AD-A283 256

I'SONT CQVXX

TITLZ: ST1'¶TY or ~(Ywwww~a rp~I &WIVt?Y AtAIN¶4T IP'r-;1AM-P-A-

PPINCIPAL INVFTIT1(ATOJR: William L. tiansorn, tPh.ox

p I A~D RE~ 5, Of P'daftaittlo-iyColle'.* tit Veterinary K*dItcineUn.vitibity of (.oorfliaAthens, fýoorqia 30(,)2 -7 387

PEPORT DATE: Octob~er JRi, 1992D T

TYPr or PEPORT: Ar,nua1

PPFPAAzo FOR. (1,5, ARM~Y MEDICAL RESEARCHI AND DEVELOPMENT comMEND)FORT LETTRICKFREDERICK, MARYLAND 21702-5012

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g (thef ng. m n "a t4l' flng theq .ta nteJeded an onoiltlor ig a nd rneflg ICC oOf 1 non edd -e .".e znts re'i-arding rIn, burden estimate or any other -ý<t Of IN%

cotecT•o•n of infor'a;tono. ncluding siaggestion' for reducing tt- ouraen to S•flfr,n;jion iffadduar'brS '..r c, Oe•-torite for tnformation Operationfi and ReOfts. ,•2 15 ,ef fe Oin

Dare faiqtrny, S•uiC 1204 Arhlngton. VA 222024302. r to tr n Offon Of fnu•u m-d 4n udfjet, Ptoer-Orr Aed -- T.n PrnCiet (0704-0f08), W.anttinitOn. DC 20503

I. AGENCY USE ONLY (Leave blank) 2. REPORT DATE 3. REPORT TYPE AND DATES COVERED

11992 October 27 lAnnual Report (9/28/91 - 9/27/92

4. TITLE AND SUBTITLE 5. FUNDING NUMBERS

Study of Compounds for Activity Against Leishmania Contract No.DAMD17-90-C-0131

6. AUTHOR(S) W. L. Hanson 62787A

V. B. Waits 3M162787A870.AM.037W. L. Chapman, Jr. WUDA335539

7. PERFORMING ORGANIZATION NAME(S) AND ADORESS(ES) 8. PERFORMING ORGANIZATIONREPORT NUMBER

The University of Georgia Research FoundationAthens, Georgia 30602

9. SPONSORING/ MONITORING AGENCY NAME(S) AND AODRESS(ES) 10. SPONSORING/ MONITORINGAGENCY REPORT NUMBER

U.S. Army Medical Research and Development CommandFort DetrickFrederick, Maryland 21702-5012

11. SUPPLEMENTARY NOTES

12a. DISTRIBUTION/ AVAILABILITY STATEMENT 12b. DISTRIBUTION CODE

Approved for public release; distribution unlimited

13. ABSTRACT (Maximum 200 words)

During this project period a total of 41 new compounds were studied forantileishmanial activity against Leishmania donovani in hamsters. Only one hadmeasurable suppressive activity and this compound was toxic.

One new compound was studied for efficacy against Leishmania braziliensispanamensis and this compound was not active and was toxic when administered via theintramuscular route.

A computer search of a total of 736 compounds that have been tested 1gainstL. b. panamensis since 1980 identified a total of 37 compounds with suppressiveactivity equal to or greater than the standard reference compound, Glucantime.rhese compounds were then studied in detail to determine the most promising drugand the most promising routes of administration and dosage regimen against L. b.panamensis. For comparativs purposes these compounds were tested simultaneouslyagainst L. donovani and L. b. panamensis.

14. SUBJECT TERMS 15. NUMBER OF PAGES

Leishmania donovani, Leishmania braziliensis panamensis, 42

Chemotherapy, 8-aminoquinolinrs, oligonucleotides, Phosphoniums 16. PRICE CODE

Sinefunain17. SECURITY CLASSIFIC.ITION IS. SECURI.Y CLASSIFICATION 19. SECURITY CLASSIFICATION 20. LIMITATION OF ABSTRAC

OF REPORTj OF THIS PAGE OF ABSTRACT

Unclassified Unclassified Unclassified Unlimited

NSN 7540.01-280-S500 S'anrdard Sorm 298 ýQov 2 119),;i3, 102

Of the 27 compo)unds identified, 32 were I-amino-quinolinos.This class of compounds contains the most potent compound*studied thus far in this laboratory for suppressiv, activityagainst visceral and cutaneous lelshmaniasis. With twoorceptlons, these compounds were several fold more active atainstLA dulnoyani than aqainat L, b. pana•gnsia. Ten of those 37compounds were sufficiontly active aqainat L, lb V n• )isa to beof interest and the most active of the group was W14041'•01 (";DS -

3.76 mg/kgj). Unfortunately this compound was not active whenadministered orally and is toxic in hamsters at a'domsA', level aslow as 26 mg/kt;, a dosaqe which suppreseas L VL b , V. n2mvleslons by only 76%.

Regarding the nther two active compounds, a phosphoniumcompound and Sinetunqin were noted to be active but the formerwas toxic and the activity of neither was remarkable.

A total of 31 oligonucleotiden was studied for inhibition ofmultiplication of promastiqotes of L, dgngyAnj. in yjtjr. Onlyone of these had any suppressive activity.

Aoeession ?or

DTIC TAB 0]Uri.nnounced 0J'As t If tcnat i!

Av"Ilrbllt7 CodesA~r,,1- aud/or

Diat Speci.al

ACKNOWLEDGEMENT

For technical assistance in carrying out this work, we wish tothank Mrs. Barbara Harris, Miss Laura A. Lamb, and Miss ShannonWaits.

tORZWORD

Opinions, interpretations, conclusions and recommendations arethose of the author and are not necessarily endorsed by the U.S. Army.

Wbhere copyrighted material in quoted, permission has been obtainedto use such materisl.

Where material from documents designated for limited distributionis quoted, permission has been obtained to use the material.

Citations of commercial organizations and trade names in thisreport do not constitute an official Department of the Army endorsementor approval of the products or services of these organizations.

I In conducting research using animals, the investioator(s) adheredto the "Guide for the Care and Use of Laboratory Animals," prepared bythe Committee on Care and Use of Laboratory Animals of the Institute ofLaboratory Animal Resources, National Research Ccuncil (NIH PublicationNo. 86-23, Revised 1985).

- IFor the protection of human subjects, the investigator(s) haveadhered to policies of applicable Federal Law 45 CFR 46.

In conducting research utilizing recombinant DNA technology, theinvestigator(s) adhered to current guidelines promulgated by theNational Institutes of Health.

PI Signature "X -. -t JA •-- Date ac l / /M'2-

3

TABLE OF CONTENTS

Acknowledgement ................................................. 1

Foreword ......................................................... 2

Introduction .................................................... 5

Materials and MethodsA. Primary Visceral Test System .......................... 7B. Primary Cutaneous Test System ......................... 8C. Comparative Antileishmanial Aztivity of Selected ..... 9

Compounds Against L. donovani and L.. braziliensispanamensis

D. Zn vitro Studies of Oligonucleotides Against ......... 9L. donovani

ResultsA. Primary Visceral Test System .......................... 11B. Primary Cutaneous Test System ......................... 11C. Comparative Antileishmanial Activity of Selected ..... 11

Compounds Against L. donovani and L. braziliensispanamensis

D, In vitr Studies of Oligonucleotides Against ......... 13L. donovani

Discussion ..................................................... 14

Conclusions .................................................... 16

Literature Cited ............................................... 17

Appendix 1 ...................................................... 19

Appendix 2 ...................................................... 41

4

TABLEZS

TABLE PAGE

I. Summary of compounds studied for suppressive 20activity against ~IJ liman.A flyAni. in theprimary visceral test system.

1I. Summary of compounds studied for uuppressive 22activity against nLjAA 2z PJA

a in the primary cutaneous test system.

III. Summary of results obtained from studies on the 23comparative activity of compounds tested againstL nia dnQvani and Leis nla brazilienais

IV. Summary of results of additional testing of 29compounds found highly suppresive against14ihmni donovan.

V. Summary of results of selected oligonucleotides 30tested for in vitro inhibition of promastigotes ofLihai aonovani.

FIGURE

I. Structures of the most active compounds found in 31the primary cutaneous test system and testedsimultaneously in the primary visceral test systemduring this contract period.

A

5

INTRODUCTION

Protozoan parasites of the genus Leishmania are widespreadthroughout the world where they cause a complex of visceral orcutaneous diseases in human beings as well as some animalsincluding dogs in numerous tropical and sub-tropical countries(1,2,3). Since the leishmaniases commonly exist as zoonoses,these diseases pose a significant potential threat to militarypersonnel as well as military dogs throughout endemic areas.Recent publicity regarding infection of personnel involved inOperation Desert Storm has reemphasized the military significanceof the leishmaniases.

Better drugs are needed for the treatment of theleishmaniases since those currently available are often notsatisfactorily effective and are potentially toxic to man andanimals.

This laboratory has been involved for several years instudies to identify new compounds for antileishmanial activityagainst both visceral (Leishmania donovani) and cutaneous(Lebs-Qnia, braziliensis panamensis) leishmaniasis. Among themost promising active compounds found against visceralleishmaniasis durinq these studies is the 8-aminoquinoline,WR06026. This compound is now undergoing clinical trials inKenyan visceral leishmaniasis patients. Screening for compoundsactive against visceral leishmaniasis has continued during thisyear ix: the event that WR06026 does not perform in the field asexpected.

Since 1980 a total of 736 compounds has been tested foractivity against cutaneous leishmaniasis. Most of thesecompounds were selected on the basis of previous activity againstvisceral leishmaniasis although a few were selected specificallyfor testing against cutaneous leishmaniasis. Generally,compounds active against visceral leishmaniasis proved to beeither inactive or much less active against cutaneousleishmaniasis. The 8-aminoquinoline, WR06026, mentioned in thepreceding paragraph is the most potent compound found thus faragainst visceral leishmaniasis but this compound has given highlyvariable results when studied against cutaneous leishmariasis andwhen active, is much less active against cutaneous leishmaniasisin hamsters than against visceral leishmaniasis in the same host.Based on available evidence, it appears possible that WR06026 maybe effective in human beings for visceral but not for cutaneousleishmaniasis. In order to ascertain the possibility of therebeing other promising compounds with possible efficacy againstcutaneous infection, a search was made of the entire cutaneousdata base to identify compounds that had been equally or morepotent against cutaneous leishmaniasis than the refference drug,Glucantime. A total of 37 compounds were identified, of which 32

6

were 6-aminoquinolines. These compounds were then studied indetail against both visceral and cutaneous leishmaniasis duringthis year to determine the most promising drug against cutaneousinfections. Since 8-aminoquinolines such as WR06026 are morepotent against visceral leishmaniasis when administered orally,it was decided to test compounds of this class both orally andintramuscularly. The remaining compounds were tested by eitherthe oral or intramuscular route.

This repot tf ummarizes the results of studies conducted forthis contract during the period September 28, 1991 - September27, 1992.

7

MATERIALS AND METHODS

A. Primary Visceral Test System

A Khartoum strain of L. doncvani (WR378) was used and thegolden hamster (Me.;ocricetus auratus), 50-70 gm, served as thehost animal. Suspensions of amastigotes for infection ofexperimental hamsters were prepared by grinding heavily infectedhamster spleens in sterile saline in a Ten Broeck tissue grinderand diluting the suspensions so that 0.2 ml containedapproximately 10 X 106 amastigotes. Each experimental hamsterwas infected via the intracardiac injection of 0.2 ml of theamastigote suspension.

The testing procmdure used was that described by Stauberand his associates (4,5,6) as modified by Hanson et al. (7). Onday 3 following infection, hamsters were divided randoml¾ intoexperimental groups consisting of a minimum of 6 animals pergroup, initial group weights were obtained, and administration oftest compounds was initiated. Each compound was tested at 2 or 3drug dosage levels dependent on the priority rating and nature ofthe compound.

The vehicle for the test compounds was 0.5% hydroxyethyl-cellulose-0.1% Tween 80 (HEC-Tween). Each test group contained

6 hamsters and received one of the desired drug dosage levels. Acontrol group of 6 hamsters received the 0.5% HEC-Tween vehicleonly and the reference compound, Glucantime was given at 3 drugdosage levels (208, 52, and 26 total mg/kg) based on antimonycontent. All test compounds were administered routinely twicedaily via the intramuscular route on days 3 through 6. Finalgroup weights were obtained on all experimental hamsters on day 7and all animals were killed, livers removed, weighed, and liverimpressions made for enumeration of amastigotes. Subsequently,the total number of parasites per liver was determined asdescribed by Stauber, et al. (4,5,6).

In addition to recording body weight changes as a generalindicator of toxicity of the test compounds experimental hamsterswere observed for such clinical signs of toxicity as nervousdisorders, roughened hair coat, and sluggish activity. Deaths ofthe animals was also considered indicative of significant drugtoxicity.

After determining the ratio of numbers of amastigotes perhost cell nucleus the weight of the organ, and initial and finalweights of the hamsters, the raw data was evaluated with an IBMPC XT microcomputer using a program which calculates percentweight change, total numbers of parasite3, mean numbers ofparasites per organ, and percent parasite suppression. Thecomputer program then performs linear and non-linear regressionanalysis and calculates a SD5 0 for each active compound from each

$

of the analyses (drug donage resulting in 50% suppression ofamastigotes). The SD5 o from the non-linear analysis is used fora comparison of the relative efficacy of the test compounds andthe efficacy Z test compounds relative to that of the referencecompound, Glu,:antime. The linear regression analysis is includedonly for comparison with the non-linear analysis.

B. Primary Cutaneous Test System

LeiDJaia brAzitliensis panfm_•fjj (WR539) was used inthese studies. Mole golden hamsters, 50-70 gm served asexperimental hosts.

Promastigotes for eittablishing experimental infections inhamsters were grown in Schneider's Drosophila Medium (Hendricks,et al., 8) and quantitated using procedures described previously(Hanson and Roberson, 9). 'n preparation for infection andweekly during the experiment, the hair was clipped on the dorsaltail head and a commercial depilatory agent applied to the areato remove the remaining hair. Each hamster was inoculated viathe intradermal route with approximately 1.5 X 107 promastigotesof L., brazilieii yangnenais near the base of the tail using a0.25 ml glass syringe equipped with a 30 gauge X 1/2" needle.Each experimental group consisted of six hamsters. Initial bodyweights were obtained and administration of therapy, generallyvia the intramuscular route, was initiated on day 19postinfection, and continued through day 22 postinfection.Glucantime was included at two dosage levels (832 and 208 totalmg Sb/kg) as the reference compound and a group of six hamstersreceived vehicle only (HEC-Tween). Test compounds wereadministered generally at 416 and 208 total mg/kg.

Lesion area of each experimental hamster was determinedwith the aid of a template made at WRAIR and calibrated accordingto the formula r 1 r 2 v where r, is the major radius of the lesionand r 2 is the minor radius (Wilson et al., .0). The mean lesionarea of each experimental group was obtained and the percentsuppression of lesion size calculated by comparing the meanlesion area of each treated group with that of the groupreceiving vehicle only with the aid of a computer program and anIBM PC XT microcomputer. The computer program performs linearand non-linear regression analysis and calculates an SD 5Q foreach active compound using both analyses. The SD 50 obtained fromthe non-linear analyses is used for a rough comparison of therelative efficacies of the test compounds and the relativeefficacy of test compounds with that of the reference compound,Glucantime. The li:;ear regression analysis is performed forcomparison with the non-linear analysis.

9

C.. Comparative Antileishmanial Activity of Selected Cowptu:LisAgainst L. donovani and L. braziliensis panamensis.

The most active compounds in the primary cu t÷ .aous trstsystem were selected for these studies from the da'.a ba-,e.Thirty-seven compounds were tested simultaneously in tlZe )-tmarycutaneous and primary visceral test systems using tie r •c..duresdescribed above in sections A and B. Since most of ti.t "-tivecompounds were 8-aminoquinolines, two routes of admini.'.,+ ation(i.e. intramuscular and oral) were used against each ,. asite.Dosage levels for the cutaneous test system were gener" ily higherthan that used for the visceral test system due to the fact thatthe reference compound Glucantime requires approximately a fourfold higher dosage level in the cutaneous system than in thevisceral test system for activity.

D. In Vitro Studies of Oligonucleotides Against L. donovani

Promastigotes of L. donovani were cultured from an infectedhamster spleen in Schneider's Drosophila Medium (Hendricks, etal., 8) and quantitated using procedures described previously(Hanson and Roberson, 9). Promastigotes from four-day cultures(fourth to twelfth subpassage) were used in this work.(Unpublished data indicates that this age culture is the best forestablishing infections in hamsters.)

Cultures were harvested by centrifugation and resultingpellets were resuspended in Schneider's Drosophila Medium to afinal concentration of 6.5 X 106 per ml. Using round bottommicrotiter plates (Dynatech), 200 Al of the parasite suspensionwas added to each well and plates incubated at 26"C (Day 0).

Approximately 24 hours later, the oligonucleotides wereadded to appropriate wells at 30 micromolar concentrations (Day1). Sets of four cultures were used for each as well as foruntreated controls. Cultures were again incubated until Day 4 atwhich time total numbers of promastigotes/ml for each well weredetermined using the procedures described by Hanson and Roberson(9).

Mean numbers of parasites per well for each treated welland for untreated wells were calculated. Percent suppression orinhibition of parasite growth was determined using the followingformula:

Percent Suppression = mean number of parasites for theuntreated controls minus the mean number of parasitesfor the test compound divided by the mean number ofparasites for the untreated control times 100.

10

Negative percent suppression indicated enhanced qrowth ofpaiksites in the treated wel1l as compared to growth in theuntreated wells.

11

RESULTS

A. Primary Visceral Test System

During this reporting period a total of 41 compounds werestudied for efficacy against Leishmania donovani infections inhamsters. One compound was administered via three differentroutes at three different dosage levels for each route, twocompounds were administered via two routes at three dosage levelsper route, one compound was administered via a single route atthree dosage levels, and all others were administered via oneroute at two dosage levels (Table I). Only one (ZP10397) of thecompounds studied in this system was considered active (greaterthan 50% parasite suppression) and this compound was toxic at thehighest dosag.- level studied. Three of the inactive compoundswere also toxic in hamsters.

B. Primary Cutaneous Test System

One compound (BM10620) was studied for efficacy against L.k. nanamensis in the primary cutaneous test system. Thiscompound was administered via the intramuscular, subcutaneous,and oral routes at three dosage levels for each route (Table II).Although this compound was toxic when administered via theintramuscular route, some suppression of parasite inducedcutaneous lesions was noted. This compound was neither activenor toxic when administered via the other routes.

C. Comparative Antileishmanial Activity of Selected CompoundsAgainst L. donovani and L_. braziliensis Danamensis

A group of compounds (Figure 1) which were selected fromthe cutaneous test system data base because they had been foundto have antileishmanial activity equal to or greater than thereference compound, Glucantime, were studied simultaneously viathe oral and intramuscular routes for efficacy against both Lsionovani and L-. b. panamensis for comparative purposes as well asto determine the compound most active against L._. & panamensis asindicated in Table III.

It was noted from the results obtained that, with twoexceptions (WR049577 and WR027794), those compounds that wereactive at all against L. b. panamensis were considerably moreactive against L. donovani. For example, four 8-aminoquinolinecompounds (WR211789, WR211666, WR223658, WR223756) were 99-100%suppressive against L. donovani at the lowest dosage levelstested (either 6.5 or 13 mg/kg) when administered either orallyor via the intramuscular route. Additional studies (Table IV)were done on these compounds to determine the SD50 forcomparative purposes. In contrast, only WR211789 and WR223658

4

12

were active against L. k.. n h and the SDs'a of each ofthese compounds were in excess of 100 aq/kg against thisparasite. All of these compounds except WR211789 showed evidenceof toxicity to hamsters when administered at dosage levels of 104or 208 mg/kg against p b n mnsils.

Ten of the 37 compounds studied in these experiments weresufficiently active against L._ b_, pnmeMlja to be of interest.Eight of these were 8-aminoquinoline compounds. Among these,WRO06007 with an SD5 q of 79.8 mg/kg was approximately seven timeslese efficacious against " kU p.an.mensi than against L.

flnvan when administered via the intramuscular route. Similarcomparative studies using the oral route of administration couldnot be done because of the insufficient quantity of this compoundavailable. WR027794 was approximately 3-4 times less potentagainst Lp kn nAmen.i than L, donoYnL and this compoundappeared to be equally effective when administered via the oralor intramuscular routes. Similarly the efficacy of WR027779 wasapproximately two fold more active against L_, dQnoXani and thiscompound was approximately equally active when administeredorally or intramuscularly. The difference in potency of WR027780against L.,. D_ namensis and L. donovai was likewiseapproximately two fold but this compound was about twice asactive when administered via the intramuscular route than via theoral route. The efficacy of both WR006877 and WR006021 was twoto three times greater against L. donovani than against L,. D.

namnis.. Although the activity of these compounds against L.Qflo-val was similar when administered either orally or

intramuscularly, these compounds were active against L, b.2anamensi only when administered via the intramuscular route.WR006881 (SD50 -77.7 mg/kg) was only slightly less potent againstL. a. mnsis than L, donovai.

The most active compound against L. b_ panani.D was the8-aminoquinoline, WR049577 (SD 50 -3.76 mg/kg). Although thiscompound was the most potent compound studied against L, D,panamensis, it was not active when administered orally and istoxic (causing weight loss in recipient hamsters) at dosagelevels as low as 26 mg/kg while suppressing lesion size by only76% at this same dosage.

Regarding the two active compounds that were not 8-aminoquinolines, one (WR122536) is a phosphonium compound whichhad an SD50 of 58 mg/kg when administered via the intramuscularroute. Unfortunately, this compound was toxic (caused weightloss in recipient hamsters) at 104 mg/kg.

The other active compound that was not an 8-aminoquinolinewas Sinefungin (WR254847). This compound has been testedpreviously in this laboratory and found to be active against bothL.. D Panamensis and L. donovani in hamsters (see Final Report,Contract No. DAMDI7-85-C-5012, October 31, 1990). The difference

13

in the activity of this compound against L. b. panamensis and L.dIonovani was greater in the current experiments than in initialstudies.

D. In Vitro Studies of Oligonucleotides Against L. donovani

Table V summarizes the results of the in vitro testing of31 selected oligonucleotides for inhibition of growth ofpromastigotes of L. donovani. One oligonucleotide (LE001.01J910806) appeared to suppress the multiplication of L. donovani intwo separate studies (91.4% and 53.0% inhibition). Due to thedifferences in percent suppression obtained in the twoexperiments, a confirmatory experiment would be desirable beforedrawing a final conclusion on the activity of this compound.

14

DISCUSSION

The main area of emphasis during this contract period wasto determine the compound with the best efficacy againstcutaneous leishmaniasis caused by L., t. anamensis. To this endcomputer analysis of the results from the testing ofapproximately 736 compounds screened since 1980 was done and as aresult 37 compounds were selected from the data base as havingefficacy against cutaneous leishmaniasis in the hamster which wasequal to or greater than that of the standard reference compound,Glucantime.

These compounds were then studied simultaneously in detailfor activity against L. D. Panamensis as well as L. donovanli.The latter parasite was included for comparative purposes. It isinteresting that of the ten compounds identified by these studiesas the most active against L b panamensis, all but two were 8-aminoquinolines.

Studies in this laboratory have historically shown that the8-aminoquinolines are the most active compounds against both L.donovani and L. b. panamensis in hamsters. In addition, asverified in these current studies, the 8-aminoquinolines havealmost always been more active against L. donovani than againstLu. b. Panamensis. The same is true for the reference compound,Glucantime.

The reasons for the higher efficacy of the 8-aminoquinolines against L. donovani are unknown but it may be dueto the fact that liver parasites are more accessible to theparent compounds and their metabolites since this class ofcompounds are metabolized in the liver. Apparently, lesscompound and/or metabolites is distributed to sites distant tothe liver, a hypothesis supported by observations in thislaboratory of less activity of these compounds against spleenicparasites than liver parasites in L. donovani infections inhamsters (see Final Report, Contract No. DAMDI7-85-C-5012,October 31, 1990).

This suggested problem of bioavailability appears to be anespecially important one in cutaneous leishmaniasis. It ispossible that this question could be addressed by regimenvariation or possibly application of the drug directly onto thelesion.

Several metabolites of the 8-aminoquinoline, WR06026, themost active drug against L. donovani in hamsters, have been foundto be active against L. donovani. It is suggested that theseshould be tested against L. b. panamensis. In addition moredefinitive studies of Sinefungin and some of its analogs, whenavailable, should be tested against L. b. panamensis.

15

Antisense RNA's have been exploited with varying success toblock the activity of specific genes to inhibit the replicationof viruses as well as various human cancer cells (11, 12)ý. Dr.R. Meyer, Microprobe, Inc., under a separate contract (DAMDI7-88-C-6201) developed the idea to apply this technology againstLeiJ31i and has synthesized a number of antisense as well assense oligonucleotides for possible inhibition of the growth ofLeishmania. A number of these preparations were studied duringthe past contract period (see Annual Report, Contract No. DAMD17-90-C-0131, October 27, 1991) and these studies have continuedduring this contract period. These oligonucleotides were suppliedto our laboratory for testing. Thus far this approach has notappeared to be especially promising although some suggestion ofinhibition of growth of Leishmania donovani in vitro wasobserved. One possible explanation for the lack of inhibitionobserved in these experiments is the fact that it is sometimesdifficult to get the oligonucleotides into cells at the righttime to block messenger RNA activities (11). Work in this areais continuing. (See report by Dr. Meyer for additional details.)

4 b

16

CONCLUSIONS

1. Since 8-aminoquinolines remain the most active compoundsscreened to date against both visceral and cutaneousleishmaniasis, it would be advisable to continue to study thisclass and especially the metabolites for activity against bothvisceral and cutaneous leishmaniasis.

2. A more definitive testing of Sinefungin and some of itsanalogs when available should be conducted.

3. Promising novel compounds as indicated by published in vitroantileishmanial studies or by reports of activity against otherorganisms should be screened for antileishmanial activity invlvo.

4. Any deoxyoligonucleotides that show in vitro activity shouldbe tested for in vivo antileishmanial activity.

5. Continued in vitro testing of selected oligonucleotides wouldbe useful.

17/

LITERATURE CITED

1. Kinnamon, K. E., E. A. Steck, P. S. Louzeaux, L. D.Hendricks, V. B. Waits, W. L. Chapman, Jr., and W. L.Hanson. 1979. Leishmaniasis: Military significance andnew hope for treatment. MiU. Med. 44(10): 660-664.

2. Tropical Disease Research, Seventh Programme Report, 1January 1983 - 31 December 1984. UNDP/World Bank/WHOImprimerie A. Barthelemy, Avignon, France 1985. Pages 7/3 -7/18.

3. Chapman, W. L., Jr. and W. L. Hanson. 1984. LeishmaniasisIn "Clinical Microbiology and Infectious Diseases of the Dogand Cat." W. B. Saunders Company, Philadelphia, pp. 764-770.

4. Stauber, L. A., E. M. Franchino, and J. Grun. 1958. Aneight-day method for screening compounds against Leishmaniadonovani in the golden hamster. J. Protozool. 1: 269-273.

5. Stauber, L. A. 1958. Host resistance to the Khartoumstrain of Leishmania donovani. The Rice Institute PamphletVoQl. XLV(1): 80-96.

6. Stauber, L. A. 1958. Chemotherapy of experimentalleishmaniasis. Proc. 6th international Concr. on Tror. Med.j "a . ZU: 797-805.

7. Hanson, W. L., W. L. Chapman, Jr., and K. E. Kinnamon.1977. Testing of drugs for antileishmanial activity ingolden hamsters infected with Leishmania donovani.Internat'l. J. Parasitol. 7: 443-447.

8. Hendricks, L. D., D. Wood, and M. Hajduk. 1978.Hemoflagellates: Commercially available liquid media forrapid cultivation. Parasitol. 76: 309-316.

9. Hanson, W. L. and E. L. Roberson. 1974. Density ofparasites in various organs and the relation to number oftrypomastigotes in the blood during acute infections ofTrynanosoma cruzi in mice. J. Protozool. j1: 512-517.

10. Wilson, H. R., B. W. Dieckmann, and G. E. Childs. 1979.Leishmania braziliensis and Leishmania mexicana:r•xperimental cutaneous infections in golden hamsters.Exptl. Parasitol. 47: 270-283.

18

11. Moffat, A. S. 1991. Making sense of antisence. Science253: 510-511.

12. Szczylik, C., T. Skorski, N. C. Nicolaides, L. Manzella, L.Malaguarnera, D. Venturelli, A. M. Gewirtz, and B.Calabretta. 1991. Selective inhibition of leukemia cellproliferation by BCR-ABL antisense oligodeoxynucleoties.Science : 562-565.

19

APPENDIX 1

20

Table I. Summary of compounds studied for suppressive activityagainst jeisflji donovani in the primary visceral testsystem.

Bottle # Route Dosel Supgresl Dose2 Suppres2 Dose3 SuPpres3

BM10620 IM 104* 38 52 18 13 29PO 104 -6 52 22 13 17SQ 104 19 52 27 13 18

BM10371 IM 208 25 52 37 ND NDBL21100 IM 208 17 52 14 ND NDBL59588 IM 208 -44 52 -42 ND NDBL56390 IM 208 -18 52 -34 13 -12BL34170 IM 208 -5 52 2 ND NDBL29759 IM 208 25 52 24 ND NDAX26839 IM 208* -3 52 19 ND NDAY97173 IM 208 36 52 33 ND NDAY97315 IM 208 10 52 35 ND NDAH90393 IM 208 16 52 33 ND NDAG66089 IM 208 12 52 12 ND NDAG50330 IM 208 -1 52 0 ND NDAD60466 IM 208 3 52 8 ND NDBM12991 IM 208 -14 52 -7 ND NDAR81714 IM 208 40 52 23 ND NDAP64866 IM 208 20 52 12 ND NDAN35100 IM 208 6 52 36 ND NDAN15359 IM 208 9 52 19 ND NDBM12508 IM 52 11 ND ND ND ND

PO 208 0 52 -6 13 -3BM12491 IM 52 -13 ND ND ND ND

PO 208 -10 52 7 13 1AE95204 IM 208 22 52 -14 ND NDZP10397 IM 208 D 52 51 ND NDAR94417 IM 208 28 52 36 ND NDZC07760 IM 208 -4 52 3 ND NDZC07751 IM 208 0 52 -15 ND NDZA01419 IM 208 15 52 -20 ND NDBL86558 IM 208 -9 52 -19 ND NDAS64898 IM 208 -17 52 -21 ND NDAQ07393 IM 208 28 52 7 ND NDAN39528 IM 208 35 52 13 ND NDAM04315 IM 208 -11 52 -12 ND NDAJ91813 IM 208 4 52 -19 ND NDAR02802 IM 208 -3 52 -10 ND NDAP86979 IM 208 -4 52 -7 ND NDZG81239 IM 208 16 52 1 ND NDAL02996 IM 208 13 52 22 ND NDAH69718 IM 208 -23 52 8 ND NDAG53859 IM 208 14 52 -12 ND NDAG53840 IM 208 25 52 16 ND NDAG53831 IM 208 22 52 27 ND ND

See following page for footnotes.

Table I. (continued)

• Toxic as indicated by death of hamsters and/or 15% or greaterloss of body weight

D: All hamsters receiving this drug dosage level diedIM: Intramuscular route of drug administrationPO: Ora± route of drug administrationSQ: Subcutaneous route of drug administrationND: Not done

22

Table II. Summary of compounds studied for suppressive activityagainst Leishmania braziliensis panamensis in theprimary cutaneous test system.

Bottle P Route Dosel Suppresl Dose2 Suppres2 Dose3 SupDres

BM10620 IM 104' 73 52 51 13 7PO 104 11 52 -7 13 -7SQ 104 15 52 -11 13 -59

* Toxic as indicated by death of hamsters and/or 15% or greaterloss of body weight

IM: Intramuscular route of drug administradionPO: Oral route of drug administrationSQ: Subcutaneous route of drug administration

23

Table III. Summary of results obtained from studies on thecomparative activity of selected compounds against bothLeishmania donovani and Leishmania braziliensispanamensis.

40 to0

co 40 44 v(

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COW) 0 0 %a) V)04 C4 IN *ni-m- -- - -

4N6n 0 0 0 0W co kaI w0 n I

C 4 0 Ný 1? 0 0 N 1> ('4 v0 (0 40 - 0) 4

o 40 0 4 0 0 4 N N ( C)IO ) C

N(o . O0.1 W)N 4l 40I)n .

-ý - - -! !0 . . * 0 .

NO N (U)~ - 0 4

CL0 . - ~oa O ~ . ( N CL-0G1 E - E L0 E 040.4 g, 0 g a -'L a 004 a

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to In0toCl nI

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25

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(CN - CIA - C4 W C'J

N 00 (D* C%-C 0) N-- CM a-.- - OC0V r o 0-

C~l CY 0c CO l 0' CDO

'0n ( 'L-- to Cl) 0 D W~~ C ) La LO

- La Nn NO C ) ~ J ~ C 'CC ~ Cl. C L)

0N~ .- Z-Z -ZCOZ c'l -~ CO

"0~~~~ Z3 V "a" 1 0 .-j II -J g i

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27

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V( OC) C J w~ Nl wO ' 1 N 10 N cii 0 C) 1

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.g0 10 r=0 E0E0 ]E0E2)9g E0

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: oj CL j .j . .i . j . j 1iL

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aCC)Q0 CDcc1 0:c C rTc

28

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Cq

c~@ 0 ) cQQU cc Nt C?

N N) C.) N. (D N-N

toU 0 U) 0W) cm 0 0

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04 to

C~0

29

Table IV. Summary of results of additional testing of compoundsfound highly suppressive against Leishmania donovani.

Bottle I Route Dosel Sumpresl Dose2 SuDPres2 Dose3 Suppres3

BK50713 IM 3.25 97 0.8 -6 0.4 -23PO 3.25 94 0.8 6 0.4 -29

BG11417 IM 3.25 99 0.8 59 0.4 29PO 3.25 99 0.8 68 0.4 28

BG21744 IM 3.25 100 0.8 12 0.4 -31PO 3.25 99 0.8 25 0.4 -5

BG22125 IM 3.25 98 0.8 42 0.4 -16PO 3.25 94 0.8 31 0.4 6

IM: Intramuscular route of drug administrationPO: Oral route of drug administration

30

Table V. Summary of results of selected oligonucleotides testedfor in vitro inhibition of promastigotes of Leishmaniadonovani.

Compound Percent Suppression

LE502 910410 34.4LE002.01 J 910415 20.8

LE002.02 J 910415 25.5

LE002.03 J 910416 22.2

LE002.04 J 910416 26.6

LE002.05 J 910716 26.3

LE002.06 J 910716 21.6

LE002.07 J 910716 26.8

LE001.01 J 910806 91.4*

LE001.02 J 910806 14.3

LE001.04 J 910808 47.0

HBV040.01 J 910806 38.8LEOO1QX -76-9*"

LE002QX -222.5

LE001SX -179.2

LE002SX -10.7

LE001.01H 17.4

LE002.01H -14.5

LEOO1.01Q -34.9LE002.01Q -32.0

LE001.01S -27.5LE002.01S 7.3LE001HX 8.3

LE002HX -75.2

LE001HY 32.8

LE002HY 27.2

LE001QY 16.0LE002QY -71.6

LE001SY -2.4

LE001.01J 27.2

LE501.01J 53.0

* Based on triplicate cultures** Negative percent suppression indicates enhancement of parasite

numbers

31

Figure 1. Structures of the most active compounds found in theprimary cutaneous test system and tested simultaneouslyin the primary visceral test system during this contractperiod.

M-7i I

32

NH 2

WR057023Co60NHCH2CH2CH2OH

CH30-WR053215

CH3

NHCH(CHr2)3NHCH(CH 3)2

WR223658C H3 0

CH3

NH(CH 2)6NHC 2H5

Cm (N~WR21I S9

CH3

33

NH(CH2)5NHCH(CH3)2

CH3 o 6D WR006877

CH30

NH(CH2)5NHCHC 2H5 WR027779

CH3 N-C66

NHCH(CH 3)CH2(CH2)2NHCH(CH3)2

CH30-o 6 WR006020

NHCH2CH2NCH2[(CH3)2CHCH2J2

CH3O-& 6 WR007296

34

NH(CH 2)5NHCHCH3

CH Dc CH3H 3)2D WR027796

NH(CH2)5NHCH (CH3)2

OH OO6 WR006881I

CH3 - o 6 WR027742

NH(CH2)3NHCH2C(CH3)3

CH30 o 6 WR027785

C2H5

NHi(CH 2)2NtHCH

CH06 ) C2H5WR027788

O-CH-----CH-M-

WR052252

C -9[N #9WR006561I

NII(CH2)5NHCH(CH 3)C3F1 7

CH30-O WRO27780

O(CH2)5CH3

CH3CH3O

0 2- CH3 WR2544 19

NHCH(CH 2)3NH 2

CH3

36

CAH

N1{(CH2) 2NkHCH ý

N CH3

CH3O0-6 0 WR027792

NH(CH 2)5NHCH 2CH(CH3)2

CH34 6 WR027793

NH(CH 2)5NH(CH2)4CH3

N

CH3O0-b 0 WR027794

NH(CH 2)5N(C2H5)2

CH3 oK W iR223756

CH3

37

CH30

WRO6 1250

N-HCH(CH 3 )CH2CH2 CH2 NH-( 1-

N11(CH2)NH(CH 2)3CH3

CH30-6 6DWRO06023

NE(CH2)6NHiCH(CH 3)2

CH3 00 &W*R21 1666

CH3

CF3

0

OCH3 WR249668

CH30 3

-#> CH3

NCH(CH3)CH 2CH2 CH2NH 2

~~~17 7~

38

NH(CH 2)SNHCH(CH 3)2

CH3 o o WRO0602 1

CH3

Nl-CH(CH2)3NHCH(CH 3)2

CH30- 6WR006027

CH3

NHCH 2CHCH-r2 'N(Ct2H5)2

N WROO7511

CH3 o6DCH3 0

NII(CH2)3N(CH 2CH3)2

CH300CH3# 6WR0069 17

CH30

39NH(CH2)6N(CH 2CH3)2

CH3 J o6D WRO06007

NH(CH 2)5NHCH(CH 3)2

OH

Br=C2 Ž WR099029

cif WR122536

40

NHCH2CH2NHCH 2CH3

CH30 o. 6 WR007561

NH(CH2)5NHCH(CH 3)2

666 WR027795

NH(CH2)6NH2

NH

H2N N 0 NH2

WR254847

OHOH HO

41

APPENDIX 2

42

PERSONNEL EMPLOYED FROM THIS CONTRACT DURING THIS REPORT PERIOD

Name and Position Percent Effort Length of Employment

Barbara Harris 100% 9/23/91 - 12/23/91Laboratory Technician II 1/9/92 - Present

Laura Lamb 16% 7/1/92 - 9/30/92Graduate Assistant

Shannon Waits 15% 9/28/91 - 6/5/92Student Laboratory Technician

Virginia Waits 100% 9/28/91 - PresentResearch Coordinator II

BIBLIOGRAPHY OF PUBLISHED WORK

None

GRADUATE DEGREES RESULTING FROM THIS CONTRACT

None