improved for

~I. GC-MS modifications in the ion-monitoring

parameters, sacrificing some resolution for sensitivity,

gave a further significant increase. Initially, reso­

lution on each ion-monitoring channel was set at 0.2 amu

with a 10% valley between adjacent peaks. By setting

the resolution at 1.0 amu and accepting a reduced valley

between peaks, the MID sensitivity could be increased up

to 10-fold. Figure 8 is a representation of this

26

improvement for 1.0 ng of LAAM drawn from two super­

imposed recordings and indicates approximately a five­

fold increase in sensitivity.

The only aspect of gas chromatography conditions

rigorously tested was column packings. Dexsil 300, OV-

17, OV-l, and SE-30 liquid phases were evaluated, all

in 1-3% concentrations. Particular examination was

made of the GC-CI-MS background, temperature stability,

and acceptable resolution of LAAM and its amide­

metabolites in a retention time period limited to 5 min.

Three percent OV-17 was superior to all the other phases,

including its temperature stability, which permitted iso­

thermal analysis at 275°C, in the operational range for

LAAM and the metabolites. Relative retention times are:

LAAM, 0.59; dinor-LAAM, 1.00: nor-LAAM, 1.30; and the

total elapsed time between serial injections is approx­

imately 2.5-3.0 min.

As a result of observing an apparent increase in

sensitivity, judged from peak heights on the MID re­

cording as serial analyses were made of plasma samples,

various techniques of GC column preconditioning were

tried. Lipids and fatty acids contained in the extracts

seemed to be critical to the phenomenon, but when a

series of fatty acids and esters were tried, only tris­

tearin improved the sensitivity to the observed levels.

Peak heights for all six components of the analysis

27

increased by greater than 50%. The column required an

injection of at least 70 ~g of tristearin. By reinject-

ing previously analyzed plasma extracts before beginning

a batch of new samples, a useful, lasting improvement

was achieved. This technique is routine practice as

part of the analytical procedure. Figure 9 shows a

multiple ion-recorded chromatogram for LAAM, the two

metabolite-amide derivatives, and their corresponding

isotope internal standards following a typical analysis.

GC-MS Data, Calculation, and Manipulation

Manual manipulation of the raw data measured

and calculated from the multiple-ion monitor recording

of six peaks, is a complex, extremely time-consuming

task. There are six data points on each chromatogram,

six recorder attentuations to be rationalized, six base-

lines to subtract background, and six peak heights to be

measured. These data must then be fed into mathematical

formulae to calculate drug concentrations in the original

sample. With several hundred analyses required in the

clinical pharmacology study, it was prudent to design

a device and build a system that could automatically

perform this task.

Only two analog-to-digital converters were needed

to handle the six GC-MS ion peaks, three each, because

the time resolution between the peaks was sufficient to

28

allow serial processing. Circuitry was built which

would calculate peak areas by total signal summation and

not by the more conventional time-sampling technique.

This provides an actual peak area value. Programs

supplied with the SP 4000 microprocessor allow for stor­

age of instrument calibration data, standard regression

curves, and appropriate calculations. In consequence,

the simultaneous analysis of LAAM and its metabolites

in a plasma sample can be carried through rapidly and

automatically from extract aliquot injection into the

gas chromatograph to print-out of the concentrations

on a teletype, and it is completely free of possible

operator error at the data reduction stages.

Similarly, the necessity for storing the very

large quantity of data and subsequently presenting it

in tabulated and various graphic forms argued for com­

puter capability. The Tektronix 4051 desk-top model with

interactive printer-plotter was ideal for the purpose.

The data and programs are stored on magnetic tapes;

programs can be written in basic, alpha-numeric language,

and there is 16 K capacity of which only 2 K is required

by the instrument for executive functions. Necessary

programs were written so that all of the data tables,

linear and semi-log concentrations, time-course graphs,

and calculated results presented in this report were

produced through the Tektronix instrumentation.

29

By experimentally testing and optimizing each of

the parameters in the complete method, chemical and

instrumental, it is possible to maintain acceptable pre­

cision and quantitative sensitivity below 5 ng/ml for

LAAM and both metabolites in a 2.0 ml plasma sample.

Standard curves, constructed following repetitive anal­

ysis of 2.0 ml fortified plasma samples, are shown in

Figures IDA, B, and C. The vertical axes are shown

equally divided but without fixed-ratio values. The

individual values can be quite different for any given

run of standard plasmas, because the ion-monitoring re­

corder voltage values for each channel are adjusted for

each sample and for each of the six constituents of each

sample, in order to record on-scale peaks of measurable

size as the run proceeds. The peak heights are not nor­

malized to a fixed voltage in the calculation of the

ratios.

Precision at 200 ng/ml is 2.0%; at 25 ng/ml,

7%; at 10 ng/ml, 15%, at 5 ng/ml, 20%.

Sensitivity to 3 ng/ml with a 2.0 ml plasma

sample is practical and routine. Below this concentra­

tion, background from the plasma becomes a contributing

factor to the quantitative analysis. This limit can

obviously be improved, if necessary, by using larger

plasma samples or dissolving the extract in a smaller

volume of methanol and injecting a larger aliquot into

the GC-MS.

Analysis of Biological Test Samples

30

Samples of plasma taken from monkeys, each given

oral doses of LAAM, were analyzed as a pilot test of the

analytical method. The drug concentration versus time

profiles was plotted, and the results were used as a

guide to the concentration ranges likely to be encountered

in the human study.

One hundred-forty plasma samples taken from

four monkeys were analyzed, along with appropriate

standards and blank monkey plasma. For the purpose of

this report, the results of 23 samples (6, 6, 6, and 5

from the four monkeys) taken following a single, acute

oral dose of LAAM at 2 mg/kg are shown in Table 4 and

Figures IlA, B, C, and D. The monkey study and plasma

samples were generously provided by Dr. David Downs,

Parke-Davis and Company, Ann Arbor, Michigan, according

to an agreed protocol for dose and blood-drawing times.

It is most striking that the plasma concentra-

tions of the drug and metabolites are very low, the

highest being at 28 ng/ml for nor-LAAM in monkeys 6733

and 6719. All other values are less than 15 ng/ml.

Many of the later data points are close to the sensitiv-

ity limit of the method for reliable quantitation. The

points were used, however, because the samples did con-

tain LAAM and metabolites. LAAM appears to be very

31

rapidly degraded in the monkey; only those samples drawn

up to 2 hr post-ingestion contained LAAM, but both

metabolites were continuously detectable. The data are

generally pharmacodynamically consistent and, although

the concentration values are different, the time-course

curves for the four animals are approximately parallel.

The experience with these samples and the ease

with which they were assayed proved the efficacy of the

total analytical procedure. In addition, it allowed an

operations time study of the procedure from sample re­

ceipt to data generation. It is convenient to analyze

'samples in batches of 40, each batch having ten stan-

dards and reagent blanks. With current equipment, com­

plete processing of a batch requires approximately 27

man-hours. It includes: 2.5 hr centrifuge time, 1 hr

for solvent extraction, 30 min incubation time, and 2.5

hr of other wet chemistry. It requires 12 hr GC-CIMS­

MID, including 2 hr for instrument set-up and "tuning";

also, about 9 hr is needed to make all the computations

necessary to generate the final concentration values and

plot the time-course curves.

Clinical Study

The human clinical-pharmacology study was a col­

laborative endeavor between Dr. Walter Ling of the

32

University of California, Los Angeles, and the Veterants

Administration Hospital, Sepulveda, California, and the

author. Dr. Ling provided the human subject volunteers

and maintained them in the hospital under medical super­

vision for the duration of the study. He was responsi­

ble for their medical care and all the clinical aspects.

The pharmacological design, including drug administra­

tion, blood sampling protocols, methods of collecting

and preserving the samples, and, finally, analysis of

the plasma and data interpretation, was the responsi­

bility of the author. There were 12 human subjects in

the collaborative study. They were all adult (mean

age, 36 years), male heroin addicts who had been main­

tained on methadone at 60-80 mg daily for at least two

years. The subjects comprised five Caucasians, four

Mexican-Americans, and three Blacks. They were all

within the normal IQ range and had normal clinical chem­

istry profiles, including liver function, and no recent

major medical problems. Blood clinical chemistry anal­

yses were performed for each subject at the beginning,

in the middle, and at the end of each LAAM study period.

No abnormalities were revealed.

Table 5 shows the dose of LAAM ingested and the

body weight of each subject. Seven subjects (001 through

005, and all and 012) received 80 rng doses irrespective

of body weight, and five subjects (006 through 010)

33

received doses of 1 mg/kg body weight. The weights of

all the subjects, excepting 003, changed during the

study period~ Although subject 012 gained eight pounds,

from 134 to 142 pounds (approximately 6%), all the other

subjects remained in the range (-4% to +4%) of their

starting weight, and none of the changes was regarded

as having a significant influence on the pharmacokine­

tic interpretation of the analytical data.

Each received ten oral doses of LAAM, given in

fruit juice at 48 hr and 72 hr intervals, over a total

period of 22 consecutive days. The first dose was given

on Friday, followed by Monday, Wednesday, Friday-­

regularly through 22 days. Table 6 shows the time of

dose administration for each subject as a function of

hours into the study. It will be seen that dose times

were uniform to within a few min for all the subjects.

Blood samples were drawn according to the fol­

lowing protocol, maintaining the draw-times as closely

as practically possible. Times are in hours following

each dose. Samples were drawn over a total period of

42 days, including 21 days following the last dose.

Zero. Dose 1:

Zero. Dose 2:

0.25, 0.5, 1.0, 1.5, 2, 4, 6, 12,

16, 24, 48, 72.

0.25, 0 .. 5, 1.0, 1.5, 2, 4, 6, 12,

16, 24, 48.

Zero. Dose 3: 0.25, 0.5, 1.0, 1.5, 2, 4, 6, 12,

16, 24, 48.

Zero. Doses 4-9: 1.0, 12, 24, 48 (72).

Zero. Dose 10: 0.25, 0.5, 1.0, 1.5, 2, 4, 6, 12,

34

24, 48, 72, 144 (6 days), 168 (7 days),

240 (10 days), 336 (14 days), 408 (17

days), 504 (21 days).

For the first three subjects, 49 plasma samples per

subject were drawn and analyzed. The blood samples

were drawn in vacutainer tubes containing fluoride,

immediately centrifuged to separate the plasma, which

was then transferred to chemically clean and inert

screw-capped tubes and stored frozen until analyzed.

This procedure minimized any sample contamination from

plasticizers or components of the vacutainer stoppers.

For subjects 004 through 012, 65 plasma samples per

subject were drawn and analyzed.

Every 12 hr throughout the study period a urine

specimen was collected from each subject, total volume

and pH were recorded, and two 20-ml samples from each

l2-hr collection were preserved for analysis. These

urine samples (710) are stored frozen and will be

35

analyzed as interpretation of the plasma data dictates,

for complete pharmacokinetic evaluation. At intervals

during the study, urine analyses were performed for

each subject to determine whether any other drugs had

been surreptitiously ingested. There were positive

findings in three subjects: imipramine was detected

in subject 010 on two occasions four days apart; pseudo­

ephedrine on two consecutive days, and cocaine on one

other day in subject 011; barbiturate on two consecu­

tive days, and pseudoephedrine, morphine, and propoxy­

phene, once each on separate days in subject 012. In

subject 010, imipramine was detected , four days prior to

the first dose. The pseudoephedrine was detected in

subjects OIl and 012 on the day of the eighth dose. The

other findings occurred substantially during the final

three weeks of the study on the day or immediately after

the last dose. They did not disturb the analytical

method or affect the assay accuracy for LAAM and its

metabolites in plasma.

RESULTS

The plasma concentrations of LAAM, nor-LAAM

and dinor-LAAM following repetitive oral doses of LAAM

over a 42 day period are given for each of the 12 human

subjects in Tables 7 through 18. Times at which blood

samples were drawn are given in the tables, both as

cumulative time in hours into the study and as times

following each dose. The data are presented graphically

as log-concentrations versus linear-time plots for each

subject. Separate graphs, Figures 12A and B through

23A and B, depict the time courses of LAAM and its

metabolites following the first dose and the last dose

of the drug. The total time-course profiles for LAAM,

nor-LAAM, and dinor-LAAM are shown in Figures l2C, D,

E, through 23C, D, and E.

By inspection and analysis of the data in the

graphs and tables, it can be seen that within the study

range of 0.73-1.51 mg/kg, the dose of LAAM ingested is

not a critical variable with respect to subsequent plas­

ma concentrations achieved and that it cannot be used

to predict either the likely maximum concentrations of

LAAM and its metabolites following single or multiple

doses or the possibility of drug and metabolite

37

accumulation in any given patient. Subjects 006-010 are

a uniform dose group, each having received 1 rng/kg LAAM.

However, from examination of their plasma concentration

profiles and their concentration ranges between first

and last doses, it is apparent that there are two sub­

groups--subjects 006, 007, and 008 and subjects 009 and

010. The former are within narrow tolerances following

first and last doses, as are subjects 009 and DID, but,

as separate groups, they are quite disparate. Differ­

ences in maximum concentrations between the two groups

for LAAM, nor-LAAM, and dinor-LAAM (first and last doses)

are in the range of 20-45%. Subjects 009 and 010 are

consistently lower. The difference in dosage was

greatest between subjects 002 and 004, at 0.73 mg/kg and

1.51 mg/kg, a decrease of 27% and an increase of 51% from

the uniform dose group, respectively; yet the differences

in the maximum concentrations between these two subjects

for LAAM and the metabolites following both first and

last doses are only in the range of 5-25%. Figure 24

illustrates these observations, particularly the simi­

larity in plasma concentration values for subjects 006,

007, and 008 and for subjects 009 and 010 and, also, the

significant differences between them as two groups, des­

pite each subject having received the same 1 mg/kg dose

of LAAM. In contrast, there are relatively minor dif­

ferences between subjects 002 and 004, despite their

38

doses differing from each other by almost 100%. In

addition, subject 002, who received the minimum dose,

did not exhibit the minimum plasma concentrations after

the first dose nor did subject 004, who received the

maximum dose, have the maximum plasma concentrations,

although he required the longest time to reach maxi­

mum concentrations of nor-LAAM (10 hr) and dinor-LAAM

{40-48 hr}. It is evident from all the foregoing re­

sults that the dose range of LAAM used in the study

was too narrow to overcome the individual biological

variations within the 12 subjects and, consequently,

predictable dose-response patterns are not revealed.

The time taken to reach maximum plasma concen­

tration following the first dose was very similar for

LAru1 in all 12 subjects, 3-6 hr with a mean of 4.4 hr.

For nor-LAAM, ten subjects were within the 4 to 7 hr

period, with a mean value of 5.6 hr. Subjects 004 and

010 required approximately 10 hr. For dinor-LAAM,

eight subjects required 5 to 7 hr, with a mean of 6.6 hr.

The remaining four subjects, numbers 002, 004, 005, and

010, required from 24 to 48 hr to reach their maximum

dinor-LAAM concentrations.

For each of the subjects, the time required to

reach maximum plasma concentrations of Lk~, nor-LAAM,

and dinor-LAAM following the tenth (last) dose was vir­

tually the same as that seen after the first dose.

39

The maximum plasma concentrations for LAAM and

the metabolites after the first dose varied over a wide

range without any apparent relation to dose. For LAAM,

values ranged from 52 to 510 ng/ml. However, six sub­

jects were between 100 and 200 ng/ml; four had less

than 100 ng/ml; two (subjects 008 and all) exhibited

extraordinary high.maximum concentrations of 268 ng/ml

and 510 ng/ml, respectively. Maximum nor-LAAM concen­

trations varied between 65 and 175 ng/ml. There were

five subjects with less than 100 ng/ml and another

five had between 100 and 150 ng/ml. Only two subjects

(number 004, at 162 ng/ml and number 008 at 175 ng/ml)

had greater than 150 ng/ml. For dinor-LAAM, the range

was 11-92 ng/ml with five subjects less than 20 ng/ml

and five between 20 and 40 ng/ml. Two subjects (011

and 012) had greater than 40 ng/ml (92 and 67 ng/ml,

respectively). Although the time-course profiles with

respect to plasma concentrations of LAAM and its metab­

olites are generally pharmacokinetically reasonable for

each subject, with some exceptions such as seen in sub­

ject 011, there are no apparent correlations between

different subjects. For example, it might be expected

that those subjects with LAAM concentrations in the

lowest range (less than 100 ng/ml) would have the

highest nor-LAAM or dinor-LAAM concentrations. This is

not the case. Similarly, the two patients (numbers all

and 008) with the highest values of LAAM are not the

same two with the highest nor-LAAM (numbers 004 and

008), or dinor-LAAM (numbers 011 and 012).

40

By inspection of the total time-course plasma

concentration curves, it is evident that some subjects

accumulated LAAM and/or its metabolites for as long as

dosing was continued. Only subjects 006, 007, and 008

continuously accumulated all three drugs, LAAM, nor­

LAAM, and dinor-LAAM. These three subjects, plus two

others (004, 005), accumulated LAAM, and these five,

plus 003, accumulated dinor-LAAM. This phenomenon was

seen by examining the total time-course curves in vari­

ous ways. The broken line shown in Figures 12C, D, E

through 23C, D, and E traces plasma concentration values

at a fixed time after each dose and clearly illustrates

the trend to plateau or accumulation. Also, by deter­

mining the plasma concentration difference at two time

points, e.g., 100 and 400 hr after the first dose, the

magnitude of increase or decrease for each subject can

be evaluated and compared. Similarly, the concentration

change between two different dose times, e.g., between

dose 3 and dose 10, can be used. Each of these tech­

niques have limitations, but the pattern of drug and/or

metabolite(s) accumulation in six of the subjects is

clear. The accumulation is not related to dose, at

least within the range used in this study, and subject

41

007 exhibited the phenomenon most dramatically of all

subjects. It is equally clear for the other six sub-

jects (001, 002, 009, 010, 011, 012) on exactly the

same dosage regimen that there was no continuous in-

crease in concentrations, and they reached a plateau

at a time in keeping with approximately four to five

half-lives of elimination. An apparent plateau was

achieved for LAAM after three to four doses (mean 3.7

doses, or 218 hr), for nor-LAAM after three to four

doses (mean 3.4 doses, or 196 hr), and for dinor-LAAM

after three to six doses (mean 4.3 doses, or 267 hr) .

Subjects 009 and 010 were strikingly similar in this

regard and, again, contrast remarkably with subjects

006, 007, and 008, the three who most obviously accumu-

lated LAAM and the metabolites, and with whom they form

the uniform dose group at 1 mg/kg.

The plasma concentrations of LAAM and the

metabolites increased between the maximum achieved

after the first dose and the concentration at plateau

or maximum after the last dose, for all subjects except

011. The factor of increase for LAAM was 1.2-2.0; for

the five subjects accumulating the drug, 1.2-2.5. For

nor-LAAM, the factor was 1.4-2.0, with three accumula-

tors at 1.7-4.1. Dinor-LAAM ranged from 3.0-4.0 with

six accumulators at 5.0-10.0. Subject 007 accumulated

ECCLES ,.. j CE llBR/iny

LAM4 2.5-fo1dj nor-LAAM, 4.l-fo1d; and dinor-LAAM,

lO.O-fold over the ten-dose study period.

42

The semi-log scale graphs of plasma concentra­

tions versus time for each of the patients describe

curves with an absorption phase, a distribution phase

occurring between 4 and 12 hr, and an elimination or

clearance phase between 24 and 72 hr after the first

dose of LAAM. A plasma elimination phase for nor-LAAM

is clearly described, but in all subjects the concen­

tration of dinor-LAAM continued to increase, or at best

plateau, between the first and second doses. Accord­

ingly, half-lives were calculated following the first

dose, for LAAM during its distribution (t~a) and elimi­

nation phases (t~8), and for nor-LAAM elimination (t~S).

Half-lives of elimination were calculated for LAAM,

nor-LAAM, and dinor-~1 following the last dose, from

506-1008 hr. These data are given in Table 19. Inas­

much as concentration values were measured frequently

over a long period of time, the equation t~ = 0.693/Ke

= 0.693/8 was used to calculate the half-life values

where Ke is the rate constant of elimination and B the

rate constant for first-order kinetics. The least

squares regression line was calculated by computer from

the graphs, and the slope and B values determined from

the equations log C = log Co - (S/2.303)t and B = slope

x 2.303.

43

For the a distribution phase of LAAM, it was

necessary to "feather" the curve to obtain the line of

first-phase residuals. This was done by computer in

which the B phase regression line was extrapolated to

zero-time and the extrapolated concentrations values

subtracted from the concentrations on the experimental

curve at the data time points. Alpha was then equal to

2.303x the slope of the residual line, and the half-life

(t~) equaled 0.693/0.. These calculations were also

made with the Tektronix 4051 mini-computer.

The mean value of t~o. for LAAM was 2.4 hr.

Seven of the subjects had values between 1.1 and 2.9 hr

with a mean of 1.5 hr, and the other five subjects

ranged from 3.0 to 4.5 hr with a mean of 3.7 hr. The

t~S for LAkM after the first dose ranged from 21.5 to

45.8 hr with a mean of 37.5 hr. The LAAM t~S after the

last dose had a greater range for the 12 subjects,

15.9-104.5 hr, but the mean at 46.8 hr was not remark­

ably different from that after the first dose. By

contrast, the S for nor-LAAM increased by approxi­

mately 63%, from a mean of 38.2 hr (range 13.5-60.2)

after the first dose to 62.4 hr (range 12.9-129.6) after

the tenth and final dose. After the first dose there

were two subjects at less than 30.0 hr and three others

greater than 50.0 hr, but the mean value was changed

only negligibly to 35.7 hr by neglecting these five

44

sUbjects. The plasma elimination half-life for dinor­

Lk~M after ten doses and following the decay curve for

about 500 hr beyond the last dose was extremely vari­

able, ranging from 22.5 hr to 429.9 hr. The mean value

was 161.9 hr. Neglecting the bizarre 22.5 hr value for

subject 011, the range was still 96.7 to 429.9 hr, and

the mean at 174.6 hr. Subjects 002 and 003 eliminated

LAAM and both metabolites at significantly slower rates

than the mean times, and subjects 005, 009, 011, and 012

exhibited faster rates for all three drug components.

The t~S for LAAM throughout the study appears to be in­

dependent of dose and generally consistent with first­

order pharmacokinetics.

DISCUSSION

Although interpretation of the analytical data

from this study is obviously limited by the fact that

only 12 subjects were involved, all of whom were male

opiate-narcotic addicts, and by the apparent range and

diversity of the results, it is nonetheless the largest

group studied and the first in which the human subjects

were completely controlled throughout the period. In

addition, the new GC-CIMS analytical method permitted

specific, accurate, quantitative analyses of the parent

drug and both metabolites in plasma to sensitivity

levels not possible previously. The reports of Billings

and McMahon (1974), Kaiko and Inturrisi (1975), and

Henderson (1976, 1977) were all severely constrained

by available analytical methodology and patient control.

Notwithstanding, many of the inferences and conclusions

in this report are consistent with or substantiate those

of these earlier investigators. A principal difference

is the LAAM t~a of 2.4 hr from this study against their

6 to 7 hr value. Only Henderson (1976) reported a S

phase elimination value for LAAM. His value of 49 hr

after 30 doses compares favorably with the results of

this study, 37.5 (first dose) and46.8 hr (last dose).

The t~ values for nor-LAAM are also similar to

Henderson's, but the dinor-LAAM values in this report

(range 162-175 hr) are the first to be calculated,

made possible by the accuracy and sensitivity of the

analytical method.

46

The appearance of nor-LAAM and dinor-LAAM in the

plasma very rapidly follows the absorption of LAAM, and

the metabolite concentrations rise to their maxima only

1.0 to 2.0 hr after the parent drug. This probably

results from a first-pass hepatic metabolism effect fol­

lowing oral administration and is in keeping with obser­

vations for similar drugs, such as propoxyphene. Despite

this rapid onset of metabolism, the t~a for LAAM is short

and the t~S, long, suggesting significant plasma protein

and tissue binding of the drug with a consequent large

volume of distribution.

From the study data, it is not possible to pre­

dict a quantitative time-course for the drug and metabo­

lites based on dose. If a greater range of doses had

been used, significant differences between patients

could have been anticipated; but this is not likely

when doses of 1 mg/kg ~25%, which achieve a satisfactory

clinical response, are administered three times per

week. For those patients who reached plateau plasma

concentrations, as seen by inspection of the total time­

course graphs, they did so after the third dose and

47

and before the fifth dose, for LAAM (218 hr), nor-LAAM

(196 hr), and dinor-LAAM (300 hr). These times approx-

imate the theoretical pharmacokinetic factor of three

to five half-lives, except for the dinor-LAAM secondary

metabolite. This is not surprising in view of its

apparent very long half-l (approximately 170 hr)

and its production being dependent upon two stages of

metabolism and other in vivo variables.

Although only three subjects accumulated LAAM

and both metabolites continuously, five subjects ac-

cumulated more than one of the drugs, and six accumu-

lated dinor-LAAM. Henderson (1977) has suggested that

LAAM will not accumulate if administered in doses less

than 0.86 mg/kg and at intervals greater than 48 hr.

The half-life values and accumulation observed in this

study support the view that at 1 mg/kg doses, three times

per week, accumulation of the metabolites is likely to

occur in at least half the subjects and is not readily

predictable for any particular individual; and, impor-

tantly, that LAAM will also accumulate in some subjects

on this regimen. Inter-subject variations in medical

health, diet, drug metabolism rates, and other pharma-

cokinetic factors will obviously influence the possibil-

ity of accumulation for any particular subject.

Although the plasma concentration ranges varied

widely between subjects, the individual time-course

profiles of the parent drug and metabolites relative

to each other express reasonable pharmacodynamics and

kinetics. As a sub-group, subjects 006, 007, and OOB

compare very closely in regard to the time taken to

reach maximum concentration and the values achieved

after the first dose. They all accumulated LAAM and

4B

the metabolites, particularly dinor-LAAM, which was

increasing steeply even between the first and second

doses. This is especially true for subject 007, who

had the longest half-life of elimination for nor-LAAM

and a very long dinor-LAAM half-life. Subjects 009 and

010 are also closely matched as a pair but are very

different from 006, 007, and OOB. From considerations

of drug dose, body weights, medical histories including

opiate narcotic use, and clinical response to LAAM dur­

ing the study, there is no obvious explanation why these

subjects should appear as more uniform relative to any

other subject in the study with the possible exception

of 011. Subject 011 achieved maximum plasma concentra­

tions of LAAM and nor-LAAM within the study mean follow­

ing the first dose and at a similar time to the other

subjects; but, in all other regards, the time-course

profiles of LAAM and metabolites were extremely erratic

and eccentric. This is especially true for dinor-LAAM,

which followed a course unlike that in any other subject

from onset to the end of the study period. The plasma

49

concentrations of the secondary metabolite plunged from

20 ng/ml at 18 hr after the first dose to zero at 24 hr,

and then back to 100 ng/ml at 48 hr before continuing

to rise to levels more than three times this concentra­

tion after the tenth dose. The nor-LAAM concentrations

fell dramatically after the tenth and final dose at a

rate unique to this subject. For the first four doses,

the course of LAAM in subject all was similar to that

in the other subjects, but thereafter it, too, was very

erratic. Just prior to the sixth, eighth, and tenth

doses, the concentration of LAAM fell to zero, and within

12 hr after the last dose it was again at zero. This

pattern was not seen in any other subject but is remin­

iscent of the metabolism profile observed in the test

monkeys, Figures llA, B, C, D. Overall, the plasma con­

centration differences between maxima after first and

last doses is remarkable. LAAM decreased by a factor of

0.36, as did nor-LAAM by 0.75. Only dinor-LAAM ulti­

mately increased by a factor of 3.0, but its terminal

half-life was incredibly short at 22.5 hr against a mean

of about 170 hr.

The total picture for subject all can be ration­

alized if the possibility of nor-LAAM having a biphasic

effect on hepatic microsomal drug metabolism is recog­

nized, first inhibitory via inactive P-450-nor-LAAM

complex formation (Franklin, 1977) J and then apparently

50

stimulatory at a later time after compensatory P-450

has been synthesized and the complex begins to degrade.

This phenomenon has been reported extensively by

Buening et al. (1976) and by Roberts et ale (1976) for

SKF 525-A and propoxyphene, drugs with structural formu­

lae and biotransformation characteristics similar to

LAAM. The initial absorption and distribution of LAAM

and its first elimination from the plasma would be seen

as normal, with a rapid hepatic first-pass production of

nor-LAAM which would then complex and inactivate a sig­

nificant proportion of the cytochrome P-450 available for

drug metabolism. Consequently, metabolism would be inhib­

ited and the appearance of dinor-LAAM would be slow and

the apparent plasma clearance of nor-LAAM rapid. Further­

more, as new cytochrome P-450 (which is not all capable

of forming a complex during metabolism) was synthesized

to compensate for the apparent loss of the initial cyto­

chrome P-450 to the inactive complex, the metabolism rate

would be steadily restored, and the metabolism of LAAM

and nor-LAAM would be noticeably stimulated. This would

result in a fast elimination of the parent drug and me­

tabolites from the plasma, as evidenced in subject 011

by the extraordinarily short half-lives and a large in­

crease in the concentration of dinor-LAAM.

51

There is also the suggestion in the data from

subjects 004 and 012 that the same mechanism may be

involved but less significantly than in 011. In subject

004, the appearance of dinor-LAAM was very erratic, and

in subject 012 the plasma clearance of nor-LAAM was

even faster than in 011 after the first dose, although

it was re-established closer to the study mean after

multiple doses of the drug. If correct, this biochem­

ical mechanism could be important for potential drug

interactions which would alter hepatic drug metabolism

in addicts treated with LAAM as clinical outpatients,

particularly as they are prone to use and abuse multi­

ple drugs.

There are a number of necessary studies to be

undertaken as this research continues, all of which are

vital to an unequivocal pharmacodynamic and kinetic

description of LAAM in man. Most importantly a single

dose by intravenous injection or infusion is required

to describe the kinetics free from absorption and any

first-pass effects. Plasma concentrations should be

folllowed for at least five half-lives. Similarly, the

half-life elimination should be determined for LAAM and

nor-LAAM, after a single oral dose at 1 mg/kg, from plas­

ma concentrations followed for as long as the analytical

method will permit. This is a very difficult clinical

undertaking because the narcotic addict patient would

52

be threatened by onset of withdrawal symptoms unless

given additional doses. Female subjects should be stud­

ied and, because of the kinetic and time-course profile

disparaties, the monkey as a primate model for the human

condition needs to be very carefully evaluated. Also,

human plasma protein binding data are needed for the

range of LAAM and metabolite concentrations seen in

this study, so that accurate volumes of distribution

can be determined experimentally. Finally, urine samples

collected during the study will be analyzed to develop

quantitative metabolite excretion data which should sub­

stantiate the plasma findings.

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Blachly, P.H. I-alpha acetylmethadol in treatment of

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Buening, M.K. and Franklin, M.R. SKF 525-A inhibition,

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54

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55

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Henderson, G.L., Weinberg, A.J., Hargreaves, W.A.,

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56

Kaiko, R.F. and Inturrisi, C.E. A gas liquid chromato­

graphic method for the determination of acetyl­

methadol and its metabolites in human urine.

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57

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58

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59

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Pharmacol. Exp. Therap. 145, 11, 1964.

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Anal. Chern .. !Q..' 1243, 1968.

FIGURES

Figure 1. Synthesis scheme for 1-a-acety1methado1

(LAAM) from methadone.

61

9~

Q

o H

II

I .....

. CH

3 H

3C

-H

2C

-C-C

-CH

2 -

C-

N

O I

'C

H3

~

CH

3

I~

o II

9 18

OH

H

H

2

I I

/C

H3

)It.

H

3C

-CH

2-C

H6

C C

H2

-9-N

, C

H

CH

3

~

3 I

#'

H3C

-C

-? \.

7 /' C

H3

(CH

3C

O)2

0.,

. H3C-H2C-CH-C-CH2-~-N'CH3

C5

H5

N

~

CH

3

U

0')

N

Figure 2. Biotransformation pathways for l-a­

acetylmethadol (LAAM).

63

........ C

H3

CH

3-C

H2-

CH

-C

-CH

2 -C

H-

N

16

I '

CH3

Q

CH

3-C

-O

~

CH

3

AC

ET

YLM

ET

HA

DO

L ~

...

I \

(1

/ N

-DE

ME

TH

YL

AT

ION

0

~

/CH

3

~

........ C

H3

CH

3-C

H2

-CH

-C-C

H2

-CH

-N

CH

3-C

H2-

CH

-C-C

H2-

CH

-N

I 6

I 'H

I 6

I '

CH3

CH

3-C

-O

'7

CH

3 HO

~

CH

3 "

I I

o ~

~

NO

RACE

TYLM

ETH

AD

OL

MET

HA

DO

L j

N

DE

ME

TH

YLA

TIO

N 1

DE

AC

ET

YLA

TIO

N

N-D

EM

ET

HY

LA

TIO

N

/H

CH

3-C

H2

-CH

-C-C

H2

-CH

-N

16

I 'H

C

H3-

C-O

~

CH

3 II

I o ~ 9

DIN

ORA

CETY

LMET

HA

DO

L

Q

....... C

H3

CH

3-C

H2

-CH

-C-C

H2

-CH

-N

16

I 'H

HO

-:

y

CH

3 I

. ~

NO

RMET

HA

DO

L 0

'\

~

65

2.0 rnl PLASMA + 1.0 rnl Int. Std. Solution + 1.0 ml Buffer (pH 9.2) + 10.0 ml n-Butyl Chloride

Shake 10' , Centrifuge 1500 rpm 5'

AQUEOUS PHASE:DISCARD

ORGANIC SOLVENT PHASE DISCARD

AQUEOUS PHASE: DISCARD

I

ORGANIC SOLVENT PHASE

Extract with 10 rnl 0.2N HCl Shake 10', Centrifuge 1500 rpm 5'

I

ACID PHASE

Adjust to pH 12-13, add 0.5 ml 5 N NaOH Incubate 30' @R.T. then finally

10 min @70°C Cool, extract with 10 ml CHC1

3

VORTEX FOR 5'

CHC1 3 PHASE

Evaporate to dryness with air ({200CC/min)

Reconstitute with { 25 VI methanol for GC-CI-MS

Figure 3. Extraction method for LAAM, metabolites, and

isotope internal standards from plasma.

66

Figure 4A. LAAM, electron impact mass spectrum.

o o

o t{)

~

o

o t{) r0

o o r0

o LO C\J

o o N

o LO(J)

N

LO

O(J) Ot{)

CD Nr<1 """CO

o t{)

67

68

Figure 4B. Dinor-L~~, electron impact mass spectrum

I 0 0 0 0 lO

~

o L{) tf)

o o rr>

o L{) N

0 0 N

0 lC)

L{)

lO

-~ tf)

lC)

0 0 --en

en OL() L{)

69

OJ

" ~

Figure SA. LAAM, chemical ionization mass spectra

using methane reagent gas.

70

o o

o 10

~

+ 2

o

o LO N

o o N

o 10

71

Figure SB. Nor-LAAM amide, chemical ionization mass

spectra using methane reagent gas.

72

o o

00 lC)~

+ r<)r<)

~ N N r<)

0 0 r<>

am lC)~ C\J N

I"-

02 0 N

I"-

0 L()

o ----------------------~O

a lC)

~

o

o L()

73

OJ

" ~

74

Figure SC. Dinor-LAAM amide, chemical ionization mass

spectra using methane reagent gas.

o o o

to

~

o

Oen L()~ NN

l'-

0° ON N

l'-

0 LO

N 00 0-

o L{)

<.0 OJ

75

Q)

"'-~

Figure 6A. LA&~, chemical ionization mass spectra

using methane-ammonia as reagent gas.

76

+ ~

o o o lO

~

o

77

¢ oL() L{)r<1 t<)

N -Ot(") 0 t<)

LO <..0 N

00 L()'\t NN

r0 N N

N ~ 00

ON ~ N CO

0 L()

Figure 6B. Nor-LAAM amide, chemical ionization mass

spectra using methane-ammonia as reagent gas.

78

o o

o o rno

----I.- CO

o t.{)

N

o t.()

N

______________________ ~o o

o L()

~

o

o l{')

79

80

Figure 6C. Dinor-LAAM amide, chemical ionization mass

spectra using methane-ammonia as reagent gas.

o o

o I,{)

~

+ ___ ..r

~

o

o l!) r0W

N r0

CO o Ot0 o r<>

81

o Q)

o " N ~

o LD

o If)

CO

82

Figure 7. Comparative GC-CI-MS, molecular ion monitor­

ing of LAAM and metabolites using methane or methane­

ammonia as reagent gas.

LAAM~ ~NOR

A-

'DINOR

- CH4: NH3 GAS

t --- CH4 GAS 1\ '\ II

: I I I I ! I I

...' I ..

83

Figure 8. M.S. recorder responses at molecular ion

for 1.0 ng LAAM, at maximum resolution (0.2 amu) and

minimum resolution (1.0 amu).

84

85

- MIN. --- MAX.

Figure 9. Multiple ion monitor recording of LAAM,

nor-LAAM, dinor-LAAM, and isotope internal standards.

86

M/e

3

54

3

57

3

40

3

43

3

26

3

29

LA

D

3

no

r

D3

d

in

D3

AM

:=

M

/e 3

54

-

LA

AM

=

M/e

35

7

-L

AA

M -

AM

IDE

=

M/e

34

0

-n

or-

LA

AM

A

MID

E

= M

/e 3

43

or

-L

AA

M-A

MID

E

= M

/e 3

26

-

din

or

-L

AA

M --

AM

IDE

= M

/e 3

29

(X)

-....J

88

Figure IDA. Standard assay calibration curve for L~1.

Concentration range zero-IOO ng/ml plasma.

o o

o 0'>

o CO

o I'-

0 to

0 L{)

0 ¢

0 r0

0 N

o

89

E

" 01 C

90

Figure lOB. Standard assay calibration curve for nor­

LAAM. Concentration range zero-IOO ng/ml plasma.

£0 00 01.1 V't:J

91

o E 1.0" 01

o c: ~

o r(')

92

Figure IOC. Standard assay calibration curve for dinor­

LAAM. Concentration range zero-IOO ng/ml plasma.

£0 00 OI..lU'tl

o o

o (j)

93

o E l!)"-..

CJ1 c

94

Figure llA. Monkey 6719. Monkey plasma concentration

time courses of LAAM, nor-LAAM, and dinor-LAAM follow­

ing a single acute 2 mg/kg oral dose of LAAM.

45

40

35

30

25

N

G/M

L 2

0

15

10

5 e 0

5 to

KEY

LA A

M =

II

nor-

LA

AM

=

0

dfno

r-L

AA

M

= <

)

-.""

'-. -..

MON

KEY

*671

9 D

ose

= 2

M

g/K

g

15

2

0

25

3

0

35

4

0

HOUR

S

45

5

0

~

96

Figure lIB. Monkey 6733. Monkey plasma concentration

time courses of LAAM, nor-LAAM, and dinor-LAAM follow­

ing a single acute 2 mg/kg oral dose of LAAM.

45

40

35

30

25

20

15

10

5 13

e 5

10

KEY

LAAH

=

A

nor-

-LA

AM

=

0

dino

r-L

AA

M

= <

>

MON

KEY

+67

33

Do

se =

2

Mg

/Kg

15

2

0

25

31

3 3

5

40

HOUR

S

45

5

0

\.0

-.

J

98

FigurellC. Monkey 6751. Monkey plasma concentration

time courses of LAAM, nor-LAAM, and dinor-LAAM follow­

ing a single acute 2 mg/kg oral dose of LAAM.

45

4e

35

30

25

N

G/M

L 2

0

15

Ie

5 e o

5 Ie

KEY

LAAM

:=

I::

l no

r-LA

AM

=

0

dino

r-L

AA

M =

0

MON

KEY

i675

1 D

ose

=

2

Mg

/Kg

15

2

0

25

3

0

35

4

0

HO

URS

45

5

0

\.0

\.

0

100

Figure lID. Monkey 6775. Monkey plasma concentration

time courses of LAAM, nor-LAAM, and dinor-LA&~ follow­

ing a single acute 2 mg/kg oral dose of LAAM.

45

413

35

30

25

N

G/H

L 2

0

15

10

5 £1 0

5 to

KEY

LA AM

=

b,.

nor-

LA

AM

=

0

dfno

r-L

AA

M =

<>

MON

KEY

+67

75

Do

se =

2

Mg/

Kg

t5

20

2

5

30

HOUR

S 3

5

413

45

5

0

6 ......,

102

Figure 12A. Subject 1. Human plasma concentration

time courses of LAAM, nor-LAAM, and dinor-LAAM follow­

ing the first dose of LAAM at zero time to 72 hr.

Ieee

t00

NG

/ML 10

1 •.

<1>

,

e 5

Ie

~ D

ose

+1

KEY

LAAM

=

~

nor-L

AA

M

==

0 df

nor-

LA

AM

=

<>

SU

BJE

CT

t t

Do

se

='

80

M

g B

od

y

weIg

ht

= 6

2

Kg

/ tl

' -_

_

= _~

15

20

2

5

30

3

5

40

4

5

S0

5

5

60

6

5

HOUR

S

70

7

5

~ I-

'

Do

se +2~

104

Figure 12B. Subject 1. Human plasma concentration

time courses of LAAM, nor-LAAM, and dinor-LAAM follow­

ing the tenth (last) dose of LAAM at 504 hr to 575 hr.

Ieee

10

e

NG/ML.~

te

KEY

LA AM

=

~

nor-L

AA

M

= 0

df

nor-

LA

AM

=

<>

SUB

JEC

T:

t D

ose

=

80

M

g B

od

y welgh~

= 6

2

Kg

1.

, '

I I

I ,

I ,

, I

, ,

I ,

,

50

0 ~05

51

0

51

5

52

0 5

25

53

0

53

5

54

0

54

5

55

0

55

5

56

0

56

5 5

70

5

75

~

Dos

e tI

e

HO

URS

~

106

Figure 13A. Subject 2. Human plasma concentration

time courses of LAAM, nor-LAAM, and dinor-LAAM follow­

ing the first dose of LAAM at zero time to 72 hr.

10

e0

tee

NG

/ML 10

1 ..

I

e 5

~ D

ose

.,

KEY

LAAM

=

II

nor~LAAM

= 0

dl

nor..

".LA

AM

=

<>

SUBJECT~

2 D

ose

=

80

M

g B

ody

welgh~

= t

eg

K

g

te

t5'

20

2

5

30

3

5

40

45

S8

55

6

a 6

5

70

75

~

~ H

OU

RS

D

ose

+

2 ~

108

Figure l3B. Subject 2. Human plasma concentration

time courses of LAAM, nor-LAAM, and dinor-LAAM follow­

ing the tenth (last) dose of LAAM at 504 hr to 575 hr.

Ieee

t 001

= a

I '0

KEY

LA AM

=

ll.

nor-

-LA

AM

=

0 dl

nor-

LA

AM

=

<>

Q

SU

BJE

CT

t 2

Do

se =

80

M

g B

ody

welg

hl

= te

g

Kg

I¢t

1 I

' I'

, ,

, ,

, ,

' I

I I

, I

50

0 ~05 5

t0 5

15

52

0 5

25

53

0 5

35

54

0 5

45

55

0 5

55

56

0 5

65

57

0 5

75

~

Dos

e tI

e

HO

URS

~

110

Figure 14A. Subject 3. Human plasma concentration

time courses of LAAM, nor-LAAM, and dinor-LAAM follow­

ing the first dose of LAAM at zero time to 72 hr.

Ieee

t00

NG

/ML

KEY

LAAM

=

~

no

r .....

LAAM

==

0

dfno

r-L

AA

M

:=

<>

SU

BJE

CT

t 3

Dos~

:=I

80

M

g B

od

y

welg

hl

=

94

K

g

1 •• I~ _

_ ~ _

_ ~ _

__

_ ~ _

__

_ ~ _

__

__

__

_ ~ _

__

_ ~ _

__

_ ~ _

_ ~~ _

_ ~ _

__

_ ~ _

__

_ ~ _

_ ~ _

__

_ ~ _

_ ~

~ 5

te

Do

se it

15

2e

25

se

3

5

4e

HO

URS

45

5e

55

60

6

5

7e

75

+

~ D

ose

+

2 :

::

112

Figure 14B. Subject 3. Human plasma concentration

time courses of LAAM, nor-LAAM, and dinor-LAAM follow­

ing the tenth (last) dose of LAAM at 504 hr to 575 hr.

Ieee

t00

NG

/ML 10

KEY

LAAM

=

~

nor-

LA

AM

=

0

dfno

r-L

AA

M

= <>

SUB

JEC

T::

3 D

ose

::

8

0

Mg

Bo

dy

we

igh

t =

94

K

g '-'--------~

t ,

I I

I I

I I

, I

, I

! ,

, ,

I

see 5

05

5t0

51

5

52

0 5

25

53

0 5

35

54

0

54

5 5

5e

55

5 5

6e

56

5 5

70

57

5

~ D

ose

+to

HO

URS

.-...

.-...

w

114

Figure l5A. Subject 4. Human plasma concentration

time courses of LAAM, nor-LAAM, and dinor-LAAM follow­

ing the first dose of LAAM at zero time to 72 hr.

Ieee

te0

NG

/ML 10

KEY

LAAH

=

6,

nor-L

AA

M

= 0

df

nor-

LA

AM

=

<>

~~_/

----

----

/1

/

SUB

JEC

T:

4 D

ose

=

8e

Mg

Bo

dy

weIg

ht

= 5

3

Kg

~ .....

. ~

1 ~ A

'<3

I'

Tt

e 5

~ D

ose

+1

te

15

2e

25

3

0

35

40

45

5

e

55

6

e 6

5

HO

URS

---...., ...

~:> <~

~~~\,

'",

7fJ

75

~

~ D

ose

1

2 ~

116

Figure l5B. Subject 4. Human plasma concentration

time courses of LAAM, nor-LAAM, and dinor-LAAM follow­

ing the tenth (last) dose of LAAM at 504 hr to 575 hr.

Ieee

t 00

. 'r-

"'U-

10

KEY

LAAM

=

8

nor-L

AA

M

= 0

df

nor-

LA

AM

=

<>

/~ ~.

SUB

JEC

T:

4 D

ose

=

8

0

Mg

Bo

dy

welg

hl

= 5

3

Kg

I.

I '

, I

I ,

, I

I I

I '

, I

I

5ee

~e5

51

e 5

15

52

e 5

25

53

e 5

35

54

e 54

5 5

5e

55

5 5

6e

56

5 5

7e

57

5 ~

Dos

e 1

10

H

OU

RS

~

118

Figure l6A. Subject 5. Human plasma concentration

time courses of LAAM, nor-LAAM, and dinor-LAAM follow­

ing the first dose of LAAM at zero time to 72 hr.

Iee

e

10

0

NG

/ML 10

1 IE

'>

'

o 5

~ D

ose

.1

KEY

LAAM

=

~

nor-

LAA

M

= 0

di

nor-

LA

AM

=

<>

SUB

JEC

T:

5 D

ose

=

8

e

Mg

Bo

dy

w

eig

ht

=

61

Kg

-------

------

~

-----~----

---- 10

15

2

0

25

3

0

35

40

45

5

0

55

60

6

5

HO

URS

70

75

~ .....

.,

Do

se

+2

~

120

Figure 16B. Subject 5. Human plasma concentration

time courses of LAAM, nor-LAAM, and dinor-LAAM follow­

ing the tenth (last) dose of LAAM at 504 hr to 575 hr.

10

00

10

0c=

-~

NG

/MLL

A

}.

10

KEY

LAAM

=

~

nor-

LA

AM

=

0

dfno

r-L

AA

M

= <>

SUB

JEC

T:

5 D

ose

=

80

M

g B

od

y

weig

ht

= 6

1 K

g

-------

I.

I !

I !

' ,

, ,

I I

' ,

! I

I

50

0

50

5

51

0

51

5

52

0

52

5

53

0 5

35

5

40

5

45

5

50

5

55

5

60

5

65

5

7e

57

5

~ D

ose

il

0

HOUR

S

~

~

~

122

Figure 17A. Subject 6. Human plasma concentration

time courses of LAAM, nor-LAAM, and dinor-LAAM follow­

ing the first dose of LAAM at zero time to 72 hr.

tee

e

100~

NG

/ML 10

1_

f 5

10

Dos

e +1

KEY

LAAM

=

!J.

nor-

LAA

M

=

0 di

nor-

LA

AM

=

<>

15

2e

2

5

30

3

5

40

HOUR

S

SUB

JEC

T:

6 D

ose

=

1

Mg

/Kg

45

50

5

5

60

6

5

70

~ 7

5

Do

se

+2

f-!

I:\J

W

124

Figure l7B. Subject 6. Human plasma concentration

time courses of L~1, nor-LAAM, and dinor-LAAM follow­

ing the tenth (last) dose of LAAM at 504 hr to 575 hr.

Ieee

KE

Y LA

AM

= ~

nor-

LA

AM

=

0 di

nor-

LA

AM

=

<>

SUB

JEC

T:

6 D

ose

=

1 M

g/K

g

100~

0;

kJ

:

*"

1.

I ,

, •

I '

I '

I ,

' ,

, I

I

50

0 ~05

51

0 5

15

52

0 5

25

53

0 5

35

54

0 5

45

55

0 5

55

56

0 5

65

57

0 5

75

~

Dos

e 11

0 H

OU

RS

126

Figure 18A. Subject 7. Human plasma concentration

time courses of LAAM, nor-LAAM, and dinor-LAAM follow­

ing the first dose of LAAM at zero time to 72 hr.

teee

10

0

NG

/ML 113

KEY

LAAM

=

~

nor-

LAA

M

= 0

di

nor-

LA

AM

=

<>

1 _

' ,

o 5

~ -

~ 1

0

15

2

0

Dos

e +1

25

31

3 3

5

40

HOUR

S

SUB

JEC

T:

7 D

ose

=

1

Mg

/Kg

45

50

5

5

60

6

5

70

75

~ D

ose

+

2

~

I\J '"

128

Figure l8B. Subject 7. Human plasma concentration

time courses of LAAM, nor-LAAM, and dinor-LAAM follow­

ing the tenth (last) dose of LAAM at 504 hr to 575 hr.

Iee

e

1130

~-..-s-,~~--~ .. --~-

NG

/ML 10

SUB

JEC

T:

7 D

ose

=

1

Mg

/Kg

'----_

_ ..Jr

-t

L:J

r---

----

----

____

__

A.

----

-.--.

v'

....--

----.

----

-~ .. --

.----~------------:II

KEY

LAAM

=

~

nor-

LA

AM

=

0

dfno

r-L

AA

M

=

<>

1L

I I

I '

, I

' I

I ,

' I

, ,

I

50

e 5

05

51

0

51

5

52

0 5

25

53

0 5

35

54

0 5

45

55

e 5

55

56

e 5

65

57

0

57

5 ~

+

~ D

ose

*

10

HO

URS

130

Figure 19A. Subject 8. Human plasma concentration

time courses of LAAM, nor-LAAM, and dinor-LAAM follow­

ing the first dose of LAAM at zero time to 72 hr.

Iee

e

10

0 ..

.

NG

/ML 10

KEY

LAAM

=

~

nor-

LAA

M

= 0

di

nor-

LA

AM

=

¢

SUB

JEC

T:

8 D

ose

:::

1

Mg

/Kg

=---.

. ---------{-~~------~.--'-----------.-.---.----~---.

1 P

H

• ..

e 5

~ 1e

15

2

e 2

5

30

3

5

40

4

5

50

5

5

60

6

5

70

7

5

+

Do

se

+t

HO

UR

S D

ose

+2

I-'

W

I-'

132

Figure 19B. Subject 8. Human plasma concentration

time courses of LAAM, nor-LAAM, and dinor-LAAM follow­

ing the tenth (last) dose of LAAM at 504 hr to 575 hr.

Ieee

10

0

NG

/ML 10

KEY

LAAM

=

b.

nor-

LA

AM

=

0 di

nor-

LA

AM

=

¢

SUB

JEC

T:

8 D

ose

= t

Mg

/Kg

"~------v'

----.---

----_ ... -

------

--.--

-~.-.

1 ,

' !

, •

, ,

! ,

, ,

, ,

I I

,

50

e

50

5

51

0

51

5

52

0

52

5

53

0

53

5

54

0

54

5

5S

0

55

5

56

0

56

5

57

0

57

5

~ G

D

ose

tIe

H

OUR

S

134

Figure 20A. Subject 9. Human plasma concentration

time courses of LAAM, nor-LAAM, and dinor-LAAJ1 follow­

ing the first dose of LAAM at zero time to 72 hr.

10

00

__

t00

NG

/ML 1e

1..

. I

o 5

tf\ D

ose

+1

KEY

LAAM

=

!;;

. no

r-L

AA

M

= 0

di

nor-

LA

AM

=

0

(7

SUB

JEC

T:

9 D

ose

=

t

Mg

/Kg

10

15

20

2

5

30

3

5

40

45

50

5

5

60

6

5

70

7

5

+

HO

URS

D

ose

+2

......

LV

U

1

136

Figure 20B. Subject 9. Human plasma concentration

time courses of LAAM, nor-LAAM, and dinor-LAAM follow­

ing the tenth (last) dose of LAAM at 504 hr to 575 hr.

Iee

e

100

NG

/ML 113

KEY

LA AM

:=

t:,

. no

r-LA

AM

=

0

dino

r-L

AA

M

=

<>

SUB

JEC

T:

9 D

ose

:=

1

Mg

/Kg

1.

I ,

, I

, ,

, •

, ,

I ,

, ,

I

50

0 5

05

51

0 5

15

52

0 5

25

53

0 5

35

54

0 5

45

55

0 5

55

56

0 5

65

57

0

57

5

~ D

ose

fi

e

HOUR

S

~

W

-..J

138

Figure 21A. Subject 10. Human plasma concentration

time courses of LAAM, nor-LAAM, and dinor-LAAM follow­

ing the first dose of LAAM at zero time to 72 hr.

Iee

e

lee

NG

/ML 10

I _

. o

5 ~

Do

se

+1

KEY

LA AM

=

I:J.

nor-

LAA

M

= 0

df

nor-

LA

AM

=

<>

~~-~

10

1

5

20

2

5

3a

3

5

40

HOUR

S

SU

BJE

CT

: 10

D

ose

=

1

Mg

/Kg

A-

----"'y"

45

5

0

55

6

0

65

7

0

75

~

Do

se

+2

I-'

W

\.0

140

Figure 21B. Subject 10. Human plasma concentration

time courses of LAAM, nor-LAAM, and dinor-LAAM follow­

ing the tenth (last) dose of LAAM at 504 hr to 575 hr.

Iee

e

t00

NG

/ML 10

KEY

LAAM

=

6-

nor-

LAA

M

= 0

dl

nor-

LA

AM

=

<>

~

<>

SUB

-.JEC

T:

10

Do

se =

1

Mg

/Kg

v

1.

I I

, I

, '

I !

, •

• •

, I

I

5ee

~e5 5

te 5

15

52

e 5

25

53

e 5

35

54

e 5

45

55

e 5

55

56

e 5

65

57

e 5

75

~

Dos

e .1

0

HO

URS

~

142

Figure 22A. Subject 11. Human plasma concentration

time courses of LAAM, nor-LAAM, and dinor-LAAM follow­

ing the first dose of LAAM at zero time to 72 hr.

1013

0

10

0

NG

/ML 10

KEY

LAAM

=

D.

nor-

LAA

M

= 0

di

nor-

LA

AM

=

<>

1 w

an>

A

r..'

; 5

10

1

5

Do

se it

20

2

5

30

3

5

40

HO

URS

SUB

JEC

T:

11

Do

se

=

80

M

g B

od

y

welg

hl

=

57

K

g

45

5

0

55

6

0

65

7

0

75

~

Do

se

+2

j--I

~

w

144

Figure 22B. Subject 11. Human plasma concentration

time courses of LAAM, nor-LAAM, and dinor-LAAM follow­

ing the tenth (last) dose of LAAM at 504 hr to 575 hr.

Ieee

10

0

NG

/ML 10

KEY

LAAM

=

~

nor-

LA

AM

=

0

dino

r-L

AA

M

= <

>

SUB

JEC

T:

11

Do

se =

se

Mg

Bo

dy

weig

hl

=

57

K

g

... ~ -...

......

'--

1. :

:=m

e,

, 'f)

• '0

'

, ,

, '0

'

, ,

I I

5ee

50

5 5

1e

51

5

52

e 5

25

5

3e

53

5 5

40

5

45

55

0 5

55

56

0 5

65

57

0

57

5 ~

~ D

ose

.1

0

HOUR

S ~

U1

146

Figure 23A. Subject 12. Human plasma concentration

time courses of LAAM, nor-LAAM, and dinor-LAAM follow­

ing the first dose of LAAM at zero time to 72 hr.

1130

13

10

0

NG/M

L 10

KEY

SUB

JEC

T:

12

Do

se

= 8

0

Mg

LAAM

=

11

nor-

LA

AM

=

0 di

nor-

LA

AM

=

0 B

ody

wei

gh

t =

61

Kg

//

,/-""

//

/"",/

/~/

~--

~~ --"

'- .... ,-

, "'-"

--'-

.

1sn

I

IT

].J

e 5

10

15

20

2

5

30

3

5

40

45

50

5

5

60

65

7

0

75

~

~ ~

~ D

ose

+1

HO

URS

Dos

e +2

148

Figure 23B. Subject 12. Human plasma concentration

time courses of LAAM, nor-LAAM, and dinor-LAAM follow­

ing the tenth (last) dose of LAAM at 504 hr to 575 hr.

Ieee

1013

NG/ML~

10

KEY

LAAM

=

l:1

nor-

LA

AM

=

0

dino

r-L

AA

M =

<>

SUB

JEC

T:

12

Do

se =

80

M

g B

od

y

weig

ht

= 6

1 K

g

.A.

1.

I I

I '

, ,

, I

, '

, ,

, J

I

50

0 5

05

51

0 5

15

5

20

52

5 5

3e

53

5 5

40

54

5 5

50

55

5 5

60

56

5 5

7e

57

5

~ D

ose

i1

0

HO

URS

I-

-' .t:>

. \.

0

150

Figure 12C. Subject 1. Human plasma concentrations of

LAAM; total time course.

65

0

60

0

550

S0e

450

400

350

NG/M

L 30

0

250

20

0

150

t00

50

0 (3

+ +1

0~

~ 20

0 ~

+00 +

SUB

JEC

T:

1 D

ose

=

8

0

Mg

LA A

M

Bo

dy

w

eig

ht

= 6

2

Kg

6 =

C

on

cen

trati

on

8

hrs

p

ost

do

se

~

= D

ose

ad

min

isle

red

400

Si0

+ ~

HdUR

S 60

0 70

0 80

0 9

00

10

00

........

Ul

........

152

Figure 12D. Subject 1. Human plasma concentrations of

nor-LAAM; total time course.

(,I) L

L. -0

0)00 ~

Q)

L Q)

l: C-'"' <tN 0 (J) « CD .-...J ...... C

fl 0 0) L E

-:Lo+Jo+J -0 ..c. C 0

~ 0) OJ .. 00 .- U Q) t- Q) c: (J) UII 300 W U Q J Q) >-m (1)""0 II n :::::> 0 0 (J) Q CD <:J -E-

CS) CS) (!l LJ) N

153

CS) (S) (0

(f) 0:

CS) E => (S) 0 LJ) :::t:

..rE-

~~ "¢'

--E-CS)

~

--E-CS) (S) N

-E-

(S)--E-(S)

-E-

CS)-E-CS) (S) (S)

U') (5)

154

Figure 12E. Subject 1. Human plasma concentrations of

dinor-LA&~; total time course.

lI) (\J (\J

~

VJ o Q.

VJ L

.L -0

0)00 ~

tl> L (J)

2: C""'" <C (\J 0 en <l: <0 .--J ....., C

II 0 0) L E

-2:~~ -0 .J:.. C 0

(S.) 0) OJ .. ro .- U (l) t- Q) C (f) U U 3 0 0 W U C J (J) >-en en -0 II II ~ 0 0 (f) C CO<:] ~

-- ........ ..... - ................. -

U? (\J Q

155

Ci.;) Q C)

(S) CS) (1')

CS) CS) ........

CS) (S) tD

(J) 0::

~<5 U) :I:

...re-

~-E-..q-

-E-CS)

~

~ CS) CS) {\J

-E-

CS)-E-Q

-E-

Q-E-

156

Figure l3C. Subject 2. Human plasma concentrations of

LAAMi total time course.

6se

6ae

5se

5ee

450

400

350

NG/M

L 30

0

250

2ea

IS0

lea

50

0 0 +

+10~

+

2

00

+ +0

0 +

SU

BJE

CT

: 2

Do

se

=

80

M

g LA

AM

B

od

y

weig

ht

=

10

9

Kg

6 =

Co

ncen

trati

on

8

hrs

p

ost

do

se

t =

D

ose

ad

min

iste

red

4013

5

i0

+ +

HduRS

60

0 70

0 80

0 g

00

10

00

j-J

U1

.......

158

Figure 13D. Subject 2. Human plasma concentrations of

nor-LAAMj total time course.

..., (J) o 0..

(J) L

L -0 0) (l) ~ CO L

a> L 0) C ""'"' <t (S.) 0 C/} <:t - .--.J .., C

II 0 0) L E

N:E~"" -0 ..c. C 0

(S) 0> Q) .. 00 .- U Q)

r- ~ C tfJ UII::tO 0 w u a J Q) )0.-en W-O U lJ => 0 0 (f)oro<J~

159

CS) CS) (s;) -CS) (s;) C)

(S) CS) co

(S) CS) .......

CS) Ci) co

(f) a:

CS) -E ::> CS} 0 lJ) :I:

--E-

~--E-~

-E-CS)

~

-E-CS) CS) (\J

-E-

(S)ooE-CS)

~

CS)-E-CS) (S) (S)

LJ) (S)

160

Figure l3E. Subject 2. Human plasma concentrations of

dinor-LAAM; total time course.

LO N N

(j) UJ o

""0

......, U) o 0-

Il) L

...c -0 0) (l) ~ CO L

Il)

LC» C-+J «0 0 ({J <:( - .--1 ......, C

II 0 0) L E

N~""">"" -0 ..r. C 0

(S) 0) (j) .. CO .- U ~ I- (J) C (f} U Jl ): 0 0 W U 0 -') 0) >-en 00-0 11 II :J 0 0 (f) 0 m <J --E-

CS) l/) l/)

N CS)

161

cs:> G) 0)

Q cs;) CO

CSi) (S) ,......

cs;) (s;) co

(f) Q:

CS) -E ::> C5:) 0 IJ) :z:

-Eo-

~-E-*'q'"

-E-(S)

~

-Eo-CS) (S) N

-E-

(S)~ CS)

-E-

QooE-

162

Figure l4C. Subject 3. Human plasma concentrations of

LAAMi total time course.

65

3

60

0

550

500

450

400

350

NG/M

L 30

0

250

200

150

100

50

0 0 ~

+10~

+ 200

+ ~0

0 +

SU

BJE

CT

: 3

Do

se =

80

M

g LA

AM

B

od

y

weig

ht

= 9

4

Kg

6 =

C

on

cen

trati

on

8

hrs

p

ost

do

se

t =

D

ose

ad

min

iste

red

40

0

5~0

+ +

HOURS

60

0

70

0

800

900

t00

a I-

-' C

!"l

W

164

Figure l4D. Subject 3. Human plasma concentrations of

nor-LAAMi total time course.

53

0

459

430

3se

3ee

NG/M

L 2

5e

200

150

lee

5e

e e ~

lee

~ ~

~ 2ee

~

~00 ~

SU

BJE

CT

: 3

Do

se =

80

M

g LA

AM

Bo

dy

w

eig

ht

= 9

4

Kg

~

=

Co

ncen

trati

on

8

hrs

p

ost

do

se

t =

D

ose

ad

min

iste

red

41 ~

5~e

HOUI~S

see

700

see

gee

Ieee

.....

.. Q

)

U1

166

Figure l4E. Subject 3. Human plasma concentrations of

dinor-LAAMi total time course.

225

220

175

150

125

NG/M

L

H3e

75

50

25

e

f le

e

~ ~

+2

00

+ ~0

0 +

SU

BJE

CT

: 3

Do

se

= 8

0

Mg

LA A

M

Bo

dy

w

eig

ht

= 9

4

Kg

6 =

C

on

cen

trati

on

8

hrs

p

ost

do

se

~ =

Do

se

ad

min

iste

red

400 + +

5'10 Hd

URS

6ee

700

800

ge

0

1000

I--'

0"1

-....J

168

Figure 15C. Subject 4. Human plasma concentrations of

LAAM; total time course.

65

0

Be0

550

500

450

400

3513

NG

/HL

3013

250

200

150

100

50

0 0 +

~104

~ 20

0 ~

+00

~

SU

BJE

CT

: 4

Dos

e =

80

M

g LA

AM

Bo

dy

we

igh

t =

5

3

Kg

6

=

Co

ncen

trati

on

1

2 h

rs

po

st

do

se

~ =

D

ose

ad

min

iste

red

40

0

5~0

~ ~

BdUR

S 6

00

70

0 8

00

9

00

10

00

I-' '" 'J)

170

Figure l5D. Subject 4. Human plasma concentrations of

nor-LAAMi total time course.

see

459

400

35

0

3ee

NG/H

L 2

50

200

150

tee

50

e

~ I

I I I

'~\

I \

I \

I \

+1

0+

, 1 I I I I , I r I I I I I I , 1

\ \ \ \ \ \ \ \ \ \

+ 200

+

\ \ \ \ \ \

\ \ \ , \ \ , \

~00 +

\ \ \ \ \ \ , \ , , \ ,\

SU

BJE

CT

: 4

Dos

e =

80

M

g LA

AM

Bo

dy

weig

ht

= 5

3

Kg

6

=

Co

ncen

trati

on

1

2 h

rs

t =

Do

se

od

min

isle

red

I I I I I I I I I I I I I 41 +

5~0

HduR

S 60

0 70

0 80

0

po

st

do

se

90e

1000

.....

. ...

..)

......

172

Figure 15E. Subject 4. Human plasma concentrations of

dinor-LAAJ1; total time course.

225

2ae

175

150

_ r , \ , 1 , ,

I ,

125

L f

I ,

I ,

, 1

, (

, ,

I

lee

L I

\ f

\ 1

, I

I I

I ,

75

L f 1 I , , ,

sa

L , , , , ,

25

1\

I r

e

; +I

e~ +

20

0 +

+00 +

I I I I I I I I I I I I I

lJ I,

SU

BJE

CT

: 4

Do

se

= 8

0

Mg

LAA

M

Bo

dy

w

eig

ht

= 5

3

Kg

6

= C

on

cen

trati

on

1

2 h

rs

po

st

do

se

t =

Do

se

ad

min

iste

red

800

ge0

1000

40

0 5

i0

~ +

HdURS

60

0 70

0 t-

' ...

.,J

w

174

Figure l6C. Subject 5. Human plasma concentrations of

LAAM; total time course.

Q) (J) o

-0

~

tJ)

o Q..

(/) L

L -0

N (1) 0>- L ~ a>

L C~ <t ...... 0 ({J <{ «) .--.J -+J C

11 0 0> L E

If) l: -+J -+J ""'0 Leo

(S) 0) Q,I "CO'- U (l)

!-- Q) C (j) U II 3 0 0 w u Ci J !l) >-CO (j) -0 II II ::J 0 0 (f) Ci aJ <J ~

('i) l.f) N

175

176

Figure 16D. Subject 5. Human plasma concentrations of

nor-LAAMi total time course.

53

0

450

40e

350

300

NG/M

L 25

0

200

150

10e

S0 e

4 ~I

e+ +

2ee +

~ee +

SU

BJE

CT

: 5

Do

se

= 8

0

Mg

LAA

M

Bo

dy

w

eig

ht

=

61

Kg

6 =

C

on

cen

trati

on

1

2 h

rs

po

st

do

se

t =

Do

se

ad

min

iste

red

4ee

+ 5+e

+

HO

URS

B0e

70

3 se

e ge

e 10

00

I-'

-...J

-...J

178

Figure 16E. Subject 5. Human plasma concentrations of

dinor-LAAMi total time course.

....., 01 o Q.

01 L

...c -0

N Q) 0)- L ~ <D

:E C~ <t..- 0 (J) <l: <0 .--.J ~ C

II 0 0) L E

lJ) ::E ~ ~ -0 ..c C 0

Ci) 0) Q) •. CO .- U <D }- IV C (J) U H 3 0 0 W U 0 J (l) > m (J)(J II 11 ::,:) 0 0 (J) C CCl <J ~

I , I

I I

t I

I I

.... ......

179

CS) Ci) C',)

Ci) (S) CO

Ci) (S) ,......

CS) (S) CO

(J) ~

(S) 1lIIIE :::J (S) 0 lJ) ::c

-Eo-

~-E-""""

-E-CS)

~

-Eo-CS) (S) (\J

......... -E-

Q-orE-CS)

-orE-

CS)--E-

lJ) (\J CS)

180

Figure 17C. Subject 6. Human plasma concentrations of

LAAMi total time course.

65

0

600

550

500

450

40

0

350

NG/H

L 3

00

250

20

0

150

, , " 10

0 I

50

(3

(3 ~

t00

20

0 te

0 ~

~ 4

+ 'l\

~

, ,

SUB

JEC

T:

6 D

ose

= 1

Mg/

Kg

LAAM

~

=

Co

ncen

trati

on

1

2 h

rs

po

st

do

se

t =

D

ose

ad

min

iste

red

, , , , 4~

+ 5~0

Hd

~RS

600

700

80

e

90

0

100e

I-

-' 0

0

I--'

182

Figure 17D. Subject 6. Human plasma concentrations of

nor-LAJL~; total time course.

183

Q) (S)

Ui Q

0 (S)

-0

oo+J Ui (S)

0 Q

0-0)

Ui L Q ..r.. (S)

-0 co N G)

2:- L « aJ « C ......., (S)

-I 0 t.n Q I'-

O)~ C ~ 0

" t. E Q

CO O)~ -0 (S)

~ C 0 co aJ

.. - 0 aJ (J) t- C fJ) Q! U Ii 0 0 ~5 w u a 1.1) :I: """') Q) co (J) II II -Eo-.::J 0 (j) a <J --E- ~-E-

oq-

--E-(S)

~

-E-Q (S) C\I

-Eo-

Q-E-Q

--E-

0-E--J

Q Q (S) (S) (S) ::I:(S) (S) Q Q 0 lI) 0 lI) ($.) '-11) Q If) Q (S;) LJ) "'It' "'It' ('r) ('r) C!>N N Lf) (S)

z

184

Figure l7E. Subject 6. Human plasma concentrations of

dinor-LAAM; total time course.

225

230

17

5

150

12

5

NG/H

L

tee

75

50

25

o o ~

+10~

+ 2

00

+

~00 +

400 ~

SUB

JEC

T:

6 D

ose

= 1

Mg/

Kg

LAAM

6

= C

on

cen

trati

on

1

2 h

rs

po

st

t =

Do

se

ad

min

isle

red

5~0

+ HdU

RS

60e

700

8e0

900

le0

e

~

00

t.

n

186

Figure laC. Subject 7. Human plasma concentrations of

LAAJ1; total time course.

(£) (.D 0

-0 ......,

(0 0 0-

(0 L.

.r:. -0

('\J Q)

e- t-o (£) o C ...,.,

-.J 0 fh

0)-.) C ~ 0 ........ '- e

f'.. O)~ -0 l: C 0

(£) .. - 0 <I) t- C (0 uno 0 W U 0 -, Q) co U) II n ~ 0 (f)Q<l~

\ \ \ \ \ \

... ....... ... ... ... .... ......

('5) lI) N

...... ... ... ..............

187

188

Figure l8D. Subject 7. Human plasma concentrations of

nor-LAAMi total time course.

500

453

400

3se

300

NG/M

L 2

50

200

15

0

100

so

e

~

, , , , , ,

+Hl~

+2

e0 +

", ,

", ,

I I

I I

I

~ee +

I I

I I

I , I , 4e+

+ 5~e

HO

URS

SUBJ

ECT:

7

Do

se =

1

Mg/

Kg

Loa

m

6 =

Concen~ro~lon

12 h

rs po

s~

do

se

~ =

Do

se od

mJnl

s~er

ed

eoe

7ee

soo

900

1000

I-'

00

\.

0

190

Figure l8E. Subject 7. Human plasma concentrations of

dinor-LruL~; total time course.

191

(l,l (S) en (S)

0 (S.)

-0 .......,

(J] CS) 0 cs;)

Q.. 0)

(J]

L ...c (S)

-0 (S)

N a> CO

:L- L « Q)

« c ......, (S)

-.J 0 U) (S) .......

0)......., C ~ 0

" L E

'" 0) ~ -0 (S)

:L C 0 (S)

(J) CO

.. - U Q)

t- C (f) (f)

U II 0 0 ~ (S) -E :::::>

W U Cl <S> 0 -:) Q) lJ) :x: ro (J) U II -E-::) 0 (f) Cl <J ...:E- (S)-Eo-

" \ (S) '\ "t'

\ '\ \

\ -E-'\

CS)

~

-E-CS) (S) (\J

-E-

(S)-E-C'li)

-Eo-

CS)-E-Lf) CS) If) CS) If) CS) (\I (.'\) r.... lI) N cs:> Lf) (S) If) C\I C\J r.... U) C\J CS)

192

Figure 19C. Subject 8. Human plasma concentrations of

LAAM; total time course.

650

600

550

50

0

450

40

0

350

NG/H

L 3

00

25

0

20

0

150

t00

50

0 0 t

+ 10~ +

20

0 +

+00 +

SUB

JEC

T:

8 D

ose

=

1

Mg

/Kg

LA

AM

6

= C

on

cen

trati

on

1

2 h

rs

po

st

do

se

t =

Do

se

ad

min

iste

red

4~ +

5+0

600

700

S0e

90

0

l00

0

HOUR

S I--

-' \D

W

194

Figure 19D. Subject 8. Human plasma concentrations of

nor-LAAM; total time course.

see

450

4ee

35e

3ee

NG/Hl

""' .

.. "

25

0

i./r

~~~~

2e

e

150

100

50

o

+ +1

0~

+ 2

00 +

~00 +

SUB

0EC

T:

8 D

ose

=

1 M

g/K

g LA

AM

6 =

C

on

cen

trati

on

1

2 h

rs

po

st

do

se

~

= D

ose

ad

min

iste

red

41 +

5 10

HOUR

S

60

0

70

0

80e

90

0

10

00

t--'

\.0

11

1

196

Figure 19E. Subject 8. Human plasma concentrations of

dinor-LAAM; total time course.

¢'I CI) o

'"'0 ....

to o 0-

'" c.. ..r:. "'0

(\J <i> e- c.. o <i> OC ....

....J 0 to CI;)......, C ~O­, c.. e

co CI;)......, "'0 ~CO

cP •• - 0 (,?

..... C to U II 0 0 W UQ J G) CD (I) II U :::> 0 C/) 0<J-E---------

LI) N C\J

('S) lt)

lJ) N

, " , , , , , , , , ,

Lf) ,...... (S) lJ) lJ) (\J (S)

197

('S) (S) 0)

(S) (S) 0)

(S) CS) ,.....

CS) (S) CO

C/) ~

~5 l/) :r:

...:e-

~...:e-"¢"

"'IIIiE-(S)

~

...e-CS) (S) (\J

...e-

(S)-E-(S)

~

('S)-E-

198

Figure 20C. Subject 9. Human plasma concentrations of

LAAM; total time course.

......, (J)

o Q..

(f) L

L

N ~­<t <t C ..J 0

...., (f)

0)......, C ~ 0 "L E (J) 0) .,..J -0 ~ C 0

CJ •• - (J ~ t- C (f) U II 0 0 W U Q ~ Q) m (/) 11 n :::> 0 (f) 0 <J ....:E-

CS) If) N

(S) (Sj N

199

<.'S) CS) CJ)

<.'S) ,(S) Q:)

<.'S) CS) 1'00

<.'S) <S) <0

(f) c:t:

~~ lJ) J:

~

~-E-• r • V I I ,

I -E-I I (S)

" ~ " \

'\ '\ -E-'\

\ <S) '\

'\ CS) '\ (\J

-E-

(S)-E-CS) -~

200

Figure 20D. Subject 9. Human plasma concentrations of

nor-LAAM; total time course.

see

450

4ee

350

3e0

NG/H

L 25

0

20e

150

t0e

50

o

+ +

10~ +

20

0 +

~00 +

SUB

JEC

T:

9 D

ose

=

t

Mg

/Kg

LA

AM

~

= C

on

cen

lrati

on

1

2 h

rs

po

st

do

se

t =

Do

se

ad

min

iste

red

400

5i0

~ +

HdURS

60

0 70

0 80

0 90

0 10

00

tv

o r-'

202

Figure 20E. Subject 9. Human plasma concentrations of

dinor-LAAM; total time course.

225

230

175

150

125

NG/H

L le

e

75

50

25

o

+ +10

+ +

200 +

+00 +

SUB

JEC

T:

9 D

ose

=

1

Mg

/Kg

LA

AM

6

= C

on

cen

trati

on

1

2 h

rs

po

st

do

se

~

= D

ose

ad

min

iste

red

400

sqa

+ +

H~URS

600

700

800

900

1000

N

o w

204

Figure 2lC. Subject 10. Human plasma concentrations of

LAAM; total time course.

650

60

0

550

500

450

400

350

NG/M

L 30

0

250

200

t50

100

50

0

f +10

+ +

200 +

~00 +

SUB

JEC

T:

10

Do

se

=

1 M

g/K

g LA

AM

L

=

C

on

cen

trati

on

1

2 h

rs

po

st

do

se

~ =

Do

se

ad

min

iste

red

4~ +

:l:RS

600

700

800

ge0

1000

tv

o U1

206

Figure 2lD. Subject 10. Human plasma concentrations of

nor-LAAM; total time course.

530

4se

400

3S0

30

0

NG/M

L 25

0

200

IS0

t0e

50

o

+ ~1

0~ ~ 2

00 +

~00 +

SUB

.JEC

T=

10

Do

se

= 1

M

g/K

g LA

AM

6

=

Co

ncen

trati

on

1

2 h

rs

po

st

do

se

t =

Do

se

ad

min

iste

red

400

s'le

~ +

HduRS

60

0 70

0 80

0 90

0 10

0e

I\.)

o ....,J

208

Figure 21E. Subject 10. Human plasma concentrations of

dinor-LAAM; total time course.

225

2ee

175

150

125

1'30

75

50

25

e

~ ~Ie+

~ 2ee

~

~ee ~

SU

BJE

CT

: 10

D

ose

= 1

Mg/

Kg

LAAM

6

= C

on

cen

lrali

on

1

2 h

rs

po

sl

do

se

t =

Do

se

ad

min

isle

red

400

500

~ ~

H~URS

600

7e0

80

0

900

1000

I:

\.)

o \..0

210

Figure 22C. Subject 11. Human plasma concentrations of

LAAM; total time course.

211

CS)

Q) (S)

w CS)

0 -0 ...., (.'S)

w CS)

0 m Q.

In (S) L CS)

.J: (X)

-0 N (1)

0)- L ~ Q) CS)

L: C -a.J (S)

«('-. 0 en ,.......

« If) .---1 ...., C

l! 0 CS) - 0) L E (S)

-:E-a.J-a.J V (0

.J: C 0 (S) 0) Q) (f)

.. 00 .- U (J.) ~

1- cl) C en (S)--E-=:J U 11 3- 0 0

., ~ 0 ,;- lJ) :::t:

W U Q , ,

-; (1) >-,

--E-, , en IJ) -0 11 II , => 0 0

, CS)-E-

(f)QQ:)<J CS) v

-E-(S)

~ , I I -E-I I CS) ,

(S) r N

-E-

CS)--E-(S)

-Eo-

....J CS)--E-

CS) CS) CS) CS) (S) CS) cs)::I:cs) (S) CS) (S) (S) If) (S.) If) CS) If) CS) I.J)'CS) I.J) CS) If) (S) (S) (0 (0 lJ) Lf) v v ('I"')(!)('I"') C\J C\J lJ) CS)

Z

212

Figure 22D. Subject 11. Human plasma concentrations of

nor-LAAMi total time course.

52

0

453

430

3S0

30e

NG/H

L 25

0

200

t50

tee

50

o

+ +1

0~ +

200 +

~e0 +

SUB

JEC

T:

It

Do

se

= 8

0

Mg

LAA

M

Bo

dy

w

eig

ht

= 5

7

Kg

6 =

Co

ncen

trati

on

1

2 h

rs

po

st

do

se

t =

D

ose

ad

min

iste

red

41 5~0

+ Hdu

RS

600

700

80

0

ge0

t000

tv

,....

.., w

214

Figure 22E. Subject 11. Human plasma concentrations of

dinor-LAAM; total time course.

225

230

175

150

125

NG/H

L 10

0

75

50

25

o

+ ~1

0~ ~ 2

00 ~

~00 ~

SUB

JEC

T:

t 1

Do

se =

80

M

g LA

AM

Bo

dy

weig

ht

= 5

7

Kg

6

=

Co

ncen

trali

on

1

2 h

rs p

ost

do

se

~

=

Do

se ad

min

iste

red

400

5'10

~ ~

HJUR

S 60

0 70

0 80

0 g

e0

10

00

tv

I--'

U1

216

Figure 23C. Subject 12. Human plasma concentrations of

LAAM; total time course.

65

e

6~0

550

500

450

40

0

350

NG/M

L 3

00

25

0

20

0

150

100

50

0 B

~ ~0

0 +

t00

~ ~

+2

00

+

, f , , I , , , , , I , , , , I , , , I , , , , , I , , , I I , I 4+ +

5+0

HOUR

S

SUB

JEC

T:

12

Dos

e =

80

M

g LA

AM

Bod

y w

eig

hl

=

61

Kg

6 =

C

on

cen

lrali

on

12

h

rs p

ost

do

se

t =

Do

se

ad

min

isle

red

600

700

800

90

0

10

00

tv

I--

-' -...

.J

218

Figure 23D. Subject 12. Human plasma concentrations of

nor-LAAM; total time course.

se0

450

430

350

330

NG/M

L 25

0

200

t50

10e

50

e

+ +10

+ +

200 +

~00 +

SU

BJE

CT

: 12

D

ose

=

80

M

g LA

AM

B

od

y

weig

hl

= 6

1 K

g 6

= C

on

cen

trati

on

1

2 h

rs

po

st

do

se

t =

Do

se ad

min

iste

red

I I I I I I I I I

400

50

0

+ +

H~URS

600

700

800

gee

10

00

tv

t-'

1..0

220

Figure 23E. Subject 12. Human plasma concentrations of

dinor-LAAM; total time course.

U') (\J C\I

OJ (J) o

-0 .....,J

(I) o a. en L

-C -0

N (J.) 0)- L ~ Q)

',E C.....,J <- 0 (J) <<0---l .....,J C

II 0 N 0> L. E -',E.....,J......, -0

-C C 0 (S) 0) Q)

··00·- 0 (J.) to- (1)C (I) U II 3 0 0 W U 0 "J Q) >-en (J) -0 11 II ::;:) 0 0 (j') a co <l .....:E-

--- - .........

(S) If)

... _- .......... --- ................ -.... -

lJ) N CS)

221

('Ii) (S) 0)

(S) CS) 0)

(S) ('Ii) r-..

(S) (S) <0

(/) 0::

CS) E ::::> (S) 0 IJ) :J::

-E-

~~ ~

~ CS)

~

-E-CS) CS) (\f

--E-

(5)--E-CS)

-E-

CS)...e-

Figure 24. Comparison of mean concentration-time

courses.

A. Following first dose: Subjects 6, 7, and 8.

B. Following last dose: Subjects 6, 7, and 8.

C. Following first dose: Subjects 9 and 10.

D. Following last dose: Subjects 9 and 10.

E. Following first dose: Subjects 2 and 4.

F. Following last dose: Subjects 2 and 4.

Black:

Red:

Blue:

LAAM

Nor-LAAM

Dinor-LAAM

222

10

00

1

00

0

1000

100

1°°1

100

NG

N

G

NG

ML'

- M

L

ML

,.

........

1°1

10

10 I'

HO

UR

S A

H

OU

RS

C

HO

UR

S

E

I-P

i i

i i

i i

i I

0 2

0

40

6

0

80

0

20

4

0

60

8

0

0 2

0

40

6

0

80

1000~

1000

-:1

10

00

IOO~1

.... 10

0-::1

.--~

loo~r ~

NGJ

NG

~

NG

--

-_f

l M

L M

L M

L 10

10

10

B

0

II. F

I P

i I

HO

UR

S

HO

UR

S H

OU

RS

i

i i

i i

i I

J I

i i

I ,

I tv

tv

50

4 5

24

54

4 5

64

58

4

50

4 5

24

54

4 5

64

58

4

50

4

52

4 5

44

56

4 5

84

w

APPENDIX

Table Page

1 COMPARATIVE EFFICIENCY OF METABOLITE-AMIDE DERIVATIVE FORMATION WITHOUT DEGRADATION OF LAAM. . .. ... 226

2 RECOVERY EFFICIENCY FOR LAAM AND METABOLITES UNDER VARIOUS CONDITIONS OF SOLVENT EVAPORATION . . . . . . . 227

3 GC-CHEMICAL IONIZATION-MS: LAAM, NOR-LAAM AND DINOR-LAAM PROTONATED MOLE­CULES AS A PERCENTAGE OF THEIR BASE PEAKS USING VARIOUS REAGENT GASES. . . . 228

4 MONKEY PLASMA CONCENTRATIONS OF LAAM AND HETABOLITES FOLLOWING SINGLE ORAL 2 mg/kg DOSE OF LAAM AT ZERO TIME. . .. 229

5 HUMAN STUDY: INDIVIDUAL DOSE OF LAAM AND BODY WEIGHT FOR EACH SUBJECT . . . . .. 230

6 TIMES OF DOSE ADMINISTRATION HOURS INTO STUDY. . . . . . . . . . . . . . . . . . 231

7 SUBJECT 001: HUMAN PLASMA CONCENTRATION OF LAAM AND METABOLITES OVER A 42-DAY PERIOD FOLLOWING REPETITIVE ORAL DOSES OF 80 mg LAAM. . . . . . . . . . . . .. 23 2

8 SUBJECT 002: HUMAN PLASMA CONCENTRATION OF LAAM AND METABOLITES OVER A 42-DAY PERIOD FOLLOWING REPETITIVE ORAL DOSES OF 80 mg LAAM. . . . . • . . . . . . .. 234

9 SUBJECT 003: HUMAN PLASMA CONCENTRATION OF LAAM AND METABOLITES OVER A 42-DAY PERIOD FOLLOWING REPETITIVE ORAL DOSES OF 8 0 mg LAAM. . . . . . . . . . . . .. 2 3 6

10 SUBJECT 004: HUMAN PLASMA CONCENTRATION OF LAAM AND METABOLITES OVER A 42-DAY PERIOD FOLLOWING REPETITIVE ORAL DOSES OF 80 mg LAAM. . . . . . . . . • . . .. 238

Table

11 SUBJECT 005: HUMAN PLASMA CONCENTRATION OF LAAM AND fvlETABOLITES OVER A 42-DAY PERIOD FOLLOWING REPETITIVE ORAL DOSES

225

Page

OF 8 a mg LAA..M. . . . . . . . . . . . .. 241

12 SUBJECT 006: HUMAN PLASMA CONCENTRATION OF LAAM AND METABOLITES OVER A 42-DAY PERIOD FOLLOWING REPETITIVE ORAL DOSES OF 73 mg LAAM. . . . • . . . . . . . •. 244

13 SUBJECT 007: HUMAN PLASMA CONCENTRATION OF LAAM AND METABOLITES OVER A 42-DAY PERIOD FOLLONING REPETITIVE ORAL DOSES OF 89 mg LAAM. . . . .. • .. .. • • . . . . 247

14 SUBJECT 008: HUMAN PLASMA CONCENTRATION OF LAAM AND !-1ETABOLITES OVER A 42-DAY PERIOD FOLLOWING REPETITIVE ORAL DOSES OF 75 mg LAAM. . . . .. . . . . . .. .. .. 250

15 SUBJECT 009: HU~Vlli PLASMA CONCENTRATION OF LAJU1 AND METABOLITES OVER A 42-DAY PERIOD FOLLOWING REPETITIVE ORAL DOSES OF 82 MG LAAM. . . . . .. .. .. • . .. . ... 253

16 SUBJECT 010: HUMAN PLASMA CONCENTRATION OF LAAM AND METABOLITES OVER A 42-DAY PERIOD FOLLOWING REPETITIVE ORAL DOSES OF 80 mg LAA..~. . . . . . . . . .. . . .. 256

17 SUBJECT 011: HUMAN PLASMA CONCENTRATION OF LAAM AND METABOLITES OVER A 42-DAY PERIOD FOLLONING REPETITIVE ORAL DOSES OF 80 mg LAA..T>.1. • . . . . . . . • . . . . 259

18 SUBJECT 012: HUMM~ PLASMA CONCENTRATION OF LAAM AND METABOLITES OVER A 42-DAY PERIOD FOLLOWING REPETITIVE ORAL DOSES OF 80 mg LAAM. . . • • • • . • 262

19 PLASMA HALF-LIVES IN HOURS FOR LAAM AND METABOLITES. • .. . . . . . . . 265

LAA

M

din

or-

LA

AM

no

r-A

mid

e

TA

BL

E

1

CO

MPA

RA

TIV

E

EF

FIC

IEN

CY

O

F M

ET

AB

OL

ITE

-AM

IDE

D

ER

IVA

TIV

E

FOR

MA

TIO

N

WIT

HO

UT

D

EG

RA

DA

TIO

N

OF

LAA

M

25

°C

-3

0

Min

7

0°C

-

70

°C

-2

5°C

-

25

°C

-7

0°C

-

50

°C

-3

0

Min

3

0

Min

1

0

Min

3

0

Min

1

0

Min

1

0

Min

H

C1

NaO

H

NaO

H

NaO

H

NaO

H

NaO

H

100%

32

%

90%

90

%

90%

85

%

0%

100%

>9

0%

>90%

>9

0%

>90%

0%

100%

50

%

65%

70

%

70%

NO

TE

: 5

.0

N N

aOH

u

sed

at

pH

13

.

50

°C

-3

0

Min

N

aOH

53%

90%

70%

N

N

0'\

LAA

M

TA

BL

E

2

REC

OV

ERY

E

FF

ICIE

NC

Y

FOR

LA

AM

A

ND

M

ET

AB

OL

ITE

S U

ND

ER

VA

RIO

US

CO

ND

ITIO

NS

O

F SO

LV

EN

T

EV

APO

RA

TIO

N

50

ng

LA

AM

D

o A

dd

ed

to B

ott

om

of

Ev

ap

ora

tio

n

Tu

be

100%

50

n

g

LAA

M

Do

Ad

ded

to

1

0

ml

Ch

loro

form

55%

50

ng

LA

AM

D

o A

dd

ed

to

10

m

l C

hlo

rofo

rm D

ried

w

ith

A

dd

itio

n o

f 2

00

cc/m

in o

f F

ilte

red

A

ir

>90%

NO

TE

: A

ll

ev

ap

ora

tio

ns w

ere

p

erf

orm

ed

in

a

70

°C w

ate

r b

ath

.

Reco

veri

es

were

dete

rmin

ed

b

y ad

din

g

50

ng

o

f LA

AM

D

3 to

th

e d

ry resi­

du

es,

a rati

o o

f LA

AM

D

o/L

AA

M

D3

= 1

.0 =

10

0%

.

l\J

l\J

-....

j

228

TABLE 3

GC-CHEMICAL IONIZATION-MS: LAAM, NOR-LAAM AND DINOR-LAAM PROTONATED MOLECULES AS A PERCENTAGE OF THEIR BASE

PEAKS USING VARIOUS REAGENT GASES

Methane

LAAM <1

nor-LAAM (amide) <2

dinor-LAAM (amide) <1

Isobutane

1

18

17

Ammonia-Methane

100

30

30

229

TABLE 4

MONKEY PLASMA CONCENTRATIONS OF LAAM AND METABOLITES FOLLOWING SINGLE ORAL 2 mg/kg DOSE

Monkey Number

6719

6733

- - - - -

6751

-

- - - - - -

6775

OF LAAM AT ZERO TIME

Sample Time (Hours)

- -

- -

zero 2 4 6

11 24

zero 2 4 6

11 24

zero 2 4 6

11 24

zero 4 6

11 24

- - - -

- - - -

- -

- -

Concentration in ng/m1

LAAM nor-LAAM dinor-LAAM

0 0 0 11 28 15

7 11 11 0 5 7 0 1 3 0 0 0

- - - ------

0 0 0 8 6 5 8 8 7 8 7 8 0 2 4 0 2 3

- - - - -- - - - - - - - -

0 0 0 3 7 15 1 2 7 0 1 4 0 0 2 0 0 1

- - - - - - - - - - - -0 0 0 0 3 3 0 2 2 0 1 2 0 0 0

230

TABLE 5

HUMAN STUDY: INDIVIDUAL DOSE OF LAAM AND BODY WEIGHT FOR EACH SUBJECT

Subject Dose (rng/kg) Body Wt (kg) Dose (rng)

002 0.73 109 80

003 0.85 94 80

006 1.00 73 73

007- 1.00 89 89

008 1.00 75 75

009 1.00 82 82

010 1.00 80 80

001 1.29 62 80

012 1.31 61 80

005 1.39 61 80

011 1.40 57 80

004 1.51 53 80

231

TABLE 6

TIMES OF DOSE ADMINISTRATION HOURS INTO STUDY

Dose Subject No.

No. 1 2 3 4 5 6

1 0.08 0.89 0.25 0.03 0.08 0.08

2 72 72.08* 72.18 72.12 72.17 72.17

3 120 120.08 120.25 120.12 120.17 120.17

4 168 168.08 168.25 168 168.08 168.17

5 240 240.08 240.25 240* 240* 240*

6 287.83 288.08 288.42 288* 288* 288.08*

7 335.92 336 336.42 336* 336* 336*

8 408* 408* 408* 408.08 408.10 408.17

9 455.92 456 456.25 456* 456* 456*

10 503.92 504 504.25 405.03 504.25 405.17

Dose Subject No.

No. 7 8 9 10 11 11

1 0.08 0.08 0.08 0.08 0.25 0.08

2 72.17 72.17 72.17 72.08 72.08 72.08

3 120.17 120.17 120.17 120.25 120.08 120.08

4 168.17 168.17 168.25 168.25 168.08 168.08

5 240* 240* 240* 240* 240* 240*

6 288.17 288.17 288.33 288.08 288.08 288.08

7 336* 336* 336* 336* 336* 336*

8 408.17 408.33 408.08 408.08 408* 408*

9 456* 456* 456* 456 456* 456*

10 504.17 504.24 504.25 405.08 504.08 504.08

*Approximate time.

232

TABLE 7

SUBJECT 001: HUMAN PLASMA CONCENTRATION OF LAAM AND METABOLITES OVER A 42-DAY PERIOD FOLLOWING

REPETITIVE ORAL DOSES OF 80 mg LAAM

--Specimen Hour(s) Hour (s) Concentration in ng/ml

No. Into Study Post Dose LAAM Nor-LAAM Dinor-LAAM

1 0 0 0 0 0

2 2 +1*

2 46 40 0

3 4 4 52 50 20

4 5 5 51 64 28

5 6 6 54 65 25

6 7 7 46 60 28

7 8 8 4E 66 30

8 10 10 29 58 32

9 12 12 23 49 17

10 16 16 21 46 32

11 24 24 13 29 24

12 48 48 9 18 32

13 72 72/0 9 10 29

14 78 +2*

6 58 74 40

15 80 8 39 68 50

16 120 +3*

48/0 23 34 55

17 126 6 88 112 89

18 128 8 68 112 162

19 168 48/0 31 41 70

20 174 +4*

6 88 115 1-3

21 176 8 84 132 130

22 240 72/0 16 36 77

23 246 + 5*

6 88 122 64

24 248 8 71 116 115

25 288 48/0 33 49 100 +6*

26 294 6 92 122 113

233

TABLE 7 (continued)

Specimen Hour(s) Hour(s) Concentration in ng/ml No. Into Study Post Dose LAAM Nor-LAAM Dinor-LAAM

27 296 6 92 122 113

28 336 48/0

29 342 +7*

6 94 109 73

30 344 8 155 95 118

31 408 72/0 56 15 75

3] 414 +8*

6 72 93 84

33 416 8 75 90 85

34 456 48/0 69 42

35 462 +9*

6 91 95 102

36 464 8 79 102 40

36 464 8 79 102 40

37 504 48/0 30 33 70

38 506 +10*

2 140 96 86

39 508 4 110 107 83

40 509 5 94 92 89

41 510 6 75 96 88

52 511 7 79 105 101

43 512 8

44 514 10 54 79 86

45 516 12 25 71 79

46 520 16 29 61 80

47 528 24 37 52 71

48 552 48 29 32 72

49 576 72 27 25 31

*Indicates time of dose. See Table 6.

234

TABLE 8

SUBJECT 002: HUMAN PLASMA CONCENTRATION OF LAAM AND METABOLITES OVER A 42-DAY PERIOD FOLLOWING

REPETITIVE ORAL DOSES OF 80 rng LAAM

Specimen Hour(s) Hour(s) Concentration in ng/m1 No. Into Study Post Dose LAAM Nor-LAAM Dinor-LAAM

1 0 +1* 0 0 0 0

2 2 2 72 83 16

3 4 4 67 III 20

4 5 5 72 69 21

5 6 6 62 72 22

6 7 7 55 72 23

7 8 8 50 72 23

8 10 10 39 62 24

9 12 12 37 67 42

10 16 16 38 65 26

11 24 24 32 67 33

12 48 38 21 83 31

13 72 +2* 72/0 37 32

14 78 6 34 205 76

15 80 8 54 105 41

16 120 +3* 48/0 20 53 43

17 126 6 90 117 54

18 128 8 67 100 56

19 168 +4* 48/0 27 60 51

20 174 6 84 105 59

21 176 8 66 117 58

22 240 +5* 72/0 14 35 44

23 246 6 76 107 56

24 248 8 76 50 76

25 288 +6* 48/0 26 77 76

26 294 6 106 182 94

235

TABLE 8 (continued)

Specimen Hour{s) Hour (s) Concentration in ng/m1 No. Into Study Post Dose LAAM Nor-LAAM Dinor-LAAM

27 296 8 76 155 94

28 336 +7* 48/0 26 72 70

29 342 6 33 138 182

30 344 8 68 125 76

31 408 +8* 72/0 21 19 70

32 414 6 125 170 112

33 416 8 92 32 44

34 456 +9* 48/0 28 80 70

35 462 6 120 147 64

36 464 8 96 155 88

37 504 +10* 48/0 43 100 88

38 506 2 76 98 76

39 508 4 100 182 88

40 509 5 82 76 53

41 510 6

42 511 7 115 165 88

43 512 8 100 133 76

44 514 10 77 133 76

45 516 12 77 129 70

46 520 16 54 50 76

47 528 24 45 133 76

48 552 48 34 94 64

59 576 72 30 76 76

*Indicates time of dose. See Table 6.

236

TABLE 9

SUBJECT 003: HUMAN PLASMA CONCENTRATION OF LAAM AND METABOLITES OVER A 42-DAY PERIOD FOLLOWING

REPETITIVE ORAL DOSES OF 80 mg LAAM

Specimen Hour(s) Hour (s) Concentration in ng/ml No. Into Study Post Dose LAAM Nor-LAAM Dinor-LAAM

1 0 +-1 *

0 0 0 0

2 2 2 54 38 13

3 4 4 68 50 16

4 5 5 55 58 16

5 6 6 55 65 20

6 7 7 47 65 20

7 8 8 47 65 21

8 10 10 39 56 20

9 12 12 31 56 22

10 16 16 21 47 21

11 24 24 15 43 20

12 48 48 8 26 23

13 72 +-2*

72/0 7 16 13

14 78 6 73 55 28

15 80 8 55 52 22

16 120 +-3*

48/0 34 50 49

17 126 6 103 100 54

18 128 8 77 102 49

19 168 +-4*

48/0 40 56 52

20 174 6 103 92 69

21 176 8 80 92 57

22 240 +- 5*

72/0 27 41 31

23 246 6 85 88 67

24 248 8 80 95 68

24 248 8 80 95 68

25 288 +-6*

48/0

26 294 6 97 105 68

237

TABLE 9 (continued

Specimen Hour(s) Hour(s) Concentration in ng/ml No. Into Study Post Dose LAAM Nor-LAAM Dinor-LAAM

27 296 8 85 105 75

28 336 -,:-7* 48/0 27 45 47

29 342 6 86 84 64

30 344 8 66 84 66

31 408 -,:-8* 72/0 22 37 62

32 414 6 95 89 76

33 416 8 95 116 100

34 456 -':-9* 48/0 23 44 66

35 462 6 106 100 108

36 464 8 84 89 71

37 504 -,:-10* 48/0 27 57 71

38 506 2 160 116 90

39 508 4 142 128 85

40 509 5 134 120 76

41 510 6 120 120 76

42 511 7 103 112 76

43 512 8 97 116 85

44 514 10 79 104 76

45 516 12 74 104 76

46 520 16 57 112 90

47 528 24 42 92 80

48 552 48 24 58 62

49 576 72 22 45 66

*Indicates time of dose. See Table 6.

238

TABLE 10

SUBJECT 004: HUMAN PLASMA CONCENTRATION OF LAAM AND METABOLITES OVER A 42-DAY PERIOD FOLLOWING

REPETITIVE ORAL DOSES OF 80 mg LAAM

Specimen Hour(s} Hour(s) Concentration in ng/ml No. Into Study Post Dose LAAM Nor-LAAM Dinor-LAAM

1 0 +1* 0 0 0 0

2 0.25 0.25 0 0 0

3 0.5 0.5 24 0 0

4 1 1 58 -62 0

5 1.5 1.5 151 85 2

6 2 2 145 109 0

7 4 4 144 105 0

8 6 6 119 136 0

9 12 12 66 162 24

10 16 16 52 135 0

11 24 24 47 134 8

12 48 48 32 101 31

13 72 +2* 72/0 22 50 10

14 72.25 0.25 28 76 8

15 72.5 0.5 46 76 8

16 73 1 III 115 8

17 73.5 1.5 140 134 7

18 74 2 194 161 10

19 76 4 176 209 5

20 78 6 151 229 18

21 84 12 104 255 23

22 88 16 79 180 15

23 96 24 80 150 32

24 120 + 3* 48/0 26 125 21

25 120.25 0.25 36 128 18

26 120.5 0.5 44 128 19

239

TABLE 10 (continued)

Specimen Hour(s) Hour(s) Concentration in ng/ml No. Into Study Post Dose LAAM Nor-LAAM Dinor-LAAM

27 121 1 98 153 21

28 121.5 1.5 153 222 41

29 122 2 139 173 34

30 124 4 144 183

31 126 6 145 247 33

32 132 12 83 184 158

33 136 16 82 222 71

34 144 +-4* 24 58 250 28

35 168 48/0 51 339 69

36 169 1 65 227 169

37 180 12 123 509 48

38 192 24 70 406 51

39 216 +- 5* 48 47 221 81

40 288 +- 6* 48/0 55 200 73

41 289 1 46 200 32

42 300 12 153 334 63

43 312 +-7* 24 88 244 60

44 408 +-8* 72/0

45 409 1

46 420 12 190 79 81

47 432 +-9* 24 153 294 76

48 504 +-10* 48/0 97 221 146

49 504.25 0.25 214 209 190

50 504.5 0.5 109 217 161

51 505 1 172 223 154

52 505.5 1.5 277 373 156

53 506 2 311 101 61

54 508 4

55 510 6 323 288 148

240

TABLE 10 (continued)

Specimen Hour(s) Hour(s} Concentration in ng/ml No. Into Study Post Dose LAAM Nor-LAAM Dinor-LAAM

56 516 12 242 297 158

57 520 16 191 286 175

58 528 24 155 298 153

59 552 48 101 226 137

60 576 72 77 298 154

61 672 168 8 35 71

62 744 240 16 24 41

63 840 336 4 12 19

64 912 408 1 4 6

65 1008 504 0 4 1

*Indicates time of dose. See Table 6.

241

TABLE 11

SUBJECT 005: HUMAN PLASMA CONCENTRATION OF LA.Al1 AND METABOLITES OVER A 42-DAY PERIOD FOLLOWING

REPETITIVE ORAL DOSES OF 80 mg LAAM

Specimen Hour(s) Hour (s) Concentration in ng/m1 No. Into Study Post Dose LAAM Nor-LAAM Dinor-LAAM

1 0 +1* 0 0 0 0

2 0.25 0.25 0 11 0

3 0.5 0.5 13 18 0

4 1 1 35 29 0

5 1.5 1.5 92 66 1

6 2 2 129 82 3

7 4 4 148 92 4

8 6 6 154 109 8

9 12 12 103 III 10

10 16 16 65 92 11

11 24 24 47 92 13

12 48 48 20 64 8

13 72 +2*

72/0 10 40 16

14 72.25 0.25 19 44 18

15 72.5 0.5 49 51 8

16 73 1 61 68 21

17 73.5 1.5 57 68 16

18 74 2 89 94 24

19 76 4 100 130 24

20 78 6 84 136 31

21 84 12 36 149 42

22 88 16 35 132 35

23 96 24 21 106 14

24 120 48/0 7 63 29 +3*

25 120.25 0.25 10 69 32

26 120.5 0.5 200 65 29

242

TABLE 11 (continued)

Specimen Hour(s) Hour (s) Concentration in ng/ml No. Into Study Post Dose LAAM Nor-LAAM Dinor-LAAM

27 121 1 44 80 31

28 121.5 1.5 52 87 71

29 122 2 74 101 36

30 124 4 95 133 43

31 126 6 101 149 46

32 132 12 63 134 42

33 136 16 52 129 52

34 144 24

35 168 +4* 48/0 15 82 37

36 169 1 36 87 37

37 180 12 82 139 78

38 192 24 39 120 21

39 216 +5* 48 21 94 20

40 288 +6* 48/0 20 81 48

41 289 1 70 105 54

42 300 12 80 136 64

43 312 +7* 24 52 142 64

44 408 +8* 72/0

45 409 1

46 420 12 168 134 99

47 432 +9* 24 41 120 39

48 504 +10* 48/0 31 90 83

49 504.25 0.25 33 108 90

50 504.5 1 99 112 81

52 505.5 1.5 207 218 62

53 506 2 139 163 107

54 508 4 144 182 106

55 510 6 129 255 120

56 516 12 121 168 115

243

TABLE 11 (continued)

Specimen Hour{s) Hour{s) Concentration in ng/m1 No. Into Study Post Dose LAAM Nor-LAAM Dinor-LAAM

57 520 16 61 206 112

58 528 24 36 180 103

59 552 48 24 132 88

60 576 72 4 40 24

61 672 168 11 38 49

62 744 244 0 20 24

63 840 336 0 0 0

64 912 408 0 0 0

65 1008 504 0 0 0

*Indicates time of dose. See Table 6.

244

TABLE 12

SUBJECT 006: HUMAN PLASMA CONCENTRATION OF LAAM AND METABOLITES OVER A 42-DAY PERIOD FOLLOWING

REPETITIVE ORAL DOSES OF 73 mg LAAM

Specimen Hour(s) Hour(s) Concentration in ng/ml No. Into Study Post Dose LAAM Nor-LAAM Dinor-LAAM

1 0 0 0 0 0 +1*

2 0.25 0 0 0 0

3 0.5 0.5 34 27 3

4 1 1 76 66 8

5 1.5 1.5 138 94 10

6 2 2 131 101 12

7 4 4 85 94 12

8 6 6 143 III 9

9 12 12 45 76 13

10 16 16 38 78 15

11 24 24 31 66 16

12 48 48 14 48 15

13 72 72/0 13 38 22 +2*

14 72.25 0.25 20 40 22

15 72.5 0.5 51 54 23

16 73 1 134 89 43

17 73.5 1.5 156 106 25

18 74 2 153 118 27

19 76 4 114 III 27

20 78 6 93 109 29

21 84 12 56 102 31

22 88 16 41 88 27

23 96 24 36 96 33

24 120 48/0 27 75 38 +3* 25 120.25 0.25 40 83 40

26 120.5 0.5 88 93 42

245

TABLE 12 (continued)

Specimen Hour(s) Hour(s) Concentration in ng/m1 No. Into Study Post Dose LAAM Nor-LAAM. Dinor-LAAM

27 121 1 149 118 43

28 121.5 1.5 215 136 44

29 122 2 180 144 46

30 124 4 137 154 47

31 126 6 00

32 132 12 75 147 47

33 136 16 56 123 41

34 144 24 56 130 51

35 168 48/0 41 113 59

36 169 1 74 116 54

37 180 12 97 052 56

38 192 24 65 157 68

39 216 48 39 94 59

40 288 48/0 21 75 75

41 289 1 75 104 91

42 300 12 104 136 78

43 312 24 65 148 89

44 408 +-8* 72/0 66 108 81

45 409 1 235 163 97

46 420 12 218 108 58

47 432 +-9* 24 151 112 "114

48 504 +-10* 48/0 96 145 92

49 504.25 0.25 105 169 108

50 504.5 0.5 55 155 235

51 505 1 156 165 92

52 505.5 1.5 213 180 101

53 506 2 115 198 102

54 508 4 343 208 100

55 510 6 339 245 114

246

TABLE 12 ( continued)

= Specimen Hour (s) Hour(s) Concentration in ng/m1

No. Into Study Post Dose LAAM Nor-LAM! Dinor-LAAM

56 516 12 227 222 107

57 520 16 149 208 102

58 528 24 129 217 104

59 552 48 96 146 90

60 576 72 81 131 91

61 672 168 30 47 74

62 744 240 21 24 61

63 840 336 16 18 30

74 912 408 16 3 17

65 1008 504 12 3 10

*Indicates time of dose. See Table 6 .

247

TABLE 13

SUBJECT 007: HUMAN PLASMA CONCENTRATION OF LAAM AND METABOLITES OVER A 42-DAY PERIOD FOLLOWING

REPETITIVE ORAL DOSES OF 89 mg LAAM

Specimen Hour(s) Hour(s) Concentration in ng/m1 No. Into Study Post Dose LAAM Nor-LAAM D i nor - LAAr-1

1 0 +1* 0 0 0 0

2 0.25 0.25 32 5 0

3 0.5 0.5 66 18 0

4 1 1 150 58 3

5 1.5 1.5 200 84 5

6 2 2 117 48 2

7 4 4 174 87 6

8 6 6 71 III 12

9 12 12 95 103 9

10 16 16 96 6 11

11 24 24 54 104 14

12 48 48 22 78 14

13 72 + 2* 72.0 22 54 19

14 72.25 0.25 50 78 18

15 72.5 0.5 101 89 22

16 73 1 251 140 23

17 73.5 1.5 285 163 24

18 74 2 288 187 25

19 76 4 213 176 25

20 78 6 187 148 22

21 84 12 106 67 26

22 88 16 94 187 32

23 96 24 61 161 31

24 120 +3* 48/0 50 131 36

25 120.25 0.25 129 36

26 120.5 0.5 91 144 36

248

TABLE 13 (continued)

Specimen Hour(s) Hour (s) Concentration in ng/ml No. Into Study Post Dose LAAM Nor-LAAM Dinor-LAAM

27 121 1 232 201 41

28 121.5 1.5 283 250 45

29 122 2 305 254 44

30 124 4 237 256 48

31 126 6 173 225 42

32 132 12 114 230 45

33 136 16 90 209 43

34 144 24 73 225 52

35 168 +4* 48/0 49 183 57

36 169 1 123 197 56

37 180 12 103 214 55

38 192 24 78 233 71

39 216 48 52 170 62 +-5* 40 288

+6* 48/0 65 198 76

41 289 1 94 201 76

42 300 12 155 307 85

43 312 +7* 24 106 295 88

44 408 +8* 72/0 116 272 88

45 409 1 193 249 69

46 420 12 347 433 106

47 432 +9* 24 219 387 92

48 504 +10* 48/0 156 327 92

49 504.25 0.25 166 345 101

50 504.5 0.5 190 369 107

51 505 1 255 390 109

52 505.5 1.5 306 384 103

53 506 2 412 407 105

54 508 4 616 488 114

55 510 6 569 478 113

249

TABLE 13 (continued)

Specimen Hour (s) Hour(s) Concentration in ng/ml No. Into Study Post Dose LAAM Nor-LAAM Dinor-LAAM

56 516 12 377 498 117

57 520 16 307 442 105

58 528 24 276 481 119

59 552 48 395 109

60 576 72 106 325 96

61 672 168 61 187 83

62 744 240 33 104 65

'63 840 336 10 53 51

64 912 408 1 22 30

65 1008 504 0 9 18

*Indicates time of dose. See Table 6.

250

TABLE 14

SUBJECT 008: HUMAN PLASMA CONCENTRATION OF LAAM AND METABOLITES OVER A 42-DAY PERIOD FOLLOWING

REPETITIVE ORAL DOSES OF 75 mg LAAM

Specimen Hours(s) Hour(s) Concentration in ng/m1 No. Into Study Post Dose LAAM Nor-LAAM Dinor-LAAM

1 0 +1* 0 0 0 0

2 0.25 0.25 0 0 0

3 0.5 0.5 33 18 3

4 1 1 141 83 9

5 1.5 1.5 227 119 12

6 2 2 268 147 15

7 4 4 265 175 23

8 6 6 182 147 23

9 12 12 97 134 25

10 16 16 69 125 27

11 24 24 47 106 32

12 48 48 24 73 34

13 72 +2* 72/0 15 48 36

14 72.25 0.25 32 51 35

15 72.5 0.5 71 64 36

16 73 1 190 104 37

17 73.5 1.5 55 154 39

18 74 2 276 166 44

19 76 4 270 182 50

20 78 6 208 170 49

21 84 12 116 160 49

22 88 16 79 169 47

23 96 24 66 143 47

24 120 +3* 48/0 52 96 51

25 120.25 0.25 68 104 53

26 120.5 0.5 89 109 53

251

TABLE 14 (continued)

Specimen Hour(s) Hour(s) Concentration in ng/rn1 No. Into Study Post Dose LAAM Nor-LAAM Dinor-LAAM

27 121 1 261 163 57

28 121.5 1.5 302 195 60

29 122 2 330 222 64

30 124 4 264 232 67

31 126 6 197 216 65

32 132 12 138 215 68

33 136 16 104 214 69

34 144 24 80 180 73

35 168 +4* 48/0 50 134 70

36 169 1 148 159 72

37 180 12 160 240 91

38 192 24 92 194 87

39 216 +5* 48 61 160 97

40 288 +6* 48/0 66 159 97

41 289 1 180 198 109

42 300 12 177 273 127

43 312 +7* 24 128 286 126

44 408 +8* 72/0 99 166 104

45 409 1 331 248 118

46 420 12 257 304 127

47 432 +9* 24 161 258 121

48 504 +10* 48/0 123 194 113

49 504.25 0.25

50 504.5 0 .. 5 187 201 113

51 505 1 222 215 116

52 505.5 1.5 305 246 124

53 506 2 365 268 125

54 508 4 413 291 131

55 510 6 418 317 142

252

TABLE 14 (continued)

Specimen Hour(s} Hour(s} Concentration in ng/ml No. Into Study Post Dose LAAM Nor-LAAM Dinor-LAAM

56 516 12 283 315 140

57 520 16 211 279 136

58 528 24 174 275 133

59 552 48 144 243 205

60 576 72 101 184 135

61 672 168 34 44 63

62 744 240 16 20 36

63 840 336 8 10 13

64 912 408 7 8 6

65 1008 504 5 8 4

*Indicates time of dose. Table 6.

253

TABLE 15

SUBJECT 009: HUMAN PLASMA CONCENTRATION OF LAAM AND METABOLITES OVER A 42-DAY PERIOD FOLLOWING

REPETITIVE ORAL DOSES OF 82 mg LAAM

--Specimen Hour (s) Hour(s) Concentration in ng/ml

No. Into Study Post Dose LAAM Nor-LAAM Dinor-LA&~

1 0 +1* 0 0 0 0

2 0.25 0.25 0 0 0

3 0.5 0.5 0 0 3

4 1 1 14 6

5 1.5 1.5 70 34 6

6 2 2 31 76 12

7 4 4 46 72 12

8 6 6 69 83 13

9 12 12 45 61 14

10 16 16 68 64 15

11 24 24 180 55 15

12 48 48 24 27 15

13 72 +2* 72/0 24 22

14 72.25 0.25 12 22 20

15 72.5 0.5 16 26 21

16 73 1 32 30 19

17 73.5 1.5 118 55 22

18 74 2 156 73 25

19 76 4 97 82 16

20 78 6

21 84 12 104 81 27

22 88 16 96 77 21

23 96 24 134 73 30

24 120 +3* 48/0 50 43 29

25 120.25 0125 54 35 28

26 120 .. 5 0.5 72 51 30

254

TABLE 15 (continued)

Specimen Hour (s) Hour(s) Concentration in ng/ml No. Into Study Post Dose LAAM Nor-LAAM Dinor-LAAM

27 121 1 43 61 35

28 121.5 1.5 56 85 37

29 122 2 60 106 35

30 124 4 197 132 32

31 126 6

32 132 12 76 117 43

33 136 16 33 92 51

34 144 24 15 101 61

35 168 +4* 48/0 50 44 30

36 169 1 101 60 32

37 180 12 131 73 32

38 192 24 59 102 41

39 216 +5* 48 37 64 36

40 288 +6*

48/0 34 52 30

41 289 1 79 77 38

42 300 12 187 120 45

43 312 +7* 24 41 112 49

44 408 +8* 72/0 20 59 29

45 409 1 96 74 28

46 420 12 171 112 49

47 432 +9* 24 65 84 43

48 504 +10* 48/0 79 68 45

49 504.25 0.25 58 65 42

50 504.5 0.5 138 119 53

51 505 1 184 122 53

52 505.5 1.5 180 117 61

53 506 2 250 121 54

54 508 4 324 173 67

55 510 6 229 129 52

255

TABLE 15 (continued)

Specimen Hour{s) Hour{s) Concentration in ng/ml No. Into Study Post Dose LAAM Nor-LAAM Dinor-LAAM

56 516 12 130 119 51

57 520 16 149 132 56

58 528 24 155 121 54

59 552 48 45 78 42

60 576 72 13 24 12

61 672 168 10 16 23

62 744 240 9 6 16

63 840 336

64 912 408

65 1008 504

*Indicates time of dose. See Table 6.

256

TABLE 16

SUBJECT 010: HUMAN PLASMA CONCENTRATION OF LAAM AND METABOLITES OVER A 42-DAY PERIOD FOLLOWING

REPETITIVE ORAL DOSES OF 80 mg LAAM

Specimen Hour (s) Hour(s) Concentration in ng/m1 No. Into Study Post Dose LAA14 Nor-LAAM Dinor-LAAM

1 0 +1* 0 0 0 0

2 0.25 0.25 0 0 0

3 0.5 0.5 0 0 0

4 1 1 21 10 2

5 1.5 1.5 30 18 3

6 2 2 67 22 3

7 4 4 104 40 6

8 6 6 140 52 7

9 12 12 74 58 9

10 16 16 105 63 12

11 24 24 59 54 15

12 48 48 30 17

13 72 +2* 72/0

14 72.25 0.25

15 72.5 0.5 26 20 16

16 73 1 55 38 18

17 73.5 1.5 180 60 19

18 74 2 181 63 19

19 76 4 124 90 25

20 78 6 123 71 14

21 84 12 103 74 14

22 88 16 68 71 23

23 96 24 13 65 31

24 120 +3* 48/0 40 30

25 120.25 0 .. 25 43 41 29

26 120.5 0.5 41 39 27

257

TABLE 16 (continued)

Specimen Hour(s) Hour (s) Concentration in ng/ml No. Into Study Post Dose LAAM Nor-LAAM Dinor-LAAM

27 121 1 58 50 31

28 121.5 1.5 158 66 32

29 ' 122 2 122 74 34

30 124 4 115 87 33

31 126 6 212 95 28

32 132 12 131 105 40

33 136 16 94 85 30

34 144 24 58 68 32

35 168 +4* 48/0 48 47 30

36 169 1 108 54 31

37 180 12 162 93 38

38 192 24 56 65 22

39 216 +5* 48 27 44 30

40 288 +6* 48/0 48 54 34

41 289 1 95 60 27

42 300 12 147 102 43

43 312 +7* 24

44 408 +8* 72/0

45 409 1 210 79 46

46 420 12 177 99 37

47 432 +9* 24 91 89 49

48 504 +10* 48/0 40 59 39

49 504.25 0.25 75 57 38

50 504.5 0.5 151 66 41

51 505 1 205 75 40

52 505.5 1.5 231 87 41

53 506 2 289 115 51

54 508 4 269 126 50

55 510 6 210 113 46

258

TABLE 16 (continued)

-------- ==============~~============ ~--======== Specimen

No.

56

57

58

59

60

61

62

63

64

65

Hour(s) Into Study

516

520

528

552

576

672

744

840

912

1008

*Indicates time

Hour (s) Post Dose

12

16

24

48

72

168

240

336

408

504,

Concentration in ng/ml LAAt-1 Nor -- LP...c\.l\1 Din 0 r - LAAN

168 113 45

118 105 34

73 84 35

62 67 43

49 61 41

54 21 29

10 9 10

50 3 8

3 3 4

30 2 3

"-----------.--<------.~

of dose. See 'I'c .. ble 6 •

259

TABLE 17

SUBJECT 011: HUMAN PLAS~ffi CONCENTRATION OF LAA}i AND METABOLITES OVER A 42-DAY PERIOD FOLLOWING

REPETITIVE ORAL DOSES OF 80 mg LAAM

Specimen Hour(s) Hour(s) Concentration in ng/m1 No. Into Study Post Dose LAAM Nor-LAAM Dinor-LAAM

1 0 +1* 0 0 0 0

2 0.25 0.25 0 8 0

3 0.5 0.5 0 10 0

4 1 1 20 14 0

5 1.5 1.5 64 24 1

6 2 2 89 38 3

7 4 4 510 84 9

8 6 6 226 108 16

9 12 12 99 88 22

10 16 16 126 71 20

11 24 24 67 80 1

12 48 48 70 92

13 72 +2* 72/0 32 18 19

14 72.25 0.25 50 19 23

15 72.5 0.5 46 23 27

16 73 1 100 37 11

17 73.5 1.5 303 79 10

18 74 2 342 102 34

19 76 4 169 78 22

20 78 6 81 42 53

21 84 12 67 70 46

22 88 16 73 42

23 96 24 90 69 23

24 120 +3* 48/0 40 39 54

25 120.25 0.25 76 80 80

26 120.5 0.5 93 33 29

260

TABLE 17 (continued)

Specimen Hour (s) Hour(s) Concentration in ng/m1 No. Into Study Post Dose LAAM N or-L.Aru1 Dinor-LAAM

27 121 1 50 59 53

28 121.5 1.5 135 72 63

29 122 2 388 82 63

30 124 4 171 126 52

31 126 6

32 132 12 225 130 70

33 136 16

34 144 24

35 168 +4* 48/0 92 53 70

36 169 1

37 180 12 103 78 72

38 192 24 113 75

39 216 +5* 48 595 80 64

40 288 +6* 48/0 0 34 60

41 289 1

42 300 12

43 312 +7* 24 108 136 66

44 408 +8* 72/0 0 8 18

45 409 1

46 420 12 94 70 23

47 432 +9* 24 5 23 33

48 504 +10* 48/0 0 8 18

49 504.25 0.25

50 504.25 0.5 34 109 32

51 505 1 97 181 32

52 505.5 1.5 230 202 51

53 506 2 256

54 508 4 183 197 47

55 510 6 41 144 49

261

TABLE 17 (continued)

Specimen Hour (s) Hour (s) Concentration in ng/ml No. Into Study Post Dose LAAM Nor-LAAM Dinor-LAAM

56 516 12 0 76 52

57 520 16 0 81 56

58 528 24 0 43 43

59 552 48 0 14 27

60 576 72 0 4 10

61 672 168 0 3 0

62 744 240 0 3 0

63 840 336 0 3 0

64 912 408 0 0 0

65 1008 504 0 0 0

*Indicates time of dose. See Table 6.

262

TABLE 18

SUBJECT 012: HU~urn PLASMA CONCENTRATION OF LAAM AND ~mTABOLITES OVER A 42-DAY PERIOD FOLLOWING

REPETITIVE ORAL DOSES OF 80 mg LAAM

Specimen Hour(s) Hour(s) Concentrations in ng/m1 No. Into Study Post Dose LAAM Nor-LAAM Dinor-LAAM

1 0 +1* 0 0 0 0

2 0125 0125 24 0 0

3 0.5 0.5 75 0 0

4 1 1 24 0 0

5 1.5 1.5 100 52 15

6 2 2 92 76 21

7 4 4 101 65 22

8 6 6 80 68 24

9 12 12 80 66 16

10 16 16 52 49 26

11 24 24 25 25 11

12 48 48 82 67

13 72 +2*

72/0 16 0 15

14 72.25 0.25 43 3 13

15 72.5 0.5

16 73 1 176 90 25

17 73.5 1.5

18 74 2 171 98 28

19 76 4 305 118 11

20 78 6 0 107 32

21 84 12 65 57 15

22 88 16 135 68 27

23 96 24 45 52 26

24 120 +3* 48/0

25 120.25 0.25 99 28 39

26 120.5 0.5 80 49 38

263

TABLE 18 (continued)

Specimen Hour(s) Hour (s) Concentration in ng/ml No. Into study Post Dose LAAM Nor-LAAM Dinor-LAAM

27 121 1 189 80 44

28 121.5 1.5 242 104 46

29 122 2 242 130 48

30 124 4 240 116 44

31 126 6 198 120 39

32 132 12 117 107 46

33 136 16

34 144 24 71 65 24

35 168 +- 4 * 48/0 45 49 49

36 169 1 190 63 32

37 180 12 20 134 36

38 192 24 93 86 53

39 216 +- 5* 48 100 51 37

40 288 +- 6 * 48/0 30 37 56

41 289 1 167 61 56

42 300 12 140 133 67

43 312 +-7* 24

44 408 ~8*

72/0 18 38 50

45 409 1

46 420 12 95 100 52

47 432 +-9* 24 90 112 79

48 504 +-10* 48/0 65 33 58

49 504.25 0.25

50 504.5 0.5 203 59 48

51 505 1 509 125 62

52 505.5 1.5 532 159 70

53 506 2 354 171 79

54 508 4 270 163 79

55 510 6 184 189

264

-'ontinued}

_' (s) Concentration in ng/m1 c Dose LAAM Nor-LAAM Dinor-LAAM

12 575 333 191

16 246 146 80

!) 24 110 140 69

59 48 98 139 72

60 72 72 85 56

61 6, ... 168 23 2 15

62 744 240 32 0 5

63 840 336 0 4

64 912 408 17 0 3

65 1008 504 18 0 4

*Indicates time of dose. See Table 6.

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NAME:

BIRTHPLACE:

BIRTHDATE:

SCHOOLS:

COLLEGE:

DEGREES:

HONORS:

VITA

Bryan Smith Finkle

Sunderland, County Durham, England

March 5, 1936

Monkwearmouth, Sunderland and Lemington, Newcastle-Upon-Tyne, England

Rutherford College, Durham Uni­versity, and

Northampton College, London Uni­versity, England

National and Higher National Certificates in Chemistry, Physics and Pure Mathetmatics (England)

Distinguished Service Award, Santa Clara County District Attorney's Office, California, 1965.

American Academy of Forensic Sciences Award of Merit for Outstanding Service to Forensic Science, 1974.

Certificate of Honor, Awarded for Research in Toxicology by the University of Ghent, Belgium, 1976.

President, International Association of Forensic Toxi­cologists, 1975 to present.

Vice-President, American Academy of Forensic Sciences, 1973-1974.

President, Executive Board, California Association of Toxicologists, 1975.

President, Forensic Sciences Foundation, 1976 to present.

267

PROFESSIONAL ORGANIZATIONS:

American Academy of Forensic Sciences--Fellow and Past Vice-President

Forensic Science Foundation--President

International Association of Forensic Toxicologists-­President

California Association of Toxicologists--Past President

Forensic Science Society of Great Britain

Society of, Toxicology

American Association for the Advancement of Science

American Association of Clinical Chemists

California Association of Criminalists

Sigma Xi

Western Pharmacology Society

PUBLICATIONS:

Sixty-three abstracts and publications at national and international scientific meetings; and the following published research papers:

Jackson, J.V. and Finkle, B.S. Occurrence of pseudo­barbiturates in post-mortem material. Nature 199: 1061-1063, 1963.

Sunshine, I. and Finkle, B.S. The necessity for tissue studies in fatal cyanide poisonings. Int. Archiv. fur Gewerbepathologie und Gewerbehygine ~:558-561, 1964.

Finkle, B.S. The identification, quantitative determina­tion, and distribution of meprobamate and glute­thimide in biological material. J. Forensic Sci. ~:509-528, 1967.

Finkle, B.S., Biasotti, A.A., and Fradfor, L.W. The occurrence of some drugs and toxic agents encount­ered in drinking driver investigations. J. Foren­sic Sci. 13:236-245, 1968.

268

Sunshine, I., Maes, R., and Finkle, B.S. An evaluation of methods for the determination of barbiturates in biological materials. Clin. Toxicol. l:28l-296, 1968.

Smith, W.C., Harding, D.M., Biasotti, A.A., Finkle, .B.S., and Bradford, L.W. Breathalizer experiences under the operational conditions recommended by the California Association of Criminalists. J. Forensic Sci. ~:58-64, 1969.

Maes, R., Hodnett, N., Landesman, H., Kananen, G., Finkle, B.S., and Sunshine, I. The gas chroamtographic determination of selected sedatives (ethchlorvynol, Paraldehyde, Meprobamate, and Carisprodol) in biological material. J. Forensic Sci. 14:235-254, 1969. --

Finkle, B.S. Drugs in drinking drivers: A study of 2,500 cases. J. Safety Res. l:179-l83, 1969.

Finkle, B.S. A progress report on a statewide computer program for analytical and case toxicology data. Fifth International Meeting of Forensic Sciences, Toronto, Ontario, Canada, 1969.

Lebish, P., Finkle, B.S., and Brackett, J.W., Jr. Deter­mination of amphetamine, methamphetamine, and re­lated amines in blood and urine by gas chromatog­raphy with hydrogen-flame ionization detector. Clin. Chern. 16:195-200, 1970.

Finkle, B.S., Cherry, E.J., and Taylor, D.M. A GLC based system for the detection of poisons, drugs, and human metabolites encountered in forensic toxicol­ogy. J. Chromatographic Sci. ~:393-4l9, 1971.

Finkle, B.S. Ubiquitous reds: A local perspective on secobarbital abuse. Clin. Toxicol. !:253-264, 1971.

Bradford, L.W., Biasotti, A.A., Finkle, B.S., Harding, D. M., and Smith, W.D. Inquiry into standards of practice of blood alcohol analysis. J. Forensic Sci. 11:127-130, 1971.

Forrest, F.M., Forrest, I.S., and Finkle, B.S. Alcohol­chlorpromazine interaction in psychiatric patients. Agressologie 13:67-74, 1972.

269

Finkle, B.S. and Taylor, D.M. A GC/MS reference data system for the identification of drugs of abuse. J. Chromatographic Sci. 10:312-333, 1972.

Finkle, B.S. and Taylor, D.M. A GC/MS reference data system for the identification of drugs of abuse. Finnigan Spectra 2, 1972.

"Statement--Secobarbital Abuse." u.S. Senate Judiciary Committee Proceedings. Senator Birch Bayh, Committee on Juvenile Delinquency. 1972.

A GC/MS system for the identification of drugs, narcotics, and poisons. Proceedings: Sixth International Meeting of International Association of Forensic Science, Edinburgh, U.K., 1972.

Secobarbital Abuse--A major factor in escalating traffic accidents in California. Proceedings: Fourth International Congress of Traffic Medicine, Paris, France, 1972.

Finkle, B.S. Forensic Toxicology of Drug Abuse: A status report. Analtyical Chern. !!:18-26, 1972.

Finkle, B.S. A comprehensive GC/MS reference data system for toxicological and biomedical purposes. J. Chromatographic Sci. 12:304-328, 1975.

Finkle, B.S. Forensic Toxicology--Relationship to anal­ytical chemistry. Forensic Science--American Chemical Society Symposium Series, 1974.

Finkle, B.S. "Will the real drugged driver please stand up"--An analytical toxicology assessment of drugs and driving. Proceedings of the Sixth Interna­tional Conference on Alcohol, Drugs and Traffic Safety, 197.4.

Finkle, B.S. GC-MS: Married Bliss or Breach of Promise. Microfilm. Journal of Legal Medicine, 1976.

Finkle, B.S. and Franklin, M.R. The formation of cyto­chrome P-450-455 nm complexes in vivo and iso­lated perfused rat liver. American Society for Pharmacology and Experimental Therapeutics, 1975.

270

Publications of Case Reports in the Bulletin of the International Association of Forensic Toxicolo­gists:

Two Placidyl (Ethchlorvynol) Deaths

Driving Under the Influence of Meproba­mate and Glutethinide

Librium and Valium Detection in Urine

Fatal Darvon (Propoxyphene) Ingestion

Brevital (Methohexital) Death

Fatal Ingestion of Viodex (Amphetamine)

Two Arsenic Deaths

Fatal M.D.A. Case

Detection of Tybamate in Urine

Barbiturate and Chloral Hydrate Death

Amphetamine-Tuinal Involvement in a Fatal Single Vehicle Accident

Fatal Multiple Drug (7) Ingestion

Low-Level Alcohol-Barbiturates Fatality

Three Cocaine Fatalities

Drugs and Driving. Four Cases

Pentazocine Fatality

Flurazepam and Alcohol Fatality

Formaldehyde-Methanol Suicide

Amitriptyline and Chlordiazepoxide Death

Fatal Ethchlorvynol

Monograph:

2 (1) , 1965

2 (1) , 1965

2 (2) , 1965

4 ( 3) , 1967

4 (3) , 1967

4 (4) , 1967

5 (1) , 1968

5 (2) , 1968

6 (3) , 1969

8 (I) , 1971

8 (1) , 1971

8 (I) , 1971

8 (1) , 1971

8 (3-4),1972

10 (16), 1974

10 (16), 1974

10(16),1974

11 (2) , 1975

11 ( 2) , 1975

11 ( 2) , 1975

Finkle, B.S., Contributor. Investigation of the problems and opinions of aged drivers. National Safety Council Research Report #5/68, 1968

271

Finkle, B.S., Co-author. Techniques of Combined Gas Chromatography--Mass Spectrometry. Applications in Organic Chemistry and Biochemistry. (McFadden, W.) Wiley & Sons, 1972.

Finkle, B.S. "A descriptive appreciation of modern laboratory instrumentation, with special empha­sis on gas chromatography and mass spectrometry." Legal Medicine Annual, 7th ed., 1975. (Cyril H. Wecht) Appleton-Centruy-Crofts.

Finkle, B.S. Contributor. Methodology for analytical toxicology. (Sunshine, I., ed. in chief) CRC, 1975.

Finkle, B.S. Contributor. Manual of Toxicology Methods. (Sunshine, I., ed. in chief) Chemical Rubber Company, Cleveland, Ohio, 1971 and 1975.