and final report i - defense technical information … i otic. fill od i (n determination of drug...

I OTIC. FILL ODII (N Determination of Drug Pharmacokinetics

and Metabolic Profile

J( Volume I

0)

I <ANNUAL AND FINAL REPORT

I

March 1, 1988TDavid R. Hawkins, Ph.D

Supported by:

U.S. ARMY MEDICAL RESEARCH AND DEVELOPMENT COMMAND,Fort Detrick, Frederick, MD 21701-5012

~DTIC Contract No.: DAMD17-83-C-3030 T CELECT h

Huntingdon Research Centre, AR 18Huntingdon, APR 2 01988Cambs,

PEI8 6ES, S. DEngland.

Approved for public release; distribution unlimited

1The findings in this report are not to be construed as an OfficialDepartment of the Army position unless so designated by other authorisedwdocuments.

I E3EST AVAILABLE COPY

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Huntingdon Research Centre (if applicable)

6C. ADDRESS (City; State, and ZIP Code) 7b. ADDRESS (City, State, and ZIP Code)

Huntingdon, Cambs

Ba. NAME OF FUNDING /SPONSORING 8b. OFFICE SYMBOL 9. PROCUREMENT INSTRUMENT IDENTIFICATION NUMBERORGANIZATION U.S.- Army Medical (if applicable)ORGNIZTIO U.S. rmyMedcal (I plale

Research & Develo ment CbmnmnI Contract No. DAMDl-83-C-3030ADDRESS(City, State, and ZIP Code) 10. SOURCE OF FUNDING NUMBERS

PROGRAM PROJECT TASK IWORK UNITELEMENT NO. NO. 3M4- NO. ACCESSION NO.Fort Detrick 3M4-I

Frederick. Maryland 21701-5012 63750A 63750D808 AA 03011. TITLE (Icde Security Classification)

Determination of Drug Pharmacokinetics and Metabolic Profile (Volume I)

* 12. PERSONAL AUTHOR(S)

David R. Hawkins, Ph.D.13a. TYPE OF REPORT 13b. TIME COVERED 14 DATE OF REPORT (Year, Month, Day) 5. PAGE COUNT

Annual and Final FROM 11/i/82 TO 9/Q86 1988 March 1 152

16 SUPPLEMENTARY NOTATIONAnnual (for the period January 1, 1984 through September 30, 1986)Final (for the period November 1, 1982 through September 30, 1986)

17. COSATI CODES 18. SUBJECT TERMS (Continue on reverse if necessary and identify by block number)

FIELD GROUP SUB-GROUP

06 15 WR 171,669, WR 171,669, WR 158,122, metabolism,

06 20 pharmacokinetics, beagle dogs, rhesus monkeys.1, ABSTRACT (Continue on reverse if necessary and identify by block number)

The pharmacokinetics and metabolism of halofantrine, WR 171,669, have been studied in

beagle dogs after oral doses of 5 to 100 mg/kg of the carbon-14 labelled drug. The routesand rates of excretion and plasma and blood concentrations of the drug and itsmetabolites were measured. A similar investigation was carried out with WR 178,460, theN-desbutyl derivative of WR 171,669, administered as a single oral dose of 21 mg/kg todogs.

Rates and routes of excretion and plasma and blood concentrations of WR 158,122 andits metabolites have been investigated in rhesus monkeys after administration of 10 mg/kgoral doses to animals with cannulated bile ducts. Metabolites were quantified afterchromatographic separation and information on their structure obtained by massspectrometry of isolated samples. -

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- UNCLASSIFIED/UNLIMITED 12 SAME AS RPT CQ DTIC USERS Unclassified

22s. NAME OF RESPONSIBLE INDIVIDUAL 22b TELEPHONE (Include Are& Code) 22c. OFFICE SYMBOLMrs. Virginia Miller 301/663-7325 SGRD-RMI-S

DD Form 1473, JUN 86 Previous editions are obsolete. SECURITY CLASSIFICATION OF THIS PAGE

JFOREWORD

I In conducting research using animals, the investigator(s) adhered to

the "Guide for the Care and Use of Laboratory Animals," prepared by the

Committee on Care and Use of Laboratory Animals of the Institute of

Laboratory Animal Resources, National Research Council (NIH Publication No.186-23, Revised 1985).

IAooession ForkNTIS GRAarIDTIC TABUnannouncedJustification

fBy ____________

Distribution/

Availability OodesAvand/or

Dot Speo ial

I1

jCONTENTSPage

INTRODUCTION 1

1. Metabolism and pharmacokinetics of14C-halofantrine hydrochloride(1 4C-WR 171,669.HCI) in the dog (pilot study) 2 - 11

2. Metabolism and pharmacokinetics of14C-halofantrine hydrochloride(14C-WR 171,669.HCl) in the dog (main study) 12 - 78

3. Metabolism and pharmacokinetics of14C-WR 178,460.HCl in the dog 79 - 91

4. The absorption, excretion and biotransformationof 1 4 C-WR 158,122 in the rhesus monkey 92 - 133

5. The identification of the major metabolites of'4C-WR 158,122 in the rhesus monkey 134 - 148

6. Metabolism and pharmacokinetics of 14C-WR 238605in beagle dogs 149 - 174

7. The metabolism and pharmacokinetics of14C-WR 238605 in beagle dogs and in therhesus monkey 175 - 261

8. Plasma concentrations and relative bioavailabilityof three pyridostigmine sustained releaseformulations after single oral doses to dogs 262 - 283

3 9. Bioavailability and pharmacokinetics ofpyridostigmine in male beagle dogs after oraland intravenous doses 284 - 327

1VOLUME I 1 - 148VOLUME II 149 - 327

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j [ INTRODUCTION

During the period of this contract from November 1982 to September1986 we have been involved in various studies on bioavailability,pharmacokinetics and metabolism of drugs in animals. This work hasincluded studies on the pharmacokinetics and metabolism of four differentcompounds WR 171,669, WR 178,460, WR 158,122 and WR 238605 all newpotential anti-malarial drugs. These studies have been carried out withbeagle dogs except for WR 158,122 which was studied in rhesus monkeys andWR 238605 where dogs and rhesus monkeys were used.

I In addition two bioavailability and pharmacokinetic studies withpyridostigmine have been completed in beagle dogs. These studies weredesigned to assess the bioavailability of pyridostigmine when administeredin tablet and syrup formulations.I

1

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METABOLISM AND~ PHARMACOKINETICS OF

1 M4 C-HALOFANTRINE HYDROCHLORIDE

(1 4 C-WR 171,669.HC1) IN THE DOG

(PILOT STUDY)

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JHRC/WRI 1/8335

SUMMAPY

I1. The purpose of this study was to carry out a pilot metabolism and

pharmacokinetic study of the new potential antimalarial drua 14C-WR

171,669.HC1 in the beagle dog, which would assist in the design and

analysis of samples in a more extensive main study. In this study the

drug was administered orally, as a suspension in a 0.40 aqueous

solution of Tween 80 containing 0.2% (w/v) methylcellulose, at a dose

level of 15 mg/kg bodyweight.1

2. Almost all the radioactive dose was excreted in the faeces, 96.4%

during 7 days. Most of this material was eliminated during the first

48 hours (72.8%). During the seventh day, only 1.1% was excreted in

the faeces. A total of only 0.1% of the dose was measured in urine.

3. Peak plasma concentrations of radioactivity occurred at 4 to 5 hours

(0.95 Ag/ml) declined up to 12 hours and then increased to a secondary

peak at 24 hours (0.71 Og/ml). Concentrations then appeared to

decline biphasically with an initial rapid phase until 7 days (t

37 hours) and a slower terminal phase (t 502 hours). Elood

concentrations of radioactivity followed a similar profile and were

always lower than plasma.

4. Almost all the radioactivity (86%) in a solvent extract of the 0-24

hour faeces sample was associated with the unchanged drug. The

remaining radioactivity was associated with several more polar

components.

A chromatographic method was developed using reverse phase hplc forthe separation of JR 171,669 and its metabolites in plasma.

N The concentration of the unchanged drug in plasma declined from0.49 pg/ml at 3 hours to 0.13 pg/ml at 30 hours. During this time the

proportion of WR 171,669 declined from 55% of total radioactivity at

3 hours to 221 at 30 hours with a corresponding increase in the

amounts of some more polar metabolites. These metabolites did not

seem to ccrrespond chromatographically with N-deshutyl WP 171,669

(WR 179,460) under the test conditions.

S: 3 :

I HRC/WRI 1/8335

TABLE 1

Excretion of radioactivity after oral administration of14C-WR 171,669.HCI to a beagle dog at a dose level of 15 mg/kg

Time Radioactivity excreted Cumulative excretion

(hours) (% dose) (I dose)

Urine Faeces Cage wash Urine Faeces Cage w3sh Total

0- 24 0.04 52.1 2.3 0.04 52.1 2.3 54.424- 48 0.03 20.7 0.6 0.07 72.8 2.9 75.848- 72 0.01 15.2 0.1 0.08 88.0 3.0 91.172- 96 0.01 4.5 0.1 0.09 92.5 3.1 95.796-120 0.01 1.6 C0.1 0.10 94.1 3.1 97.3120-144 <0.01 1.2 0.1 0.10 95.3 3.2 98.6144-168 0.01 1.1 (0.1 0.11 96.4 3.2 99.7

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HRC/WRI 1/8335

TABLE 2

Concentrations of radioactivity in plasma and whole-blood afteroral administration of 14C-WR 171,669.HC1 to a beagle dog

at a dose level of 15 mg/kg

Time - -g equivalents % dose/litre

WR 171,669.HCI/ml

Plasma Whole-blood Plasma Whole-blood

15 mins <0.08 C0.05 <0.05 v0.0330 mins 0.12 0.08 0.07 0.0545 mins 0.24 0.17 0.15 0.111 hrs 0.45 0.34 0.28 0.212 hrs 0.72 0.53 0.45 0.333 hrs 0.90 0.68 0.57 0.434 hrs 0.94 0.72 0.59 0.455 hra 0.95 0.73 0.59 0.467 hrs 0.83 0.69 0.52 0.43

12 hrs 0.60 0.53 0.38 0.3324 hrs 0.71 0.55 0.44 0.3530 hrs 0.61 0.52 0.38 0.332 days 0.54 0.42 0.34 0.263 days 0.33 0.24 0.21 0.154 days 0.20 0.12 0.12 0.085 days 0.15 0.09 0.10 0.066 days 0.11 0.07 0.07 0.047 days 0.07 <0.06 0.05 <0.048 days 0.07 <0.06 0.04 <0.04

10 days 0.05 <0.06 0.03 <0.0412 days 0.05 <0.06 0.03 CO.0414 days 0.04 <0.06 0.02 <0.0419 days 0.03 <0.06 0.02 <0.04

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

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HRC/WRI 1/8335

TABLE 3

Proportions of radioactivity in methanol extracts of faeces samplesup to 72 hours after oral administration of 11C-WR 171,669.HC1

to a beagle dog at a dose level of 15 mg/kg

Results are expressed as % faecal radioactivity

.-------------------.---------------- .. ------------Time period Extract 1 Extract 2* Total

(hours)

0-24 68.0 15.9 83.9

24-48 54.8 10.4 65.2

48-72 27.6 21.9 49.5

* Methanol diethylamine (9 1, v/v)

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HRC/WRI 1/8335

TABLE 4

Radioactivity in I minute fractions of hplc eluate after injectionof extracts of faeces collected during 24 hours after oral

administration of 14C-WR 171,669.HCl to a beagle dog at a dose levelof 15 mg/kg

Fraction numbers % eluted Total % dose

(inclusive) radioactivity (Extracts 1 and 2)

Extract 1 Extract 2

2- 9 7.0 21.7 4.3

16-25 6.6 - 2.3

27-351 86.0 J 78.3 37.0

# Corresponds to elution of 14C-WR 171,669

7

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HRC/WRI 1/8335

TABLE 5

Concentrations of 14C-WR 171,669 free base# in plasma after oraladministration of 14C-WR 171,669.HCl to a beagle dog at a dose

level of 15 mg/kg

Time (hrs) Concentration (1ig/ml)* % eluted radioactivity

3 0.46 54.5

7 0.39 50.1

12 0.18 32.5

30 0.12 21.6

Refers to radioactivity eluted from hplc column with thesame retention time as authentic 14C-WR 171,663See Table 6. Results are not corrected for recovery

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HRC/WRI 1/8335

TABLE

Radioactivity in 1 minute fractions* ot±hplc eluate after injectionof extracts of plasma collected after oral administration of14C-WR 171,669.HCl to a beagle dog at a dose level of 15 mg/kg

Results are expressed as % eluted radioactivity

Fraction number Sampling time (hours)

3 [7 12 30

1 + + + +2 + + + +3 2.9 + 4.0 +4 13.4 16.6 34.9 47.95 19.3 19.9 8.4 13.76 5.5 9.1 20.3 16.87 2.2 + + +8 2.4 4.2 + +9 + + + +

10 14.1 + + +11 37.6 12.1 + +12 2.7 32.3 27.2 12. 813 + 3.1 5.3 8.814| + 2.6 + +15L + + +

* Fraction collection and measurement was continuedup to 30 minutes after injection

+ Below limit of accurate measurement: 3 hrs <2.2%7 hrs <2.5%

12 hrs <3.3%30 hrs <3.6%

Radioactivity in fractions 10-14 was taken to be associatedwith WR 171,660

: 9 :

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yIuI1HRC/WRI 1/8335

FIGURE 1

Concentrations of radioactivity in plasma (o-o) and whole blood (0-0)after oral administration of

14C-WR 171,669.HC1 to a beagle dog

1.0

0.9

0.8

0.7

0.6

E

S0.5

S0.4

V0.3

c

0*

U

0.1

01

-.4

'I.l

|0.l4 8 2 2448 7

HRC/WRI 1/8335

FIGURE 2

Concentrations of radioactivity in plasma (o-o) and whole blood (0-0)after oral administration of l4c-WR 171,669.HCl to a beagle dog

1.0

0.50

U

~0

-

o 0.0.

.02

210 1 . 1 61

0ie(as

!

METABOLISM AND PHARMACOKINETICS OF

14C-HALOFANTRINE HYDROCHLORIDE

(1 4C-WR 171,669.HC1) IN THE DOG

(MAIN STUDY)

19 : L2 :

!HRC/';RI 1/84132

SUMMARY

1. The purpose of this study was to investigate the metabolism and

pharmacokinetics in the beagle dog of the new potential anti-

malarial drug, WR 171,669.HC1. Single oral doses of14C-WR 171,669.HCl have been administered to six dogs, each dog

receiving the drug at a different dose level within the range 5 to

100 mg/kg. The drug was administered in suspension in 1% aqueous

sodium carboxymethylcellulose solution. The radiolabelled drug was

shown to be stable under the conditions of dosing. No clinical or

other drug-related effects were observed in the dogs after dosing.

2. Excretion of radioactivity in faeces accounted for between 75.3% and

89.2% dose during seven days after administration. Urinary

excretion during seven days ranged from 0.03% dose (100 mg/kg dose)

to 0.31% dose (5 mg/kg dose).

3. Peak plasma concentrations of radioactivity (0.39 - 2.01 Lg equiv.

free base/ml) occurred at times from 1 to 30 hours after dosing.

Radioactivity could be detected in the plasma of all animals at

10 days after dosing and at 24 days in the plasma from animals dosed

at 40 or 60 mg/kg. Total areas under plasma radioactivity

concentration-time curves increased in an approximately linear

fashion with dose level from 5 to 60 mg/kg, but the result obtained

from the 100 mg/kg dose level was 5 times lower than expected.

4. Whole-blood radioactivity concentrations similar to or higher than

in plasma occurred at some sampling times after dosing at 5, 40 or

100 mg/kg, (maximum ratio 1.9), but not after dosing at 10, 20 or 60

mg/kg.

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HRC/WRI 1/84132

5. Peak concentrations of unchanged WR 171,669 in plasma (0.30 -

1.39 tg/ml) occurred at 1 to 2 hours (5, 10, 20, 100 mg/kg) or 5 - 6

hours (40 and 60 mg/kg). Second peaks occurred at 30 and 24 hours

after the 10 and 60 mg/kg doses. The area under the

concentration-time curve showed an approximately linear increase

with dose level up to 60 mg/kg but was about 5 times lower than

expected at 100 mg/kg. Estimated half-lives for the decline in

concentrations were in the range 4.3 - 8.0 hours for animals showing

a single concentration maximum and 20 and 25 hours for the other two

animals.

6. The proportion of plasma radioactivity associated with metabolites

of WR 171,669 in plasma increased with time after dosing.

Metabolites were eluted with shorter retention times than WR 171,669

under reverse phase hplc conditions. A minor metabolite had the

same retention time as desbutyl WR 171,669 (WR 173,460).

7. Unchanged WR 171,669 was the major component in extracts of

0-24 hour faeces but a minor component in most later extracts. In

extracts up to 72 hours, WR 171,669 accounted for 17% dose (5 mg/kg)

to >40% dose (40, 60, 100 mg/kg). A minor radioactive component in

faecal extracts corresponded chromatographically (hplc and tlc) to

WR 178,460. Thin-layer chromatography showed the presence of a

number of other radioactive metabolites in faeces extracts. A

similar pattern of radioactive metabolites was found in extracts of

faeces from an animal which had received 1 4 C-WR 178,460.HC1

suggesting the existence of common pathways of metabolism for the

two compounds.

14

HRC/WRI 1/84132

TABLE 1

Excretion of radioactivity by dogs after single oral doses of14C-WR 171,669.HCl at various levelsResults are expressed as % administered radioactivity

Sample Time period Nominal dose level (mg/kg)(hours) ------------------------------------------

5 10 20 40 60 100

Urine 0 - 6 <0.01 <0.01 <0.01 NS <0.01 <0.016 - 24 0.09 0.06 0.05 0.03 0.02 0.02

24 - 48 0.09 0.08 0.03 0.02 0.03 0.0148 - 72 0.04 0.03 0.02 0.01 0.01 <0.0172 - 96 0.03 0.04 0.06 0.01 0.02 <0.0196 - 120 0.03 0.01 0.01 <0.01 0.01 <0.01

120 - 144 0.02 0.01 0.01 <0.01 0.01 <0.01144 - 168 0.01 <0.01 0.01 0.01 <0.01 <0.01

Total 0. 31 0. 23 0.19 0.08 0.10 0.03

Faeces 0 - 24 43.7 22.8 55.3 55.5 60.0 84.324 - 48 15.5 24.8 12.1 15.2 7.1 1.748 - 72 13.3 11.9 7.3 3.8 NS 0.972 - 96 6.1 6.4 4.2 3.7 1.8 0.796 - 120 5.6 4.8 2.2 2.5 6.2 0.6

120 - 144 2.8 3.3 2.2 2.2 3.5 0.5144- 168 2.2 1.3 NS 1.1 1.5 0.3

Total 89.2 75. 3 83. 3 84.0 80. 1 89.0

Cagewash 0 - 24 0.26 0.32 0.06 0.24 0.08 0.2024 - 48 0.06 0.23 0.09 0.09 0.08 0.0948 - 72 0.14 0.17 0.24 0.07 0.08 0.04

|72 - 96 0.08 0.15 0.18 0.02 0.02 <0.0196 - 120 0.09 0.08 0.06 0.03 0.09 0.01

120 - 144 0.04 0.02 0.06 0.03 0.05 <0.01144 - 168 0.03 0.03 0.02 0.05 0.03( <0.01

Total 0.70 1.00 0.71 0.53 0.43 0.34

Overall total 90.2 76.5 84.2 84.6 80.6 | 89.4

NS No sample

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HRC/WRI 1/84132

TABLE 2

Cumulative excretion of radioactivity by dogs after single oral doses of?4C-WR 171,669.HCl at various levels

Results are expressed as % administered radioactivity

Sample Time period Nominal dose level (mg/kg)(hours) ------- -----------------------------------

5 10 20 40 60 100

Urine 0 - 6 <0.01 <0.01 <0.01 NS <0.01 <0.010 - 24 0.09 0.06 0.05 0.03 0.02 0.020 - 48 0.18 0.14 0.08 0.05 0.05 0.030 - 72 0.22 0.17 0.10 0.06 0.06 0.030 - 96 0.25 0.21 0.16 0.07 0.08 0.030 - 120 0.28 0.22 0.17 0.07 0.09 0.030 - 144 0.30 0.23 0.18 0.07 0.10 0.030 - 168 0.31 0.23 0.19 0.08 0.10 0.03

Faeces 0 - 24 43.7 22.8 55.3 55.5 60.0 84.30 - 48 59.2 47.6 67.4 70.7 67.1 86.00 - 72 72.5 59.5 74.7 74.5 67.1 86.90 - 96 78.6 65.9 78.9 78.2 68.9 87.60 - 120 84.2 70.7 81.1 80.7 75.1 88.20 - 144 87.0 74.0 83.3 82.9 78.6 88.70 - 168 89.2 75.3 83.3 84.0 80.1 89.0

Cagewash 0 - 24 0.26 0.32 0.06 0.24 0.08 0.200 - 48 0.32 0.55 0.15 0.33 0.16 0.290 - 72 0.46 0.72 0.39 0.40 0.24 0.330 - 96 0.54 0.87 0.57 0.42 0.26 0.330 - 120 0.63 0.95 0.63 0.45 0.35 0.340 - 144 0.67 0.97 0.69 0.48 0.40 0.340 - 168 0.70 1.00 0.71 0.53 0.43 0.34

Overall total 90.2 76.5 84.2 84.6 80.6 89. 4-- vrltta----9------- -------------------------------

NS No sample

16

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HRC/WRI 1/84132

TABLE 3

Concentrations of radioactivity in plasma of dogs after singleoral doses of 1 4C-WR 171,669.HCl at various levels

Results are expressed as Lg equivalents WR 171,669 free base/ml

Time Uominal dose level (mg/kg)---- ...------------I? IIN ;-5 10 201 40 60 100

0.5 hour 0.08 0.19 0.71 0.10 0.09 0.251 0.23 0.37 1.62 0.32 0.30 0.582 0.39 0.46 1.52 0.95 0.69 0.823 0.39 0. 52 1.41 1.26 1.09 0.934 0.38 0.53 1.29 1.76 1.43 0.855 0.38 0.53 1.21 1.99 1.55 1.006 0.38 0.54 1.09 2.01 1.58 0.937 0.37 0.52 1.04 1.87 1.48 0.91

12 0. 23 0.65 0.90 1.35 1.26 0.6724 0.17 0.36 0.59 0.89 1.83 0.5130 0.16 0.70 0.50 0.72 1.36 0.45

2 days 0.14 0.56 0.50 0.69 1.00 0.323 0.11 0.43 0.43 0.67 0.86 0.264 0.11 0.32 0.32 0.66 1.04 0.235 0.08 0.22 0.24 0.51 0.71 0.206 0.05 0.14 0.17 0.33 0.63 0.197 0.04 0.10 0.13 0.25 0.53 0.168 0.03 0.08 0.10 0.21 0.43 0.16

10 0.02 0.05 0.07 0.14 0.26 0.1212 <0.02 0.04 0.05 0.11 0.19 0.1115 <0.02 0.03 0.04 0.08 0.12 0.0818 <0.02 <0.02 0.03 0.06 0.09 0.0521 <0.02 <0.02 <0.02 0.05 0.05 10.0424 <0.02 <0.02 <0.02 0.05 0.04 <0.0428 <0.02 <0.02 <0.02 <0.04 <0.04 <0.04

1

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HRC/WRI 1/84132

TABLE 4

Concentrations of ralioactivity in plasma of dogs after singleoral doses of 4 C-WR 171,669.HC1 at various levels

Results are expressed as % dose/litre

Time Nominal dose level (mg/kg)

5 10 20 40 60 100

0.5 hour 0.13 0.15 0.30 0.02 0.01 0.0221 0.35 0.30 0.68 0.06 0.04 0.0502 0.61 0. 37 0.64 0. 19 I0.1i0 0. 0713 0.61 0.42 0.59 0.26 0.16 0.0814 0.59 0.42 0.54 0.36 0.21 0.0745 0.58 0. 43 0.51 0.40 0.23 O. 0876 0.59 0.43 0.46 0.41 0.23 0.0817 0.57 0.42 0.44 0.38 0.22 0.079

12 0. 36 0. 52 0. 38 0.27 0. 18 0. 05824 0.27 0.29 0.25 0.18 0.27 0.04430 0.25 0.56 0.21 0.15 0.20 0.0402 days 0.21 0. 45 0.21 0.14 0.15 0. 0283 0.18 0.34 0.18 0.14 0.12 0.0234 0.17 0.25 0.13 0.13 0.15 0.0205 0. 12 0. 17 0.1I0 0.1i0 0.1I0 0.0176 0.08 0.11 0.07 0.07 0.09 0.0167 0.07 0.08 0.06 0.05 0.08 0.0148 0.05 0.06 0.04 0.04 0.06 0.0140 0.03 0.04 0.03 0.03 0.04 0.011

12 <0.03 0.03 0.02 0.02 0.03 0.01015 <0.03 0.02 0.02 0.02 0.02 0.00718 <0.03 <0.02 0. 01 0.01 0.01 0. 00421 <0.03 <0.02 <0.01 0.01 0. 008 <0.00424 <0.03 <0.02 <0.01 0.009 0.006 <0. 004

28 <0.03 <0.02 <0.01 <0.008 <0.006 <0.004L--------------------------------------- ------ -------------------- ------ -------

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HRC/WRI 1/84132

TABLE 5

Concentrations of radioactivity in whole-blood of dogs aftersingle oral doses of 1

4C-WR 171,669.HCI at various levelsResults are expressed as ±g equivalents WR 171,669 free base/ml

Time Nominal dose level (mg/kg)

5 10[20 40 [60 10--------- ----------------------------------------

0.5 hour 0.05 0.12 0.52 <0.10 <0.10 0.171 0.17 0.25 1.12 0.23 0.20 0.442 0.32 0.33 1.16 0.72 0.52 0.693 0.33 0.38 1.12 0.97 0.84 0.824 0.31 0.37 1.03 1.20 1.14 0.725 0.31 0.35 0.90 1.47 1.16 0.806 0.33 0.38 0.87 1.53 0.84 0.797 0.29 0.37 0.80 1.38 1.08 0.76

12 0.24 0.28 0.68 1.09 0.83 0.5524 0.17 0.25 0.50 0.76 1.46 0.4730 0.16 0.52 0.36 0.88 1.14 0.532 days 0.13 0.43 0.34 0.81 0.86 0.373 0.11 0.32 0.30 0.61 0.73 0.354 0.09 0.21 0.26 0.44 0.81 0.335 0.07 0.15 0.16 0.21 0.60 0.286 <0.05 0.09 0.11 0.28 0.44 0.257 <0.05 0.07 0.10 0.21 0.41 0.238 <0.05 0.07 0.07 0. 18 0. 32 0.26

10 <0.05 <0.05 <0.05 0.11 0.20 0. 1912 <0.05 <0.05 <0.05 <0.10 0.12 0.1615 <0.05 <0.05 <0.05 <0.10 0.10 0.1518 <0.05 <0.05 <0.05 <0.10 <0.10 <0.1021 <0.05 <0.05 <0.05 <0.10 <0.10 <0.1024 <0.05 <0.05 <0.05 <0.1O <0.10 <0.1028 <0.05 <0.05 <0.05 <0.10 <0.10 <0.10

19I

!IRC/WRI 1/84132

TABLE 6

Concentrations of radi activity in whole-blood of dogs after single

oral doses of ?4C-WR 171,669. HCl at various levels


-------------------------------------------------Time Nominal dose level (mg/kg)

5 10 20 40 60 j100-- ------- -------------; - ------0.5 hour 0.08 0.10 0.22 <0.02 <0.02 0.015

1 0.27 0.20 0.47 0.05 0.03 0.0382 0.50 0.26 0.49 0.15 0.08 0.0603 0.51 0.30 0.47 0.20 0.12 0.071

4 0.48 0.30 0.44 0.24 0.17 0.0625 0.49 0.28 0.38 0.30 0.17 0.0706 0.51 0.31 0.37 0.31 0.12 , 0.0697 0.46 0.30 0.34 0.28 0.16 0.066

12 0.37 0.22 0.29 0.22 0.12 0.04824 0.26 0.20 0.21 0.15 0.21 0.04130 0.25 0.41 0.15 0.18 0.17 0.0462 days 0.20 0.34 0.15 0.16 0.13 0.0323 0.17 0.25 0.13 0.12 0.11 0.0314 0.14 0.16 0.11 0.09 0.12 0.029

5 0.10 0.12 0.07 0.C4 0.09 0.0246 <0.09 0.07 0.05 0.06 0.06 0.0227 <0.09 0.05 0.04 0.04 0.06 0.0208 <0.09 0.06 0.03 0.04 0.05 0.023

10 <0.09 <0.05 <0.03 0.02 0.03 0.01612 <0.09 <0.05 <0.03 <0.02 0.02 0.01415 <0.09 <0.05 <0.03 <0.02 0.02 0.01318 <0.09 <0.05 <0.03 <0.02 <0.02 <0.010

21 <0. 09 <0. 05 <0.03 <0.02 <0.02 <0. 01024 <0.09 <0.05 <0.03 <0.02 <0.02 <0.01028 <0.09 <0. 05 <0. 03 <0. 02 <0.02 <0.010

20

...i . .. . . .. . . .. . . . .. - . . .

HRC/WRI 1/84132

TABLE 7

Total areas under plasma radioactivity concentration -time curves after single oral doses

of 14C-WR 171,669.HCl to dogsat various levels

Dose level Area Area/dose level(mg/kg) (4g h.ml- 1) (jg h ml- ' mg- I kg)

5.01 23.46 4.710.2 72.19 7.119.6 86.21 4.440.8 152.29 3.761.4 227.52 3.7102.7 82.74 0.80

I.21

L------------L-------------------------------J

HRC/WRI 1/84132

TABLE 8

Concentrations of WR 171 669* free base in plasma of dogsafter single oral doses of 1 C-WR 171,669. HCl at various levels

Results are expressed as 4g/ml

Time Nominal dose level (mg/kg)(hours) ---5 -----. 10 20 40 60 100

0.5 m NM 0.66 1M M4 NII1 0.23 0.34 1.27 0.06 0.07 0.532 0.30 0.25 1.19 0.77 0.50 0.733 0.25 0.22 0.96 1.01 0.71 0.714 0.20 0.20 0.80 1.27 0.93 0.585 0.21 0.17 0.65 1.39 0.89 0.646 0.19 0.20 0.55 1.33 0.95 0.607 0.18 0.17 0.32 1.25 0.83 0.64

12 0.06 0.08 0.28 0.72 0.45 0.2624 <0.03 0.08 0.09 0.27 1.02 0.1130 NM 0.17 0.04 0.19 0.59 0.0748 M 0.07 <0.03 0.07 0.25 NM72 R4 <0.03 <0.03 <0.05 0.10 WM96 NM <0.03 <0.06 <0.05 0.06

120 <0.04 <0.06 <0.05 <0.06 NM144 4 M M 4 <I <0.06 NM168 4 , M NMM N <0.07 NW

,I Not measured* Refers to radioactivity eluting from an hplc column

with the same retention time as WR 171,669. Resultsare not corrected for recovery

Calculated limits of detection are based on the limitof detection for a single fraction of hplc eluate(Appendix 5)

22

HRC/WRI 1/84132

TABLE 9

Total areas under plasma concentrations of WR 171,669 free base

against time curves after single oral doses of

of 14C-WR 171,669.HCl to dogs

at various levels

(a) Observed curves

Dose level Area4 Area/dose level

(mg/kg)* (4g h.ml-1) (4g h.ml-

1 mg-

I kg)

4.67 2.07 0.44

9.51 5.96 0.6318.3 9.70 0.5338.0 21.04 0.5557.2 35.00 0.6195.7 9.12 0.095

Calculated as WR 171,669 free base

To time of last detectable measurement

(b) Fitted curves (see Table 19)

Dose level .rea# Area/dose level(mg/kg)* (4c 11..h11-) (ag h.ml- 1 mg- I kg)

4.67 2.48 0.53

9.51 6.49 0.6818.3 8.40 0.4638.0 21.11 0.5657.2 34.11 0.6095.7 9.64 0.10

To infinite time

23

i

HRC/WRI 1/84132

TABLE 10

Concentrations of radioactivity in plasma of dogs (after oral doses of14C-WR 171,669.HCl) associated with a component eluting from an holc

column with the same retention time as WR 178,460

Results are expressed as Lg equivalents WR 178,460 free base/mil

Time Nominal dose level (mg/kg)(hours)1T [5 10 j 20 40 60 100

0.5 tl N4 + NM M4 M41 + + + + + +2 0.02 0.02 0.10 0.04 + 0.043 0.02 0.04 0.12 0.05 0.12 0.094 0.03 0.06 0.14 0.11 0.15 0.125 0.04 0.07 0.14 0.18 0.21 0.17

6 0.05 0.08 0.14 0.21 0.27 0.177 0.06 0.10 0.19 0.20 0.31 0.23

12 0.05 0.05 0.14 0.21 0.32 0.1524 0.02 0.05 0.13 0.22 0.29 0. 1030 NM 0.04 0.11 0.16 0.29 0.1148 1M 0.04 0.07 0.16 0.24 NM72 NM + 0.02 0.10 0.15 tl96 NM + + + 0.09 NM

120 NM + + + 0.05 M4144 U14 M NM NM + NM168 Nr N W M4 WN j + NM

M4 Not measured+ Below limit of detection (0.02 - 0.06 .g/ml)

24

HRC/WRI 1/84132

TABLE 11

Radioactivity in 30 second fractions of hplc eluate after injectionof extracts of plasma collected after oral administration of14C-WR 171,669.HCI at a dose level of 5 mg/kg


Time Fractions(hours) - - - - -

6 - 9 14 -15 17 - 194 L 23 - 26*

1 + + + 1002 17.6 + 5.3 77.13 30. 3 + 5.8 63.94 37.6 + 10.3 52. 15 33.3 + 11.9 54.86 33.6 + 16.0 50.57 33.6 + 18.4 48.0

12 48.5 + 25.0 26.524 88.5 + 11.5 J

+ Not detected, see Appendix 5 for limits ofdetection* Corresponds to WR 171,669

Corresponds to WR 178,460

25

I

HRC/WRI 1/84132

TABLE 12

Radioactivity in 30 second fractions of hplc eluate after injectionof extracts of plasma collected after oral administration of

14C-WR 171,669.HCI at a dose level of 10 mg/kgResults are expressed as % eluted radioactivity

Time Fractions(hours) - -

5 - 12 14 - 15 17 - 194 23 - 26*

1 9.3 + + 90.72 39.6 + 5.1 55.33 49.2 + 7.8 43.04 49.2 + 12.8 38.15 53.6 + 14.8 31.76 46.8 + 15.7 37.57 44.0 + 22.5 33.5

12 74.6 4.4 8.7 12.324 59.6 + 16.7 23.630 65.6 4.2 6.7 23.648 70.0 8.5 8.3 13.172 94.7 + + 5.396 100.0 + + +

120 100.0 + + +

+ Not detected, see Appendix 5 for limits ofdetection

* Corresponds to WR 171,669Corresponds to WR 178,460

26

i ITIRC/WRI 1/84132

TABLE 13

Radioactivity in 30 second fractions of hplc eluate after injectionof extracts of plasma collected after oral administration of14C-WR 171,669.HCI at a dose level of 20 mg/kg


Time FractionsS( hours) -- - - - - - - - - -- - - - - - - - - -

6 - 10 14 - 15 17 - 194 23 - 26*

0.5 6.6 + + 93.41 21.4 + + 78.62 14.1 + 7.6 78.2

3 22.2 + 9.4 68.44 26.1 + 12.2 61.75 33.3 + 13.2 53.56 33.3 1.5 14.6 50.67 46.0 3.1 20.6 30.3

12 50. 3 2.0 17. 1 30. 624 55.3 4.9 24.6 15.230 55.8 11.3 24.4 8. 548 75.0 9.0 16.0 +72 82.2 11.6 6.2 +96 77.5 10.0 + 12.5

120 100.0 + + +

+ 'lot detected, see Appendix 5 for limits ofdetection


2

I I

HRC/WRI 1/84132

TABLE 14


14C-WR 171,669.HCI at a dose level of 40 mg/kgResults are expressed as % eluted radioactivity

Time Fractions(hours) .5 - 10 14 - 15 17 - 194 22 - 26*-- ---------- -- ----- ------ - ----

1 40.4 + + 59.62 14.0 + 4.9 81.23 15.1 + 4.7 80.24 20.9 + 7.1 72.05 20.1 + 10.2 69.66 21.7 + 11.9 66.37 21.1 + 12.3 66.6

12 34.3 + 17.6 48.124 42.4 + 27.8 29.830 48.9 + 24.6 26.748 63.6 + 26. 7 9.772 70.5 6.4 17.0 6. 196 100.0 + + +

120 100.0 + + +------------------- --------------------- ---------+ Not detected, see Appendix 5 for limits of

detection* Corresponds to WR 171,669

Corresponds to WR 178,460

FIRC/WRI 1/84132

TABLE 15

Radioactivity in 30 second fractions of hplc eluate after injectionof extracts of plasma collected after oral administration of1

4C-WR 171,669.HC1 at a dose level of 60 mg/kgResults are expressed as % eluted radioactivity

Time Fractions(hours)

6 - 10 14 - 15 17 - 194 22 - 26*

1 23.0 + + 77.02 27.7 + + 72.33 22.5 + 12.5 65.04 22.8 + 12.1# 65.1#5 27. 5 + 15. 3 57. 26 20.2 + 19.6 60.37 19.9 + 23.7 56.4

12 35.8 + 28.3 36.024 22.6 3.4 18.1 55.930 30.0 2.7 24.0 43.448 41.6 6.8 27.1 24.572 60.2 8.0 19.9 11.996 72.6 11.1 10.1 6.1

120 78.9 12.6 8.5 +144 89.3 10.7 + +168 100.0 + + +



* Fractions 19,20 and 26-28

- 2,1IJ

HRC/WRI 1/84132

TABLE 16


'4C-WR 171,669.HCl at a dose level of 100 mg/kgResults are expressed as % eluted radioactivity

Time Fractions(hours) r-7 - 9 [ 14 - 15 - 17 - 204 22 - 27*

1 9.4 + + 90.62 5.2 + 5.5 89.33 12.4 + 11.1 76.54 15.3 + 16.0 68.75 17.5 + 18.7 63.86 14.1 + 21.0 64.97 + + 29.2 70.8

12 35. 5 + 25.7 38.824 44.4 9.7 23.9 22.030 44.6 13. 5 26.9 15.0



41

I

HRC/WRI 1/84132

E

C.

co ' ' 0 Qm m m- Mtn 0 4 c - a

0 i4 - 00 00 nCn C N ~ D N NI -

1 1-4 -I-1-

1 0C I1 I4 10 0 N 0 W WO' Z ItNrI n-t -VU DNi

1 (N %0 I 0) 4 ,41-00 % t MM C-4 -4 C I 1I4 - 4-

u I0 m 0 IT %0w ~ vN D 0 N 0 r, r- I

>, I 1 I I -01 o0% QNN NvI DO

8- I -4I I0 0 U 0ITL 0L nU N 0 Q- n U4- r4- -I I -4 -4 -4C -. 4

4JIJ

1V 0- I 3 V D-r\D0'f) C %'TV NCq0L oVC --4I Oilm I I, ~0~~ 0 0r-otnm 0 M0a C - 1 0NM -

0 > I

o4 10 -4 Dt - )C DU ON (I) 00 r- 4oLn v ,4 - ~ fE x0 III i I I I

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VU 10 -4 M M M I4NN

Q) C 0 4 V M - m' C4 C) CN r Dr t DO r nN 1IT4 ITI na NC41 )0 ML o-0NU I I I

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41 I I- 1

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1 101 a\- \I l o1 -W r-1'r N0r C'JO'Z ZZ ZZ I QI) I4 I n I w w Mw m m NN 1-4

.I 0- N Cn 0'4 (-440V% %-00oe fI, I 4 I

V I I I 4 U ~ W ~ .-.-31

VI I I I

HRC/WRI 1/84132

TABLE 18

Parameter estimates for the fitted models for totalradioactivity concentrations in plasma

Parameter Nominal dose level (mg/kg)(units)

5 10 20 40 60 100

T (h) 0.28 0 0.40 0.38 0.35 0.22k (h-') 0.655 0.752 5.99 0.566 0.355 0.959;2 (h-1 ) 0.233 0.0110 0.0994 0.0122 0.0078 0.0575A3 (h-1) 0.0099 0 0.0080 0.0016 0 0.0042

A2 (dpm/ml) 1433 1397 2461 1593 2013 943A 3 (dpm/ml) 519 33 1294 128 26 401

h, (h) 1.06 0.92 0.12 1.22 1.95 0.72h 2 (h) 2.97 63.1 6.97 56.7 89.3 12.1h 3 (h) 70.1 - 87.1 433 - 167

32

HRC/WRI 1/84132

N r-NCN IIn I r- kDI nL n

I I I I 1 o m C ;I ;L

Inc 4O T0v D14r r ON co 00r C IIn r0 1 r-. InI N1. Q

C; 1-4 C; In 8 ; ; rC . C) -C I

0 r- w co m co n.r- ~-' lz N-40 0I

I~~~~~~~ v I ~ I 0 0 0 nl

+1 + 1

IL C) CN ON~~'( - O N 1

w 00000000040000000 000 0 11

IQ ID In a% ID 0 I *00C Zr- INC4ciC m 00000

I.. I I ONl-00'~0II,00 0 -00 0 mU110 Ir- r- a- loll........................................................

to2 ID 0 -4 1 0000000 0 0000 0 0 0000cq 1- 0 I00

U) I I I V VI

0 e I w 1 0 Cl ow 000 0 1* 1

w I I ! 0 N I OCI- 0 0 00

CL I- 0 04-4-4-0000000

o r- N' Io I 00 l

I2 D N C CC) Io U) en 0-0Nl IN 0 000 0 'rC '- (n 0N 0m 1

C; I CIn I 8 8 00 - -I 4 ;00 0 0 0 OOI -00

LW I s-

-T 1,-S N 0 00 0 -C 0r -( IIPMm C -E Q. CN 1210 ______________ 0_0_ 00_00_N_0 4.1U-I I

. C)1 I; I; 0r*N orIal is-0 In I 0 n - ' n I I N 0v~mn nI '0 -IIC- IZ nCIN000rN4m ON'4.

o 0I 0I l0 4 4 0 00 00 000- 0~0

I 1 00 W122~ ~ ID m Io IT 0__ _ __ _ _ _ __ _ _ _ ___ .1

m4~ 02 In [ 1 To(nC ' m - 'j-TI~~Z ID In MN4 . 0 0 ~ N . 4 -4~

2 ~ ~ I I II'2 I......................... . . w0 I I I I 0000 0000000 00 I'O o Ut~

-4 I II :1 I I n - N -V 0 w N I o

In IN m0... IT__ _ _ __ _ _ _ _ _ 4-I 4-IE. 4 S1

4~I I I I no ~ ? tll3

HRC/',RI 1/84132

TABLE 20

Weighted residual mean squares for the fitted curves(weights were scaled to have an average value of unity)

Nominal Unchanged Totaldose level compound radioactivity(mg/kg)

5 0.00065 133.710 0.00354 1917.420 0.00513 1182.040 0.00194 1153.960 0.01255 484.0100 0.00256 120.6

* 34

r.

HRC/WRI 1/84132

TABLE 21

Extraction* of radioactivity from faeces samples up to 72 hoursafter single oral doses of 1 4C-WR 171,669. HCl to dogs at various levels


Nominal dose Time period Extract Total*l e v e l ( h o u r s ) ......

(mg/kg) 4.1 2 3

5 0-24 15.2 26.9 22.4 64.5 (28.2)24-48 16.0 25.1 15.6 56.7 ( 8.8)48-72 67.5 10.6 5.1 83.2 (11.0)

10 0-24 59.2 24.3 8.9 92.4 (21.1)24-48 29.2 27.9 14.9 72.0 (17.9)48-72 6.6 28.3 18.7 53.6 ( 6.4)

20 0-24 42.2 26.8 14.9 83.9 (46.4)24-48 28.7 29.9 11.6 70.2 ( 8.5)48-72 18.0 29.1 16.5 63.6 ( 4.6)

40 0-24 41.8 31.2 15.7 88.7 (49.2)24-48 13.3 24.2 27.9 65.4 (10.0)48-72 23.1 33.5 17.3 73.9 ( 2.8)

60 0-24 22.2 24.7 26.0 72.9 (43.7)24-48 12.9 30.7 19.6 63.2 ( 4.5)

100 0-24 9.6 13.6 29.5 52.7 (44.4)24-48 22.2 25.7 19.2 67.1 ( 1.1)48-72 15.9 28.4 15.9 60.2 ( 0.5)

* Figures in brackets are total extracted expressed as

% doseExtract 1, methanol; Extracts 2,3, methanol diethylamine

(9:1, v/v)

I : :535

. . ... ......

HRC/WRI 1/84132

TABLE 22

Radioactivity in 30 second fractions of hplc eluate afterinjection of extracts of faeces collected up to 72 hours

after oral administration of 14C-WR 171,669.HCIto a beagle dog at a nominal dose level of 5 mg/kg

Time interval Zone No.* Fraction numbers % eluted % dose(hours) (inclusive) radioactivity

0-24 1 4 - 11 32.1 2.1(extract 1) 2 22 - 25 11.4 0.8

3 35 - 39 55.4 3.7Others 16 1.2 0.08

0-24 1 4 - 15 32.6 7.0(extracts 2&3) 2 18 - 21 11.4 2.5

3 27 - 31 55.9 12.0

24-48 1 4 - 10 79.6 7.02 22 - 23 15.7 1.43 33 4.7 0.4

48-72 1 4 - 11 76.0 8.42 19 - 22 15.0 1.73 31 - 33 9.0 1.0

L ------- J------------- -------------------------------- I--------Zone 3 corresponds to WR 171,669

36

HRC/WRI 1/84132

TABLE 23


after oral administration of 1 4C-WR 171,669.HC1to a beagle dog at a nominal dose level of 10 mg/kg

Time interval Zone No. A Fraction numbers % eluted % dose(hours) (inclusive) radioactivity

0-24 1 4 - 18 7.9 1.1(extract 1) 2 22 - 27 3.4 0.5

3 33 - 43 87.8 11.9Others 29,30, 46-48 0.9 0.1

0-24 1 5 - 12 13.3 1.0(extracts 2&3) 2 19 - 20 3.3 0.3

3 27 - 31 82.3 6.3Others 14,23 1.2 0.09

24-48 1 4 - 18 39.5 7.12 20 - 24 9.7 1.73 31 - 36 50.8 9.1

48-72 1 4 - 11 77.0 4.92 19 - 22 11.2 0.73 30 - 32 10.0 0.6

Others 16 1.8 0.1

Zone 3 corresponds to WR 171,669

37

__I _ ____ ____

HRC/WRI 1/84132

TABLE 24


after oral administration of 14C-WR 171,669.HCl

to a beagle dog at a nominal dose level of 20 mg/kg

Time interval Zone No. $ Fraction numbers % eluted % dose(hours) (inclusive) radioactivity

0-24 1 4 - 21 13.1 3.1(extract 1) 2 22 - 27 4.5 1.0

3 32 - 42 81.3 18.9Others 28-30, 45-46 1.1 0.3

0-24 15 - 18 15.3 3.5(extracts 2&3) 2 19 - 24 4.7 1. 1

3 26 - 35 80.0 18.5

24-48 1 5 - 16 80.1 6.82 19 - 22 11.0 0.93 30- 32 9.0 0.8

48-72 1 4 - 11 85.3 3.92 20 - 22 6.4 0.33 31 - 32 5.6 0.3

Others 15 - 16 2.8 0.1


3!

I

I38 m

I

HRC/WRI 1/84132

TABLE 25


after oral administration of 14C-WR 171,669. HCIto a beagle dog at a nominal dose level of 40 mg/kg

Time interval Zone No. * Fraction numbers % eluted % dose(hours) (inclusive) radioactivity

0-24 1 5 - 14 9.5 2.2(extract 1) 2 18 - 25 4.6 1.1

3 27 - 34 85.8 19.9

0-24 1 4 - 14 5.0 1.3(extracts 2&3) 2 17 - 24 3.8 1.0

3 25 - 34 91.1 23.7

24-48 1 4 - 11 87.0 8.72 16 - 18 10.8 1.13 23 - 26 2.2 0.2

48-72 1 4- 9 88.1 2.52 17 - 19 11.9 0.3

------------------- ----------- ----------------------------------------Zone 3 corresponds to WR 171,669

39

I

L 2HRC/WRI 1/84132

m TABLE 26


after oral administration of 14C-WR 171,669. HC

to a beagle dog at a nominal dose level of 60 mg/kg

Time interval Zone No. $ Fraction numbers % eluted % dose(hours) (inclusive) radioactivity

0-24 1 6 - 11 5.5 0.7(extract 1) 2 18 - 21 2.9 0.4

3 27 - 34 90.4 12.0Others 14,25,26 1.2 0.2

0-24 1 4 - 11 4.8 1.5(extracts 2&3) 2 17 - 20 2.7 0.8

3 25 - 31 92.0 28.0Others 22 0.5 0.2

24-48 1 4 - 11 49.7 2.22 16 - 18 15.9 0.73 23 - 26 33.3 1.5

48-72 No faeces sample for this time period


40 .

HRC/WRI 1/84132

TABLE 27


after oral administration of 1 4C-WR 171,669. HCIto a beagle dog at a nominal dose level of 100 mg/kg

Time interval Zone No. * Fraction numbers % eluted % dose(hours) (inclusive) radioactivity

0-24 1 5 - 15 3.2 0.3(extract 1) 2 19 - 22 2.1 0.2

3 27 - 36 92.8 7.5Others 18,23-26 1.8 0. 1

0-24 1 4 - 13 2.6 0.9(extracts 2&3) 2 16 - 20 2.7 1.0

3 25 - 33 93.7 34.0Others 21 - 24 1.0 0.4

24-48 1 5 - 10 67.4 0.72 17 - 18 15.0 0.23 24 - 25 17.6 0.2

48-72 1 5 - 8 88.3 0.42 17 11.7 0.06


41

HRC/WRI 1/84132

TABLE 28

Proportions of radioactivity in extracts of faecesassociated with WR 171,669 after single oral doses of14C-WR 171,669.HCl to dogs at various levels

(1) Results expressed as % extracted radioactivity

Time period Nominal dose level (mg/kg)(hours) 5 1 0 _ 20 -. . .40 ... 60 i0O1

0 - 24 (extract 1) 55.41 87.8 81.3 85.8 90.4 92.80 - 24 (extracts 2,3) 55.9 82.3 I80.0 91.1 92.0 93.7

24- 484 4.7 50.8 9.0 2.2 33.3 17.648- 724 9.0 10.0 5.6 ND MS ND

(2) Results expressed as % dose

Time period Nominal dose level (mg/kg)(h ours ) ------ ------ ------ ---- -- ------ ------

5 10 20 40 60 100

0 - 244 15.7 18.2 37.4 43.6 40.0 41.524 - 484 0.4 9.1 0.8 0.2 1.5 0.248 - 72! 1.0 0.6 0.3 ND NS ND

Total 0- 72 17.11 27.9 38.5 43. 41.5 41.7--- -- -- -- -- ---- - -- -----.-........ . . .-

ND Not detectedNS No sample

All 3 extracts for each time period

42

HRC/WRI 1/84132

0

'0

U.(D 0

ra' 1. 440 1

c

4-1 0'0

U00-4 a4

-4 -10041 22a) r_

u0

x ()

14

J 43

H-RC/WRI 1/84132

FIGURE 2

Concentrations of radioactivity in the plasma of dogsup to 72 hours after single oral doses

of 14C-WR 171,669. HCl

Key: 5 mg/kg, 0 20 mg/kg, 0 60 mg/kg*10 mg/kg, @ 40 mg/kg, a 100 mg/kg

2

c 0.50

4

o 0.2

0.1

0.05

0.020 6 12 18 24 30 236 42 48 54 60 66 72

Time (hours)

- 44

'iRC/VIRI 1/84132

FIGURE 3

Concentrations of radioactivity in the plasma of dogsafter single oral doses of 14 C-WR 171,669.HCI

(Values before 6 hours not shown)

Key. A 5 mg/kg, 0 20 mg/kg, 0 60 mg/kgA130 mg/kg, 6 40 mg/kg, a 100 mg/k-g

5

2

E

0-- 4

4-J

0 0.2U

0

0.1

0.05

0.0210 2 4 6 8 10 12 14 16 18 )-0 24 24

Time (days)

J . 45

HRC/WRI 1/84132

FIGURE 4

Concentrations of radioactivity in the plasma andwhole-blood of a dog after a single oral dose of1 4C-WR 171,669. HCl at a level of 5 mg/kg

KET: A-PLSMR t-BLO

.5

.2

0,L)

t' .10

.05

.02 , I , I ! I I !

0 6 12 18 24 30 36 42 48 S4 60 66 ?2

T 4 (h, r.

:46

HRC/WRI 1/84132

FIGURE 5

Concentrations of radioactivity in the plasma and whole-blood of a dogafter a single oral dose of 1C-WR 171,669.HCl at a level of 5 mg/kg


KEY: A-PLRSMR d-BLOOD

5

2

-- .2

C.1

0

.05

.02

0 2 4 6 8 10 12 14 16 18 20 22 24

Time (days)

47

____________________________________________

HRC/WRI 1/84132

FIGURE 6

Concentrations of radioactivit in the plasma and whole-blood of a dogafter a single oral dose of C-WR 171..669.HiCl at a level of 10 mg/kg

KEY: &-PLR3MR 4-BLOOD

2

C

-- .2

5-

C

0 2 1 4 3 6 4 8 5 0 F6 7

Tim (hors

04

H-RC/WRI 1/84132

FIGURE 7

Concentrations of radioactivit in the plasma and whole-blood of a dogafter a single oral dose of 1C-WR 171,669.HCl at a level of 10 mg/kg


KET: A-PLRSMR O-BLOOD

5

.2

- .2

05- 0 2 1 6 is 2 2 2

T1m dy

04

HRC/WRI 1/84132

FIGURE 8

Concentrations of radioactivity in the plasma and

whole-blood of a dog after a single oral dose of14C-WR 171,669. HC1 at a level of 20 mg/kg

KEY: A-PLRSMR 4-BLOOD

.2

CC

0C

0

05

.020 6 12 16 24 30 36 42 46 54 60 66 72

Time (hours)

5o)

HRC/WRI 1/a4132

FIGURE 9

Concentrations of radioactivity in the olasma and whole-blood of a dog

after a single oral dose of 1 C-WR 171,669.HC1 at a level of 20 mg/kg


KET: A-PLRSMR O-BLOOD

5

2

* .5

o

C0

C

.05

0 2 4 6 6 10 12 14 16 16 20 22 24

TLme ( s

I

HRC/WRI 1/84132

FIGURE 10

Concentrations of radioactiVitY in the plasma and whole-blood of a dog

after a single oral dose of 1 C-WR 171,669.HCl at a level of 40 mg/1kg

KEY: &-PLRSMR -0 -BLOJOD

5

2

0

.2

C

C .1 A

0

.02

a 6 12 to 24 30 36 42 48 54 60 66 72

Time (hou.rs)

52

HRC/WRI 1/84132

FIGURE 11

Concentrations of radioactivity in the plasma and whole-blood of a dogafter a single oral dose of 1 C-WR 171,669.HCl at a level of 40 mg/kg


KET: A-PL;SMR O-BLOOD

5

2

N.5

C0

- .2

5-.

0.1

0

.02

0 2 4 6 8 10 12 14 16 18 20 22 24

Time (dys)

II : 53

HRC/WRI 1/84132

FIGURE 12

Concentrations of radioactivity in the plasma andwhole-blood of a dog after a single oral dose of

14C-WR 171,669.HCl at a level of 60 mg/kg

KEY: &-PLRSMP O-BLOOD

5

2

0,

C0

.2

6 2 1 2.-3 V 4 4 0 6

.054

HRC/WRI 1/84132

FIGURE 13

Concentrations of radjoactiviy in the plasma and whole-blood of a dogafter a single oral dose of C-WR l71..669.HC1 at a level of 60 ny/kg


KEY: A-PLRSMR BL D

5

2

C

0)

C

0

.0

0 2 4 6 a 10 12 14 16 is 20 22 24

TL me da~y s

HRC/WRI 1/84132

FIGURE 14

Concentrations of radioactivity in the plasma and whole-blood of a dogafter a single oral dose of

1 4 C-WR 171,669.HCl at a level of 100 mg/kg

KEY: A-PLRSMR <-BLOOO

2

C:0

-~.2

0.1

0

.05

.02

0 6 12 16 24 30 36 42 48 54 60 66 72

Time (hours)

IRC/WRI 1/84132

FIGURE 15

Concentrations of radioactivity in the plasma and whole-blood of a dogafter a single oral dose of 1 4C-WR 171,669.HCl at a level of 100 mg/kg


KEY: A-PLASMR *-BLOOD

5

2

C0

.2

C

0

.05

.02 .7 '0 2 4 6 10 12 14 16 16 20 22 24

Tt me t

I. 57 :

I

HRC/WRI 1/84132

FIGURE 16

Concentrations of WR 171,669 tree base in plasma aftersingle oral doses of f4C-WR 171,669.HCI

KEY: A-5 MG/KG 0-20 MG/KG M-60 MG/KG

2

.2

0

0s-

c..

05

.02

0 6 12 16 24 30 36 42 46 54 60 66 72

T1me (hou rs)

II

: 5 :

HRC/WPI 1/84132

FIGURE 17

Concentrations of WR 171,669 free base in plasma aftersingle oral doses of 1

4 C-WR 17l,669.HC1

KE'T: A-1O MG/KG 0-40 MG/KG 03-100 MG/KG

2

.5

2

0,

C0

.0

* 05

HRC/WRI 1/84132

FIGURE 18

Concentrations of total radioactivity and of WR 171,669 free base

in plasma of a dog after a single oral dose of4C-WR 171,669.HC1 at a level of 5 mg/kg

KET: 0-14C-171669 A -TOTRL

2

.5

E

.2

0

C0L).

.02

0 6 12 18 24 30 36 42 48 S4 60 66 72

Ti me (hours)]

60

HRC/WRI 1/94132

FIGURE 19

Concentrations of total radioactivity and of WR 171,669 free basein plasma of a dog after a single oral dose of


KET: O-I 4,C-17 1669 &-TOTRL

E

.2C0

C0

L)

.05

0 6 12 18 24 30 36 42 48 54 60 66 72

Time (hours)

.1

HRC/WRI 1/84132

FIGURE 20


4C-WR 171,669.HCI at a level of 20 mg/kg

KEC: 0-14C-171669 A-TOTRL

2

.S

E

.2C0

C "

C:0U

.020 6 12 18 24 30 36 42 48 S4 60 66 72

Time (hours)

62

HRC/WRI 1/84132

FIGURE 21

Concentrations of total radioactivity and of WR 171,669 free base

in plasma of a dog after a single oral dose ofT4C-WR 171,669.HCl at a level of 40 mg/kg

KEY: *-14C-171669 A-TOTRL

2

1 t.S

E

.2

o

c .1

C

C

L-

.05

.02

0 6 12 18 24 30 36 42 46 54 60 66 72

TLme (hours)

63

1

HRC/WRI 1/84132

FIGURE 22

Concentrations Of total radioactivity and of WR 171,669 free base

in Tlasma of a dog after a single oral dose of"C-WR 171..669. MC1 at a level of 60 mg/kg

KEY: 0-14C-171669 A -ToFrRL

2

.5

E

.2

0,

.02

0 2 1 4 3 6 2 4 4 6 6 7

Tie (hus

NRC/WPI 1/84132

FIGURE 23


1"C-WR 171,669. iC1 at a level of 100 mg/kg

EY: 1-I4C-171669 &-TOTRL

2

E

.2C

o

c

05

.02 _ _ _ _ _ _ _ _. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

0 6 12 I8 24 30 36 42 46 54 60 66 72

T me (hours]

6 5

: -I

HRC/WRI 1/84132

FIGURE 24

Observed points (I) and fitted curves for concentrations of totalradioactivity in plasma after a single oral dose of1


.5

.2

E

C0) cA

C

0

C AS

L-

c .05S0

0 24 48 22 96 120 144 166 192 216 240I

Time (hours)

66

II

HRC/WRI 1/84132

FIGURE 25

Observed points (L) and fitted curves for concentrations of totalradioactivity in plasma after a single oral dose of


E .2

CA

.5--

oA

0 "

C

0-

oA

VC

.020 36 ?2 106 144 160 216 252 288 324 360

Time (hours)

67

_1

HRC/WRI 1/84132

FIGURE 26

Observed points (6) and fitted curves for concentrations of totalradioactivity in plasma after a single oral dose of1 C-WR 171,669.HCI at a level of 20 mg/kg

E2

0 .2

CA

0

CoA

.02

.010 36 72 106 144 180 216 252 288 324 360 396 432

Time (hours)

68

HRC/WRI 1/84132

FIGURE 27

Observed points (A) and fitted curves for concentrations of total

radioactivity in plasma after a single oral dose of

' C-WR 171,669.HCl at a level of 40 mg/kg

2

EA

CA

0,

C

C0

.02

0 48 96 144 192 240 288 336 384 432 480 528 576

Time (hours)

.69:

HRC/WRI 1/64132

FIGURE 28

Observed points (L) and fitted curves for concentrations of totalradioactivity in plasma after a single oral dose of


2

.5

.2C

C

C "

0U

.0

.02

0 48 96 144 192 240 288 336 384 432 480 528 576

Ti me (hour s)

70

IHRC/WRI 1/84132

FIGURE 29

Observed points (A) and fitted curves for concentrations of totalradioactivity in plasma after a single oral dose of

14C-WR 171,669.HC1 at a level of 100 mg/kg

C0 .2

C

0

.OS A

0 36 72 108 144 18o 216 252 288 324 360 396 432

TLme (hours)

71

HRC/WRI 1/84132

FIGURE 30

Observed points (A) and fitted curves for concentrations of

WR 171,669 free base in plasma after a single oral dose

of L4CWR 171,669.HC1 at a level of 5 mg/kg

.5

.2 A

E .1

C

.05C0

*02

0 6 12 18 24 36 42 48

Time (hours)

72

HRC/WRI 1/84132

FIGURE 31

Observed points (A) and fitted curves for concentrations ofWR 171,669 free base in plasma after a single oral dose

of 14C-WR 171,669.HCI at a level of 10 mg/kg

.5

A

A

.2

A A A

E

N

C0

00

02

.01

0 6 12 18 24 30 36 42 48

Time (hours)

73

I

HRC/WRI 1/84132

FIGURE 32


of 14C-WR 171,669.HCl at a level of 20 mg/kg

2

.5

A

S.2

Co0

CD0A

d

C0o .OS

oAU

.02

.01

0 6 12 1 24 30 36 42 48

Time (hours)

74

H-RC/WRI 1/84132

FIGURE 33I


of 1 4C-WR 171,669.HC1 at a level of 40 mg/kg

2

.AA

E

.2A

C

A

C

0

.02

.01a 6 12 18 24 30 36 42 48

Time (hourns)

75

HRC/WRI 1/64132

FIGURE 34


of 14C-WR 171,669.HCl at a level of 60 mg/kg

2

A AA

.5A

E

2

CA

0 .05

W

.02

.01

a 12 24 36 48 60 72 84 96

Ti me (hours)l

: 76 :

HRC/WRI 1/84132

FIGURE 35


of 14C-WR 171,669.HCI at a level of 100 mg/kg

2

- AE

.2 -

C

o .050

.02

C:0I

.01

0 6 12 18 24 30 36 42 48

Time (hours)

77

HRC/WRI 1/84132

FIGURE 36

Thin-layer chromatogram autoradiograph of extracts of faecesSolvent system: chloroform : methanol : 35% ammonia (70:30:1, v/v)

KEYA Dog 1, 0-24 hours, extract 1, after administration of1

4C-WR 178,460.HCl at 20 mg/kgB Dog 1, 24-48 hours, extracts 1,2,3, after administration of

14C-WR 178,460.HCI at 20 mg/kgC Dog 1, 0-24 hours, extract 1, after administration of1

4C-WR 171,669.HCI at 20 mg/kgD Dog 1, 24-48 hours, extracts 1,2,3, after administration of1

4C-WR 171,669.HCI at 20 mg/kgE Dog 3, 0-24 hours, extract 1, after administration of

14C-WR 171,669.HC1 at 60 mg/kg

F Dog 3, 24-48 hours, extracts 1,2,3, after administration of14C-WR 171,669.HCI at 60 mg/kg

i WR 178,460. HCI2 WR 171 ;9Cq.HCl

78

METABOLISM AND PHARMACOKINETICS OF

14 C-WR 178,46O.HC1 IN THE DOG

79

HRC/WRI 2/83612

SUMMARY

1i. The purpose of this study was to carry out a pilot investigation of

the metabolism and pharmacokinetics in the beagle dog, of the compound14 C-WR 178,460.HCI, a potential pharmacologically active metabolite ofthe anti-malarial drug WR 171,669.HC1. I" C-WR 178,460.HCI wasadministered orally, as a suspension in 1% aqueous carboxymethyl-

cellulose solution, to a single dog at a dose level of 21 mg/kg.

2. Excretion of radioactivity in faeces accounted for 72.4% dose duringthe first 24 hours after dosing and for 97.2% dose in seven days. Atotal of only 0.1% dose was excreted in urine.

3. Plasma concentrations of radioactivity reached a plateau at 2 hoursand remained within the range 0.58 to 0.73 pg equivalents WR 178,460free base/ml (vg/ml) until 2 days. Concentrations then appeared todecline biphasically with an initial more rapid phase (t 52.6 h ±9.5 h S.E.) followed by a slower terminal phase for which a half-life

could not be precisely determined. By 21 days the plasmaconcentration had declined to 0.02 Lg equiv./ml.

4. Whole-blood concentrations of radioactivity were slightly higher thanthe corresponding plasma concentrations during the period from 5 to 24hours after dosing (maximum blood : plasma ratio 1.15) but then

declined more rapidly to be below 0.06 Mg/ml at 8 days.

. 30

HRC/WRI 2/83612

TABLE 1

Excretion of radioactivity after oral administrationof 14C-WR 178,460.HCI to a beagle dog at a dose

level of 21 mg/kg

Results are expressed as % dose

Time Radioactivity excreted Cumulative excretion(hours) ..... ..Urine Faeces Cage wash Urine IFaeces Cage wash iTotal

0- 24 0.05 72.4 0.20 0.05 72.4 0.20 72.624- 48 0.02 8.4 0.12 0.07 80.8 0.32 81.248- 72 0.02 7.2 0.12 0.09 ' 88.0 0.44 88.572- 96 0.009 4.9 0.09 0.10 I 92.9 0.53 93.696-120 0. 007 2.1 0 .11 0.11i 95.0 0.64 95.8120-144 0.006 1.2 0.03 0.12 96.2 0.67 97.0

144-168 0.0 0 0 4 1. 0 0.01 0 . 12 97.2 0.68 98.0---------------------------- -------------------------- ----------

L8

HRC/WRI 2/83612

TABLE 2

Concentrations of radioactivity in plasma and whole-bloodafter oral administration of 1

4 C-WR 178,460.HCl to abeagle dog at a dose level of 21 mg/kg

------------------ ----------------------kg equivalents % dose/litre

Time WR 178,460"/ml

Plasma Whole-blood/ Plasma Whole-blood4---------------------------- ----------- ---15 min 0.07 0.07 0.03 0.0330 min 0.20 0.18 0.08 0.081 hr 0.30 0.29 0.12 0.122 hrs 0.64 0.52 0.26 0.213 hrs 0.66 0.62 0.27 0.254 hrs 0.68 0.66 0.28 0.275 hrs 0.66 0.69 0.27 0.286 hrs 0.64 0.69 0.26 0.287 hrs 0.61 0.70 0.25 0.29

12 hrs 0.58 0.67 0.24 0.2724 hrs 0.71 0.72 0.29 0.2930 hrs 0.73 0.70 0.30 0.292 days 0.66 0.57 0.27 0.233 days 0.48 0.31 0.20 0.134 days 0.30 0.24 0.12 0.105 days 0.20 0.14 0.08 0.066 days 0.14 0.08 0.06 0.037 days 0.10 0.07 0.04 0.038 days 0.09 < 0.06 0.04 <0.03

10 days 0.05 - 0.06 0.02 40.0312 days 0.05 <0.06 0.02 < 0.0314 days 0.04 < 0.06 0.02 < 0.0318 days 0.03 < 0.06 0.01 - 0.0321 days 0.02 4 0.06 0.009 < 0.03

L -- L--:_ -------------------------------* Calculated as free base

A value of 1.052, for the specific gravity ofdog blood, was used in calculation of theseresults from data in Appendix 6

82

HRC/WRI 2/83612

TABLE 3

Extraction of radioactivity from faeces samples up to 72 hoursafter oral administration of 14C-WR 178,460.HC1 to a

beagle dog at a dose level of 21 mg/kg


Time period Extract 1 Extract 2 Extract 3 Total*

(hours)

0 - 24 24 31 21 75 (54.6)

24 - 48 19 23 14 57 (4.8)

48 - 72 9 30 17 55 (4.0)

* Total is calculated from individual results to onedecimal place and then rounded to nearest whole number

Figures in brackets are % doseExtract 1: methanol, Extract 2, 3; methanol % diethylamine

9 : 1, v/v

.33

: 3

HRC/WRI 2/83612

TABLE 4

Radioactivity in one minute* fractions of hplc eluate after injectionof extracts of faeces collected up to 72 hours after oraladministration of 1 4C-WR 178,460.HCl to a beagle dog at a

dose level of 21 mg/kg

Time interval Fraction numbers % eluted radioactivity Total % dose(hours) (inclusive) ------------------------ (all 3 extracts)

Extract 1 Extract 2 + 3----------------------------------------------------------------

0 - 24 3 - 6 14.4 7.7 5.49 - 10 3.9 1.6 1.3

12 - 13* 81.6 89.1+ 47.0Others - 1.6 0.9

24 - 48 3 - 6 74.6 76.9 3.69 - 10 12.0 6.7 0.4

12 - 13* 13.4 9.8 0.5Others - 6.6 0.2

48 - 72 3 - 6 83.1 87.2 3.49 - 10 16.9 5.5 0.3

12 - 13' <5.0 4.24 0.1 - 0.2Others - 3.1 0.1

-------- ---------------- ---------- ------------- -----------------* Corresponds to WR 178,460

Includes 1.5% from fractions 14 and 15Fractions 11, 12 (not 13)

* For results in detail see Appendix 3

84

HRC/WRI 2/83612

TABLE 5

Concentrations of WR 178,460* free base in plasmaafter oral administration of 1

4 C-WR 178,460.HC1to a beagle dog at a dose level of 21 ma/kg

Time (hours) Concentration Proportion of total(Pg/ml) plasma radioactivity (%)

1 0.15 50.92 0.37 57.63 0.39 59.54 0.40 59.55 0.40 61.06 0.37 57.17 0.34 55.0

12 0.32 54.624 0.23 32.230 0.18 24.748 0.08 12.972 <0.04 <8.3

* Refers to radioactivity eluting from an hplc columnwith the same retention time as WR 178,460

Results are not corrected for recovery

85

I

HRC/WRI 2/83612

TABLE 6

Radioactivity in one minute fractions* of hplc eluate afterinjection of extracts of plasma collected up to 72 hoursafter oral administration of 14C-WR 178,460.HCI to a

beagle dog at a dose level of 21 mg/kg

Results are expressed as I eluted radioactivity

Time (hours) Fractions 3 - 5 Fractions 8 - 10 Fractions 11 - 13*

1 49.1 <9.6 50.9

2 36.2 6.2 57.63 35.2 5.3 59.64 34.3 6.2 59.55 34.0 5.0 61.06 37.8 5.1 37.17 39.5 5.6 55.0

12 35.9 9.5 54.6424 59.8 8.0 32.230 65.5 9.8 24.748 78.9 8.2 12.972 100 <8.3 <8.3

* For results in detail see Appendix 4Fractions 13 - 14

* Corresponds to WR 178,460

86

HRCWRI 2/83612

03

I..

0

r, -.C; D

C.ilse a-;0V9 Ms~aP~b t) OjJ~3

'CB7

HRC/WRI 2/83612

-4 r

0.

31 0

I'4V

b4 0 -ckk

>.0

1

-. J

0 A-

41

0-- C 0i

C4 ! C Ca ,z Da

IT/g30.;1tOL 4suTAnebi)UTv~*u:

HRC/WRI 2/83612

134

04

m 0

04

00

U-4

m c

I cN

- -4

0

L0

o 0 o ,I n

CC

0 00 0 00 0 0 (D- .

(Tw/aseq 99i O9V'eLT HM qU9TdATflba bri jo bii) UOT4PI4uauoD

89

Ir

HRC/WRI 2/83612

..4

U

0 41

4

0

0 4 w..4

414

w Or-

> .. 4

1ww

0

00

jJC;

0

000

HRC/WRI 2/83612

44

0

0

0- 04

0eu

0-0 >w

-4

Q EmN

c4L C0 0 C

((/ ")2,j~~a U :

THE ABSORPTION, EXCRETION AND

BIOTRANSFORMATION OF 14C-WR 158,122

IN THE RHESUS MONKEY

HRC/WRI 3/84457

SUMMARY

1. The purpose of this study was to investigate the pharmacokinetics and

metabolism of WR 158,122 a new potential anti-malarial drug in rhesus

monkeys. Single i0 mg/kg oral doses of 1 4 C-WR 158,122 have beenadministered in suspension in 2% aqueous sodium carboxymethylcellulose

solution to both intact monkeys and monkeys with cannulated bile

ducts. The effects of incorporating bile salt (2% sodium taurocholate)

in solution in the dosing suspension have also been studied.

2. In the intact monkeys excretion of radioactivity in faeces of one male

and one female animal accounted for 88.0% and 91.7% respectively of

the dose during 6 days after oral administration of 14C-WR 158,122.

Most of the drug (87.5% and 91.3% dose respectively) was excreted

during the first 48 hours after dosing. Urinary excretion during the

6 days after dosing accounted for 1.07% (male) and 1.95% of dose

(female).

3. After oral administration of 14C-WR 158,122 to monkeys with cannulated

bile ducts excretion of radioactivity in the faeces accounted for

41.5% (male) and 71.2% of dose (female) during the 3 days after

dosing. Excretion in urine accounted for 24.5% (male) and 6.42% of

dose (female), and in bile accounted for 19.0% (male) and 3.07%

(female) during 3 days after dosing. Most of the radioactivity was

eliminated in bile, urine and faeces during the first 2 days after

dosing.

4. In intact animals peak concentrations of radioactivity in plasma of

0.17 &g/ml (male) and 0.22 ig/ml (female) were reached at 5 hours.

Concentrations declined to 0.12 ig/ml (male) and 0.18 [g/ml (female)

at 6 hours, and rose again to 0.13 ig/ml (male) and 0.37 g/ml (female

peak level) at 24 hours, after which concentrations declined to 0.05

ig/ml (male) and 0.14 Lg/ml (female) at 30 hoirs and were below the

limit of accurate measurement (0.05 4g/ml) at 48 hours.

Concentrations of radioactivity in whole-blood were lower than

corresponding plasma concentrations up to 6 hours after dosing. At 30

hours whole-blood concentrations were greater than those in plasma.

93

HRC/WRI 3/84457

5. In animals with cannulated bile ducts, concentrations of radioactivity

in plasma were greater than corresponding plasma concentrations in the

intact animals. Peak concentrations of 3.42 4g/ml (male) and 0.70

4g/ml (female) were reached at 5 hours after dosing. There was no

secondary peak at 24 hours when concentrations were 1.37 g/ml (male)

and 0.29 4g/ml (female). Concentrations continued to decline to 0.13

4g/ml in the male at 54 hours and 0.04 4g/ml in the female at 48

hours. Concentrations in the male at 72 hours and in the female at 54

hours were below the limit of accurate measurement (0.03 4g/ml).

Concentrations of radioactivity in whole-blood were lower than

corresponding plasma concentrations at all times where concentrations

were measurable.

6. Comparison of concentrations of radioactivity in plasma between

monkeys receiving single oral doses of 14C-WR 158,122 with and without

bile salt included in the dosing medium indicated that the addition of

bile salt to the dose had no effect on the extent of absorption.

Without bile salt incorporated in the dosing medium a peak of mean

concentrations of 0.63 Lg/ml was reached at 4 hours after dosing,

after which mean concentrations declined to 0.45 gg/ml, 0.07 g/ml and

0.03 pg/ml at 6, 24 and 30 hours respectively. With bile salt

incorporated in the dosing medium a peak of mean concentrations of

0.33 pg/ml was reached at 4 hours after dosing. Mean concentrations

declined to 0.29 pg/ml, 0.10 4g/ml and 0.07 4g/ml at 6, 24 and 30

hours respectively.

Mean areas under plasma radioactivity-time curves up to 24 hours after

dosing were 0.67 g.h.ml-1.mg-l.kg without bile salt and 0.43

g.h.ml-1.mg-1 .kg with bile salt added to the dosing medium.

94

1.HRC/WRI 3/84457

7. Most of the radioactivity in urine whs associated with two radioactive

components corresponding to 14% and 50% of extracted urinary

radioactivity. Most of the residual radioactivity was associated with

3 components which underwent partial hydrolysis on incubation with

P-glucuronidase/arylsulphatase. Metabolite patterns in bile weresimilar to those in urine but different in proportion. The two major

metabolites present in urine were also present in bile but mainly in a

conjugated form. After enzymic hydrolysis these two metabolites

accounted for 5% and 40% respectively of the extracted biliary

radioactivity (compared with 5% or less for both components beforehydrolysis). A third, more polar metabolite remained after enzymic

hydrolysis and accounted for 10-20% of the biliary radioactivity. In

extracts of faeces almost all the radioactivity was associated with

unchanged WR 158,122 with small traces of the two main urinary

metabolites.

9

I

. 9

I

I

HRC/WRI 3/84457

TABLE 1

Excretion of radioactivity in the urine, faeces, cage debris and cage washof rhesus monkeys after single oral doses of 1 4C-WR 158,122

Results are expressed as % dose/stated time interval

Time (h) Monkey No. Mean....---. ....----

C502d B518?

Urine

0- 6 0.00 0.67 0.346- 24 0.90 1.08 0.99

24- 48 0.13 0.14 0.1448- 72 0.02 0.03 0.0372- 96 0.01 0.01 0.0196-120 0.00 0.01 0.01

120-144 0.01 0.01 0.01-------------.....-----------

Faeces

0- 24 64.45 76.60 70.5324- 48 23.05 14.70 18.8848- 72 0.47 0.37 0.4272- 96 0.01 0.02 0.0296-120 ND ND 0.00120-144 0.00 0.00 0.00

Cage debris

0- 24 0.28 1.78 1.0324- 48 0.23 0.09 0.1648- 72 0.06 0.01 0.0472- 96 0.01 ND 0.0196-120 0.01 ND 0.01

120-144 0.01 ND 0.01-- - - -- L ----- --. .------------

Cage wash

0- 6 0.01 0.06 0.046- 24 0.06 0.08 0.07

24- 48 0.01 0.02 0.0248- 72 0.01 0.00 0.0172- 96 0.00 ND 0.0096-120 0.00 ND 0.0096-120 0.17 0.12 0.15120-144 0.01 ND 0.01144-150 0.10 ND 0.05

ND Not detected (<2 x background)

. 96

' ... -

[IRC/WRI 3/84457

TABLE 2

Cumulative excretion of radioactivity in the urine, faeces, cage debris and

cage washings of rhesus monkeys after single oral doses of 14 C-WR 158,122


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

Time (hours) Monkey No. Mean

C502d B518?-------------------------------------

Urine

0- 6 0.00 0.67 0.340- 24 0.90 1.75 1.330- 48 1.03 1.89 1.460- 72 1.05 1.92 1.490- 96 1.06 1.93 1.500-120 1.06 1.94 1.500-144 1.07 1.95 1.51

Faeces

0- 24 64.45 76.60 70.530- 48 87.50 91.30 89.400- 72 87.97 91.67 89.820- 96 87.98 91.69 89.840-120 87.98 91.69 89.840-144 87.98 .91.69 89.84

Cage debris

0- 24 0.28 1.78 1.030- 48 0.51 1.87 1.190- 72 0.57 1.88 1.230- 96 0.58 1.88 1.230-120 0.59 1.88 1.240-144 0.60 1.88 1.24

Cage washings

0- 6 0.01 0.06 0.040- 24 0.07 0.14 0.110- 48 0.08 0.16 0.120- 72 0.09 0.16 0.130- 96 0.09 0.16 0.130-120 0.09 0.16 0.130-120 0.26 0.28 0.270-144 0.27 0.28 0.280-150 0.37 0.28 0.33

97

IIRC/WRI 3/84457

TABLE 3

Excretion of radioactivity in the bile, urine and faeces ofrhesus monkeys with cannulated bile ducts after single oral doses of

14C-WR 158,122



C502d B518------- -- ---- ----- - --- --- - -- --

Bile

0- 2 0.09 0.08 0.092- 4 1.32 0.28 0.804- 6 2.13 0.27 1.206- 8 1.96 0.31 1.148-24 9.43 1.52 5.48

24-30 1.77 0.31 1.0430-48 1.85 0.25 1.0548-54 0.25 0.03 0.1454-72 0.20 0.02 0.11

Urine

0- 2 0.01 0.16 0.092- 4 2.91 0.39 1.654- 6 0.16 0.47 0.326- 8 NS 0.12 0.068-24 12.29 4.16 8.2324-30 1.99 0.50 1.2530-48 4.51 0.43 2.4748-54 1.48 0.08 0.7854-72 1.11 0.11 0.61

Faeces

0-24 15.82 51.73 33.7824-48 23.55 18.74 21.1548-72 2.17 0.70 1.44

NS No sample

. 9

RC/WRI 3/84457

TABLE 4

Cumulative excretion of radioactivity in the bile, urine and faeces ofrhesus monkeys with cannulated bile ducts after single

oral doses of L4C-WR 158,122



C502d B5189

Bile

0- 2 0.09 0.08 0.090- 4 1.41 0.36 0.890- 6 3.54 0.63 2.090- 8 5.50 0.94 3.220-24 14.93 2.46 8.700-30 16.70 2.77 9.740-48 18.55 3.02 10.790-54 18.80 3.05 10.930-72 19.00 3.07 11.04

Urine

0- 2 0.01 0.16 0.090- 4 2.92 0.55 1.740- 6 3.08 1.02 2.050- 8 3.08 1.14 2.110-24 15.37 5.30 10.340-30 17.36 5.80 11.58

0-48 21.87 6.23 14.050-54 23.35 6.31 14.830-72 24.46 6.42 15.44

Faeces

0-24 15.82 51.73 33.780-48 39.37 70.47 54.920-72 41.54 71.17 56.36

99

HRC/WRI 3/84457

TABLE 5

Concentrations of radioactivity in the plasma of rhesus monkeys aftersingle oral doses of C-WR 158,122

Results are expressed as jg eqivalents/ml


C502d B51M

o ND ND 0.000.5 ND ND 0.001 ND ND 0.00

2 0.05 ND 0.033 0.08 0.09 0.094 0.16 0.20 0.185 0.17 0.22 0.206 0.12 0.18 0.15

24 0.13 0.37 0.2530 0.05 0.14 0.1048 ND ND 0.00

ND Not detected (<0.05 jLg/ml)

ND assumed as zero in calculationof the means

1: 100

HRC/WRI 3/84457

TABLE 6

Concentrations of radioactivity in the plasma of rhesus monkeys aftersingle oral doses of C-WR 158,122



C502d B518

0 ND ND 0.000.5 ND ND 0.001 ND ND 0.002 0.08 ND 0.043 0.13 0.23 0.184 0.28 0.53 0.415 0.29 0.58 0.446 0.21 0.47 0.3424 0.22 0.95 0.5930 0.08 0.36 0.2248 ND ND 0.00

ND Not detected (<0.08 % dose/litre)


101

HRC/WRI 3/84457

TABLE 7

Concentrations of radioactivity in the whole-blood of rhesus monkeysafter single oral doses of 1 4 C-WR 158,122

Results are expressed as 4g equivalents/g

-------------------------- ---------- --------Time (hours) Monkey No. Mean

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

C502d B51S----------------------------------

0 ND ND 0.000.5 ND ND 0.001 ND ND 0.002 ND ND 0.003 ND ND 0.004 0.09 0.12 0.115 0.10 0.13 0.126 ND 0.11 0.06

24 0.12 0.30 0.2130 0.21 0.24 0.2348 ND ND 0.00

--------------------------------- -------------ND Not detected (<0.08 Lg/g)


102

I

HRC/WRI 3/84457

TABLE 8

Concentrations of radioactivity in the whole-blood of rhesus monkeysafter single oral doses of 14 C-WR 158,122

Results are expressed as % dose/kg

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


C502d B51X--------------------------------

0 ND ND 0.000.5 ND ND 0.001 ND ND 0.002 ND ND 0.003 ND ND 0.004 0.15 0.30 0.235 0.17 0.34 0.266 ND 0.28 0.14

24 0.20 0.78 0.4930 0.36 0.63 0.5048 ND ND 0.00------------------------ ------

ND Not detected (<0.16 % dose/kg)


103

HRC/WRI 3/84457

TABLE 9

Concentrations of radioactivity in the plasma of bile duct cannulatedrhesus monkeys after single oral doses of 1

4C-WR 158,122

Results are expressed as Lg equivalents/ml


C502d B518

0 ND ND 0.000.5 0.20 0.09 0.151 0.74 0.23 0.492 1.53 0.40 0.973 2.45 0.56 1.514 3.07 0.66 1.875 3.42 0.70 2.066 3.27 0.66 1.97

24 1.37 0.29 0.8330 0.75 0.15 0.4548 0.21 0.04 0.1354 0.13 ND 0.0772 ND ND 0.00

ND Not detected (<0.03 Lg/ml)


104

I

HRC/WRI 3/84457

TABLE 10

Concentrations of radioactivity in the plasma of bile duct cannulated

rhesus monkeys after single oral doses of 1 4C-IWR 158,122



C5024 B518W

0 ND ND 0.000.5 0.34 0.24 0.291 1.28 0.61 0.952 2.64 1.06 1.853 4.23 1.49 2.864 5.28 1.75 3.52

5 5.90 1.88 3.896 5.63 1.75 3.69

24 2.35 0.77 1.5630 1.30 0.41 0.8648 0.37 0.10 0.2454 0.22 ND 0.1172 ND ND 0.00

ND Not detected (<0.06 % dose/litre)


105

HRC/WRI 3/84457

TABLE 11

Concentrations of radioactivity in the whole-blood of bile ductcannulated rhesus monkeys after single oral doses of 1 4C-WR 158,122

Results are expressed as Lg equivalents/g


C502d B518

0 ND ND 0.000.5 0.12 ND 0.061 0.46 0.16 0.312 0.96 0.25 0.613 1.54 0.36 0.954 1.99 0.45 1.225 2.23 0.47 1.356 2.14 0.45 1.30

24 0.85 0.18 0.5230 0.49 0.09 0.2948 0.15 ND 0.0854 NS ND 0.0072 ND ND 0.00

ND Not detected (<0.07 .ig/g)NS No sample


106

HRC/WRI 3/84457

TABLE 12

Concentrations of radioactivity in the whole-blood of bile ductcannulated rhesus monkeys after single oral doses of 14C-WR 158,122

Results are expressed as % dose/kg


C502d B518

0 ND ND 0.000.5 0.21 ND 0.111 0.78 0.41 0.602 1.65 0.66 1.163 2.66 0.97 1.824 3.42 1.19 2.315 3.84 1.26 2.556 3.69 1.19 2.44

24 1.46 0.49 0.9830 0.84 0.25 0.5548 0.26 ND 0.1354 NS ND 0.0072 ND ND 0.00

ND Not detected (<0.14 % dose/kg)NS No sample


10

I

:107

HRC/WRI 3/84457

TABLE 13

Concentrations of radioactivity in the plasma of rhesus monkeys afteroral doses of 14 C-WR 158,122 with and without incorporation of bile salt in

the dose suspension

Results are expressed as jg equivalents/ml

(a) Dose suspended in carboxymethylcellulose solution-----------------------------------------------------Time (hours) Monkey No. Mean SD

C498 C505 343

---------------------------- ------------------------0 ND ND ND 0.00 0.000.5 ND ND ND 0.00 0.001 0.06 i 0.08 0.05 0.06 0.022 0.23 0.50 0.24 0.32 0.153 0.21 0.69 0.66 0.52 0.274 0.20 0.74 0.95 0.63 0.395 0.17 0.61 0.81 0.53 0.336 0.15 0.50 0.69 0.45 0.27

24 0.07 0.08 0.05 0.07 0.0230 0.05 0.05 ND 0.03 0.0348 ND ND ND 0.00 0.0072 ND - ND - ND 0.00 0.00

(b) Dose suspended in carboxymethylcellulose solutionincorporating bile salt

Time (hours) Monkey No. Mean SD+

C498 C505i 343V0 -- ND ND ND 0.00 0.000.5 ND ND ND 0.00 0.001 0.05 ND ND 0.02 0.03

2 0.09 0.09 0.33 0.17 0.143 0.09 0.11 0.48 0.23 0.224 0.10 0.10 0.80 0.33 0.405 0.09 0.08 0.76 0.31 0.396 0.09 0.09 0.68 0.29 0.34

24 0.06 0.13 0.11 0.10 0.0430 0.05 0.08 0.07 0.07 0.0248 ND ND ND 0.00 0.00

ND Not detected (<0.04 4g/ml)SD Standard deviation

ND assumed as zero in calculation of the means

108

I

HRC/WRI 3/84457

TABLE 14

Concentrations of radioactivity in the plasma of rhesus monkeys afteroral doses of 14C-WR 158,122 with and without incorporation of bile salt in

the dose suspension


(a) Dose suspended in carboxymethylcellulose solution

Time (hours) Monkey No. Mean SD

C498i C505 343o ND ND ND 0.00 0.000.5 ND ND ND 0.00 0.00

1 0.10 0.13 0.06 0.10 0.042 0.37 0.83 0.28 0.49 0.303 0.34 1.15 0.78 0.76 0.414 0.32 1.23 1.12 0.89 0.505 0.28 1.02- 0.97 0.76 0.416 0.25 0.82 0.82 0.63 0.3324 0.12 0.13 0.06 0.10 0.0430 0.09 0.08 ND 0.06 0.0548 ND ND ND 0.00 0.00'72 ND ND ND 0.00 0.00

L ------------ ------ ------------------ -----

(b) Dose suspended in carboxymethylcellulose solutionincorporating bile salt

-------------------------------------------Time (hours) Monkey No. Mean SD

C498V C505* 343V--------------------------------------------

0 ND ND ND 0.00 0.000.5 ND ND ND 0.00 0.001 0.08 ND ND 0.03 0.052 0.14 0.15 0.41 0.23 0.153 0.15 0.19 0.59 0.31 0.244 0.16 0.17 1.00 0.44 0.485 0.14 0.12 0.95 0.40 0.476 0.14 0.15 0.84 0.38 0.40

24 0.10 0.22 0.14 0.15 0.0630 0.08 0.13 0.08 0.10 0.0348 ND ND ND 0.00 0.00

------------------------------------------ND Not detected (<0.06 % dose/litre)SD Standard deviation

ND assumed as zero in calculation of the means

1

:109:

HRC/WRI 3/84457

TABLE 15

Areas under plasma radioactivity concentration time curves up to 24 hoursafter single oral doses of 1 4 C-WR 158,122 to rhesus monkeys

Animal No. Dose level Area Area/dose level( ,g-h.ml - l ) ( Lg.b.ml - l . mg - l .kg)

Intact animals

C502d 10.3 2.77 0.27B518* 9.8 5.55 0.57

Bile duct cannulated animals

C502d 10.1 54.52 5.40B518 9.5 11.42 1.20

Intact animals (No bilesalt in dosing medium)

C498 10.0 2.91 0.29C505* 10.0 8.07 0.81343 10.5 9.70 0.92

Mean ± SD 0.67 ± 0.34

Intact animals (Bile saltadded to dosing medium)

C498 9.9 1.80 0.18C505 10.5 2.41 0.233434 11.0 9.82 0.89

Mean ± SD 0.43 ± 0.40----------------------------------------------- ------------ --------------------SD Standard deviation

110

HRC/WRI 3/84457

TABLE 16

Observed (0) and expected (E) values for fitted models for totalradioactivity concentrations in plasma from intact and bile duct

cannulated monkeys

Results are expressed as dpm/ml

Time (hours) Intact monkeys Bile duct cannulated

monkeys

C502d B5189 C502d B518

0 E 0 E 0 E 10 E..... O ~ ~ ~ -- ----- --- ..........EO-

| .. °-----

0.5 - - - 620 616 280 2861 - - - 2328 2373 713 6652 155 149 - - 4793 5221 1243 12653 235 278 278 283 7675 7322 175 16934 513 365 638 482 9588 8823 2053 19865 538 422 700 636 10705 9843 2200 21736 388 457 563 754 10220 10481 2053 227824 405 253 1125 568 4273 4493 905 85430 150 184 435 374 2358 2731 483 50948 - - - - 670 545 115 9954 - - - - 395 313 - -

Model

parameters

T (h) 1.1635 1.8874 0.3399 0.1661%I (h-1 ) 0.0538 0.1093 0.0935 0.1986X2 (h-1 ) 0.2898 0.1093 0.1754 0.0930A (dpm/ml) 871.8225 7561224 48014.8789 -8514.8516h, 12.9 6.34 7.41 3.49h 2 2.39 6.34 3.95 7.45

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

111

II

HRC/WRI 3/84457

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116

HRC/WIRI 3/84457

TABLE 22

Proportions of radioactivity in extracts of faeces of monkeys withcannulated bile ducts after oral doses of 14C-WR 158,122

Solvent system: chloroform:methanol:water:formic acid (80:25:3:3, v/v)

(a) Results are expressed as a proportion (%) of the total radioactivity ineach chromatogram

--- ----- --------------------------------------------------Animal No. C502d B5189-----------------------------------------------------------Rf value 0-24 hours 24-48 hours 0-24 hours 24-48 hours

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

0 0.7 <0.8 <0.8 <0.70.20 <0.7 1.6 <0.9 <0.70.24 2.4 8.3 <0.8 2.10.59+h 88.8 83.0 96.8 93.50.63 1.4 0.9 0.9 1.20.72 - 1.2 - -

Others 6.2 4.3 1.7 3.1

(b) Results are expressed as % dose*

Animal No. C5024 B5152

Rf value 0-24 hours 24-48 hours 0-24 hours 24-48 hours

0 0.11 <0.19 <0.40 <0.120.20 <0.11 0.37 <0.40 <0.120.24 0.38 1.9 <0.40 0.360.59+ 14 19 48 160.63 0.22 0.21 0.45 0.210.72 - 0.28 - -

Others 0.98 1.0 0.84 0.53-----------------------------------------------

*Calculated as proportion (%) of radioactivity inchromatogram x % dose in sample x proportion extracted

100+ Radioactive zone corresponding to IVR 158,122

117

HRC/WRI 3/84457

FIGURE 1

Cumulative excretion of radioactivity in urine and faeces of monkeysafter oral doses of 14C-WR 158,122

AL-C5O2d Urine A-B5184 Urine WD-C5O2d Faeces

*..B518* Faeces

100

90 E9E 9E

8. 0

0

Co 60

0

> 0

0

2 0

0

0

Time (hours)

HRC/WRI 3/84457

FIGURE 2

Cumulative excretion of radioactivity in urine, bile qjd faeces ofmonkeys with cannulated bile ducts after oral doses of C-WR 158,122

,&-C5O2d Bile A-516 Bile (D-502 4 Urin~e

4D-B518* Urine [D-5023 Faecas KI-518 Faeces

60

70

~'60

00

40

30

0

10

0

0 12 24 36 48 60 72

T~me (hourg]

119

HRC/WRI 3/84457

FIGURE 3

Concentrations of radioactivity in the plasma and whole-blood of monkeysafter oral doses of "C-WR 158,122

E-C502 s Plasma A-85 18 Plasma *-C02s Whole-blood

A-8518 % Whole-blood

.2

S

E

0d

0C A

0

5- .o

U

02

a 6 12 18 24 30 36 42 48

Time (hours)

* Concentrations of whole-blood are measured in 4g/g

120

_ f - -

HRC/WRI 3/84457

FIGURE 4

Concentrations of radioactivity in the plasma of monkeys with cannulate'

bile ducts and in the plasma of intact monkeys after oral doses of14C-WR 158,122

E]-C502, Intact A-8518y Intact O-C502d Cannulated

QD-851V8 Cannulated

2

E

C0

.2

C

V .1

0

.02 "

0 6 12 1 24 30 36 42 -4 54

Time (hours)

1

121 :

HRC/WRI 3/84457

FIGURE 5

Concentrations of radioactivity in the plasma and whole-blood of monkeys

with cannulated bile ducts after oral doses of 14C-WR 158,122

O-C502d Plasma 0-8518 Plasma *-C502d Vhhole-blood

S-8518 Whole-blood

s

E

C0

• .2

5-0C

Uc .1

0

.05

.02 I

0 6 12 18 24 30 36 42 48 S4

Time (hours)

* Concentrations of whole-blood are measured in 4g/g

1

: 1L22

I

HRC/WRI 3/84457

FIGURE 6

Mean concqqtrations of radioactivity in the plasma of monkeys after oraldoses of "C-WR 158,122, with and without incorporation of bile salts in

the dose suspension

- No bile salt - Bile salt

.S

E .2

C

0C

C0

05

02

a 6 12 16 24 30 36 42 48

Time (hours)

123

HRC/WRI 3/84457

FIGURE 7

Observed and fitted curves f or concentrations of total radioactivity inplasma of a male monkey (intact, CSO2d) after a single oral dose of14C-WR 158,122

0B-Observed &-Expected

.2

E

C0,

d.05

C0

U

.020 6 12 1824 30

Time (hours)

1~24

HRC/WRI 3/84457

FIGURE 8

Observed and fitted curves for concentrations of total radioactivity inplasma of a female monkey (intact, B518W after a single oral dose of

14C-WR 158,122

~-Observed A- Expected

.S

C0,

C

0

L)

02

.125

HRC/WRI 3/84457

FIGURE 9

Observed and fitted curves for concentrations of total radioactivity inplasma of a male monkey (bile duc 4 cannulated, C5023) after a single

oral dose of C-WR 158,122

9-Obao *va A-Expacted

2

E

.S

C

0

C

oI

0 .2

II

C0

.0

0 6 12 18 24 30 36 42 48 54

TIme .(hours)

126

HRC/WRI 3/84457

FIGURE 10

Observed and fitted curves for concentrations of total radioactivity in

plasma of a female monkey (bile duct cannulated, B518*) after a singleoral dose of 1

4C-WR 158,122

ED-Observed A-Expected

.S

E .2

0)

C

a

.02

a 6 12 Is 24 30 36 42 40

Ti me (h o ur s

127

4-L

HRC/WRI 3/84457

FIGURE 11

Observed and fitted concentrations of radioactivity in the plasma of amonkey (C498*) after a single oral dose of C-WR 158,122 with no

incorporation of bile salts in the dose suspension

Q1-Ob srved A-Expact d

.5

.2

E

.1

C0

C

C .050

.020 6 12 18 24 30

Time (hours)

128

I

HRC/WRI 3/84457

FIGURE 12

Observed and fitted concentrations of radioactivity in the plasma of a

monkey (C505 ) after a single oral dose of 14 C-WR 158,122 with no


E-Obseved A-Expecied

.5

E .2

0

1291

:5-2

4-7

C0

HRC/WRI 3/84457

FIGURE 13

Observed and fitted concentrations of radioactivity in the plasma of amonkey (343*) after a single oral dose of 14C-WR 158,122 with no


W-Observed &-Expecied

.5

C

C

0

U

S.02

0

.02, ,,,06 12 18 24 30

Thme (hours)

I : 130:

1

- -- -__________ -_ _ -- - - - - - -- ---- -- -- - -

HRC/WRI 3/84457 IFIGURE 14

Observed and fitted concentrations of radioactivity in the plasma of amonkey (C4989) after a single oral dose of 14C-WR 158,122 with

incorporation of bile salts in the dose suspension I

III-6berved &-Expected

4 I

E

" 0 5

0

U

.02

06 12 18 24 30

Time (hours)13

: 3N

HRC/WRI 3/84457

FIGURE 15

Observed and fitted concentrations of radioactivity in the plasma of amonkey (C505+) after a single oral dose of 14C-WR 158,122 with


KET: WD-Observed A-ExpecI ed

.2

E

C0

.05

C

C0

.02 ,,,

0 6 12 18 24 30

Time (hours)

132

HRC/WRI 3/84457

FIGURE 16

Observed and fitted concentrations of radioactivity in the plasma of a

monkey (343*) after a single oral dose of 14 C-WR 158,122 with


El-Observed s-Expected

.5

E:N

o .2

0C0

.OS06 12 1624 30

Time thouLrs)

1.33

THE IDENTIFICATION OF THE MAJOR

METABOLITES OF 14 C-WR 158,122 IN THE

RHESUS MONKEY

I 134

HRC/WRI 3/85773

SUMMARY

1. The major metabolites of WR 158,122 in urine and bile of rhesus

monkeys receiving 5 mg/kg oral doses have been isolated and purified

using a combination thin-layer and high performance liquid

chromatography.

Bile metabolites were present mainly in the form of conjugates and

samples were incubated with O-glucuronidase/sulphatase prior to

isolation.

2. The isolated metabolites were investigated by mass spectrometry as

their trimethylsilyl derivatives. The major metabolite in urine and

bile was assigned as a dihydrodiol presumably resulting from

epoxidation of the naphthalene ring. A second minor component of

similar chromatographic polarity appeared to be an isomeric

dihydrodiol giving an almost identical mass spectrum. This isomer

could be formed via epoxidation at a different position in the

naphthalene ring.

A more polar metabolite also isolated from bile and urine has been

assigned to a diphenolic compound which could be formed by

dehydrogenation of the dihydrodiol. A further related diphenolic

metabolite, only isolated from bile, was tentatively assigned to a

4 compound derived by oxidative deamination to give a quinazolindione.

1

: 135:

HRC/WRI 3/85773

FIGURE 1

Thin-layer autoradiograph of isolated urinary and biliary metabolitesobtained after oral administration of I'C-WR 158,122 to amale rhesus monkey. The developing solvent system was

isopropanol : 35% w/v aqueous ammonia (8 2,v/v)

WRI 3 502m 8-24hr 18-6-84 Solvent system a

MI MIA M2 mn~n l MIA M2 43 %urine metelolitem urine P M'I" , bile metaboites

Approx. 0.51 0.23 0.5 0.51 0.24 0.17Rf values

136

HRC/WRI 3/85773

FIGURE 2

Thin-layer autoradiographs of isolated urinary and biliary metabolitesobtained after oral administration of "C-WR 158,122 to amale rhesus monkey. The developing solvent system was

chloroform : methanol : water : formic acid (80: 25 3 3,v/v)

0 WRI 3 S02m 8-24hr 18-6-84 Solvent wetem b

MI MIA M2 untr untr tone " M1 MIA M2 M3urine metabolites urine bile bile bile metabolites

Approx. 0.25 0.22 0.11 0.25 0.22 0.11 0.07Rf values

iI1 137:

I

HRC/WPI 3/85773

FIGURE 3

High performance liquid chromatograms (u.v. detection 250 nm) ofmetabolite 'Ml' after oral administration of 14C-WR 158,122 to a

male rhesus monkey. (a from urine; b from bile; c from bileand urine combined). The eluting solvent system was

methanol water (1 l,v/v) and the retention time for 'Ml' was 4.5 mins

1 4 3 4 5 6 7 8Retention time (minutes)

Ii . 138

Ir

HRC/WRI 3/85773

FIGURE 4

High performance liquid chromatograms (u.v. detection 250 nm) ofmetabolite 'M1A' after oral administration of 14C-WR 158,122 to a

male rhesus monkey. (a from urine; b from bile; c from bileand urine combined). The eluting solvent system was

methanol water (1 l,v/v) and the retention time for 'MlA' was 3.8 mins

f

1 2

Retention time (minutes)

139

Lr

HRC/WRI 3/85773

FIGURE 5

High performance liquid chromatograms (u.v. detection 250 nm) ofmetabolite 'M2' after oral administration of 14C-WR 158,122 to amale rhesus monkey. (a from urine; b from bile; c from bile

and urine combined). The eluting solvent system wasmethanol water (1 3,v/v) and the retention time for 'M2' was 7.2 mins

3 4 5 b 7 8 9


I~140

I

HRC/WRI 3/85773

FIGURE 6

High performance liquid chromatograms (u.v. detection 250 nm) ofbiliary metabolite 'M3' after oral administration of ''C-WR 158,122 to a

male rhesus monkey. The eluting solvent system wasmethanol water (1 4,v/v) and the retention time for 'M3' was 6.5 mins

~/1

1 12 3 4 5 6 7 8 9 1


141

HRC/WRI 3/85773

FIGURE 7

Electron impact mass spectrum of the trimethylsilyl derivative ofmetabolite 'Ml' isolated from urine following oral administration of

14C-WR 158,122 to a male rhesus monkey

-i C;5

u~ an

LLa

LCO

Lin La

L.La

Cau

CN,

L- .n~-M C

=CCuCu

CCu

* C

11 CM

a~am

CCu

SO 0cc11 C 2 1 . 4 -

142-

HRC/WIRI 3/85773

FIGURE 8

Electron impact mass spectrum of the deutero-trimethylsilyl derivative ofmetabolite 'Ml' isolated from urine following oral administration of114C-WR 158,122 to a male rhesus monkey

aL

C'- - ,

a

I

L.4

0

cm ta .-

Sa-

- = 7

: 143 :

HRC/WIRI 3/85773

FIGURE 9

Electron impact mass spectrum of the trimethylsilyl derivative ofmetabolite 'M1A isolated from urine following oral administration of


C"

CCu

u' Ca

Cu C"

L..n

~- .- ~uaC~ L..i

=O

=~ C,,

CaU

C-3

C"I~

L..CS "

r" .P LM 5 .

OC , U , U ,- ,- U ,- U

144M

_____

HR(/WRI 3/85773

FIGURE 10

Electron impact mass spectrum of the deutero-trimethylsilyl derivative ofmetabolite 'MlA' isolated from urine following oral administration of

14C-WR 158,122 to a male rhesus monkey {

U-1

- c3I

r 'j 0

T '

I

-- )

01LaSr -Un

=Vj

Ci!

tE -S

1451

HRC/V;RI 3/85773

FIGURE 11

Electron impact mass spectrum of the trimethylsilyl derivative ofmetabolite 'M2' isolated from urine following oral administration of


= U

CM,

QC C1,

LU-)

-

_0

~~CM

Lin L.)C32

L.2

1.-I

= L.

Cu -

Cu CM

+ ~ C C

C.,,

.--

C= CuC

-a-

C: C .,C

C , I-. CW

C L..J

C,9C 0,'I ' , 9 19 II 1./ U U

-COCOm19U amC 94-_U

146

HRC/VJRI 3/85773

FIGURE 12

Electron impact mass spectrum of the trimethylsilyl derivative of

metabolite 'M3' isolated from bile following oral administration of1 4 C -WR 158,122 to a male rhesus monkey

I-.-

U,) UnC=C.

C.0

Li

UCM

U, C

C~j CU,CID CM

C~a,

_a,

ID

_L..jLW C

r-* rn

_= C"V

~OCO

CMC

OI= C',

COI C.M Cu

~Co

'i- xr CM___ _ _

cn , - ,

CO C.147

FIGUE 13HPC/WI 3/85773

Electron impact mass spectrum of the trimethylsilyl derivative of14C-IWR 158,122

CM

c'cc

CD~

rn -4

LMUCO cc=l ~jC

CM

Ln,

CM~~ C=

CJw

CP. CO 0

CaCI, LeC, C

CC

=C"CM~ " CM

M C-Cuj

C%# C, -n

Cc U4

* ~ d - LaCz00' amt .U't" " C M c

14

DISTRIBUTION LIST

12 copies DirectorWalter Reed Army Institute of ResearchWalter Reed Army Medical CenterATTN: SGRD-UWZ-CWashington, DC 20307-5100

1 copy CommanderUS Army Medical Research and Development CommandATTN: SGRD-RMI-SFort Detrick, Frederick, Maryland 21701-5012

12 copies Defense Technical Information Center (DTIC)ATTN: DTIC-DDACCameron StationAlexandria, VA 22304-6145

1 copy DeanSchool of MedicineUniformed Services University of the

Health Sciences4301 Jones Bridge RoadBethesda, MD 20814-4799

1 copy CommandantAcademy of Health Sciences, US ArmyATTN: AHS-CDMFort Sam Houston, TX 78234-6100

III

and final report i - defense technical information … i otic. fill od i (n determination of drug...

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