INTERNATIONAL JOURNAL OF LEPROSY^

Volume 51, Number IPrinted in the U.S.A.

Disposition of Prothionamide in Ratsand Armadillos'

John H. Peters, G. Ross Gordon, and John F. Murray, Jr.'

The thioamides, ethionamide (ETH) andprothionamide (PTH), are well establishedas bactericidal drugs against Mycobacte-rium leprae in animals and man (4, 20, 29, 31) .

ETH and PTH are virtually interchangeableand give cross resistance with each other( 31 ). Thus, the finding ( 12) that ETH was effective in mice infected with M. leprac re-sistant to dapsone (DDS) and rifampin(RFM) suggests that PTH would be equallyactive against organisms resistant to theseprimary antileprosy drugs. The minimal in-hibitory concentration (MIC) of ETH andPTH was estimated to be about 0.05 µg/ml,and it has been estimated that in man anoral dose of 375 mg would yield serum con-centrations in excess of the MIC during 24hr ( 3 '). However, these estimates may beconservative because they do not considerthe contribution of the circulatory S-oxidemetabolites of ETH and PTH, which havebeen found previously to exhibit antimy-cobacterial activities equal to their parentdrugs ETH ( 2. 16) and PTH ( 17). These con-siderations indicated the need for an ana-lytic method for study of these drugs thatwould measure both the thioamide and itsS-oxide metabolite at sensitivities adequateto detect levels in the range of the MICs of0.05 µg/ml of plasma or serum. Because atthe time these studies were initiated it hadbeen concluded that ETH was being re-placed by PTH due to the apparent lowersystemic toxicity of the latter drug ( 4), weconducted all our studies on PTH.

Previously published methods of insuf-ficient sensitivities (4, 5• 6, IS)) did not providethe bases for development of a method ca-pable of detecting levels as low as 0.05 )ig,/ml, and we chose to develop a high-perfor-

' Received for publication on 30 August 1982; ac-cepted for publication on 27 October 1982.

2 J. H. Peters, Ph.D., Director, Biochemical Phar-macology Program; G. R. Gordon, M.S., SeniorBiochemist; J. F. Murray, Jr., B.S., Biochemist (de-ceased), Life Sciences Division, SRI International,Menlo Park, California 94025, U.S.A.

mance liquid chromatographic (HPLC)technique because of our success with thisapproach for quantitating the other antilep-rosy drugs, DDS (' 8), RFM ( 22), and clofaz-imine ( 24), in biological fluids at sensitivi-ties at or below their respective MICs. Aftercompletion of our work, Jenner and Ellard( 13) reported an HPLC method for deter-mining ETH and PTH with sensitivities ofabout 0.01 .tg/ml of plasma, but these au-thors did not measure the S-oxide metab-olites. Also, Rossi and Rubsamen ( 27 )

described a quantitative thin-layer chro-matographic technique for measuring PTHand prothionamide-S-oxide (PTHSO) in bi-ologic fluids, but the limit of sensitivity oftheir method was about 0.2 mg/mi.

In this report, we present our HPLCmethod for the measurement of PTH andPTHSO in biological fluids, and we sum-marize our studies on the metabolic dis-position of PTH and PTHSO in rats andarmadillos.

MATERIALS AND METHODSDrugs and animals. The sources and

characteristics of PTH, PTHSO, and 2-pro-pylisonicotinamide (PINA) used in thesestudies were described previously ( 17) as werethose of DDS and MADDS (21 ). ETH wasobtained from the Pasteur Institut, Paris,France. Other reagents were of the highestpurity available commercially.

Mature male and female Lewis ratsweighing 200-300 g were obtained locally(Simonsen Laboratories, Gilroy, California,U.S.A.) and acclimated to our animal roomsfor at least one week prior to any experi-ment. They were maintained on laboratorychow and water ad libitum. For the acutetests, the rats were fasted 24 hr prior totreatment and during the duration of thestudies. In the chronic studies, the rats werenot fasted. For both intravenous (IV) andoral administrations, PTH and PTHSO weredissolved in polyethylene glycol-200 (J. T.Baker Chemical Co., Phillipsburg, New Jer-

54

51, 1^Peters, et al.: Disposition of Prothionamide^ 55

sey, U.S.A.) to obtain concentrations of 38mg/ml and 42 mg/ml, respectively. All ratsreceived 1.0 ml of the drug solution/kg ofbody weight. The dose of PTH (38 mg/kg)was equivalent to 500 mg PTH given to a70 kg man when the doses are expressed asmg/m 2 of body surface area ( 7). The dose ofPTHSO (42 mg/kg) was equimolar to thePTH dose.

Heparinized blood was collected by ter-minal heart puncture from four ether-anes-thetized rats of each sex at 1, 2, 4, 6, and 8hr after PTH treatment. Plasma was pre-pared immediately and stored frozen. Stud-ies of the disposition of PTHSO given IVand orally followed the same pattern, exceptthat only three animals were sacrificed ateach time post treatment. Also, to examinethe effects of chronic doses of PTH, we com-pared levels of PTH and PTHSO in un-fasted rats that were given a single and eightoral doses of 38 mg PTH/kg. In this case,we also employed three rats of each sex pertime period.

To examine the disposition of PTH andPTHSO in armadillos, we employed onemale and one female animal from our col-ony. These animals had been subjects ofstudies on DDS disposition completed sev-eral months earlier (") and were maintainedon moistened laboratory chow and water adlibitum. They had been purchased from C.P. Chase Co., Inc., Miami, Florida, U.S.A.,and on arrival were tested for infection inDr. A. H. Fieldsteel's laboratories at SRIInternational, Menlo Park, California,U.S.A. No acid-fast bacteria were detectedin ear snips of these animals by the Nu-cleopore filter technique ( 28) prior to thestudies with DDS. In the studies of PTHand PTHSO in these armadillos, we ad-ministered the drugs IV (femoral vein) afterthe animals had been anesthetized with anintramuscular dose of 50 mg ketamine hy-drochloride/kg (Parke, Davis & Co., De-troit, Michigan, U.S.A.). Heparinized bloodsamples were taken by heart puncture afteradministration of a second identical dose ofketamine. To minimize the trauma associ-ated with ketamine, drug administration,and the blood collection, only one bloodsample from the armadillos was taken onone day. Data sufficient for determining thehalf-times of disappearance of PTH andPTHSO were obtained after four or five sep-

arate administrations of drugs over a two-week period, during which time a singleblood sample was obtained at 1, 1.4, 2, 3,or 4 hr after each administration. The ar-madillos were given a two- or three-day restbetween each administration of drugs cou-pled with the single bleeding. For admin-istration, we dissolved appropriate amountsof PTH or PTHSO in polyethylene glycol-200 to obtain doses of 16 mg PTH/kg and18 mg PTHSO/kg when giving the vehicleat 0.5 ml/kg. These doses were equivalentto the doses of PTH and PTHSO used inthe rats when calculated on the basis of mg/m 2 of surface area using surface area param-eters for the rabbit ( 7). Heparinized plasmawas obtained and stored frozen as in the ratstudies.

Preliminary studies revealed that PTHand PTHSO were stable in rat plasma storedat —20°C for 42 days. Others ( 27) reportedno loss of PTH and PTHSO in human plas-ma stored frozen for 12 months.

Extraction procedure and liquid chro-matography. Aliquots (1.0 ml) of experi-mental plasma or control rat plasma con-taining 0, 0.05, 0.10, 0.50, 1.0, 5.0, and10 kig of added PTH or PTHSO were mixedwith 0.10 ml of 1 M phosphate buffer, pH7.0, and then extracted by vigorous me-chanical shaking with 5.0 ml of CHCI 3 for20 min. The mixture was centrifuged to sep-arate the phases, and the aqueous phase wasaspirated off and discarded. An aliquot (4.0ml) of the CHC1, phase was transferred toa tube containing 1.0 ml of 0.1 N HCI. PTHand PTHSO were extracted into the acidphase by vigorous agitation for 10 min. Thephases were separated by centrifugation, and0.90 ml of the acid phase was transferredto a tube containing 0.20 ml of 1 M phos-phate buffer, pH 8.0. The mixture was vig-orously extracted with 5.0 ml of CHCI 3 for20 min. After phase separation by centrif-ugation, the aqueous phase was aspiratedoffand 4.5 ml of the CHCI 3 phase was trans-ferred to a clean tube. Following evapora-tion of the solvent at 40°C under a streamof high purity nitrogen, we dissolved theresidue obtained in 150 yl of the HPLC mo-bile phase, which was chloroform : metha-nol : water (187:14:1). Aliquots (100 Al) wereinjected onto a 4.6 X 250 mm column of5-pcm LiChrosorb SI-60 (Altex Scientific,Inc., Berkeley, California, U.S.A.) using a

56^ International .Journal of Leprosy^ 1983

100-ol fixed loop injection valve (Rheo-dyne, Inc., Cotati, California, U.S.A.). Themobile phase was pumped at a flow rate of1.0 ml/min using a controlled volume mini-pump (Milton Roy Company, RivieraBeach, Florida, U.S.A.), and the column ef-fluent was monitored at 340 nm using an8-pl flow cell (Model 153 Detector, AltexScientific, Inc.). Quantitation was by peakarea using an electronic integrator (Model3380A, Hewlett-Packard, Avondale, Penn-sylvania, U.S.A.). We employed absorptionat 340 nm for quantitation, even thoughPTH and PTHSO absorb maximally inchloroform at 295 nm and 360 nm, respec-tively ( 17), to obtain maximum sensitivitiesfor both compounds and to reduce inter-ference by endogenous materials which ab-sorb less at 340 nm than at lower wave-lengths.

Calculations. Standard statistical meth-ods were employed to calculate the variousparameters used to evaluate the results ofthese studies (8 ).

RESULTSChromatograms showing the elution times

of PTH (4.2 min) and PTHSO (11.8 min)are shown in The Figure. The upper drawingshows the recovery of PTH and PTHSOadded to control rat plasma; the lower one,the peaks of PTH and PTHSO from a ratplasma taken 2 hr after administration ofPTH. Both chromatograms show that en-dogenous materials that absorb at 340 nmare eluted at about 2 min. No other peakswere noted in experimental extracts. Pos-sible interference by other compounds wasexamined in separate experiments. Inter-ference by 2-propyl isonicotinic acid (PINA)was eliminated because it does not absorbat 340 nm. Also, using absorption at 280nm for monitoring column eluates, we es-tablished that PINA was well resolved fromPTH and PTHSO, exhibiting a retentiontime of 8.2 min. ETH was completely re-solved from PTH and elutes at about 6.0min. Thus, as others have done ( 13), ETHcould be used as an internal standard in oursystem also. Finally, using absorption at 254nm for detection, we found that DDS (elu-tion at 3.4 min) and MADDS (elution at6.8 min) were resolved from PTH andPTHSO, even though they do not absorb

PTO

1 1 1I^I 1 1^1 I 1 1

PT1150

PTH

1^I I 1^1 1 l 1 1 1 110 12 1'.

TIM. (min)

THE FIGURE. Elution profiles of (A) an extract ofcontrol rat plasma with 10 pg PTH and 10 pg PTHSOadded per ml; and (B) an extract of rat plasma 2 hrafter administration of PTH.

strongly at 340 nm and are thus noncontri-buting to the assays for the thioamides.

The recoveries of added PTH and PTHSOfrom control rat plasma are shown in TableI. We obtained an average of 97% of addedPTH (range, 88% to 110%) and an averageof 88% of added PTHSO (range, 76% to98%). HPLC elution profiles of extracts ofcontrol plasma containing no added PTHor PTHSO yielded no extraneous peaks or

TABLE 1. The recovery of PTH andPTHSO from rat plasma.

PTH^PTHSO

Found Rccov-^Found Recov-(pg/ml) ery' (%)^(pg/ml) cry' (%)

0^<0.01 <0.010.050^0.045 90 0.048 960.10^0.088 88 0.088 880.50^0.55 110 0.49 981.0^0.97 97 0.84 845.0^5.2 104 4.2 84

10^9.4 94 7.6 76Mean recovery 97 88

Standard deviation 8.4 8.2Coefficient of

variation 8.7 9.4

a The recovery was calculated from directly injectedstandards of PTH and PTHSO.

A

3

1

B

3

o-1-11^1^1^1

Level ofcompound

(pg/ml)

Route of Time afteradminis-^treatmenttration^(hr)

Mean (±S.D.) PTH level (pg/mI)^Mean (±S.D.) PTHSO level (pg/m1)

Male^Female^Male^Female

51, 1^Peters, et al.: Disposition of Prothionamide^57

TABLE 2. Plasma levels of PTH and PTHSO in rats receiving PTH or PTHSO.

38 mg PTH/kg administered (N = 4)IV^1^8.82 ± 2.78

2^1.55 ± 0.744^0.17 ± 0.126^0.03 ± 0.028^0.02 ± 0.02

Oral^1^7.53 ± 2.402^2.81 ± 1.264^0.08 ± 0.066^<0.018^<0.01

17.8 ± 4.868.73 ± 5.140.43 ± 0.360.11 ± 0.080.02 ± 0.02

10.1 ± 4.483.77 ± 2.120.45 ± 0.440.34 ± 0.140.02 ± 0.02

^

7.13 ± 2.82^6.37 ± 1.00

^

3.74 ± 0.14^5.30 ± 1.56

^

0.90 ± 0.36^1.33 ± 0.54

^

0.25 ± 0.16^0.86 ± 0.28

^

0.11 ± 0.10^0.14 ± 0.10

^

9.08 ± 0.54^3.90 ± 0.82

^

5.86 ± 0.88^3.27 ± 0.74

^

0.86 ± 0.48^1.52 ± 0.68

^

0.06 ± 0.04^1.65 ± 0.28<0.01^0.29 ± 0.20

42 mg PTHSO/kg administered (N = 3)IV^

1^0.78 ± 0.432^0.22 ± 0.034^<0.016^0.04 ± 0.078^<0.01

Oral^1^1.23 ± 0.882^0.22 ± 0.164^<0.016^<0.018^<0.01

4.79 ± 2.600.77 ± 1.040.04 ± 0.050.01 ± 0.02

<0.01

2.06 ± 3.051.39 ± 1.230.05 ± 0.02

<0.01<0.01

^

6.04 ± 1.37^7.78 ± 0.97

^

2.30 ± 0.16^2.72 ± 1.38

^

0.20 ± 0.21^0.63 ± 0.38

^

0.39 ± 0.28^0.24 ± 0.17

^

<0.01^0.12 ± 0.16

^

4.83 ± 1.11^2.84 ± 2.03

^

2.06 ± 0.88^2.21 ± 1.75

^

0.37 ± 0.07^0.63 ± 0.19

^

<0.01^0.31 ± 0.19

^

0.07 ± 0.04^0.07 ± 0.12

tailing in the region of elution of either drug.From the noise level of the HPLC traces,we estimate that the practical limit of sen-sitivity is 0.01 pg of either compound perml of plasma. This was about five-fold lowerthan our target limit sensitivity of 0.05 Ag/ml.

The results of all studies in fasted ratsreceiving single doses of 38 mg PTH or 42mg PTHSO per kg are summarized in Table2. The first five lines show the mean plasmavalues of PTH and PTHSO found in themale and female rats receiving PTH IV. ThePTH disappeared very rapidly in both sexes,and we calculated t1/2 values of 0.74 hr and0.69 hr, respectively, for the male and fe-male rats (Table 4). As shown in Table 2,the corresponding plasma levels for PTHSOdeclined more slowly in both sexes, and thisdelayed disappearance was reflected in thet1/2 values of 1.2 hr found for both sexes asshown in Table 4. In both sexes, the slopesof the regression lines for PTHSO were sig-nificantly greater than the correspondingslopes for PTH (p < 0.005). Also, compar-isons of the areas under the curves (AUCs)for PTH and PTHSO in the two sexes (firsttwo lines of Table 5) show that the amount

of PTH in the plasma during 8 hr was sub-stantially less in the male than in the female.This was apparently a result of the moreextensive conversion of PTH to PTHSO bythe male compared to the female. Thus, ofthe total of PTH and PTHSO of 25.6 (pg-hr/m1) in the male rat shown in the lastcolumn of Table 5, 55% was due to PTHSO;whereas the corresponding fraction due toPTHSO in the female rat was 35%. Also,the larger amount of unchanged PTH in thefemale was reflected in the total of PTH andPTHSO being about twice that in the malerat.

The plasma levels of PTH and PTHSOobserved in the two sexes after oral admin-istration of the same dose of PTH are shownin the second entry of Table 2. Again PTHlevels declined more rapidly than PTHSOlevels in both sexes, and this was reflectedin significantly shorter (p < 0.001) t1/2 val-ues for PTH than for PTHSO (second entryof Table 4). Also, after oral dosing, the t1/2values for both PTH and PTHSO were sig-nificantly shorter (p < 0.001) in the malerats than in the female rats. Finally, theAUCs of Table 5 indicate again that themale rat formed PTHSO to a greater extent

58^ International Journal of Leprosy^ 1983

TABLE 3. Plasma levels of PTII and PTHSO in imfasted rats receiving acute and chronicoral doses.

Number oftreatments

Time afterlast Mean (±S.D.) PTH level (pg/ml) Mean (±S.D.) PTHSO level (pg/m1)

treatment(hr)

Male Female Male Female

1 1.20 ± 1.04 5.96 ± 0.82 4.70 ± 1.77 5.31 ± 0.502 0.83 ± 0.87 1.99 ±^1.11 3.20 ± 1.96 3.42 ± 0.194 0.02 ± 0.02 0.08 ± 0.02 0.85 ± 0.03 0.95 ± 0.216 0.02 ± 0.03 0.50 ± 0.54 0.60 ± 0.21 1.73 ± 1.208 <0.01 0.05 ± 0.07 0.24 ± 0.24 0.62 ± 0.61

8 1 1.25 ± 0.68 4.06 ± 4.40 4.43 ± 0.81 2.95 ± 0.920.34 ± 0.24 1.52 ± 2.58 2.51 ± 0.90 1.80 ± 2.36

4 0.03 ± 0.05 0.50 ± 0.61 0.69 ± 0.45 1.11^± 0.246 0.11 ± 0.02 0.18 ± 0.22 1.21±0.170.17 0.79 ± 0.618 0.01 ± 0.02 0.09 ± 0.07 0.33 ± 0.26 0.65 ± 0.29

than did the female rat, although the totalsof PTH and PTHSO were nearly identicalin the two sexes receiving oral PTH.

Comparisons of the slopes of the regres-sion lines from oral and IV treatments withPTH indicated that the 0/2 values for bothPTH and PTHSO were significantly shorterfollowing oral than IV treatments in the malerat. However, in the female rat, the oppositewas observed with the oral route yielding aslightly but nonsignificantly longer t1/2 val-ue for PTH after oral versus IV treatment;the t'/2 value for PTHSO was significantly(p < 0.02) longer following oral than IVtreatment with PTH.

The lower half of Table 2 lists the meanvalues of PTH and PTHSO found in ratsreceiving a dose of PTHSO equimolar tothe PTH given. It is immediately apparentthat PTHSO was readily converted to PTHin both sexes receiving PTHSO by eitherroute. The t'/2 values calculated from thesedata are shown in the fifth and sixth entriesof Table 4. Following IV treatment withPTHSO, our comparisons of the slopes ofthe regression lines for PTH and PTHSO inboth sexes indicated no significant differ-ences in any of the four possible compari-sons. Similarly, following oral treatmentwith PTHSO, the two sexes were not sig-nificantly different in the t1/2 values for PTHand PTHSO, respectively. However, in bothsexes receiving oral PTHSO, we found thatthe t1/2 values for PTH were significantlyshorter (p < 0.05) than those for PTHSO,again emphasizing that PTHSO was re-tained longer in the rat than was PTH. Allfour comparisons possible between IV and

oral treatments with PTHSO in regard tosex and compound did not yield significantdifferences between the slopes of the regres-sion lines.

The AUCs derived from these data (lasttwo lines of Table 5) are revealing from sev-eral points of view. First, the values fromboth routes of administration of PTHSOindicate that the female rat reduced PTHSOto PTH more efficiently than did the malerat. Also, as in the results after IV dosingwith PTH, we found that the total of PTHand PTHSO was higher in the female ratthan in the male rat after IV dosing withPTHSO. Again, this sex difference was notfound following oral treatment with PTHSO,as was the case with PTH. Perhaps the mostimportant observation was that the total ofthe two compounds was substantially lesswhen PTHSO was given as compared toadministering PTH, even though the t'/2values were not different. Thus, givingPTHSO would be less efficient therapeuti-cally than giving PTH, even though PTHSOis retained in the body longer.

To test whether PTH would induce itsown metabolism, we compared the plasmadecay patterns of PTH and PTHSO in un-fasted rats receiving a single and eight dailydoses of 38 mg PTH/kg (Table 3). No strik-ing differences were noted among the levelsafter these two regimens. The t'/2 valuescalculated from the data are presented inthe third and fourth entries of Table 4. Testsfor significant differences between the slopesfor all eight possible combinations of sex,compound, and number of treatments didnot yield any significant differences. Also,

51, 1^Peters, et al.: Disposition of Prothionamide^59

TABLE 4. Hall:times of disappearance (PM of PTH and PTHSO in the rat studies ofPTH and PTHSO.

Drug given andstudy conditions' Sex

PTH PTHSO

N t1/2 (hr) r N t1/2 (hr)

PTH, IV Male 18 0.74 -0.930 20 1.2 -0.953Female 19 0.69 -0.954 20 1.2 -0.938

PTH, oral Male 13 0.43 -0.979 15 0.70 -0.979Female 19 0.86 -0.928 20 1.9 -0.832

PTH, oral, unfasted Male 10 0.78 -0.726" 15 1.4 -0.865Female 14 1.0 -0.780 15 2.0 -0.766

PTH, oral X 8, unfasted Male 10 1.5 -0.861' 12 2.1 -0.852Female 14 1.4 -0.667b 14 3.3 -0.666b

PTHSO, IV Male 8 1.2 -0.688" 12 1.1 -0.774'Female 10 0.84 -0.851' 15 1.0 -0.924

PTHSO, oral Male 6 0.40 -0.841" 10 1.3 -0.898Female 9 0.73 -0.798b 12 1.7 -0.763'

All studies were acute and all rats were fasting unless otherwise noted." p < 0.01; all others were <0.001 unless otherwise noted.

p < 0.005.d p < 0.05.

in either sex, no substantial differences werenoted in the AUCs for PTH, PTHSO, orthe total of PTH and PTHSO (third andfourth entries of Table 5). Again, a substan-tial sex difference was noted with the malerats forming relatively more PTHSO thanthe female rats, and the latter exhibitinghigher totals, especially after only one doseof PTH. Also important was the observa-tion that the AUCs for PTH, PTHSO, andthe total of both were substantially less inthese unfasted rats compared to the rats re-ceiving a single oral dose after a 24 hr fast.

Thus, this study not only demonstrated thatPTH did not induce its own metabolism inthe rat, it also demonstrated that the ab-sorption of the compound was less in non-fasting animals.

Finally, Table 6 summarizes our limitedstudies of the disposition of intravenousPTH and PTHSO in armadillos. We foundthat this species also oxidized PTH toPTHSO, and that the rate of plasma declineof PTH was significantly (p < 0.01) morerapid than that of PTHSO. However, fromthe calculations of the AUCs it is apparent

TABLE 5. Areas under the plasma curves (AUC) in the rat studies of PTH or PTHSO.

Drug given and study conditions' SexAUC (µg-hr/ml) for 0-8 hr

PTH PTHSO" Total"

PTH, IV Male 11.6 14.0 25.6Female 32.0 17.3 49.8

PTH, oral Male 11.9 18.1 30.0Female 17.4 14.1 31.5

PTH, oral, unfasted Male 2.5 11.6 14.1Female 10.2 15.1 25.3

PTH, oral X 8, unfasted Male 2.0 11.3 13.3Female 7.8 9.3 17.1

PTHSO, IV Male 1.2 9.8 11.0Female 6.0 12.6 18.6

PTHSO, oral Male 1.6 8.0 9.6Female 4.2 7.4 11.6

All studies were acute and all rats were fasting unless otherwise noted.b Expressed as PTH equivalents.

60^ International Journal of Leprosy^ 1983

TABLE 6. Plasma levels and kinetic parameters in artudillos receiving 16 mg PTH/kgor 18 mg PTHSO/kg intravenously.

Drug administered Time after treatment(hr)

Mean (±AD)" level (pg/ml)

PTH^ PTHSO

PTH^ 11.4

34

AUC (µg-hr/ml)t1/2 (hr)

pPTHSO^

11.423

AUC (µg-hr/ml)t1/2 (hr)

p

3.20 ± 0.202.98 ± 0.341.50 ± 1.040.14 ± 0.06

0.155.10.52

-0.894<0.005

0.210.28 ± 0.120.34 ± 0.11

0.050.581.0

-0.610>0.1

1.43 ± 0.011.41 ± 0.211.10 ± 0.060.64 ± 0.120.31 ± 0.11

3.1b1.3

-0.932<0.00 1

3.231.88 ± 0.081.56 ± 0.120.46 ± 0.12

4.3b0.73

-0.955<0.001

Mean of two observations ± average deviation from the mean.b Expressed as PTH equivalents.

that the total levels of PTH exceeded thoseof PTHSO. When an equimolar dose ofPTHSO was given, our finding of PTH levelsindicates that the armadillo also reducesPTHSO to PTH as does the rat, even thoughin both species the oxidation pathway ap-parently predominates. The t1/2 value of0.73 hr for the administered PTHSO wassignificantly shorter (p < 0.05) than the val-ue of 1.3 hr for PTHSO formed followingPTH administration. These t1/2 values areof the same magnitude as we found in rats,but the AUC values for armadillos are sub-stantially less than for rats (Table 5) eventhough the doses were equivalent on a mg/m 2 basis. It should be noted, however, thatno 8 hr plasma levels were measured in thearmadillo.

DISCUSSIONOur findings that PTH and PTHSO were

interconvertible in rats and armadillos arein agreement with earlier studies of ETH inanimals and man (1, 9, 14

). In addition, Rossi

and Rubsamen ( 27) recently reported plasmalevels of PTHSO in patients that were ap-proximately equivalent to levels of PTH ob-served during 6 hr following treatment withPTH. Our estimates from their plasma de-cay curves for PTH and PTHSO suggest t 1/2values of approximately 3 hr and 4 hr, re-

spectively. It would appear that PTHSO isretained longer than PTH in man as wefound in the rats and armadillos. Thesecombined results coupled with the knowl-edge that the antibacterial activities of thesethioamides reside solely in the parent drugsand their sulfoxides (2, 6. 17

) emphasizes theneed to consider the sum of these activecompounds rather than the parent drugsalone when considering therapeutic effica-cies. Therefore, peak plasma concentrationsof active drugs in man following thioamideadministration of 2 pg/ml to 3 µg/m1 (". 3")should be approximately doubled to 4 µg/ml to 6 kig/m1when estimating ratios of peakconcentrations of these compounds to theirMIC of 0.05 µg/ml. Under these circum-stances, the peak ratios would be 80 to 120instead of the range of 40 to 60 estimatedpreviously ( 31 ). However, t1/2 values in manfor the thioamides and their S-oxides of 2hr to 4 hr ( 13 . 27) would still limit the esti-mated time that plasma levels would be ex-pected to exceed the MICs to about 24 hr.Thus, daily treatment with the thioamideswould still appear advisable.

The mechanism of the antibacterial ac-tion of the thioamides is unknown, but nu-merous reports indicate that these com-pounds exert their antibacterial effectsdifferently from other drugs. Thus, Al. tu-

51, 1^Peters, et al.: Disposition of Prothionantide^61

berculosis resistant to isoniazid, strepto-mycin, p-aminosalicylic acid, viomycin, cy-closerine, or thiacetazone are sensitive toETH ( 26); Al. leprae resistant to DDS andRFM are sensitive to ETH ( 12); and com-binations of ETH with clofazimine, DDS,or RFM act additively in mice infected withM. leprae ( 29). It is apparent that the thioam-ides are excellent candidates for combina-tion drug regimens in the chemotherapy ofeither leprosy or tuberculosis.

The in vivo interconversion of PTH andPTHSO also parallels the more extensivelystudied hepatotoxic thioamides, thioacet-amide ( 25 ) and thiobenzamide ( 3). These lat-ter thioamides are activated to toxic S-oxidemetabolites by mammalian microsomal ox-idases. It is not known whether ETH or PTHare acted on by these oxidases or by thosein mammalian microsomes known to oxi-dize thioether moieties of numerous pesti-cides to sulfoxides (")). In the current stud-ies, no obvious toxicities of PTH or PTHSOwere noted in either the rats or the arma-dillos, although the experiments were notdesigned to assess the relative toxicities ofthe compounds. Because ETH, PTH, andtheir S-oxides are equally active against cul-tivable bacteria and because PTH andPTHSO were equally active against Al. lep-rat, in mice, it seems unlikely that ETH andPTH and their S-oxides exhibit markedlydifferent toxicities as do the thioacetamideand thiobenzamide S-oxides from their par-ent compounds.

Our finding that male rats oxidize PTHto PTHSO more readily than do female ratsis apparently another ramification of thewell-known sexual dimorphism in rats forthe oxidation of many xenobiotics ("). Also,our inability to detect any differences inplasma patterns of PTH and PTHSO in ratsreceiving multiple doses from those foundafter a single dose indicates that, using thecurrent doses, PTH did not induce its ownmetabolism.

SUMMARYA new method for the sensitive and se-

lective measurement of prothioamide (PTH)and its S-oxide metabolite (PTHSO) in bi-ological fluids was described. The limit ofsensitivity was approximately 0.01 pg ofdrug/ml of plasma. Endogenous materials,

2-propylisonicotinamide, ethionamide,dapsone, or monoacetyl dapsone did notinterfere or contribute. Rats receiving PTH,intravenously or orally, showed a sexual di-morphism in the ability to oxidize PTH toPTHSO, with males exhibiting greater ca-pacities for this conversion. Both sexescleared the administered PTH more rapidlyfrom the plasma than the metabolite,PTHSO. Following oral or intravenousadministration of equimolar doses ofPTHSO, both sexes exhibited an ability toreduce the administered PTHSO to PTH,with the female showing greater capacitiesfor this conversion. Clearances after oralPTHSO administration were again morerapid for PTH than for PTHSO in bothsexes. However, the total of PTH andPTHSO in the plasma during 8 hr followingPTHSO administration was consistently lessthan following PTH dosing. Therefore, al-though PTHSO is retained longer than PTHafter either PTH or PTHSO administration,giving PTHSO yielded less total active drugin the circulation. Comparison of plasmapatterns of PTH and PTHSO in unfastedrats receiving one oral or eight daily oraldoses of PTH did not indicate that PTHinduces its own metabolism. Limited stud-ies in armadillos receiving PTH and PTHSOintravenously led to the same general con-clusions as those we derived from the ratstudies regarding the disposition of PTH andPTHSO.

RESUMENSe describe un nuevo mOtodo, sensible y selectivo,

para la mediciOn de protionamida (PTH) y de su me-tabolito sulfOxido (PTHSO) en liquidos biolOgicos. Lasensibilidad del método es de aproximadamente 0.01

pg de droga por ml de plasma. Otros materiales en-dOgenos (2-propil-isonicotinamida, etionamida, dap-sona y monoacetil-dapsona) no interfieren en la deter-

minaciOn. Las ratas que recibieron PTH por la via oralo intravcnosa mostraron un dimorfismo sexual en

cuanto a su capacidad para oxidar la PTH a PTHSO;los machos mostraron la mayor capacidad oxidativa.Ambos sexos eliminaron mas rapid() de su plasma a

la PTH que a la PTHSO. Ambos sexos tuvieron lacapacidad de reducir al sulfOxido (PTHSO) a PTH,

pero las hembras mostraron la mayor capacidad re-

ductora. En ambos sexos, la capacidad de depuraciOndespues de la administraci6n oral de PTHSO fue otra

vez mas rapida para la PTH que para la PTHSO. Sin

embargo, el total de PTH y PTHSO fue consistente-

mente menor que cuando se administr6 PTH. Por lo

6 ")^ International .Journal of Leprosy^ 1983

tanto, aunque la PTHSO se retiene mas tiempo que laPTH despues de la administraciOn de PTH o de PTHSO,la administraciOn de PTHSO produce menos drogaactiva total en circulaciOn. La comparaciOn de los ni-veles plasmdticos de PTH y PTHSO cn ratas que re-cibieron I u 8 dosis orates do PTII no indicaron quela PTH induzca su propio metabolismo. Algunos es-tudios en armadillos que recibieron PTH y PTHSOintravenosamente, condujeron a las mismas conclu-siones generates que las derivadas de los estudios conratas en cuanto at manejo do PTH y PTHSO.

RESUMEOn decrit ici une nouvelle methode miss au point

pour mesurer de maniere, II la Ibis sensible et selective,le prothioamide (PTH) et son metabolite S-oxyde(PTHSO) dans des liquides biologiques. La limite dela sensibilite de la methode se situait approximative-mcnt a 0.01 Ag par ml de medicament dans le plasma.Des produits endogenes, de meme que le 2-propyli-sonicotinamide, l'ethionamide, la dapsone, ou lamonoacetyl dapsone, n'interferaient pas avec ces me-sures. Des rats recevant de la PTH, soil par voie in-traveineuse, soit par voie orate, ont temoigne d'un di-morphisme lie au sexe quant a leur capacite a oxyderle PTH en PTHSO, la capacite de conversion slantplus prononcee chez les males. Les deux sexes elimi-naient plus rapidement du plasma, la PTH adminis-tree, que cela n'êtait le cas pour le metabolite, le PTHSO.A la suite de l'administration de doses equimolairesde PTHSO, par voles orate ou intraveineuse, l'un etl'autre sexe ont temoigne d'une capacite a reduire lePTHSO administre en PTH, cette capacite a convertirIc produit en son metabolite &twit plus prononcee chezla femelle. L'elimination emit egalement plus rapidepour la PTHSO, apres administration orate de PTHSO,et ceci dans les deux sexes. Neanmoins, le contenu totalde PTH et de PTHSO dans Ic plasma au cours des 8heures suivant l'administration do PTHSO etait re-gulierement plus faible que la quantitó mesuree apresl'administration de PTH. Des lors, on constate quel'administration de PTHSO libere une quantite totalede medicament actif plus faible dans la circulation,malgre Ic fait que le PTHSO soit retenu plus longtempsque le PTH apres l'administration de l'un ou l'autredes produits. La comparaison des profits plasmatiquesde PTH et de PTHSO chez des rats non soumis aujeane et recevant one ou huit doses orates journalieresde PTH, ne permet pas de conclure que le PTH entraineson propre metabolisme. Des etudes limitees chez lestatous recevant du PTH et de PTHSO par voie intra-veineuse a conduit aux memes conclusions generatesque celles tirées a partir des etudes menses sur les rats,en ce qui concerne Ic PTH et le PTHSO.

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 AI-08214.

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