Vol. 16, No. 4ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, OCt. 1979, p. 475-4850066-4804/79/10-0475/11$02.00/0

Antimalarial Properties of Floxacrine, a DihydroacridinedioneDerivativeL. H. SCHMIDT

The Kettering-Meyer Laboratory, Southern Research Institute, Birmingham, Alabama 35205

Received for publication 17 July 1979

Evaluations of the activities of floxacrine [7-chloro-10-hydroxy-3-(4-trifluoro-methylphenyl)-3,4-dihydroacridine-1,9(2H, 10H)-dione] in owl monkeys infectedwith trophozoites of a chloroquine-quinine-resistant strain of Plasmodium falci-parum, a strain of this plasmodium resistant to both of these quinolines andpyrnmethamine, or a strain of P. vivax resistant only to pyrimethamine showedthat: (i) this compound regularly effected temporary clearance of parasitemia atdaily doses of 1.25 to 2.5 mg/kg; (ii) doses required for the cure of establishedinfections were larger by factors of 6 to 64 than those that effected parasiteclearance; (iii) there was more than a 10-fold difference in doses required for thecure of infections with the different strains; and (iv) resistance to floxacrinedeveloped rapidly. Evaluations of the activities of floxacrine in rhesus monkeyschallenged with sporozoites of P. cynomolgi showed that: (i) 0.625-mg/kg dosesadministered daily throughout the incubation period provided complete protec-tion against infection; (ii) single 40.0-mg/kg doses delivered 2 h before sporozoitechallenge were without prophylactic activity; and (iii) daily doses of 40.0 mg/kg,the maximum tolerated level, productive of a hemorrhagic syndrome in somesubjects, would not cure established infections. These observations suggest thatthe potential contribution of floxacrine to malaria therapy would be limited tothe prophylactic area and for practical reasons would be restricted there by therequirement for daily dosage.

Floxacrine (Fig. 1) is one of a group of dihy-droacridinediones which have exhibited signifi-cant activity in various Plasmodium bergheimouse models. These compounds were origi-nally synthesized by W. Durckheimer and H.Seliger, Hoechst AG, Frankfurt-am-Main, intheir search for novel and effective antibacterialdrugs (German patent application no. 2337474).The results of the evaluations of the antimalarialproperties of floxacrine, pursued by W. Raether,Hoechst AG, and E. Fink, Institute of AppliedChemistry, University of Erlangen, Nurnberg,Germany, were brought to our attention in Au-gust 1974 (H. Wagner, Hoechst AG, personalcommunication). A summary report by Raetherand Fink was subsequently presented to theDeutschsprachiger Tropenmedizinischer Ge-sellschaften at its meeting, 24 to 26 March 1977,in Lindau, Bodensee, Germany. A full report isin press.

Investigations by Raether (W. Raether and E.Fink, Ann. Trop. Med. Parisitol.) in mice in-fected with trophozoites of various strains of P.berghei showed that: (i) dose for dose, the ca-pacity of floxacrine to cure infections with theparent drug-susceptible strain was from two tothree times that of chloroquine; (ii) essentially

o 011 U

N0 CF3OH

FIG. 1. Structure of floxacrine [7-chloro-10-hy-droxy- 3- (4- trifluoromethylphenyl) -3,4- dihydroacri-dine-1,9(2H, 10H)-dione].identical doses of this dihydroacridinedione de-rivative were required for the cure of infectionswith the drug-susceptible strain and strains re-sistadnt to chloroquine, pyrimethamine, and sul-fadoxine; (iii) floxacrine was approximatelytwice as active when administered subcutane-ously as when delivered orally; and (iv) irrespec-tive of route of administration, the compoundhad a favorable therapeutic index; i.e., the ratioof the 50% lethal dose to the dose required tocure 50% of infections (LD5o/CD5o) was in excessof 30. Studies by Fink showed that a single 1.0-mg/kg dose, administered intraperitoneally 24 hbefore intravenous challenge with sporozoites ofP. berghei yoelii, gave complete protection to 43of 46 mice. A study by Raether in three rhesusmonkeys infected with trophozoites ofP. cynom-

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476 SCHMIDT

olgi indicated that in this model the blood schi-zonticidal activity of floxacrine approximatedthat of chloroquine.The above observations suggested that flox-

acrine might have potentials both for treatmentof active infections with drug-susceptible anddrug-resistant strains of the human plasmodiaand for prophylaxis or radical cure of naturallyacquired P. vivax infections. Further explora-tions of these potentials were undertaken in ourlaboratories. The first was examined in owl mon-keys infected with trophozoites of a chloroquine-quinine-resistant, pyrimethamine-susceptiblestrain of P. falciparum, a multidrug-resistantstrain of this plasmodium, or a pyrimethamine-resistant, chloroquine-susceptible strain of P.vivax. The second was evaluated in rhesus mon-keys either challenged or actively infected withsporozoites of the B strain of P. cynomolgi. Thedimensions and results of these explorationshave been summarized in this report.

MATERIALS AND METHODSProcedures pertinent to evaluations of the ac-

tivities of floxacrine against infections with P.falciparum and P. vivax in owl monkeys. Thechloroquine-quinine -resistant, pyrimethamine -sus-ceptible Vietnam Oak Knoll and chloroquine-quinine-pyrimethamine-resistant Vietnam Smith strains of P.falciparum, and the chloroquine-quinine-susceptible,pyrimethamine-resistant Vietnam Palo Alto strain ofP. vivax were used in this study. The patient originsof these strains, the procedures employed in adaptingthem to growth in owl monkeys with intact spleensand maintaining them at constant virulence, thecourses of infections in untreated monkeys, and theresponses of standardized infections to treatment withchloroquine, quinine, and pyrimethamine have beendescribed elsewhere (5-7).A total of 44 owl monkeys (Aotus trivirgatus gris-

eimembra) imported directly from Barranquilla, Co-lombia, was used in the current experiments. Thisgroup included approximately equal numbers ofsubadult or adult males and females, ranging from 700to 1,040 g in weight at the time of assignment tospecific studies. The procedures followed in importingthese monkeys, adapting them to a caged environmentand laboratory diet, maintaining them in a healthystate, and handling them during conditioning and ex-perimental study periods were identical with thosedetailed previously (5, 6).Of the 44 monkeys, 25 with no previous malaria

experience were used to evaluate the activities offloxacrine against infections with the Oak Knoll andSmith strains of P. falciparum. This subgroup included22 treated subjects and 3 untreated controls. Theremaining 19 monkeys, all previously infected with theOak Knoll strain of P. falciparum and cured via treat-ment with a 4-quinolinemethanol or 4-pyridineme-thanol (11, 13), were used for studies with the PaloAlto strain of P. vivax. Fifteen of the 19 (14 treatedand 1 untreated control) were employed in the primary

ANTIMICROB. AGENTS CHEMOTHER.

assessment of the activity of floxacrine. Four (threetreated and one untreated) were used in determiningwhether resistance to this compound was responsiblefor treatment failures encountered in the primary eval-uation.The blood schizonticidal activities of floxacrine were

evaluated by procedures essentially identical to thosedetailed previously (8). In brief, infections were in-duced by intravenous inoculation of 5 x 106 erythro-cytic parasites derived from monkeys in the passagelines of the respective strains. Measurements of para-sitemia on thick and thin blood films, prepared fromthe marginal ear vein and stained with Giemsa, wereinitiated 3 days after inoculation and were repeateddaily thereafter until densities of 10 to 50 parasites per104 erythrocytes were attained (approximately 5,000to 25,000 parasites per mm3 of blood). Treatment withfloxacrine, in doses ranging from 0.156 to 40.0 mg/kgof body weight, was initiated at this time and contin-ued on a once-daily schedule for 7 consecutive days.The effects of treatment on parasitemia and parasitemorphology were assessed on Giemsa-stained thickand thin blood films. Such films were prepared justbefore administration of floxacrine on each of the 7treatment days and daily thereafter until thick filmswere parasite negative for at least 4 consecutive days.Preparation and examination of films were then re-duced to a twice-weekly schedule (either Monday andThursday or Tuesday and Friday) for 2 consecutiveweeks and, if results were uniformly negative duringthis interval, to a once-weekly schedule for 10 addi-tional weeks. Infections were considered cured andmonkeys were discarded for other uses if blood filmswere uniformly negative for 90 or more days posttreat-ment.

If parasitemias persisted at the initial or lower levelsor increased during the delivery of floxacrine, or ifparasites reappeared after an apparent blood-negativeinterval, a second treatment course was initiated, al-most invariably at a dose twofold to fourfold largerthan that administered during the initial course. Thisprocedure was repeated until the infection was curedor a dose level of 80.0 mg/kg had been reached withouteffecting cure. This retreatment practice not only ex-pands information that can be obtained via study ona single monkey, but as shown in this report and others(5, 8), may also signal the emergence of parasitesresistant to the compound under investigation.As detailed elsewhere (7), responses to treatment

were classified as: (i) none, when the course of theparasitemia in the treated subject was essentially thesame as in the untreated controls or fell within theboundaries of historical controls infected with thesame strain; (ii) suppressed, when parasitemia per-sisted throughout the treatment period, but was eitherless than at the start of treatment or no greater than1/50 the levels in untreated controls; (iii) clearancewith recrudescence, when thick blood films becameparasite negative during the latter days of treatmentor immediately thereafter and remained so for a mini-mum of 7 days before reestablishment of parasitemia;and (iv) cured, when thick films became parasite neg-ative during or immediately after treatment and re-mained so during a follow-up period of 90 days orlonger.

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ANTIMALARIAL PROPERTIES OF FLOXACRINE 477

Procedures pertinent to evaluations of theprophylactic and radical curative activities offloxacrine in rhesus monkeys inoculated withsporozoites of P. cynomolgi. The B strain of P.cynomolgi was used in these studies. Reference shouldbe made to earlier reports (10, 18-20) for detaileddescriptions of: (i) the origin of this strain and itsroutine maintenance in this laboratory; (ii) proceduresemployed in obtaining batches of infected Anophelesfreeborni and preparing, measuring, and delivenngsporozoite inocula; (iii) methods used for routine de-tection and quantification of parasitemias; (iv) char-acteristics of untreated sporozoite-induced infections;(v) responses of established infections to standardblood schizonticidal and tissue schizonticidal drugs;and (vi) procedures employed in assessing prophylac-tic and radical curative activities of new agents.A group of 48 subadult rhesus monkeys (Macaca

mulatta), imported directly from New Delhi, India,and conditioned in our animal quarters, was used inthe current study. All monkeys were tuberculin nega-tive upon receipt and, as judged by results of routinemonthly tests for tuberculin skin hypersensitivity, re-mained so throughout residence in the colony. Of theabove subjects, 42 without previous malaria experi-ence were used in evaluating the prophylactic andradical curative activities of floxacrine. This subgroupincluded 24 males and 18 females, ranging from 3.7 to5.0 kg in weight at the time of assignment to anexperiment. Another four monkeys, infected with tro-phozoites of the B strain for other purposes, were usedfor a preliminary appraisal of the blood schizonticidalactivities of floxacrine, an evaluation required to guidethe dosage regimen employed in assessing radical cu-rative activity. Finally, two monkeys, discarded ascured from an unrelated malaria therapy experiment,were used in a preliminary study of the capacity offloxacrine to produce hypoprothrombinemia. The rea-son for and scope of this study are detailed in theResults section.The prophylactic activity of floxacrine was evalu-

ated in four experiments. In three, involving 19 treatedand 3 untreated control monkeys, each inoculatedintravenously with from 2 x 105 to 2.5 x 106 sporozoites(approximately 105 to 106 infective doses), this com-pound was administered so as to cover the entire 8-day incubation period. Doses ranging downward from40.0 to 0.0098 mg/kg of body weight were administered24 h before sporozoite challenge, 2 h before challenge,and once daily thereafter for 7 consecutive days. Thefourth experiment, concerned with the duration ofprotection accorded by a single, comparatively largedose of floxacrine, rested on the results of a prelimi-nary pharmacokinetic study which showed that thiscompound had a half-life of 20 h in the rhesus monkey(personal communication, H. Wagner). In this experi-ment, four pairs of monkeys were given a 40.0-mg/kgdose of floxacrine 168, 96, 48, or 2 h before sporozoitechallenge. These eight subjects and an untreated con-trol were inoculated intravenously at the same settingwith 1.2 x 105 sporozoites.

In each of the above experiments, examinations ofthick and thin blood films stained with Giemsa wereinitiated on day 7 after sporozoite challenge and re-peated daily thereafter for 15 consecutive days, then

twice weekly for at least an additional 35 days, unlessparasitemia developed. If thick films were uniformlyparasite negative throughout this period, the monkeyswere rechallenged with approximately 105 sporozoitesto verify their susceptibility to infection.The evaluation of the radical curative activity of

floxacrine, carried out with well-established proce-dures (18, 20), involved work with 17 monkeys. Elevenwere infected specifically for this appraisal, receivinginocula ranging from 1.0 x 106 to 3.0 x 106 sporozoites.The six remaining monkeys were derived from variousprophylactic experiments where they had receiveddoses of floxacrine that were without effect on theincubation period or subsequent evolution of parasit-emia. Floxacrine was administered once daily for 7consecutive days in all assessments of radical curativeactivity. When doses of 2.5 mg/kg or lower were em-ployed, chloroquine was administered concomitantlyat a daily dose of 2.5 mg of base per kg of body weight.This combination regimen was used to ensure eradi-cation of blood schizonts, for as the results of thepreliminary study referred to earlier showed, dailydoses of 5.0 mg of floxacrine per kg were required forthe cure of trophozoite-induced infections. Doses of5.0 mg/kg or greater were administered alone; how-ever, when parasitemia reappeared, infections wereretreated with chloroquine, at 5.0 mg of base per kgdaily for 7 days. This retreatment procedure is aroutine for discriminating between recrudescence dueto persistence of small numbers of blood schizonts andrelapse due to continued multiplication of tissueschizonts (18, 20). Parasitemias were monitored onGiemsa-stained thick and thin blood films prepared onalternate days throughout the evaluation of curativeactivity.

Administration of floxacrine and chloroquine.A single lot of floxacrine [HOE-991, S 72 8991 (17 346/3)] was used in the experiments presented in thisreport. This compound, which is relatively water in-soluble, was always administered orally. The quantityof agent required for treating a group of owl or rhesusmonkeys was placed in a glass mortar, ground to apaste with 1/10 its weight of Tween 80, and thenground to a fine, relatively stable suspension withsuccessive additions of distilled water until the appro-priate concentration of floxacrine was attained. Thevolumes of suspension required for individual animalswere transferred to Erlenmeyer flasks and diluted toa final volume of 10.0 ml for owl monkeys and 30.0 mlfor rhesus monkeys. The contents of each flask weredelivered to the appropriate subject by stomach tube,followed by a 3.0-ml distilled-water rinse of vessel andtube for an owl monkey and a 20.0-ml rinse for arhesus monkey. Suspensions were made up fresh dailyand administered within 1 h of preparation.

Preparation of chloroquine solutions and their de-livery via the oral route, either alone or in combinationwith floxacrine, were identical with those describedelsewhere (15, 20).

RESULTSActivities of floxacrine against infections

with P. falciparum and P. vivax in owl mon-keys. The capacities of floxacrine to control

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parasite development and cure infections in owlmonkeys inoculated with blood schizonts of theSmith and Oak Knoll strains of P. fakciparumand the Palo Alto strain of P. vivax have beensummarized in Table 1. Utilizing the data incolumns 2 to 6 of this summary, the total-coursedoses of floxacrine (seven times the daily doses)required for cure of 50 and 90% of establishedinfections have been calculated (4), whereverresults permitted, and are set forth as CD,o's andCDso's in Table 2.The data in Table 1, columns 2 to 6, show that

floxacrine effected temporary clearance of par-

asitemia at comparatively low doses and that,with respect to this property, it was essentiallyequally active against infections with each of thetest strains. Daily doses of 1.25 mg/kg (equiva-lent to a course dose of 8.75 mg/kg) effectedclearance of parasites of the multidrug-resistantSmith strain of P. falciparum regularly and,except for a single repeated treatment failure(encountered in monkey Atr 7879; partial datain columns 3 and 4 of Table 1), were uniformly

effective in clearing parasites of the pyrimetha-mine-resistant Palo Alto strain of P. vivax. Ex-cept for a second repeated treatment failure(encountered in Atr 7994; partial data in column4 of Table 1), daily doses of 2.5 mg/kg, the lowestadministered, effected temporary clearance ofparasitemia in monkeys infected with the chlo-roquine-quinine-resistant Oak Knoll strain of P.falciparum. These observations indicate thatthe capacity of floxacrine to block schizogonywas not prejudiced by resistance to chloroquine,quinine, or pyrimethamine and was of similardimensions in infections with P. falciparum andP. vivax.The data in Table 1, column 6, together with

those in Table 2, show that the capacity offloxacrine to cure established infections did notmeasure up to its capacity to effect temporaryclearance of parasitemia. Moreover, unlike thelatter capability which was of similar magnitudefor the various test strains, the curative activityof floxacrine varied strikingly from strain tostrain. Thus, the total-course CD5o and CDso for

TABLE 1. Primary assessments ofthe activities offloxacrine in owl monkeys infected with the Vietnam Smithand Oak Knoll strains of P. falciparum and the Vietnam Palo Alto strain of P. vivax

Response to treatment (no. of subjects)h Mean day (range) from:

Infecting strain dose (mg/Effect on parasitemia Initial dose Last dose to re-kg)' Sup- Clearance with recru- Cured to parasite crudescenceNone .clearancepressed descence

Smith (P. falci- 0.156 -e 2 (P)parum) 0.312 - - 2 (R) - 9 (8-10) 10.5 (10-11)

0.625 - 2 (P) - _ _1.25 - - 2 (R) - 5.5 (5-6) 10 (9-11)2.5 - - 3 (P) 1 (P) 9.5 (7-13) 13 (12-14)5.0 - - 4 (R2) 3 (R) 5.7 (5-6) 20.8 (13-43)

10.0 - - 5 (2P, 3R3) 2 (P, R3) 6.4 (4-9) 18.4 (11-34)20.0 - - 1 (R4) 3 (R,, 2R) 6.3 (4-9) 5540.0 - - - 4 (P) 7.5 (7-8) -

Oak Knoll (P. 2.5 - - 2 (P) - 7.5 (7-8) 10 (9-11)falciparum) 5.0 - - 2 (R,) - 7 (6-8) 15 (12-18)

10.0 - - 3 (2P, R2) 2 (P, R2) 6.6 (6-8) 10.7 (7-15)20.0 - 1 (R3) 2 (R,) - 4.5 (4-5) 24.5 (21-28)40.0 - - 2 (P, R4) 3 (P, 2R2) 5.6 (5-6) 16.5 (16-17)80.0 - 1 (R5) - 1(R,) 4

Palo Alto (P. vi- 0.156 2 (P) - - - -vax) 0.312 - 1 (R,) 1 (R)" - -

0.625 - 1 (P) 2 (P) - 7 12.5 (12-13)1.25 - - 1 (R) - 6 62.5 - - 3 (P) - 4.7 (4-6) 16.7 (13-24)5.0 - - 6 (4R,, 2R2) 1 (R,) 4.4 (3-5) 20.7 (16-27)

10.0 - - 3 (P) - 6.0 (4-8) 16 (13-19)20.0 - - 6 (2R,, 2R2, 2R3) 3 (RI, 2R2) 4.8 (4-6) 23.2 (11-38)40.0 - - 3 (P) 1 (R3) 5.8 (4-7) 19 (14-27)80.0 1 (R3) 1 (R2) 6 (3R, 2R%, R3) 3 (R2, 2R) 5.1 (3-7) 15.3 (10-28)

aDose administered once daily for 7 consecutive days.'Items in parentheses refer to treatment status of monkeys in each response group. Thus, P indicates initial treatment, R,

indicates first retreatment, and H2, etc., indicate second and further retreatments. Each value preceding P or R indicates thenumber of monkeys in the various treatment categories.

-, Implies no monkeys in the respective category.dThis monkey died (nonmalarial death) 57 days after last dose of floxacrine. Infection may have been cured.

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ANTIMALARIAL PROPERTIES OF FLOXACRINE 479

TABLE 2. Approximate CD5o's and CDgo's offloxacrine for infections with the Vietnam Smithand Vietnam Oak Knoll strains ofP. falciparumand the Vietnam Palo Alto strain of P. vivax

Total-course dose (mg/kg)"

Infecting Initial treatment All casesstrain cases

Approx. Approx. Approx. Approx.CD!,o CDsn CDr,o CDso

Smith 56 154 42 112Oak Knoll 175 NA 210 NAPalo Alto >560 NA >560 NA

n NA, Not available.

previously untreated infections with the Smithstrain, the most responsive of the three understudy, were, respectively, 56 and 154 mg/kg(Table 2), doses approximately .6-fold and 17-fold greater than the 8.75-mg/kg course doserequired for regular clearance of parasitemia.The total-course CD50 for previously untreatedinfections with the Oak Knoll strain, 175 mg/kg,was at least 10-fold greater than the course doserequired for regular clearance ofparasitemia and3-fold greater than the CD50 for infections withthe Smith strain. The CD50 for infections withthe Palo Alto strain, the most difficult of thethree to cure, was >560 mg/kg, more than 64-fold greater than the dose required for clearanceof parasites of this strain, and more than 10-foldgreater than the CD50 for infections with theSmith strain. Because of low curative activitiesat the doses delivered in these studies, CD9o's forinfections with the Oak Knoll and Palo Altostrains could not be determined. These differ-ences in strain susceptibility to floxacrine wereobviously not related to resistance to chloro-quine, quinine, or pyrimethamine, for this com-pound was most active against infections withthe multidrug-resistant Smith strain and leastactive against the pyrimethamine-resistant,chloroquine-quinine-susceptible Palo Altostrain.The two drug treatment failures referred to

previously and recorded in the lower sections ofthe Oak Knoll and Palo Alto compartments ofTable 1 are of special interest. Initial treatmentof Atr 7994, the subject infected with the OakKnoll strain, with floxacrine at daily doses of 2.5mg/kg and successive retreatment of the firstand second recrudescences with doses of 5.0 and10.0 mg/kg effected prompt clearance of parasit-emia (Table 3). Treatment of the third recru-descence with doses of 20.0 mg/kg reduced butdid not clear parasitemia. Treatment of thefourth recrudescence with doses of 40.0 mg/kgeffected parasite clearance but was not curative.

Treatment of the fifth recrudescence with 80.0-mg/kg doses not only failed to effect parasiteclearance, but considering the immunity whichAtr 7994 should have acquired during previousparasitic episodes, was probably without effecton parasite development. This failure to respondto a dose of floxacrine at least 32-fold greaterthan that which effected clearance of parasit-emia in previously untreated infections could beinterpreted as the result of the emergence ofresistant parasites or, alternatively, induction ofenzymes which converted floxacrine from anactive antimalarial agent to an inactive metab-olite. The acute shortage of previously unin-fected monkeys precluded examination of thefirst alternative via direct experiments with the"Atr 7994R5 parasites." The magnitude of prob-lems related to the isolation and chemical andbiological characterization of floxacrine metabo-lites discouraged exploration of the second alter-native.The above alternatives were resolved via stud-

ies on Atr 7879, the treatment failure among themonkeys infected with the Palo Alto strain of P.vivax, and on its subinoculees. Initial treatmentof Atr 7879 with floxacrine at daily doses of 40.0mg/kg and the fit retreatment with 80.0-mg/kg doses effected prompt although temporaryclearance of parasitemia (Table 3). Two addi-tional treatment courses, each at a dosage of80.0 mg/kg, were without significant effects onparasite numbers or morphology. To delineatethe role of the parasite in this failure, each of agroup of four previously used owl monkeys wasinoculated with 5 x 106 trophozoites obtainedfrom Atr 7879 just before delivery of the fourthtreatment course. Administration of floxacrinein doses of 20.0, 40.0, or 80.0 mg/kg was withouteffect on the parasitemias of these subinoculees(Table 3). This result strongly supports the con-clusion that emergence of parasites resistant tothis compound was responsible for the treatmentfailure in Atr 7879. The failure encountered inAtr 7994 probably had a similar basis.Prophylactic and radical curative activi-

ties of floxacrine in rhesus monkeys inoc-ulated with sporozoites of P. cynomolgiLFloxacrine exhibited significant prophylactic ac-tivity (Table 4). Doses of 0.625 mg/kg or greateradministered on the day before challenge, 2 hbefore challenge, and daily thereafter through-out the 7-day incubation period provided com-plete protection against infection with relativelymassive numbers of sporozoites. A dosage of0.156 mg/kg completely protected two of fourrecipients and led to 3- and 6-day delays in theonset of parasitemia in the remaining two. Dos-ages of 0.039 and 0.0098 mg/kg were neither

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TABLE 3. Events suggestive ofdevelopment ofresistance to floxacrine during treatment of infections with theVietnam Oak Knoll strain of P. falciparum and the Vietnam Palo Alto strain ofP. vivax

Parasitemia (no. of parasites/104 erythrocytes)'

Arn.adDaily dose PosttreatmentDasfotreatment 24 h after dose: dose 7 to re-coursea (mg/kg)' Before (h after doea 7) crudescence;'dose 12 4 7 48 96 144

7994Pe 2.5 34 30 20 <1 <1 Neg Neg 117994R, 5.0 64 77 4 Neg Neg Neg Neg 187994R2 10.0 8 4 <1 Neg Neg Neg Neg 77994R3 20.0 1 16 2 <1 <1 <1 <1 NA7994R4 40.0 2 <1 <1 Neg Neg Neg Neg 167994R5 80.0 2 4 <1 5 20 58 Tf NA

800le 0 42 1,900 4,300 D" NA

7879ph 40.0 12 10 <1 Neg Neg Neg Neg 147879R, 80.0 20 14 <1 Neg Neg Neg Neg 117879R2 80.0 1 1 <1 1 10 15 12' NA7879R3 80.0 12 33 33 64 70 70 106 NA

7531h 0 12 56 172 225 180 165 122 NA

7725P' 20.0 6 8 33 6 13 28 20 NA7723pi 40.0 4 18 66 48 13 12 12 NA7711P' 80.0 15 48 24 78 57 76 100 NA

7942' 0 3 32 64 42 57 40 39 NA'P, Previously untreated; RI, first retreatment; R2 through R5, second through fifth retreatments.' Administered once daily for 7 consecutive days.eNeg, Negative thick blood film.dNA, Not available.eAtr 7994 and Atr 8001 infected with Vietnam Oak Knoll strain of P. falciparum.f T, Treated with amopyroquine (20.0 mg of base equivalent/kg) daily for 7 days and cured.5D, Subject died on day 5 of treatment.hAtr 7879 and Atr 7531 infected with Vietnam Palo Alto strain of P. vivax.'Owl monkeys infected with parasites derived from Atr 7879 at time indicated in column 9 of Atr 7879 R2

line.

TABLE 4. Capacity of floxacrine, administered throughout the incubation period, to protect rhesus monkeysagainst infections with sporozoites of the B strain of P. cynomolgi

No. of monkeys:

Daily dose (mg/kg)' . Days to patency after Days delay in onset ofChallenged with Developing patent challenge patency"

sporozoites infections

0 3 3 8,8,80.0098 2 2 8,8 0,00.039 2 2 8,8 0,00.156 4c 2 11, 14 3, 60.625 2c 0 -d2.5 4c 0

10.0 2c 040.0 2c. e 0

a Dose administered once daily, the day before inoculation, 2 h before inoculation, and for 7 consecutive daysthereafter.

b Relative to day of patency in untreated controls.c-, Implies no monkeys developed patent infections at these doses.d All monkeys that failed to develop patent infections within 59 or 60 days after the initial sporozoite challenge

were rechallenged with 5 x 10' sporozoites on either day 59 or 60. All exhibited patent infections on day 8 afterreinoculation.

'One additional recipient of daily doses of 40.0 mg/kg of body weight died approximately 24 h after dose 8,probably of drug toxicity. See text for details.

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protective nor did they effect a delay in theappearance of parasitemia.The results of a preliminary study of the phar-

macokinetics of floxacrine, coupled with theabove assessments of prophylactic activity, sug-

gested that a single dose of 40.0 mg/kg wouldprovide protective levels of this agent for 5 to 7days. The results of a direct study of this sug-gestion (Table 5) showed that administration ofsuch a dose 4 or more days before sporozoitechallenge provided no protection. Dosage 48 or2 h before inoculation did not prevent infection,but did delay the onset of parasitemia for 2 and3 days in the 48-h group and for 9 and 11 daysin the 2-h group. Parasitemias evolved nornallyin all recipients of floxacrine once patency wasestablished.The capacity of floxacrine to effect radical

cure of established sporozoite-induced infectionswas examined in two studies. In one, the com-pound was delivered over a dosage range of0.0098 to 2.5 mg/kg, daily for 7 days, in combi-nation with 2.5-mg/kg doses of chloroquine. Inthe second, it was administered over a dosagerange of 5.0 to 40.0 mg/kg daily for 7 days,followed by dosage with chloroquine at 5.0 mg/kg daily for 7 days when parasitemia recurred.The results of these evaluations showed that,when administered at doses up to the maximumtolerated level, floxacrine exhibited no capacityto cure established sporozoite-induced infectionsand had no discernible effect on the viability ofthe established tissue schizonts of P. cynomolgi.

All 21 infections treated with floxacrine indoses of 0.0098 to 2.5 mg/kg, in combinationwith chloroquine, relapsed within 4 to 9 days ofcompletion of the dosage regimen (Table 6).Such relapse intervals are encountered regularlywhen chloroquine is employed alone for controlof parasitemia in primary attacks induced byheavy sporozoite inocula (15). In keeping withthe results of the preliminary appraisal of theactivity of floxacrine against trophozoite-in-duced infections, this compound effected para-site clearance in all 17 recipients of doses of 5.0

to 40.0 mg/kg when administered alone; how-ever, parasitemias reappeared 7 to 12 days aftercompletion of treatment (Table 7, columns 3 and6). Retreatment of the recurring infections withchloroquine was followed in every instance by arelapse 5 to 16 days after delivery of the lastdose of this drug (Table 7, column 8). Suchrelapse intervals are encountered routinely whenchloroquine is used in the treatment of secondand third attacks (15). Their occurrence in thecurrent setting indicates that floxacrine is devoidof activity against the mature persisting tissueschizonts.Untoward reaction of rhesus monkeys to

floxacrine. Although the current investigationwas not concerned directly with the toxicity offloxacrine, note should be taken of an unex-pected, potentially serious side effect first en-countered in a rhesus monkey assigned to theinitial appraisal of prophylactic activity. One ofthree recipients of 40.0-mg/kg doses died ap-proximately 24 h after dose 8 (Table 4). Nountoward reactions were recognized at deliveryof that dose, but 18 h later the animal was inshock and exhibited purpuric lesions over theventral aspect of the chest, upper abdomen,axillae, and contiguous areas of the upper ann.Necropsy, performed within 30 min of death,revealed massive subcutaneous hemorrhage atthese sites and, in addition, extensive hemor-rhage in the mediastinum, omentum, and con-nective tissue about the kidneys, ureters, anduterus. Spilled blood failed to clot during the 60min covered by the necropsy.

Since the dose partner of the monkey de-scribed above showed no untoward reactions,the syndrome was considered to be non-drugrelated, and a third monkey was inoculated withsporozoites and treated with 40.0-mg/kg dosesof floxacrine as a replacement for the subjectthat died. On day 6 postchallenge, at delivery ofdose 8, this replacement exhibited diffusely dis-tributed petechiae over the face and thighs, andpurpuric lesions over the chest and biceps. Rel-atively complete hematological and biochemical

TABLE 5. Duration ofprotection accorded by a single dose offloxacrine against infections with the B strainof P. cynomolgi

Time (h) of administration of No. of infected monkeys/ Days to patency after Days delay in onset offloxacrine" no. challenged challenge patency^

No floxacrine 1/1 8-168 2/2 8, 8 0,0-96 2/2 8,8 0,0-48 2/2 10, 11 2, 3-2 2/2 17, 19 9, 11

a Single doses of floxacrine (40.0 mg/kg of body weight) were administered at the hours indicated beforechallenge with sporozoites.

b Relative to day of patency in untreated control.

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studies on peripheral blood drawn at this timerevealed a markedly elevated prothrombin time(22), 42 s as compared with 12 to 14 s in normalcontrol rhesus monkeys. Other moietiesmeasured were within normal limits. Assumingthat the hemorrhagic syndrome and hypoproth-rombinemia were related and might reflect a

vitamin K deficiency, therapy with menadiolsodium diphosphate was started with an initialdose of 37.5 mg, followed by 20.0-mg doses dailyfor 7 days. The petechiae and purpuric lesionsregressed promptly, and the prothrombin timereturned to normal on day 4 of the menadiolregimen, despite continuation of the scheduledfloxacrine dosage. Essentially the same syn-drome, with prolongation of prothrombin time,was encountered in one of the two recipients of40.0-mg/kg doses of floxacrine in the radicalcurative study (Table 7) and again was con-

TABLE 6. Capacity offloxacrine, administered incombination with chloroquine, to effect radical cureof established infections with sporozoites of the B

strain of P. cynomolgiNo. of infections: Days from last

Daily dose of doses of flox-floxacrine Treated Re acrine and chlo-(mg/kg)' (status)" lapsed roquine to re-

lapse0.0098 3 (P) 3 6,6,80.039 4 (P) 4 4,6,6,80.156 5(4P,RI) 5 4,4,8,9,60.312 5 (RI) 5 7,7,7,8,80.625 2 (P) 2 8,82.5 2 (P) 2 8,8

"Dose of floxacrine indicated was administered once dailyfor 7 days concomitantly with chloroquine at 2.5 mg of baseper kg of body weight.

b Letters in parentheses refer to attack treated: P, primary;R,, first relapse.

trolled by a dosage regimen ofmenadiol identicalwith that described above.These observations led to a limited study of

the effects of floxacrine on prothrombin time ina setting uncomplicated by recent introductionof mosquito tissue; as test subjects two excep-

tionally healthy rhesus monkeys, discarded ap-

proximately 4 months earlier as cured of theirmalarial infections, were used. Floxacrine wasadministered to each subject at a dose of 40.0mg/kg once daily for 9 days. Complete bloodcounts and measurements of prothrombin timeand glucose-oxalacetic acid-transaminase activ-ity in serum were made daily beginning 4 daysbefore the first dose and continuing for 14 daysafter dosage had been completed. Althoughthere were no overt reactions to treatment, bothsubjects exhibited elevated prothrombin times.These increased stepwise from day 4 of treat-ment, rising from 12.1 and 13.0 s in the respectivesubjects pretreatment to peaks of 55.0 and 30.0s on day 2 posttreatment, with a spontaneousreturn to normal levels of 12.2 and 13.5 s on day9 posttreatment. Glucose-oxalacetic acid-trans-aminase activities and numbers of formed ele-ments in peripheral blood were within normallimits throughout the observation period.

Together, the above observations show that,when administered to rhesus monkeys in appro-priate doses, floxacrine can produce a hypo-prothrombinemia and related hemorrhagic syn-drome of significant dimensions. Such reactionswere not noted in owl monkeys treated with40.0- or 80.0-mg/kg doses of floxacrine. Thisnegative result in the owl monkey may reflect *aspecies difference in tolerability, perhaps relatedto amounts of vitamin K synthesized by theintestinal flora, or it may be due to the regular

TABLE 7. Capacity of floxacrine, administered alone, to effect radical cure of established infections withsporozoites of the B strain of P. cynomolgia

Follow-up treatment withInitial treatment with floxacrine ch,loroquinedEffect on parasitemia

No. of

Daily dose No. of infections No. with No. of relapses Days from last dosemgkgb treatd*(stt apparent Days from first relapses Days from last dose after , ,.

clear- dose to parasite or re- to parasite reappear- treat-r

clearance' crudes- ance' mentance cences

5.0 8(5R,,3R2) 8 5,7,7,7,7,3,5,5 8 8,8,9,9,9,7,10,12 8 5,6,7,9,9,7,9,1010.0 7 (4R,, 3R2) 7 3, 7, 7, 7, 3, 5, 5 7 8, 8,9,12, 7, 8, 9 7 6, 7, 9,16, 7,9, 940.0 2 (P) 2 4,5 2 8,8 2 8,8

a With follow-up administration of chloroquine.'Dose indicated administered once daily for 7 consecutive days.'Letters in parentheses refer to attack treated: P, primary; R,, first relapse; R2, second relapse.d Chloroquine was administered in a dose equivalent to 5.0 mg of base per kg of body weight, once daily for 7 consecutive

days.'Listing of days parallels treatment status: R, data listed first, followed by R2.

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weekly administration of 7.5 mg of menadiolsodium diphosphate, which, because of a bleed-ing tendency encountered in my early work onthis subject and in work by others (21), has beenan integral part of the health maintenance pro-gram for the owl monkey colony since 1969.

DISCUSSIONAssessments in owl monkeys of the capacities

of floxacrine to suppress and eradicate infectionswith blood schizonts of a chloroquine-quinine-resistant, pyrimethamine-susceptible strain of P.falciparum, a strain ofthis plasmodium resistantto all three drugs, and a strain of P. vivaxresistant to pyrimethamine only have shown thefollowing. (i) With respect to suppressive capa-bilities, floxacrine was equally active against in-fections with the above strains, effecting tem-porary clearance of parasitemia at total-coursedoses ranging from 8.75 to 17.5 mg/kg. (ii) Flox-acrine was far less effective (by factors of 6 tomore than 64) in eradicating blood schizonts andcuring established infections than it was in re-ducing parasitemias temporarily below micro-scopically detectable levels. (iii) As measured bytotal-course CD50, there was more than a 10-folddifference in the capacities of floxacrine to cureinfections with the three test strains. These dif-ferences did not correlate with resistance to chlo-roquine, quinine, or pyrimethamine. (iv) Resist-ance to floxacrine developed rapidly duringtreatment of infections with the pyrimethamine-resistant Vietnam Palo Alto strain of P. vivaxand probably evolved during treatment of infec-tions with the chloroquine-quinine-resistantVietnam Oak Knoll strain of P. falciparum.

Assessments in rhesus monkeys of the capac-ities of floxacrine to prevent and cure infectionswith sporozoites of P. cynomolgi have shown thefollowing. (i) Floxacrine has a high order ofprophylactic activity, with doses of 0.625 mg/kgdelivered daily throughout the incubation periodproviding full protection against challenge with105 to 106 infective doses of sporozoites; doses of0.156 mg/kg fully protected two of four recipi-ents. (ii) A single large dose of 40.0 mg/kg deliv-ered 2 h before sporozoite challenge did notprevent development of infections, although itdid prolong the incubation period significantly.(iii) Floxacrine was devoid of the capacity tocure established infections. The maximum tol-erated daily dose (40.0 mg/kg), which produceda hemorrhagic syndrome in three of five recipi-ents, effected clearance of blood schizonts, butneither prevented nor delayed relapse.The divergence between the capacities of flox-

acrine to prevent and cure infections, referred toin i and iii above, merits special attention. Such

a separation of prophylactic and radical curativeproperties has not been encountered before,either in studies in monkeys inoculated withsporozoites of various strains of P. cynomolgi (9,15, 18, 20; L. H. Schmidt, unpublished data) orin human volunteers inoculated with sporozoitesof the Chesson strain of P. vivax (personal com-munication, the late A. S. Alving, University ofChicago). In both systems, compounds that ex-hibited one activity exhibited both, with dosesrequired for prevention either the same orslightly larger than those required for radicalcure. Whereas the separation of such activitiesencountered with floxacrine is unique, it is notan unreasonable finding. There are major differ-ences between the morphology of the developingtissue schizonts and the morphology of fullymature tissue schizonts and persisting tissuestages associated with relapse (1-3). It would beremarkable if all classes of chemotherapeuticagents were equally active against forms of suchstructural diversity.The failure of floxacrine to fully protect

against infections with P. cynomolgi when ad-ministered in a single 40.0-mg/kg dose 2 h beforesporozoite challenge is also noteworthy. Assum-ing that floxacrine has a half-life of 20 h (H.Wagner, personal communication), such a doseshould have provided residues in excess of thoseequivalent to doses of 0.625 mg/kg for 5 days,0.325 mg/kg for 6 days, and 0.156 mg/kg for 7days. Failure to obtain full protection with suchdosage coverage is perplexing. It raises a ques-tion as to whether what was measured in thephannacokinetic studies was floxacrine or a mixof that compound with inactive metabolites.

Since the studies recorded in this report werestimulated by the observations of Raether inmice infected with trophozoites of P. bergheiand those of Fink in mice challenged with spo-rozoites of P. berghei yoelii, it is pertinent toexamine the areas of agreement and disagree-ment in the results of these two groups of inves-tigations. They are in agreement with respect tothe finding that the capacity of floxacrine toeffect temporary clearance of parasitemia is notprejudiced by resistance to chloroquine or pyri-methamine. They are also in harmony with re-spect to the prophylactic activity of floxacrine.The relations between the temporarily suppres-sive and curative doses of floxacrine and therelative activities of this compound and chloro-quine differed significantly in the rodent andhuman plasmodium models. Whereas the dataof Raether in infections with P. berghei indi-cated that there was no more than a twofoldspread between the doses required for tempo-rary clearance of parasitemia in 90% of the miceand the CD5o, observations in owl monkeys in-

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fected with human plasmodia showed that therewas a 6-fold to 64-fold spread between dosesrequired to effect clearance of parasitemia reg-ularly and the CDso. Likewise, floxacrine wastwo to three times more active than chloroquineagainst infections with P. berghei. In contrastthe activity of floxacrine was less than 1/40 thatof chloroquine in infections with the chloro-quine-susceptible Palo Alto strain of P. vivax inowl monkeys (7). No comparison could be madeof such highly important drug parameters asstrain susceptibility and radical curative activity.The former was not examined in the rodentmodel, whereas the latter could not be studiedbecause of the absence of persisting tissue stagesin infections with P. berghei yoelii.Inasmuch as the current studies were designed

to appraise the potential of floxacrine in thetreatment of the human malarias, it is appropri-ate that this subject be touched on before con-cluding this report. The performance of floxa-crine in owl monkeys infected with P. falci--parum or P. vivax indicates that this compoundhas significant shortcomings as a blocd schizon-ticide. First, the range of doses required for cureof established infections with various strains islarge. To be broadly useful, especially whenadministrative control of drug application ismirnimal, any treatment regimen has to coverinfections with strains of the least susceptibility.Routine use of sizeable doses of floxacrine re-quired to cover all contingencies might wellevoke toxic reactions. Second, resistance to flox-acrine emerges rapidly, at a rate not less thanthat of proguanil or pyrimethamine (16, 17),compounds that have presented resistance prob-lems in field use. Third, by comparison, floxac-rine is a weak competitor of a group of aminoal-cohols which have been studied in depth in thesame animal models (11-14). Some have alreadyexhibited promise in limited clinical trials (11,12). These compounds are from two to threetimes as active as chloroquine against infectionswith chloroquine-susceptible strains, are equallyactive against drug-susceptible and multidrug-resistant strains, are effective in single-dose reg-imens, and, to this point, have not been associ-ated with evolution of resistance. The perform-ance of floxacrine in rhesus monkeys inoculatedwith sporozoites of P. cynomolgi has shown thatthis compound also has shortcomings as a tissueschizonticide. First, it appears to be totally lack-ing in radical curative activity. Secondly, al-though floxacrine has significant prophylacticactivity, the requirement for daily dosage, ifimmutable, will be a serious handicap to generaluse. This is unfortunate, since dose for dose, thiscompound is slightly more active than prima-

quine as a prophylactic and much superior basedon comparative therapeutic indexes (9, 18, 20).All qualities considered, it seems unlikely thatfloxacrine could attain more than a limited rolein malaria therapy. This pessimistic assessmentof its utility could be tempered by the results offurther studies.

ACKNOWLEDGMENTSI am indebted to Linda Allen, Ruth Crosby, and Jane Rasco

for assistance with the various experimental studies and es-pecially to Lee McGuire who collaborated in compiling dataand preparing this manuscript.

The experimental components of this report were sup-ported by a research contract between Hoechst AG, Frankfurt-am-Main, West Germany, and the Southern Research Insti-tute. Manuscript preparation was supported in part by thisInstitute and in part by a grant from the Ann Arbor BiologicalCenter, Inc., Ann Arbor, Mich.

LITERATURE CITED1. Bray, R. S. 1954. The tissue phase of malaria parasites.

J. Trop. Med. Hyg. 57:4145.2. Garnham, P. C. C. 1966. Plasmodium cynomolgi and

subspecies, bastienellii, cyclopis, and ceylonensis, p.182-186. In P. C. C. Garnham (ed.) Malaria parasitesand other Haemosporidia. Blackwell Scientific Publi-cations, Oxford.

3. Hawking, F., W. L M. Perry, and J. P. Thurston.1948. Tissue forms of a malaria parasite Plasmodiumcynomolgi. Lancet i:783-789.

4. Litchfield, J. T., Jr., and F. Wilcoxon. 1949. A simpli-fied method of evaluating dose-effect experiments. J.Pharmacol. Exp. Ther. 96:99-108.

5. Schmidt, L. H. 1973. Infections with Plasmodium falci-parum and Plasmodium vivax in the owl monkey-model systems for basic biological and chemothera-peutic studies. Trans. R. Soc. Trop. Med. Hyg. 67:446-474.

6. Schmidt, L H. 1978. Plasmodium faiciparum and Plas-modium vivax infections in the owl monkey (Aotustrivirgatus). I. The courses of untreated infections. Am.J. Trop. Med. Hyg. 27:671-702.

7. Schmidt, L H. 1978. Plasmodium falciparum and Plas-modium vivax infections in the owl monkey (Aotustrivirgatus). II. Responses to chloroquine, quinine, andpyrimethamine. Am. J. Trop. Med. Hyg. 27:703-717.

8. Schmidt, L H. 1978. Plasmodium falc4xzrum and Plas-modium vivax infections in the owl monkey (Aotustrivirgatus). III. Methods employed in the search fornew blood schizonticidal drugs. Am. J. Trop. Med. Hyg.27:718-737.

9. Schmidt, L H., S. Alexander, L Allen, and J. Rasco.1977. Comparison of the curative antimalarial activitiesand toxicities of primaquine and its d and I isomers.Antimicrob. Agents Chemother. 12:51-60.

10. Schmidt, L H., D. V. Cramer, R. N. Rossan, and J.Harrison. 1977. The characteristics of Plasmodiumcynomolgi infections in various old world primates. Am.J. Trop. Med. Hyg. 26:356-372.

11. Schmidt, L H., R. Crosby, J. Rasco, and D. Vaughan.1978. Antimalarial activities of various 4-quinoline-methanols with special attention to WR-142,490 (meflo-quine). Antimicrob. Agents Chemother. 13:1011-1030.

12. Schmidt,L H., R. Crosby, J. Rasco, and D. Vaughan.1978. Antimalarial activities of various 9-phenanthrene-methanols with special attention to WR-122,455 andWR-171,669. Antimicrob. Agents Chemother. 14:292-314.

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13. Schmidt, L H., R. Crosby, J. Rasco, and D. Vaughan.1978. Antimalarial activities of various 4-pyridine-methanols with special attention to WR-172,435 andWR-180,409. Antimicrob. Agents Chemother. 14:420-435.

14. Schmidt, L. H., R. Crosby, J. Rasco, and D. Vaughan.1978. Antimalarial activities of the 4-quinolinemetha-nols WR-184,806 and WR-226,253. Antimicrob. AgentsChemother. 14:680-689.

15. Schmidt, L. H., R. Fradkin, D. Vaughan, and J.Rasco. 1977. Radical cure of infections with Plasmo-dium cynomolgi: a function of total 8-aminoquinolinedose. Am. J. Trop. Med. Hyg. 26:1116-1128.

16. Schmidt, L. H., and C. S. Genther. 1953. The antima-larial properties of 2,4-diamino-5-p-chlorophenyl-6-ethylpyrimidine (Daraprim). J. Pharmacol. Exp. Ther.107:61-91.

17. Schmidt, L. H., C. S. Genther, R. Fradkin, and W.Squires. 1949. Development of resistance to chlor-guanide (Paludrine) during treatment of infections withPlasmodium cynomolgi. J. Pharmacol. Exp. Ther. 95:382-398.

18. Schmidt, L. H., J. Harrison, R. Ellison, and P.Worcester. 1970. The activities of chlorinated linco-mycin derivatives against infections with Plasmodiumcynomolgi in Macaca mulatta. Am. J. Trop. Med. Hyg.19:1-11.

19. Schmidt, L. H., R. N. Rossan, and K. F. Fisher. 1963.The activity of a repository form of 4,6-diamino-1-(p-chlorophenyl)-1,2-dihydiro-2,2-dimethyl-s- triazineagainst infections with Plasmodium cynomolgi. Am. J.Trop. Med. Hyg. 12:494-503.

20. Schmidt, L H., R. N. Rossan, R. Fradkin, J. Woods,W. Schulemann, and L, Kratz. 1966. Studies on theantimalarial activity of 1,2-dimethoxy-4-(bis-diethyl-aminoethyl)-amino-5-bromobenzene. Bull. W. H. 0. 34:783-788.

21. Voller, A., C. M. Hawkey, W. H. G. Richards, and D.S. Ridley. 1969. Human malaria (Plasmodium falci-parum) in owl monkeys (Aotus trivirgatus). J. Trop.Med. Hyg. 72:153-160.

22. Wintrobe, M. W. 1967. Blood platelets and coagulation,p. 328-329. In M. W. Wintrobe (ed.), Clinical hematol-ogy, 6th ed. Lea and Febiger, Philadelphia.

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