INTERNAL IONAL JOURNAL OF LEPROSY^ Volume 49, Number 3Printed in the U.S.A.

Prothionamide and Prothionamide-S-Oxide inExperimental Leprosy'

Yoshiyasu Matsuo, Hiroshi Tatsukawa, John F. Murray, Jr., andJohn H. Peters2 . 3

Ethionamide (ETH) and prothionamide(PTH) are now well established as highlybactericidal drugs against Mycobacteriumleprue in animals and man (2 •"). Combina-tions of ETH with clofazimine, dapsone(DDS), or rifampin (RFM) have been foundto act additively in mice infected with M.leprue (I 2 ), and ETH was effective in treat-ing mice infected with M. leprue resis-tant to DDS and RFM (•). Thus, the thio-namides are promising adjunctive drugs forcombination chemotherapy with DDS andRFM.

Earlier studies ( 1 —I.") performed shortlyafter ETH was introduced in the late 1950sas a drug for tuberculosis showed that, inanimals and man, the first biotransforma-lion of ETH was oxidation to ETH-S-oxide(ETHSO). It was subsequently found thatthis metabolite was reduced back to ETHin vivo, but it is not known whether theoxidation reaction is reversible or whethertwo separate enzymatic steps operating cy-clically are involved. ETHSO is also oxi-dized metabolically to an amide and sub-sequently hydrolyzed to an isonicotinicacid derivative. In man, metabolism ofETH is very extensive as shown from stud-ies wherein only 1% of the administeredETH was found in urine ( 12 ). The S-oxidemetabolite is particularly interesting be-cause, of all known metabolites of ETH, italone exhibited antimycobacterial activity

' Received for publication 28 April 1981; acceptedfor publication on 4 June 1981.

2 Y. Matsuo, M.D., Ph.D., Professor and Chairman;H. Tatsukawa, D.D.S., Research Fellow. Departmentof Bacteriology, Hiroshima University School of Med-icine, Hiroshima 734, Japan; J. F. Murray, Jr., B.S.,Biochemist; J. H. Peters, Ph.D., Director, Biochemi-cal Pharmacology Program, SRI International, MenloPark, California 94025, U.S.A.

This material was presented in part by Dr. Petersat the 24th Joint Leprosy Research Conference, NewOrleans, Louisiana, 26 September 1979.

(

2. ) We subsequently found that the prin-cipal circulatory metabolite of PTH in ratsand armadillos was the corresponding S-ox-ide, PTHSO (I). These results suggest thatthe therapeutic activity of thioamides aftertheir administration may result from the ac-tion of both the parent drugs and their S-oxides.

In this report, we summarize results oftests of the comparative activities of PTH,PTHSO, and the compound, 2-propyliso-nicotinamide (PINA) in vitro against M. tu-berculosis H37Rv and the comparative ac-tivities of PTH and PTHSO against M.leprae in mice.

MATERIALS AND METHODSThe PTH used in these studies was a gift

from May and Baker Ltd., Dagenham, En-gland. It was converted to PTHSO by ad-aptation of a procedure for the synthesis ofETHSO from ETH ( 7 ). PTH in pyridine so-lution was oxidized at 20-25°C by the drop-wise addition of 32% hydrogen peroxide.The PTHSO was isolated from the reactionmixture by chromatography on a column ofsilica. Yellow crystals melting at 120—I22°C (literature value I17°C (")) wereobtained by crystallization from a 1:1 ben-zene : hexane mixture (v/v). Thin-layerchromatography on silica gel 60 of thePTHSO and PTH, using development withethyl acetate, yielded single compact ultra-violet-absorbing spots at R1 values of 0.13and 0.65, respectively. Ultraviolet absorp-tion maxima for PTH and PTHSO in chlo-roform were at 295 nm (E = 8 x HP) and360 nm (E = 7 x 102), respectively. Massspectrometric analysis of PTH and PTHSOyielded parent ions at molecular weights180 and 196, respectively, the latter corre-sponding to the addition of one oxygen toPTH. Purity of the synthesized PTHSOwas estimated to be _̂ -95%. PINA was a giftof Dr. H. Franz, Saarstickstoff-Fatol, Pohl-

302

49, 3^Matsuo, et al.: Prothionamide and its S-Oxide^303

TABLE 1. COMparatiVe activities" ofPTH, PTHSO, and PINA on the growth ofM. tuberculosis 1137Rv.

Level ofcompoundin medium

(µg/ml)

PTH PTHSO PINA

Test I Test 2 Test I Test 2

10 ++5 ++1 .5 - - ++1.25 - - ++0.625 ++0.312 ++0.156 + +0.078 ++ ++0.039 ++ ++ + ++0 ++ ++ ++ ++

" Tests were performed by Dr. Welch, USPHSHospital, San Francisco, CA, during January (Test 1)and March (Test 2) 1978. Symbols used are: - nogrowth; + some growth; ++ growth comparable tocontrols.

heim, Germany. Our mass spectrometricanalysis of this compound yielded resultsthat testified to the authenticity of the as-signed structure.

Tests of the activities of PTH, PTHSO,and PINA in vitro were performed usingM. tuberculosis H37Rv in Dubos broth cul-ture (7H9 without malachite green). Dupli-cate samples containing 0 to 10 ,ag of thecompounds per ml of medium were incu-bated with assessments of growth made at8 and 14 days.

The antileprosy activities of PTH and

PTHSO were determined by the kineticmethod ("). Strain CFI mice were inocu-lated in the foot pads with 5 to 10 x 10 3 M.leprae obtained from the infected tissues ofuntreated patients or from nude mice withdeveloped lepromatoid lesions. PTH andPTHSO, dissolved in 50% aqueous propyl-ene glycol (Wako Pure Chemical IndustriesLtd., Japan), were given by gavage in mgper kg of body weight or fed in the dietduring the late lag or early log phase of bac-terial multiplication at the doses shown inthe tables.

RESULTS AND DISCUSSIONThe results of tests of the activities of

PTH, PTHSO, and PINA against M. tu-berculosis H37Rv are shown in Table 1.PTH exhibited an in vitro minimal inhibi-tory activity (MIC) of 0.156 and 0.312 ttg/ml in the two tests, not a significant differ-ence. PTHSO exhibited an in vitro MIC of0.078 µg/ml. Clearly, PTHSO was at leastequal in activity to PTH and may be moreactive. These observations for PTH andPTHSO agree, in principle, with earlierfindings that ETH and ETHSO exhibitedapproximately equal antimycobacterial ac-tivity (2 . 8 ). PINA was found to be inactivein this test.

Tests of the antileprosy activity of PTHat doses of 5, 10, or 20 mg/kg 6 days perweek for 30 days, starting on day 51 post-inoculation, are shown in Table 2 (Experi-ment 1). A repeat test using 2 and 20 mg/kg

TABLE 2. Effects of graded doses of PTH on the growth of M. leprae in mouse footpads. a

Experi- Treatmentment

No. of AFB (x105 ) in pooled foot pads on day Growth

105 140 175 210 245delay"(days)

None 0.24 0.66 3.1 8.2 14.2PTH-20 mg/kgPTH-I0 mg/kg

--"•

0.160.25

0.561.4

3.85.3

8.28.8

3016

PTH-5 mg/kg 0.13 0.27 1.9 6.4 11.6 11

None 0.30 0.78 1.4 8.0 11.3PTH-20 mg/kg 0.32 0.45 1.1 4.6 45PTH-2 mg/kg 0.46 0.66 9.8 18.6 3

Treatment was 6 days per week.Estimated by graphical comparison with the control group.Drug administration was started 51 days post-inoculation and continued for 30 days.

" Drug administration was started 71 days post-inoculation and continued for 35 days.No AFB detected.

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6 days per week for 35 days was also car-ried out (Table 2, Experiment 2). In theseexperiments, PTH caused growth delays ofM. leprue that were less than the periodsof drug administration in most cases. Inonly one treatment schedule (20 mg/kg inExperiment 2) was the growth delay slight-ly more than the period of drug administra-tion.

In mice receiving 10 mg PTH or PTHSOper kg for 60 days, we found that the drugtreatment caused approximately equalgrowth delays of 86 and 78 days, respec-tively (Table 3, Experiment 3). In this ex-periment, lower doses of PTHSO were rel-atively ineffective. Experiment 4 of Table3 also compares the activities of dietaryPTH and PTHSO. Again, the same dosesof dietary PTH and PTHSO yielded nearlyidentical growth delays. It is apparent fromthese experiments that the antileprosy ac-tivities of PTH and PTHSO are equivalentwhether given by gavage or in the diet.

Increasing the dietary level of PTH to0.1% yielded a growth delay of 218 days,which was 134 days greater than the periodof drug administration (Table 3, Experi-ment 5). This observation of a bactericidaleffect at this dose of PTH agreed with con-clusions of others ("•") on the bactericidaleffects of PTH in mice infected with M.leprae. These authors also reported thatPTH was only bacteriostatic at lower doses,as we have found.

The equivalence of activities of PTH andPTHSO both against M. tuberculosis andagainst M. leprae suggests that therapeuticactivities of PTH in infected animals or manresult from a summation of action of bothPTH and PTHSO.

SUMMARYPTHSO was at least equal in activity to

PTH, the parent drug, against M. tuber-culosis in vitro, but the isonicotinamide de-rivative of PTH was completely inactive.Using the kinetic method with the mousefoot pad model, PTH and PTHSO givenorally at the same doses were found to haveapproximately equal activity against M.leprae.

RESUMENSe encontai que la droga PTHSO resultO cuando

menos igualmente activa que la PTH, la droga pro-

genitora, contra el M. tuberculosis, in vitro. El deri-vado isonicotinamidico de la droga fue completamenteinactivo. Usando el metodo cinetico en el modelo delcojinete plantar del ratOn, se encontr6 que la PTH yIa vrHso, administradas oralmente y a las mismasdosis, mostraron aproximadamente Ia misma actividadcontra el M. leprue.

RESUME

Le PTHSO s'est revele au moins aussi actif que lePTH, le compose medicamenteux dont it est tire,contre M. tuberculosis in vitro, alors que le deriveisonicotinamidique du PTH s'est revele completementinactif. En utilisant une methode cynetique basee surle modele d'inoculation au coussinet plantaire de Iasouris, on a observe que le PTH et le PTHSO, parvole orale, a des doses identiques, avaient approxi-mativement une activite equivalente contre M. leprae.

Acknowledgments. This work was supported in partby grants from the Chemotherapy of Leprosy (THE-LEP) component of the UNDP/World Bank/WHOSpecial Programme for Research and Training in Trop-ical Diseases and by NIH Grant Al-08214. We grate-fully acknowledge Dr. David Thomas of SRI for per-forming the mass spectrometric analyses of thecompounds and Miss Junko Oka for technical assis-tance.

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