t /AD AObZ 300 MIAMI LillY Fl- A L~ O RANt RESEARCH LAB F/S 6/15CICUOTHERAPY OF ‘TRYPANOIOMA RHODESIENSE ’,flJ)UP 75 0 S RAIt DAOAI7—72—C—21A42UNCLASSIFIED NI. p,I~I

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REPORT NUMBER THREE

CHEMOTHERAPY OF TRYPANOSOMA RHODES IENSE

FINAL REPORT

DORA S. RANE

For the period of June 1 , ~974 to Septembe r 30, 1975

Suppor ted by

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U. S. ARMY MEDICAL RESEARCH AND DEVELOPMENT COMMANDWashington , D. C. 20314

~~~ C: Contract No. DADA 17-72-C-2142

Dr. Leo Rane Research LaboratoryUn i versity of Miam i

Miami , Florida 33142

DDC AVA ILABILITY STATEMENT

Approved for public release ; distribution unli mited .

D D C

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1978

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Rept. no. 3 (Final ), 1 Jun Th-30 Sep 7~j• SECURITY CLASS1F1CA flO N OF THIS PAGE (W1~.,e bat. Es,t.r.D~~ E I ~~ 1 r~(1,’IIueIJ’rA’rInu D A I ~~~ READ INSTRUCTIONSI~~L %JI% I U’J’~.UMLI~ I ~~ I I’..PI~ ~ BEFORE COMPLETING FORM

I. REPORT NUMBER 2. GOVT ACCESSION NO 3. RECIPIENT’S CATALOG NUMBER

Three ____________________________4. TITLE (ond Subllti.) 5. TYPE O~F ~EPO~~T’* -PE~ IOD COVEREDr - Fi nal Report

~~~ j CHEMOTHERAPY OF TRYPAN 0S 0MA~~~0D E SI EN S E , / June 1 , 1974 - Sept. 30, 15 56. PERFORMING ORG. REPORT NUMBER

1. AUTHOR(.) —-. S. CONTRACT OR GRANT NUMSER(.)

(~ f~ora 5./Rane] ~~~~ ~~~DADAJ7~ 72~ C_2i42/ .

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• 6. PERFORMING ORGANIZATION NAM E AND ADDRESS 10. PROGRAM ELEMENT, PROJ ECT, TASKAREA & WORK UNIT NUMBERSDr. Leo Rane Research Laboratory

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Un i versi ty of MiamiM i am i , FL 33142 ~~~~~~~~~ 3~~ ~~~Cj~ 75 f

II. CONTROLLING OFFICE NAM E AND ADDRESS rg~ NLPORT dATEU.S. Army Med i cal Research and September 30, 1975

Development Comand 13. NLJMB ERO F PAGESWashing ton , D. C. 20314 6

14. M O N I T O R I N G AGENCY N A M E & AODRESS(U d if f d r wt from Controlling Of f i c e ) 15. SEC RITY CLASS. (of tAle report)

IS.. D AJFI~~ATION/DOWNGRADINGSCHEDULE

~S. DISTRIBUTION STATEMENT (a! Ala Ripe n )

Approved for public release; distribut i on Unlimited .

Il . DISTRIBUTI ON STATEMENT (of A. .b.tnact entered in Block 20, if different from Report)

IS. SUPPLEMENTARY NOTES

IS. KEY WORDS (C.nWi.a. on r v ~~e aid. if nec..a~~ end identify by block number)

I. rhodeslense blood-induced infection in mice - based on mortality.

2$ ABSTRACT ~~~~~~~~~~~ — . v.u.e ~~~~~~ =..~~~~~~~~ ~~~ ~~~~~~~ by block numb.,)

Primary screen - quantitative evaluation of poten t i a l t rypanosom icida lactivity.

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SECURITY CL. *367 FICAIION OF ThtS PAGE (IStan Date Ens e r d )

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Foreword

In conducting the research described in this report ,the investigator(s) adhered to the ~Guide forLaboratory Anima l Facili ties and Car& , aspromulgated by the Committee on the Guide for LaboratoryAni mal Resources , National Academy of Sciences-NationalResearch Council.

DX Butt Sedion ~JUNANWOUMCED 0JUSTIFICA TION

ABILITY CODES01st. AVAIL and/or SPECIAL

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CONTINUAT I ON OF THE SCREEN I NG PROCEDURE FOR THE EVALUAT I ON OFTRYPANOSOMI C IDAL ACTIVITY OF CANDIDATE COMPOUNDS USIN G TRYPANOSOMA

RHODES IENSE INFECTI ONS IN M ICE

The test system described herein was developed specificall yto eval uate the trypanosomicida l activity of large numbers ofcandidate compounds.* Based on blood-induced Trypanosomarhodesiense infections in mice , it performs as a primary screenor as a secondary screen and confirmatory test and gives precisequantitative eval uations of chem i cal compounds that demonstratepotentiall y useful therapeutic and/or prophy lactic activity in 1•rhodesi ense infections. Consequently, it is also a helpfu l guide-line in the synthesis of new active agents.

These agents include: (1) chemicals structurall y related tocompounds of known va l ue in the treatment or prevention of T.rhodesi ense infections; (2) chem i cals structurall y unrelated to com-pounds of known va l ue in the treatment or prevention of I. rhodesienseinfections and; (3) structura l analogues of compounds that havedemonstrated activity in our screen i ng procedure and represent novelchemica l groups.

All candidate compounds were obtained from the Department ofMedicina l Chemistry at the Walter Reed Institute of Research.

Table I summarizes the number of compounds tested and the num-ber of mice used from August 1 , 1972, through September 30, 1 975.

Our own colony of ICR/HA Swiss mice provided all the testanimals needed in this operation . Using mice of a given age, sexand wei ght and a standard i nocu l um of the Wellcome CT— strain of I.rhodesiense, It has been possible to produce a consistently un i formdisease fatal to 100 percent of untreated animals within 4—6 days.

Test compounds were administered either parenterall y or orall yin a single dose on the day of Infection .

Activity was determined by responses to cand i date compounds byI. rhodesiense infections in mice as expressed in comparisons of themaximum survival time of the treated trypanosome-infected animals andthe surviva l time of the untreated trypanosome-Infected controls.To be classified as active , a compound must suppress the disease andproduce an increase of at least 100 percent In the life span of thetreated animals over that of the untreated controls.

* Designed , developed and operated by Dr. Leo Rane until 1973, thenoperated by Mrs . Dora Rane until 1976.

TABLE I

COMPOUNDS TESTED AND M i CE UT i LIZED

AUGUST 1 , 1972 - SEPTEMBER 30, 1 975

NUMBER OF NUMBER OFYEAR COMP OUND S M I CE

AUGUST 1 , 1972 - MAY 31 , 1973 3,030 51 ,405

JUNE 1 , 1973 — MAY 31 , 1974 1 ,581 25,360

JUNE 1 , 1974 — MAY 31 , 1975 1 ,826 33,850

JUNE 1 , 1975 - SEPT. 30, 1975 593 11 ,260

TOTAL 7,030 121 ,875

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Acceptance of a test compound ’s activity was also pred i cated onthe margin between the maximum tolerated dose (MTD) and the minimumdose producing a significant effect (MED). A max i mum tolerateddose is defined as the highest dose causing no more than one offive an i mals to die and a minimum effective dose as the minimumdose increasing the life span of treated an i mals 100~ over thelife span of untreated controls.

METHODS

An ima l Hosts. Our own breeding colony of ICR/HA Swiss micehas supplied all the an i mals used in this screen i ng procedure.Test an imals weigh 30-32 grams , weight variations in any givenexperimental or control group being carefully limited to 3 grams .In all tests an imals have been of the male sex and approximatelyof the same age.

Animals on test are housed in metal topped p lastic cages, feda standa rd l aboratory diet and g i ven water ad lib .

Once the mice have been given a drug they are kept in a roommaintained at 84° F (+ 2° F) and a relative humidity of 66~ (+ 2~).

Test Procedure. Test an i mals recei ve an intraperitonea l injectionof 0.5 cc of a 1:50,000 dilution of heparinized heart blood drawnfrom a donor mouse infected 3 days earlier.

The donor line is maintained by 3 day blood passes , each animalreceiving 0.1 cc of a 1:500 dilution of heparinized heart blooddrawn from a 3 day donor. Donors , like test an i mals , weigh 30-32grams , weight variations for each pass be i ng limited to 3 grams.

To check factors such as changes In the Infectivity of our 1.rhodesiense strain , or in the susceptibility of the host one groupof Infected untreated mice are included as negative controls. Inorder to determine the effect a drug exerts on a trypanosome Infectiontwo parameters are measured ; the first bei ng an increase in sur-vival time , and the second concerns curative action . For comparativepurposes standard compounds, stilbam idine lsethionate and 2hydroxystilbam idine Isethionate are administered at one leve l each(26.5 mgs/kg) to separate groups of 10 mice which serve as positivecontrols producing definite increases In surviva l time and curativeeffects. Another function of these two positive controls I nvolvesa check on whether three procedures are performed correctly; thedrug weighing , the preparation of drug solut i ons or suspensions andthe administration of drugs.

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Drug Administration. Test compounds are dissolved or suspendedin peanut oil and prepared in three or more graded doses. Atleast three different doses of each test compound are included inan experiment. Groups of 5 mice per dose level of drug are utilized .

Treatment consists of a single dose administered subcutaneousl yor orally on the day of infection . Deaths that occur before thefourth day , when untreated controls begin to die , are regarded asthe result of a compound’s toxic effect and not as the result ofaction by the infecting parasite.

Increases in the dose levels of highl y active compounds usuallyare followed by increases in the surviva l time of the treated mice .However, if an active drug is toxic for the host, the toxicity ofthis compound may become a limiting factor to changes in doselevels.

Treated animals alive at the end of 30 days are consideredas cured.

Drug Activity . An increase of 100* in surv i va l time is con-sidered the minimum significantl y effective response for a candidatecompound. Clearly i nactive compounds are rejected after one test ,borderline compounds after two tests.

Active compounds are subjected to a test to determine a doseresponse curve (6 or 9 different doses) so that the spread betweenthe maximum tolerated dose (MTD) and the minimum dose producinga significant effect (MED) may be established.

COMPOUNDS WITH DEFI N ITE CHEMOTHERAPEUT IC ACTIVITY AGAIN STTRYPANOSOMA RHODES I ENSE INFECT I ONS IN MICE

During the opening period of this project, June 1 , 1972 -May 31 , 1973, our screening procedure was deve l oped and itsreliability established . 3,030 selected compounds were screenedinclud ing a number of agents known to be effective in 1’.rhodesi ense infected mice .

1 ,581 compounds were tested in the period June 1 , 1 973 -May 31 , 1 974. Of these, 185 demonstrated a degree of activitysufficient to produce at lease 100 percent increases in thesurv i va l time of treated

~ rhodesiense infected mice , 92 were

active subcutaneous l y and 93 orally.

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1 ,826 compounds were tested in the period June 1 , 1 974 -May 31 , 1975. Of the 298 recognized as active compounds , 225 wereactive subcutaneousl y and 73 orall y.

593 compounds were tested in the period June 1 , 1 975 —September 30, 1975. Of the 123 recogni zed as act i ve compounds,109 were active subcutaneousl y and 14 orally.

This breakdown is si gnificant since : (I) activity eva l uationsprovided in our screening procedure are precise and quantitat i ve;(2) dose response curves of active compounds administered sub-cutaneously show a wider spread between the maximum tolerated dose(MTD) and the minimum dose producing a significant effect (MED) thandose response curves of active compounds administered orall y and;(3) these dose responses also reveal a wider spread of toxic effectswhen active compounds toxic for the host are administered sub-cutaneously rather than orall y.

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DISTR IBUTION LIST

if copies HQDA (SGRD-RP)WASH DC 20314

12 copies Defense Documentation Center (DDC)ATTN : DDC-TCACameron StationAlexandria , Virginia 2231k

1 copy SuperintendentAcademy of Health Sciences ,US ArmyATTN: AHS-COMFort Sam Houston , Texas 78234

1 copy DeanSchool of MedicineUn i formed Serv ices, Un ivers i ty ofthe Health SciencesOffice of the Secretary of Defense6917 Arlington RoadBethesda, MD 20014

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