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DRUG EVALUATION IN THE PLASMODIUM

FALCIPARUTM-AOTUS MODEL

ANNUAL REPORT

Richard N. Rossan

J11DTICJune 14, 1989 S ELECTE

JUL251989

Supported by Sal

U. S. ARMY MEDICAL RESEARCH AND DEVELOPMENT COMMANDFort Detrick

Frederick, Maryland 21701-5012

Contract No. DAMD17-87-C-7163

Gorgas Memorial Institute of Tropical &

Preventive Medicine, Inc.9650 Rockville PikeBethesda, MD 20814

Approved for public release; distribution unlimited

The views, opinions, and/or findings contained in this report arethose of the author and should not be construed as an officialDepartment of the Army position, policy, or decision, unless sodesignated by other documentation.

89 7 25 038,, -i I

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PROGRAM PROJECT TASK WORK UNITFt. Detrick ELEMENT NO. NO. 3Mi- NO. ACCESSION NO.Frederick, MD 21701-5012 62770A 12770A870 AJ 003

11. TITLE (Include Security Classification)Drug Evaluation in the Plasmodium falciparum - Aotus Model

12. PERSONAL AUTHOR(S)Richard N. Rossan

13a. TYPE OF REPORT 13b. TIME COVERED 14. DATE OF REPORT (Year, Month, Day) 1S. PAGE COUNT

Annual FROM5/I 5/TO5aZLI8 1989 June 14 4816. SUPPLEMENTARY NOTATION

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

FIELD GROUP SUB-GROUP Plasmodium falciparum;reversal of chloroquine06 -3resistance; Aotus lemurinus lemurinus; calcium06 45 channel blocker; artemisinin derivativeny RAI

19, ABSTRACT (Continue on reverse if necessary and identify by block number)

lood-induced infections of the multidrug resistant Vietnam Smith/RE strain oPlasmodium falciparum in Aotus lemurinus lemurinus were used as a model forantimalarial drug evaluation studies. Trials to reverse chloroquine resis-tance in vivo with WR 256287, an analog of tiapamil , plus chloroquine, re-sulted only in suppression of parasitemia. Similar trials with WR 149244,desipramine, plus chloroquine, were partially successful in that primarytreatments cleared parasitemias, but infections were not cured. Parasiteclearance is the first indication that in vivo reversal of chloroquine-resis-tance may eventually be feasible in human patients. However, the toxicity ofthe combined drug regimen must be ameliorated.Four derivatives of artemisinin, the active antimalarial principal of qinghaowere evaluated for their antimalarial activity in the P. falciparum - Aotusmodel. Two of these drugs, WR 255131 (arteether) and WR 254986 (artemether),

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19. are oil soluble and administered intramuscularly in three doses,

ranging'

from 0.25 to 64.0 mg/kg, q.12h. Arteether cleared parasitemias in a total -

of 23 of 25 treatments, and cured 16 of 24 infections. Artemether clearedparasitemias in a total of 20 of 23 treatments and cured 16 of 23 infections.

The two water soluble derivatives ,evaluated were WR 255663 (artelinate)and WR 256283 (artesunate).'! These drugs were administered intravenously orintramuscularly, at doses of 64.0 and 96.0 mg/kg (X3), q.6h, q.12h, or q.24h.Toxicity has been associated with these regimens. To date, artelinate clearedparasitemias in 14 of 15 treatments, and cured 4 of 15 infections. Artesunatecleared parasitemias in 3 of 3 treatments, and cured 1 of 3 infections.

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1. III

SUMMARY

The objective of this contract is to evaluate experimentalantimalarial drugs, alone or in combination, against experi-mentally induced trophozoite infections of Plasmodium falciparumin the Panamanian owl monkey (Aotus lemurinus lemurinus) . Forthe studies reported herein, the Vietnam Smith/RE strain wasused, resistant to maximally tolerated doses of chloroquine,pyrimethamine, and quinine.

In vivo trials were continued to reverse chloroquineresistance in P. falciparum by the concom-itant administrationof a calcium channel blocker plus chloroquine. Initiation ofsuch trials, summarized in the previous Annual Report for thiscontract, was based upon successful in vitro reversal of chloro-quine resistance. The explanation of this phenomenon was basedupon the hypothesis that the channel blocker prevents the activeefflux of chloroquine by the parasite, allowing chloroquine toachieve a parasiticidal level.

Additional in vivo reversal trials with WR 256287, astructural analog of verapamil, administered orally 3X/day forseven days plus chloroquine for either 3 or 5 days, resultedin suppression of parasitemia only.

Desipramine (Norpramin), WR 149244, is a tricyclicpsychotropic drug. Some drugs in this class have weak anti-malarial activity and are calcium antagonists. Trials toreverse chloroquine-resistance in vivo with desipramine showedthat a three day course of treatment with chloroquine clearedthe parasitemia in 7 of 13 monkeys, but without infection cure.A seven day treatment course of desipramine plus chloroquinecleared parasitemia in 5 of 7 monkeys, and 2 of 5 infectionswere cured, but after repeat treatments. Seven monkeys diedof drug toxicity. Although the combination of desipramineplus chloroquine reverses chloroquine resistance in vivo, atleast to the extent of clearing primary parasitemias, itsusefulness in human infections must be qualified until a non-toxic regimen is identified.

Four derivatives of artemisinin, the active antimalarialprincipal of the Chinese herb qinghao, were selected forevaluation in the P. falciparum-Aotus model. Two of thesederivatives, WR 25T131 (arteether), and WR 254986 (artemether),are oil soluble; WR 255663 (artelinate) and WR 256283 (artesunate)are water soluble.

Limited toxicity evaluation, based upon overt reactionand body weight gain or loss, indicated that a dose of 64.0mg/kg (X3), IM, q.12h of arteether and artemether, was well-

- 2-

tolerated by Aotus. Drug tolerance problems were associatedwith the intravenous administration of artelinate at a doseof 64.0 mg/kg (X3), q.6h, in a 30 mg/ml stock solution. Lowerconcentrations of stock solution, drug administration q.12h,and intramuscular injection of the drug, have reduced toxicity,but not entirely. Similar host tolerance difficulties wereassociated with artesunate, thus limiting the antimalarialevaluation.

For antimalarial evaluation, the two oil solublederivatives, arteether and artemether, were administeredintramuscularly q.12h, at doses ranging from 0.25 mg/kg (X3)to 64.0 mg/kg (X3), as both primary and repeat treatments.Arteether, WR 255131, cleared parasitemias in a total of 23 of25 treatments, and cured 16 of 24 infections. Artemether,WR 254986, cleared parasitemias in a total of 20 of 23 treat-ments and cured 16 of 23 infections.

The two water soluble artemisinin derivatives, artelinateand artesunate were administered intravenously or intramuscular-ly, q.6h, q.12h, or q.24h. Doses were 64.0 and 96.0 mg/kg (X3).Artelinate, WR 255663, cleared parasitemias in a total of 14of 15 treatments, and cured 4 of 15 infections. Artesunate,WR 256283, cleared parasitemias in a total of 3 of 3 treatments,and cured 1 of 3 infections.

The antimalarial activity of the two oil soluble artemisininderivatives, arteether and artemether, are similar and both aremore effective and less toxic than the water soluble derivatives,artelinate and artesunate.

3 3.-

FOREWORD

In conducting the research described in this report, theinvestigator adhered to the "Guide for the Care and Use ofLaboratory Animals," prepared by the Committee on Care and Useof Laboratory Animals of the Institute of Laboratory AnimalResources Commission of Life Sciences, National Research Council(NIH Publication No. 86-23, Revised 1985).

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TABLE OF CONTENTS

Page

SUMMARY 1

FOREWORD 3

TABLE OF CONTENTS 4

EXPERIMENTAL PROCEDURES 7

IN VIVO TRIALS TO REVERSE CHLOROQUINE RESISTANCE

IN PLASMODIUM FALCIPARUM BY THE CONCOMITANT

ADMINISTRATION OF CALCIUM CHANNEL BLOCKERS OR

SIMILAR ACTING DRUGS

A. Introduction 10

B. WR 256287AB (BN: BL 51153) 11

C. WR 149244AD (BN: BL 54261) 12

D. Conclusions 13

COMPARISON OF THE ANTIMALARIAL EFFICACY OF

FOUR ARTEMISININ DERIVATIVES IN THE

PLASMODIUM FALCIPARUM - AOTUS MODEL

A. Introduction 26

B. Limited toxicity evaluation of 27

four artemisinin derivatives

1. WR 255131AE (BN: BL 48816)WR 254986AB (BN: BL 26767)

2. WR 255663AG (BN: BL 54038)

WR 255663AH (BN: BL 55866)

3. WR 256283AA (BN: BL 28556)

C. Antimalarial activity of four 32

artemisinin derivatives

1. WR 255131AE (BN: BL 48816), arteether

2. WR 254986AB (BN: BL 26767), artemether

3. WR 255663AG/AH (BN: BL 54038/55866),artelinate

4. WR 256283AA/AB (BN: BL 28556/BL 35613),

artesunate

D. Conclusions 34

-5-

TABLE OF CONTENTS (CONT'D.)

Page

LITERATURE CITED 44

DISTRIBUTION LIST 45

Figure

1. Schema for drug evaluation against Plasmodium 9falciparum - induced infections in Aotuslemurinus lemurinus

Tables

1. Detailed activity of WR 256287AB plus 14WR 001544AB, chloroquine

2. Summary of the activity of WR 256287AB plus 15WR 001544AB, chloroquine

3. Detailed activity of WR 149244AD, desipramine, 16in combination with WR 001544AB, chloroquine

4. Detailed activity of WR 149244AD, desipramine 19in combination with WR 001544AB, chloroquine

5. Summary of the activity of WR 149244AD, 21desipramine, in combination with WR 001544AB,chloroquine

6. Summary of the activity of WR 149244AD, 23desipramine, in combination with WR 001544BM

7. Summary of the activity of WR 149244AD, 24desipramine, in combination with WR 001544BM,chloroqu ine

8. Evaluation of acute toxicity of WR 149244AD, 25desipramine, in combination with WR 001544BM,chloroquine

9. Toxicity evaluation of WR 255131AE, arteether, 29and WR 254986AB, artemether

10. Toxicity evaluation of WR 255663AG 30artelinate, and WR 256283AA, artesunate

11. Further evaluation of the toxicity of 31

WR 255663AG, artelinate

12. Detailed activity of WR255131AE, arteether 35

-6-

TABLE OF CONTENTS (CONT'D.)

13. Summary of the activity of WR 255131AE, 36arteether

14. Detailed activity of WR 254986AB, 37artemether

15. Summary of the activity of 38WR 254986AB, artemether

16. Pilot evaluation: detailed 39activity of WR 155663AG/AH, artelinate

17. Pilot evaluation: summary of the 40activity of WR 255663AG/AH, artelinate

18. Pilot evaluation: detailed activity 41of WR 256283AA/AB, artesunate

19. Pilot evaluation: Summary of the 42activity of WR 256283AA/AB, artesunate

20. Activity of four artemisinin 43derivatives

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EXPERIMENTAL PROCEDURES

The monkey-adapted Plasmodium falciparum strain, VietnamSmith/RE (resistant to maximally tolerated doses of chloroquine,pyrimethamine, and quinine) was used to induce experimentalmalaria infections in Aotus lemurinus lemurinus for the evalua-tion of the antimalarial efficacy of candidate drugs. Infectedblood, with sodium citrate (2.5%) as the anticoagulant, fromuntreated Aotus was diluted appropriately with chilled saline(0.85%), such that each milliliter contained 5,000,000 parasites,and this amount was injected into the saphenous vein of experimen-tal and control monkeys.

Blood films, prepared and examined daily beginning on thefirst post-inoculation day, were stained with Giemsa. Parasitemiaswere evaluated as follows: negative, if no parasites were detectedon a thick blood film after examination for at least 5 minutes;<10 parasites per cmm, if positive only on the thick blood film;parasite enumeration was by the Earle-Perez method and reportedas the number of parasites per cmm.

Blood films from untreated Aotus, serving as passage and/orcontrol subjects, were prepared and examined daily during theprimary patent period, and daily thereafter for at least threeconsecutive days after parasites could last be detected on thickblood films. When parasitemia had cleared, films were made andexamined twice weekly until a total of 100 negative days hadbeen recorded. If a recrudescence occurred, blood films wereobtained again on a daily basis.

The schema depicted in Figure 1 represents the design of atypical drug evaluation study. Parasitemias were evaluated dailyduring the treatment period and until blood films were negativefor at least seven consecutive days. The frequency of smearingwas then reduced to two times per week (Monday and Thursdays orTuesdays and Fridays). If no recrudescences occurred during a100 day examination period, the infection was considered to havebeen cured.

Drug doses were calculated as mg base per kg of body weight.Stock solution of water soluble compounds, at appropriate concen-trations, were prepared with distilled water and stored at 8*Cfor the treatment period. If a compound was water insoluble, asuspension of the requisite amount of drug was prepared dailywith 0.3% methylcellulose (in distilled water).

Oral administration of drugs was effected by gastric intuba-tion with a 14 French catheter. The total amount of fluid admin-istered, drug solution or suspension, and rinse was 14 ml.

i8 I

As in cated in the appropriate sections, some water solubledrugs were administered intravenously or intramuscularly; otherwater insoluble drugs were diluted in sesame oil and administeredintramuscularly.

-9-

FIGURE 1

SCHEMA FOR DRUG EVALUATION AGAINST

PLASMODIUM FALCIPARUM

INDUCED INFECTIONS IN ACTUS LEMURINUS LEMURINUS

In ;ect

----------------------- J------ --------------- I

Expcri4,,ental Control

r ----------r-------i

D4s A Dose!B Dos4 C-------.--- I-----------------------

1. Parasitemia' II. Parasilteinia III. Parasitemliacleared. If cleared and not affected.negative for recrudesces. Re-Rx with100 days -s Re-Rx with higher dosecured higher dose or another

or new drug. drug.

Repeat II or III as indicated

1 10 -

IN VIVO TRIALS TO REVERSE CHLOROQUINE RESISTANCEIN PLASMODIUM FALCIPARUM BY THE CONCOMITANTADMINISTRATION OF CALCIUM CHANNEL BLOCKERS

OR SIMILAR ACTING DRUGS

A. Introduction

Data associated with numerous trials to reverse chloroquineresistance in vivo were presented in a previous Annual Report (1).The genesis of these trials was based upon reports of in vitroreversal of chloroquine resistance in P. falciparum by verapamil(a calcium channel blocker) plus chlorocquine (2, 3). Infectionsof the Vietnam Smith/RE strain of P. falciparum in Aotus wereused for in vivo trials. In a total of 26 combined treatmentsduring the primary patent period, suppression of parasitemiaoccurred in 17 monkeys. Verapamil plus chloroquine cured theinfection in one monkey. In a total of 28 repeat treatments,infections were cured in 6 Aotus. The infection parameters incured monkeys were identical to those of infected, untreatedAotus exhibiting self-cure, thus making it difficult to differen-tiate drug activity from acquired immunity.

Continuation of trials to reverse resistance to chlorouineare reported herein.

- II-

B. WR 256287AB (BN: BL 51153)

This Hoffman La Roche drug is a structural analog oftiapamil, related to verapamil. Although not as potent acalcium channel blocker in humans as verapamil, it was hopedthat an analog without the cardiodynamic effects of verapamilmight prove less toxic in combination with chloroquine. Also,WR 256287 is 4X more effective in vitro than verapamil inreversing chloroquine resistance. Previous in vivo trials (1)indicated that WR 256287 administered orally 3X/day for 7 daysat a dose of 20.0 mg/kg plus chloroquine administered daily forthree days significantly suppressed parasitemias of thechloroquine resistant Vietnam Smith/RE strain. Additionaltrials with this drug combination were undertaken.

Data presented in Tables 1 and 2 indicate that WR 256287administered orally 3X/day for seven days at a dose of 20.0mg/kg plus chloroquine (20.0 mg/kg, daily) for either 3 or 5days suppressed parasitemias, but without parasite clearance.Some suppression of parasitemia was observed when chloroauine(20.0 mg/kg, daily) alone was administered.

- 12 -

C. WR 149244AD (BN: BL 54261)

WR 149244, desipramine (Norpramin), is a tricyclic psycho-tropic drug. Some drugs in this class have weak antimalarialactivity and are calcium antagonists. In vitro reversal ofchloroquine resistance in P. falciparum by desipramine wasreported (4) at concentrations similar to those in patientstreated for depression.

Data associated with trials of desipramine to reversechloroquine resistance of P. falciparum infections in Aotusare detailed herein. As shown in Tables 3-6, WR 149244 wasadministered orally, once, twice, or three times daily, foreither three or seven days. Neither desipramine nor chloroquine,administered alone, had significant antimalarial activity againstparasitemias. A three-day course of treatment with desipramineplus chloroquine (Tables 3 and 5) cleared the parasitemias in 7of 13 monkeys. Two of the seven treatments were primary, andno infections were cured. Additionally, six monkeys died within2 to 3 days after initiating treatment with desipramine pluschloroquine.

The data presented in Tables 4 and 6 show that a 7-daycourse of treatment with desipramine plus chloroquine clearedparasitemias in 5 of 7 monkeys, and 2 of 5 infections werecured after repeat treatments. One Aotus died of drug toxicityon day 4 of treatment.

Overall, 13 of 24 (54.2%) parasitemias were cleared and 2of 21 (8.3%) infections were cured (Table 7). A total of sevenmonkeys died of causes attributable to drug toxicity, i.e. acombination of desipramine plus chloroquine. An evaluation inuninfected Aotus of the acute toxicity of desipramine pluschloroquine, administered in different dose and regimens isindicated in Table 8. There were no deaths associated withthese drug regimens.

Mmm

-13-

D. CONCLUSIONS

The desideratum of in vitro chloroquine reversal is combinedtreatment with a calcium channel blocker plus chloroquine duringthe ascending phase of parasitemia resulting in parasite clearanceand cure of infection. This sequence of events would be entirelythe result of drug action. Infection cures subsequent to com-bined retreatment course may be attributable to both drug actionand acquired immunity. The trials of in vitro reversal of chloro-quine resistance presented in the previous Annual Report (1)showed that parasite clearance was not obtained with primary drugtreatments, but only by repeat treatments.

Results of studies with desipramine plus chloroquine indicatethat a primary drug treatment will clear parasitemias of thechloroquine-resistant Smith/RE strain of P. falciparum. Infectioncures, however, were obtained only after repeat drug treatments.The potential use of desipramine plus chloroquine in humanpatients must be tempered by the toxic effects of this combinationin Aotus. Additional drug evaluation in the monkey model may yielda non-toxic, curative regimen for appropriate use in patientsinfected with chloroquine-resistant malaria.

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-24 -

TABLE 7,

SUMMARY OF THE ACTIVITY OF WR 149244AD (BL 54261),DESIPRAMINE, IN COMBINATION WITH WR 001544BM (AR 20613),

CHLOROQUINE, AGAINST PLASMODIUM FALCIPARUM INFECTIONS

MALARIA DOSE mg/kg PRIMARY TREATMENTS REPEAT TREATMENTS TOTAL TREATMENTS

STRAIN TOTAL DAILY CLEARED CURED CLEARED CURED CLEARED CURED

ietnam 225 75a 0/1 0/1 0/1 0/1mith/RE

300 75a 0/1 0/1 0/1 0/1

20 5b 0/1 0/1 0/1 0/1

60 20b 0/1 0/1 0/1 0/1

36 12a 1/1 0/1 1/1 0/160 20b

72 24a 1/2 0/2 1/2 0/260 20b

75 25a 0/2 0/230 lOb 0/02

144 48a60 20b I/1 0/1 i/i 0/1

150 50a 0/2 0/2 1/1 0/1 1/3 0/330 lOb

250 25a60 25b 2/2 0/2 2/2 0/260 20b

238 96a60 20b 2/2 0/2 2/2 0/2

350 50a70 50b 5/5 2/5 5/5 2/5

525 75a 0/1 0/1 0/1 0/1 0/2 0/235 5b

a WR 149244b Chloroquine

- 25 -

TABLE 8

EVALUATION OF ACUTE TOXICITY OF WR 149244AD(BL 54261), DESIPRAMINE, IN COMBINATION WITH WR 001544BM

(AR 20613), CHLOROQUINE

Aotus WR 149244 WR 001544 Body Wt.gmsNo. Dose X Daily No. Dose X Daily No. Pre-Rx Post-Rx

mg/kg Days mg/kg Days

11773 25.0 1 3 10.0 1 3 824 828

11453 25.0 2 3 10.0 1 3 836 828

12150 5.0 1 7 10.0 1 7 774 795

11373 i0.0 1 7 10.0 1 7 885 864

11476 25.0 1 7 10.0 1 7 779 784

11475 5.0 2 7 10.0 1 7 860 829

12151 10.0 2 7 10.0 1 7 807 795

11775 25.0 2 7 10.0 1 7 858 869

- 26 -

COMPARISON OF THE ANTIMALARIAL EFFICACY OF FOURARTEMISININ DERIVATIVES IN THE PLASMODIUM

FALCIPARUM - AOTUS MODEL

A. INTRODUCTION

An herb, qinghao (Artemisia annua L.), has been usedin China for more than 400 years against the chills and feverof malaria (5). The active antimalarial principal of the herbhas been identified as a 15-carbon sesquiterpene lactone endo-peroxide and named artemisin. Studies in China with patientsinfected with P. falciparum or P. vivax showed that artemisin,an oil soluble derivative (artemether), and a water solublederivative (artesunate) possessed significant antimalarialactivity. Synthesis and selection of new artemisin derivativesyielded an oil soluble ethyl ether derivative, arteether, anda water soluble derivative, sodium artelinate. These two newlysynthesized derivatives, and artemether and artesunate wereselected for comparison of their antimalarial efficacy againstinfections of the multi-drug resistant Vietnam Smith/RE strainof P. falciparum in Aotus. Subsequent sections will delineatethese studies.

All drugs, sesame oil, and sodium bicarbonate, wereprovided by the Division of Experimental Therapeutics, WalterReed Army Medical Center.

-27 -

B. Limited toxicity evaluation of four artemisininderivatives

Since no studies with these drugs have been done in Aotus,it was considered necessary to evaluate the toxicity of atleast the highest projected dose in this monkey. Animals curedof a malarial infection were used and drug toxicity was monitoredby body weight, and overt symptoms.

1. WR 255131AE (BN: BL 48816), arteetherWR 254986AB (BN: BL 26767), artemether

Each of these compounds, soluble in sesame oil, wereadministered at a dose of 64.0 mg/kg (IM)X3, q.12h.The data presented in Table 9 show that the monkey(12007) administered WR 255131, arteether evidencedsome loss of body weight beginning 14 days post-treatment, but that one month post-treatment the pre-treatment body weight had been regained. The bodyweight loss in Aotus 12294, administered WR 254986,artemether, was attributed to an intestinal amoebainfection. No drug toxicity was associated withartemether, per se.

2. WR 255663AG (BN: BL 54038)WR 255663AH (BN: BL 55866), sodium artelinate

This water soluble artemisin derivative, sodiumartelinate, was administered intravenously. Thefirst monkey to receive artelinate was administereda dose of 64.0 mg/kg at 8:00 AM, using a 30 mg/mlstock solution. Following the second and third doses,each at 6 hour intervals, the animal became hypotonic,a condition that persisted for about 10 minutes. Themonkey died of drug toxicity on day 2 post treatment.It was suggested that the concentration of the stocksolution may have contributed to the toxic reaction,causing a precipitation of the drug in the vascularsystem. "Subsequent concentrations of stock solutionsof artelinate were reduced to either 10 mg per ml or15 mg per ml. As shown in Table 10, Aotus 11805 wasadministered a 64.0 mg/kg (X3), q.6h of artelinate.Hypotonia was again noted after doses 2 and 3. A lossof body weight was noted beginning on day 6 post treat-ment, but the pre-treatment body weight was regainedby day 26 post treatment.

Results of an additional toxicity evaluation ofartelinate are presented in Table 11. Doses of 4.0,16.0, 32.0, and 64.0 mg/kg (X3), IV, q.6h, wereadministered to a total of four Aotus. During a four-month post treatment observation period, the lowest

- 28-

body weight loss was associated with the 4.0 mg/kgdose, while the monkey experiencing the highest meanbody weight loss received the 16.0 mg/kg dose. Therewas, however, no overt manifestation of drug toxicity.

3. WR 256283AA (BN: BL 28556), artesunate

Sodium artesunate, a water soluble artemisin derivative,was converted to artesunic acid, 10 minutes beforeintravenous administration, by the addition of sodiumbicarbonate (5% solution). As shown in Table 10, onemonkey was administered a 64.0 mg/kg X3 dose, q.6h,for toxicity evaluation. Some body loss (about 2%)occurred between days 8 and 16 post treatment, with noother adverse symptoms. Problems associated withadministration of artesunic acid will be indicated ina subsequent section of this report.

- 29-

TABLE 9

TOXICITY EVALUATION OF WR 255131AE (BL 48816),ARTEETHER, AND WR 254986AB (BL 26767),

ARTEMETHER

Aotus Drug, Dose, Notes Days Post-Rx Body Weight-gmsNo.

12007 WR 255131AE, 64.0 mg/kg (x3), IM, -2 849q. 12h 2 814

5 8228 844

11 84314 81416 82519 82822 81829 840

12294 WR 254986AB, 64.0 mg/kg (x3), IM, -2 725q. 12h 2 715

5 7108 707

11 70714 68616 677

Intestinal amoebae 19 670Rx Tinidizol, 22 677250 mg/kg for 3 days 29 677

30-

TABLE 10

TOXICITY EVALUATION OF WR 255663AG (BL 54038),SODIUM ARTELINATE AND WR 256283AA (BL 28556),

SODIUM ARTESUNATE

Aotus D rug, Dose, NotesNo. Days Post-Rx Body Weight-gms

11805 WR 255663AG 64.0 mg/kg (x3), IV, -2 773q. 6h. 2 795Monkey became flaccid after 4 790administration of doses 2 and 3 6 755

8 73110 71812 71914 73126 774

11806 WR 256283AA, 64.0 mg/kg (x3), IV, -2 879q.6h. 2 866

4 8666 8708 856

10 85712 86014 85326 871

- 31-

TABLE 11

FURTHER EVALUATION OF THE TOXICITY OFWR 255663AG (BL 54038), SODIUM ARTELINATE

BODY WEIGHT (GMS)

MONKEY NO.

DAYS POST Rx 12324 11972 12316 11335

-5 797 934 870 898

0 Rx 4.0 mg/kg* 16.0 mg/kg* 32.0 mg/kg* 64.0 mg/kg*

2 800 920 848 877

5 790 904 849 877

8 785 893 829 843

11 788 872 821 847

14 799 849 820 859

17 774 823 792 823

20 780 811 800 844

23 754 805 789 831

36 782 759 805 852

121 801 796 874 815

mean body 12 91 47 51weight loss

* (x3), IV, q.6h

32 -

C. Antimalarial activity of four artemisinin derivatives

1. WR 255131AE -(BN: BL 48816), arteether

Evaluation of arteether against infections of the VietnamSmith/RE strain of P. falciparum is detailed inTable 12 and summarized in Table 13. Three doses ofthe drug were administered intramuscularly at 8:00 AM,8:00 PM, and 8:00 AM. A dose of 0.25 mg/kg (X3)suppressed parasitemia in each of two Aotus. Twoprimary treatments and two retreatments at a dose of1.0 mg/kg (X3) cleared parasitemia in 4 of 4 monkeys.The infection in 1 of 4 monkeys may be cured.

Parasitemias in nine Aotus were cleared with a doseof 4.0 mg/kg (X3); infections were cured in twomonkeys, recrudescence occurred in three monkeys, andpost treatment observation is continuing in four Aotus.

A dose of 16.0 mg/kg (X3) cured the infection in 4 of4 monkeys; the curative activity of retreatment withthis dose has not been determined in one Aotus.Parasitemias were cleared in 5 of 5 monkeys with adose of 64.0 mg/kg (X3), and infections were cured infour of these animals. The fifth monkey died on day51 post-treatment of gastric dilatation, which wasnot considered attributable to arteether.

2. WR 254986AB (BN: BL 26767), artemether

The detailed antimalarial activity of artemetheragainst Vietnam Smith/RE infections is shown in Table14 and summarized in Table 15. Parasitemias weresuppressed in each of two Aotus administered a doseof 0.25 mg/kg (X3).

A dose of 1.0 mg/kg (X3) suppressed primary parasite-mia in one Aotus and cleared parasitemia in one monkey;retreatment with this dose cleared parasitemia in 2 of2 Aotus. Infection cure remains to be determined inone monkey.

In nine Aotus administered a dose of 4.0 mg/kg (X3),the infection was cured, to date, in 2 of 6 monkeysfollowing primary treatment. Post-treatment examina-tion is continuing in five Aotus.

Infections were cured in 4 of 4 monkeys with a doseof 16.0 mg/kg (X3), and 5 of 5 monkeys with a doseof 64.0 mg/kg (X3).

- 33-

3. WR 255663AG (BN: BL 54038)WR 255663AH (BN: BL 55866), artelinate

Two drug lots of sodium artelinate were used for anti-malarial evaluation, as detailed in Table 16 andsummarized in Table 17. Four Aotus, infected with theVietnam Smith/RE strain of P. falciparum, received a64.0 mg/kg (X3) dose, adninistered intravenously, q.6h.The parasitemia was cleared (with recrudescence) in onemonkey, suppression of parasitemia resulted in oneanimal, and two monkeys died of drug toxicity, on day1 and day 6 post treatment, respectively. A 64.0 mg/kg(X3) dose, administered intravenously q.12h, clearedthe parasitemia in 2 of 2 Aotus, but the infection wasnot cured.

Intramuscular administration of a dose of 64.0 mg/kg(X3), q.12h cleared the parasitemia in 2 of 2 monkeys;however, the infections were not cured. Five monkeyswere administered intravenously a 64.0 mg/kg (X3) doseof artelinate, q.24h. The parasitemia was cleared infour of these Aotus; one animal died of drug toxicityon day 2 post treatment. The infection was cured in1 of 4 treated monkeys. Retreatment with a dose of96.0 mg/kg (X3) was as follows: two animals administeredthe drug intravenously, q.12h, with parasite clearancein both, and cured the infection in one monkey; twosubjects received the drug intramuscularly, q.12h,resulting in parasite clearance and blood films remainnegative for, 57 days; this dose, administered intra-venously q.24h to one monkey, cleared the parasitemia,with recrudescence. The highest dose evaluated in thisstudy, 128.0 mg/kg (X3), intravenously, q.6h, was toxicas the monkey died on day 2 post treatment.

4. WR 256283AA (BN: BL 28556)WR 256283AB (BN: BL 35613), artesunate

Results of pilot evaluation studies with artesunate(two drug lots) are shown in Table 18 and 19. Intra-venous adminstration (q.6h) of 64.0 mg/kg (X3) ofartesunate suppressed parasitemia in one monkey, thatdied of an intercurrent infection on day 9 post treat-ment. A dose of 64.0 mg/kg (X3) administered intra-venously q.12h cleared the parasitemia, with recrudes-cence, in one monkey; one animal died of drug toxicityon day 1 post treatment. Intramuscular administration(q.12h) of 64.0 mg/kg (X3) cleared parasitemia in oneAotus (with probable infection cure) and one monkeydied of drug toxicity.

A 96.0 mg/kg (X3) dose administered intramuscularly(q.12h) cleared parasitemia in one monkey, but did notcure the infection.

- 34-

D. CONCLUSIONS

Limited toxicity evaluation of four artemisininderivatives show that the two oil soluble derivatives,arteether and artemether, are well tolerated followingintramuscular administration in Aotus. Intravenousadministration of the water soluble derivative, sodiumartelinate, is tolerated when the stock solution concen-tration is 10 to 15 mg/ml. Toxicity problems associatedwith sodium artesunate, administered as artesunic acid,remain to be resolved.

Antimalarial evaluation of arteether and artemether,against infections of the multi-drug resistant VietnamSmith/RE strain of P. falciparum indicate a similaractivity by both drugs. Both drugs clear parasitemiaswhen administered at doses of 1.0 mg/kg (X3), and> 66%of the infections are cured with doses of. 4.0 mg/kg (X3).

Primary or repeat treatments with artelinate orartesunate, at doses of 64.0 or 96.0 mg/kg (X3) clearedparasitemias, but only 27% of the infections were cured.Neither of the water soluble artemisinin derivatives isas effective as the oil soluble derivatives, arteether orartemether.

-35

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TABLE 20

ACTIVITY OF FOUR ARTEMISININ DERIVATIVESAGAINST INFECTIONS OF PLASMODIUM FALCIPARUM

MALARIA DOSE mg/kg PRIMARY TREATMENTS REPEAT TREATMENTS TOTAL TREATMENTS

STRAIN TOTAL DAILY CLEARED CURED CLEARED CURED CLEARED CURED

VietnamSmith/RE WR 255131AE (BL 48816), arteether

0.75 0.25 0/2 0/2 0/2 0/23.0 1.0 2/2 1/2(?) 2/2 0/2 4/4 1/4(?)

12.0 4.0 6/6 3/6(?) 3/3 3/3(?) 9/9 6/9(?)48.0 16.0 4/4 4/4 1/1 1/1(?) 5/5 5/5(?)

192.0 64.0 5/5 4/4 5/5 4/4

WR 254986AB (BL 26767), artemether

0.75 0.25 0/2 0/2 0/2 0/23.0 1.0 1/2 0/2 2/2 1/2(?) 3/4 1/4(?)

12.0 4.0 6/6 4/6(?) 2/2 2/2(?) 8/8 6/8(?)48.0 16.0 4/4 4/4 4/4 4/4

192.0 64.0 5/5 5/5 5/5 5/5

WR 255663AG/Ali (BL 54038/BL 55866), artelinate

192.0 64.0 6/7 0/7 3/3 1/3 9/10 1/10288.0 96.0 5/5 3/5(?) 5/5 3/5(?)

WR 256283AA/AB (BL 28556/BL 35613), artesunate

192.0 64.0 2/2 1/2(?) 2/2 1/2(?)288.0 96.0 1/1 0/1 1/1 0/1

- 44 -

LITERATURE CITED

1. Rossan RN. 1988. Annual Report "Drug Evaluation in thePlasmodium falciparum - Aotus Model," Army Contract DAMD17-87-C-7163, 15 May 1987 - 14 May 1988.

2. Martin SK, Oduola AMJ, Milhous WK, 1987. Reversal ofchloroquine resistance i:. Plasmodium falciparum by verapamil.Science. 235: 899-901.

3. Krogstad DJ, Gluzman IY, Kyle DE, Oduola AMJ, Martin SK,Milhous WK, Schlesinger PH. 1987. Efflux of chloroquine fromPlasmodium falciparum: mechanism of chloroquine resistance.Science. 238: 1283-1285.

4. Bitonti AJ, Sjoerdsma A, McCann PP, Kyle, DE, Oduola AMJ,Rossan RN, Milhous WK, Davidson DE Jr. 1988. Reversal ofchloroquine resistance in malaria parasite Plasmodium falciparumby desipramine. Science. 242: 1301-1303.

5. Klayman DL. 1985. Qinghaosu (Artemisinin): An antimalarialdrug from China. Science. 228: 1049-1055.

- 45-

DISTRIBUTION LIST

5 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- Pt.jI- SFort Detrick, Frederick, Maryland 21701-5012

2 copies Defense Technical Information Center (DTIC)ATTN: DTIC-FDACCameron 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