Serum concentrations and antituberculosis activity of thiacetazone

Tubercle (1974), 55,41

SERUM CONCENTRATIONS AND ANTITUBERCULOSIS ACTIVITY OF THLKETAZONE By G. A. ELLARD,JEANM. DICKINSON,PATRICIAT. GAMMONand D. A. MITCHISON from the MRC Unit for Laboratory Studies of Tuberculosis, Royal Postgraduate Medical School, Ducane Road, London, WI2 OHS

SUMMARY New ultraviolet, calorimetric and fluorimetric methods are described for the determination of thiacetazone concentrations down to 0.3-0.6 pg/ml in serum and urine. Similar results were obtained when the different methods were applied to sera from patients being treated with thiacetazone-containing regimens. Peak thiacetazone serum concentrations were approximately proportional to dosage when single doses of 150 to 600 mg thiacetazone were given. The thiacetazone serum concentrations of Fhinese, Indian and Malay tuberculous patients from Singapore who were being treated with daily regimens containing 150 mg thiacetazone were similar and were unaffected by concomitant administration of a vitamin and antihistamine supplement or by streptomycin. The urinary excretion of unchanged thiacetazone by tuberculous patients in Singapore and Kenya averaged about 20 per cent of the dose. Significant deacetylation of the drug was not detected. Slide culture sensitivity tests showed that strains from 12 Kenyan patients were considerably more sensitive to thiacetazone than strains from 107 Hong Kong patients. However, a concentration of 0.4 pg/ml completely inhibited all of the Kenyan strains and partially inhibited 77 per cent of the Hong Kong strains. From consideration of the results of clinical trials of regimens of isoniazid and thiacetazone, the minimal concentration of thiacetazone in the lesions necessary to prevent the emergence of isoniazid resistance appeared to be about O-4 pg/ml in E. Africa and Hong Kong. It is suggested that the efficacy of thiacetazone-containing regimens is dependent not only on the characteristics of tubercle bacilli (sensitivity and virulence) but also on the defence mechanisms of the patient. Before such a regimen is introduced for wide scale use in a new area, its efficacy should first be established by means of a controlled clinical trial.

RBSUMI? De nouvelles methodes ultraviolette, colorim&rique et fluoromCtrique pour la mesure des concentrations sCriques et urinaires de thiacttazone jusqu’h 0,3 et 0,6 pg/rnl sont d&rites. Des rCsultats similaires ont CtCobtenus quand ces diffkrentes m&hodes ont CtCappliqutes g des serums de malades trait& par des r@mes thdrapeutiques contenant de la thiadtazone. Le pit des concentrations sCriques de thiacttazone a et6 g peu p&s proportionnel au dosage quand des doses individuelles de 150 zi 600 mg de thiacdtazone ont 6tB don&es. Les concentrations skriques de thiagtazone trouvhes chez des malades tuberculeux Chinois, Indiens et Malais de Singapour, trait& par des r&imes quotidiens contenant 150 mg de thiac&azone ont CtC semblables et ne furent pas a&&es par l’administration

42

I-LI.AKl>

AhI>

Ol-HkKS

concommittente d’un supplement en vittamine et antihistamine ou par la streptomycrnc. L’excretion urinaire de thiacetazone intacte par les malades tuberculeux h Singapour et significative de In au Kenya representait en moyenne 20”;, de la dose. Une desacetylation drogue n’a pas et6 d&eke. Des tests de sensibilite par cultures sur lames ont montre que les souches de I2 malades du Kenya Ctaient beaucoup plus sensibles a la thiacetazone que les souches de I07 malades de Hong-Kong. Cependant une concentration de 0,4 :*g/ml a completement inhibe la totalite des souches du Kenya et seulement partiellement 77?/<, des souches de HongKong. L’etude des resultats dessais cliniques avec des regimes comportant isoniazide et thiacetazone suggere que la concentration minimale de thiacetazone dans les lesions

necessaire pour prevenir l’emergence de la resistance a l’isoniazide semble &tre 0,4 ;*g/ml en Afrique de l’est et a Hong-Kong. Ceci suggere que I’efficacite des regimes therapeutiques contenant de la thiacetazone depends non seulement des caracteristiques des bacilles tuberculeux (sensibilite ou virulence) mais aussi des mecanismes de defence du malade. Avant qu’un tel regime therapeutique soit introduit sur une large Cchelle dans une nouvelle

zone, son efftcacite devrait &tre Ctablie par un essai clinique

contrBlC.

RESUMEN Se describen nuevos metodos ultravioletas, colorimetricos y fluorimetricos para determinar la concentration de thiocetazona en suero y orina por debajo de 0,3-0,6 pg/ml. Se obtuvieron resultados similares cuando les diferentes mttodos se aplicaron en sueros de pacientes tratados con esquemas que contenian thioacetazona. La concentration pica de thioacetazona en el suero era aproximadamente proportional a la dosis cuando se daba thioacetazona entre 150 a 600 mg en dosis unica. Las concentraciones sericas de thioacetazona en pacientes tuberculosos chinos, indios y malayos de Singapur trados con esquemas diarios que contenian 150 mg de thioacetazona eran similares y no se modificaban por la administration concomitante de vitaminas, antihistaminas o estreptomicina. La excretion urinasia de thioacetazona integra por pacientes tuberculosos en Singapur y Kenya oscilo en el 20% de la dosis. No se detect6 una desacetilacion manifiesta de la droga. Los test de sensibilidad en lamina mostraron que las cepas de 127 pacientes de Kenya eran mas sensibles a la thioacetazona que las cepas de 107 pacientes de Hong Kong. Sin embargo, una concentration de 0,4 pg/ml inhibia completamente todas las cepas de Kenya e inhibia parcialmente el 77 %, de las cepas de Hong Kong. De acuerdo con 10s resultados de ensayos clinicos en esquemas con isoniazida y thioacetazena la concentraci6n minima de thioacetazona a nivel de1 foeo necesaria para prevenir la aparici6n de resistencia a la isoniazida parece ser alrededer de 0,4 pg/ml en Africa Oriental y Hong Kong. Se sugiere que la eficacia de 10s esquemas que contienen thieacetazona depende no ~610 de las caracterfstieas de1 bacilo (sensibilidad y virulencia) sino tambitn de 10s mecanismos de defensa de1 paciente. Antes de introducir un esquema de este tipo para uso en gran escala en una nueva regi6n es necesario establecer primer0 su eficacia por medio de un ensayo clinico controlado.

SERUM CONCENTRATIONS OF THIACETAZONE

43

ZUSAMMENFASSUNG Beschrieben werden neue kolorimetrische und fluorimetrische Methoden sowie solche mit UV-Licht zur Bestimmung der Thiazetazonkonzentration im Serum und Urin im Bereich von 0,3-0,6 pg/ml. Gleiche Ergebnisse wurden erhalten, wenn mit den verschiedenen Methoden die Seren von Patienten untersucht wurden, die mit Regimen unter Einschlug von Thiazetazon behandelt wurden. Die maximale Serumkonzentration von Thiazetazon war ungefahr proportional zu Einzeldosen von 150 bis 600 mg Thiazetazon. Die Serumkonzentration von Thiazetazon war bei Chinesen, Indern und Malaien mit Tuberkulose gleich hoch, wenn sie mit 150 mg Thiazetazon enthahenden Regimen behandelt wurden. Zusatzliche Gaben von Vitaminen, Antihistamin oder Streptomyzin blieben ohne Einflug. Im Urin wurden bei Patienten in Singapur und Kenia ungefahr 20 % der verabreichten Dosis unverandert ausgeschieden. Eine Deazetylierung nennswerten Umfangs liet3 sich nicht festellen. Sensibilitatsprtifungen mit Objekttragerkulturen ergaben, da5 die Stamme von 12 Patienten aus Kenia wesentlich starker gegen Thiazetazon empfindlich waren als die von 107 Patienten aus Hongkong. Mit einer Konzentration von 0,4 pg/ml wurden alle Stamme aus Kenia vollstandig und 77% der Stamme aus Hongkong partiell gehemmt. Wenn man von den Ergebnissen klinischer Studien mit Isoniazid und Thiazetazon ausgeht, so dtirfte die minimale Thiazetazonkonzentration, mit der das Auftreten einer Isoniazidresistenz verhindert werden kann, bei etwa 0,4 kg/ml sowohl in Ostafrika als such in Hongkong liegen. Es wird die Auffassung vertreten, da5 die Wirksamkeit eines Regimes mit Thiazetazon nicht nur von den Eigenschaften der Tuberkulosebakterien (Empfindlichkeit und Resistenz) abhangt sondern such von der Abwehrlage des Patienten. Ehe ein derartiges Regime auf breiter Basis in einem neuen Bereich eingeftihrt wird, sollte seine Wirksamkeit zuvor durch eine kontrollierte Therapiestudie erwiesen werden.

Iutroduetlon The efficacy of the inexpensive combination of 150 mg thiacetazone (thioacetazone, p-acetylamino-benzaldehyde thiosemicarbazone) plus 300 mg isoniazid for the daily treatment of pulmonary tuberculosis, with or without an initial supplement of up to 2 months daily treatment with 1 g streptomycin, has been demonstrated in several controlled clinical studies (East African/ British Medical Research Council Investigations, 1963a; 1966; Tuberculosis Chemotherapy Centre, Madras, 1966; Hong Kong Anti-tuberculosis Association and Government Tuberculosis Service/British Medical Research Council Investigation, 1968; International Union Against Tuberculosis, 1970). The incidence of side-effects due to thiacetazone has been studied in two international co-operative investigations (Miller and others, 1966; 1970; 1972; Ferguson and others, 1971). A study has also been made of the adverse reactions encountered after giving high individual doses of thiacetazone combined with high dosage isoniazid (14 mg/kg) twice a week (Fox and others, 1974). Such a combination if therapeutically effective would as an entirely oral regimen be particularly convenient to supervise. This report describes the measurements of thiacetazone serum concentrations that were undertaken in conjunction with several of these investigations and secondly the estimation of the minimal concentration of thiacetazone in lesions that would prevent the emergence of isoniazid resistant tubercle bacilli during treatment with regimens of isoniazid and thiacetazone. Thiacetazone can be determined in serum by extracting into a suitable organic solvent and measuring its ultraviolet absorption (maximum near to 333 nm) (Burke & Titus, 1950; Spinks, 1949; 1951; Wernitz & Tornuss, 1952). Alternatively, thiacetazone can be hydrolysed to p-amino-

44

I I.1

ARI)

ANI)

OI’III

RS

benzaldehyde which can then be determined calorimetrically b! diazotisation and coupling (Hur!,c 1950; Hendricks and other\ & Titus. 1950; Chatterjee & Bose. 1953: Heilmeyer & Heilmeyer. 1950: Short 1961 ; Wernitz & Tornuss, 1952: Wollenberg, 1950). None of these methods. however. is suficiently set-um when

sensitive

to determine

accurately

the concentrations

of thiacetuone

present

111rhc

normal therapeutic: dose5 of the drug are given. Furthermore drugs such ah pyrazinamide and rifampicin interfere with the determination of thiacetazone by ultraviolet methods, and /I-amino-salicylic acid when it is estimated calorimetrically. Interference can also be anticipated from several potential metabolites of thiacetazone. although little is known concerning the actual routes by which thiacetazone is metabolised in man. Estimation of the minimal inhibitory concentration of thiacetazone from ;/I Gtro titration5 presents difficulties

not encountered

with

other

antituberculosis

drugs. Since it is a bacteriostatic

drug, unstable

in culture media. allowance must be made for its deterioration during incubation of the titrations (Rist, Gals $r Jullien. 1951 ; Lcat 61 Marks. 1970). There is also no clear-cut endpoint of inhibition; instead. increasing inhibition of growth of tubercle bacilli extends over a wide range of concentrations. Finally, strains may contiin a proportion of naturally resistant organism?. varying according to the region of the world from which the strain was obtained (Grosset & Benhassine. 1970). The sensitivity of strain5 of tubercle bacilli was therefore studied in slide culture sensitivity tests in which short incubation observation of the inhibitory action of the drug.

periods

can be Llsed and which

allow

direct

Methods Detetmination

qf’thiacetazone

in SPIW~I und urim

Four different methods were used to determine thiacetazone. The first method was slightly modified from that originally devised by Wernitz & Tornuss (1952). The other three methods were novel. Method I (Ultraviolet). A sample (3 ml) of serum or urine was extracted by shaking in a small separating funnel with I ml M K,HPO, and 8 ml choloroform/amyl alcohol (4:1 by volume). The lower phase was then dried by filtering through anhydrous sodium sulphate and the absorption of the extract measured at 333 nm using a Beckman DBG spectrophotometer. The absorption of the extract was also scanned on a Unicam SP 800 Spectrophotometer to check that the extracts were free from abnormal ultraviolet-absorbing compounds. Method 2 (Ultraviolet). This method was identical to the first with the exception that amyl alcohol was replaced by propan-2-01. Method 3 (Calorimetric). Thiacetazone was first extracted into chloroform/propan-2-01 (4: I by volume) as described in Method 2. After filtering through anhydrous sodium sulphate, 4 ml of the extract was slowly dried in a small centrifuge tube by heating at 85°C. An aliquot (2 ml) of a freshly prepared mixture of n-heptanol/methanol/concentrated HCI (2:1 :I by volume) was then added and the mixture heated for 1 hr at 100°C to convert thiacetazone to p-amino-benzaldehyde. After cooling, 0.5 ml 2N HCI and 6 ml n-heptane were added and the mixture agitated for 15 set on a vortex mixer. This procedure led to the extraction of the liberated p-amino-benzaldehyde into 2N HCI, a portion (0.4 ml) of which was then reacted by a modification of the Bratton and Marshall (1939) procedure after the addition of 0.1 ml ethanol. Diazotisation was achieved by adding 0.01 ml aqueous 1 per cent sodium nitrite, excess nitrite being destroyed 5 min later by the addition of 0.01 ml aqueous 10 per cent ammonium sulphamate. After a further 5 min, 0.01 ml of 2 per cent N-I-N-naphthylethylene-diamine-dihydrochloride in acetone/water (1 :I by volume) was added. and after standing in the dark for 30 min the optical density was determined at 550 nm in a Unicam SP 600 spectrophotometer using micro-cells.

SERUM

CONCENTRATIONS

OF

THIACETAZONE

45

Method 4 (Fluorimetric). Thiacetazone was extracted as in Method 2 and a 4 ml portion of the extract dried as in Method 3. Thiacetazone was then oxidised to p-acetylamino-benzoic acid by adding 1 ml O*lN NaOH and 0.1 ml O.lM KMNO,, agitating for 1 min on a vortex mixer and standing for a further 10 min at room temperature. After destroying excess permanganate by adding 0.5 ml aqueous 1 per cent hydroxylamine hydrochloride, the solution was acidified with 1.1 ml N H&SO, and p-acetylamino-benzoic acid hydrolysed top-amino-benzoic acid by heating for 30 min at 100°C. After cooling, 3 ml of pH 5.8 citric acid/phosphate (Mcllvaine) buffer was added and the mixture extracted by shaking with 4 ml ethyl acetate. Finally, p-amino-benzoic acid was recovered by shaking 3 ml of the ethyl acetate extract with 1 ml O*lN NaOH and its fluorescence measured at 280/340 nm in an Aminco-Bowman spectrophotometer after adding O-5 ml ethanol to O-5 ml of the O.lN NaOH extract. Extracts with fluorescence greater than that given by 0.5 pg/ml p-aminobenzoic acid in this solvent system were diluted appropriately to avoid errors due to concentration quenching of the fluorescence. Stand&c - Serum samples were analysed in batches of approximately 75 together with standards in quadruplicate consisting of 3 ml aliquots of water and of normal blank serum containing 0 and 5 ilg/ml(3 pg/ml for the fluorimetric method) thiacetazone respectively. The samples and standards were coded and randomised before extraction. The standards were prepared from a stock solution containing 1 mg/ml thiacetazone in ethanol, which could be stored for many months at -20°C without appreciable decomposition. When urinary concentrations of thiacetazone were determined an aqueous standard containing 10 ,ug/ml thiacetazone was employed. Concentrated urine samples were diluted appropriately before extraction. Determination qfp-aminobenzaldehyde-thiosemicarbazone

and p-acetylamino-benzoic acid in urine

These compounds are potential metabolites of thiacetazone. The former compound was extracted into chloroform/amyl alcohol as described in Method 1 for thiacetazone. Four ml of the extract was then extracted by shaking with 1 ml O.lN HCl. After adding O-2 ml 2N HCl and O-4 ml ethanol to 0.2 ml of the O*lN HCl extract, p-aminobenzaldehyde-thiosemicarbazone was determined calorimetrically by diazotisation and coupling by the procedure described in Method 3. The absorption of the product was measured at 580 nm 5 min after reaction. Concentrations of p-acetylamino-benzoic acid were determined fluorimetrically after acid-hydrolysis to p-aminobenzoic acid by the procedure described in Method 4. Collection of samples First study (Kenya) - Serum samples were obtained from 24 tuberculous patients who were under treatment for at least 2 weeks previously with daily doses of 150 mg thiacetazone plus 300 or 400 mg isoniazid. Ten of these patients were also receiving daily doses of Ig streptomycin. Blood was taken 0, 2, 4, 6 and 24 hr after giving a dose of thiacetazone and a second set was collected in the same manner a week later. Twenty-four-hour urine collections were also obtained from 6 of the patients. The completeness of the urine collections was checked by determining their creatinine contents using the method of King & Wooton (1956). Twelve samples of normal (blank) serum were also obtained from other patients who were being treated with isoniazid plus streptomycin. Second study (Singapore) - Serum samples were collected from 78 tuberculous patients during the third week of their participation in the second international co-operative investigation into thiacetazone side-effects (Miller and others, 1970). In this study patients were allocated at random to treatment with daily doses of thiacetazone (150 mg) plus isoniazid (300 mg) plus streptomycin (I g), to thiacetazone plus isoniazid or to isoniazid plus streptomycin. Half of the patients within each regimen received, by random allocation, an additive supplement of vitamins and antihistamine, and the remainder received a placebo supplement. Blood was collected 0, 2, 4, 6, and 24 hr after the oral medicament had been given. Complete urine collections were also obtained on 2 consecutive days from 29 of the patients. The identity of the patients and of the drugs they had received was

46

t:LLARD

AND

OTHERS

not revealed until after the thiacetazone analyses had been completed. Fifty-two of the 78 patients studied had received thiacetazone and of these patients 20 were Chinese, 16 were lndian and 16 were Malay. Of the 52 patients who had received thiacetazone plus isoniazid. 27 had also received streptomycin and 24 the vitamin and antihistamine additive. Third study (Kenya) - Serum samples were collected from 18 tuberculous patients. Each patient received 2 single doses of thiacetazone separated by 2-3 weeks. The 2 doses were chosen at random from among 4 possible sizes of 150, 300, 450 or 600 mg. The patients were receiving concomitantly tuberculosis chemotherapy consisting of daily doses of 300 mg isoniazid and Ig streptomycin. Blood was collected 0, 2, 4, 6, 24 and 48 hr after each thiacetazone dose. Twenty-four hour urine collections were also obtained before the first thiacetazone dose had been given (blank) and for the first 3 days after each thiacetazone dose. Fourth study (Kenya) - Serum samples were obtained from 20 tuberculous patients who were under treatment for at least 2 weeks previously with daily doses of 150 mg thiacetazone plus 300 mg isoniazid. Blood was taken 0 and 4 hr after giving a dose of thiacetazone. Analysis

qf samples

At the time of collection 24 hour urines were diluted to 2.41 and, in order to reduce possible bacterial contamination, one drop of streptomycin (500 pg/ml) and one drop of penicillin (I ,OOO,OOO units/ml) were added to aliquots of urine (20 ml) and to all blood samples. Serum and urine samples from the first three studies were then stored in the dark at -20°C for periods ranging from l-14 weeks (mean 6 weeks) until analysis in London by one or more of the 4 methods described above. Samples from the first 2 studies were analysed by Method 1. In the third study serum samples were first extracted with chloroform/propan-2-01 and thiacetazone determined by Method 2. Duplicate aliquots (2 ml) of the extracts were then dried to dryness and thiacetazone determined both colorimetrically (Method 3) and tluorimetrically (Method 4). The thiacetazone concentrations of urine samples in this study were determined as in the ultraviolet Method 2, but carrying out the extraction using double quantities. Duplicate aliquots of the choroform/propan-2-01 extracts containing 4-8 pg thiacetazone were then dried to dryness and the drug determined calorimetrically and fluorimetrically. In the fourth study serum samples were analysed in London within 4 days of their collection in Kenya. using Methods 2 and 3. Slide culture sensitivity tests Slide culture sensitivity tests of M. tuberculosis to thiacetazone were done on single sputum specimens obtained before chemotherapy from each of 12 African patients in Nairobi and from each of 107 Chinese patients in Hong Kong (Hong Kong Tuberculosis Treatment Services/British Medical Research Council Investigation, 1972), using the method described in the report. End points were taken either as the lowest concentration of thiacetazone that completely inhibited multiplication of the bacilli to form microcolonies or that which produced a just detectable decrease in microcolony size. Results Stability of thiacetazone Thiacetazone was shown to be stable in aqueous solution and in urine when stored in the dark at -20°C for periods of up to a year. Preliminary experiments suggested it was also stable in serum under these conditions. However, occasionally significant breakdown of thiacetazone occurred after a few weeks storage in serum. Regression analyses were therefore undertaken for the first 3 studies to determine whether there was a significant correlation between serum thiacetazone concentrations and the number of weeks the samples had been stored prior to analysis. No such correlation was demonstrated. In order to investigate the stability of thiacetazone further, each

SERUM

CONCENTRATIONS

47

OF THIACETAZONE

serum sample from the fourth study was divided in half. One set was then analysed immediately, identical results being obtained whether Method 2 (ultraviolet) or Method 4 (fluorimetric) was employed. Thiacetazone was then added to the other set of 0-hr samples to a concentration of 20 pg/ml and these and the 4-hr samples were stored in the dark for a further 7 weeks at -20°C before analysis. Over this period the mean fall in thiacetazone concentrations averaged about 2 per cent per week. Other investigations demonstrated that once thiacetazone had been extracted into chloroform/amyl alcohol or chloroform/propan-2-01 it was stable almost indefinitely at -20°C. Thiacetazone serum concentrations

The weights of the patients investigated in the four studies were similar, averaging 51,49, 52 and 49 kg, respectively. The results obtained from patients under treatment with daily regimens containing 150 mg thiacetazone are summarised in Table I. In the first study (Kenya) serum samples were obtained from each patient on 2 occasions separated by a week. The 2 sets of results did not differ significantly. The second study showed that the thiacetazone serum concentrations of the Singapore patients were not influenced by their racial origin (Chinese, Indian or Malay) or by concomitant treatment with streptomycin or the vitamin and antihistamine supplement (detailed results not tabulated). As had been expected the serum concentrations of thiacetazone obtained immediately before the test dose (0-hr) and those 24 hours later did not differ significantly. There were however significant differences between patients in the minimum thiacetazone serum concentrations, which averaged approximately a third of the peak (6-hr) values. The similarity of the serum results obtained in the two Kenyan studies also indicated that significant decomposition of the drug had not occurred during the period (mean 7 weeks) for which the samples from the first study were stored prior to analysis, The serum concentration achieved in the third study (Kenya) after giving single doses of thiacetazone ranging from 150 to 600 mg to patients previously untreated with the drug are summarised in Table II. Since significantly different results were not obtained by the three different methods (2-4) employed, the mean results of all the assays are presented in this table. The maximum thiacetazone concentrations encountered were approximately proportional to the dose given although the time of which maximal concentrations of thiacetazone occurred increased from about 4-5 hr after 150 mg (Tables I and II) to 6 hr or later when 450 or 600 mg thiacetazone was given (Table II). Urinary excretion of thiacetazone and its metabolites

The urinary excretion of thiacetazone by the African patients in Kenya and the Chinese, Indian and Malay patients in Singapore during daily treatment with regimens containing 150 mg thiaceta-

TABLE I.-SERUM

CONCENTRATIONS OF THIACETAZONE &G/ML) AFTER DAILY DOSAGEWITH 150 MG THIACETAZONE

Period after dose (hr.) Study 1 Kenya? 2 4

Singapore? Kenyas

~

No. of subjects

0

24

0.5 + 0.4*

52

0.8 kO.6

20

0.4f@3

/

~ ,

1

4

1.4*0.7

~

1.6kO.9

1.2+09

1.7kO.9

~

2.3 + 1.2

2.3k1.3

2

I

1

1.2kO.7

*Mean + Standard deviation individual results. tMethod 1. IMean results using Methods 2 and 4.

6

-

i 1

24 0.6kO.4 0.8 + 0.7

I

IL,L,4KL~

4x TABLF

I[.~--StKCIM

~ON(.tYWRA3

IONS 01 THIACElA/ONE

AND

OTHI

(!&/ML)

I’,41 tt YIS (3Ri)

KS

AF’I~K

SlV(sl.t- kStS

OF THt4(+lA/Oht

IU 18 E;t R’I A’.

swl~y:“)

Dow thiocrtoxtw (111x)

150 300 450 600

*Mean, SMean

TABLE

Ill.-

methods 2-4 (see te\tt). + standard deviation individual

URINARY

st1rN’y

EXCRETION

results.

OF UNCHANGED

No. of

DO.Yflg~~

.\lh,kTfS

I

Kenya

I50 mg daily

2

Singapore

I50 mg daily

3

Kenya

Single doses of 150-600

“Mean

i

standard

THIACETAZONE

~ mg

deviation

“(, dose excreted uttchunged

6

19,4+

5.8’

29

23.2&

9.6

I8

20.8 + IO.9

individual

results.

zone determined in studies 1 and 2 averaged about 20 per cent (range 9-43 per cent) of the dose (Table III). The excretion of thiacetazone by the Singapore patients was unaffected by their racial origin or by whether they were also being treated with streptomycin or the vitamin and antihistamine supplement. The urinary excretion of unchanged thiacetazone by the female patients was found to be approximately a third greater than that of the male patients. Similar results were obtained when the alternative ultraviolet method (Method 2) was employed in the third study. After giving single doses of 150-600 mg thiacetazone to the Kenyan patients about 15 per cent of the dose was excreted unchanged during the first day, 5 per cent during the second and I per cent during the third day. The total excretion of unchanged thiacetazone averaged 21 per cent of the dose, irrespective of its actual size (150-600 mg). By comparison the total excretion of thiacetazone estimated by the calorimetric and fluorimetric methods (Methods 3 and 4) averaged 30 per cent and 36 per cent of the doses given respectively. Scans of the ultraviolet absorption of the extracts from the urines of the Singapore patients investigated in the second study clearly demonstrated the presence of the drug in collections from patients who had been prescribed the drug and its absence among those who were not. Significant concentrations of p-aminobenzaldehyde-thiosemicarbazone were not detected in the urine of patients being treated with thiacetazone and it was concluded that less than I per cent of the dose could have been excreted as this potential metabolite. Similarly it was estimated that less than 5 per cent of the dose could have been eliminated as p-acetylamino-benzoic acid. Sensitivity

of’methods used to determine

thiacetazone

in serum and urine

The extraction of thiacetazone into chloroform/amyl alcohol (Method 1) or chloroformjpropan-2-01 (Method 2-4) was virtually quantitative and the recovery from serum was at least 95 per cent that

SERUM

CONCENTRATIONS

OF

THIACETAZONE

49

from aqueous solution. The mean optical densities of the extracts of blank sera were equivalent to 0.6 ~0.3 yg/ml thiacetazone whether the ultraviolet Methods 1 (studies 1 and 2) or 2 (study 3) was employed. A variance analysis of data obtained from the Singapore patients investigated in the second study demonstrated that the serum blanks of individual patients did not differ significantly, and the magnitude of the serum blanks was not influenced by the racial origin of the patient. the time at which the blank serum was collected and whether or not the patients were receiving the vitamin and antihistamine supplement. The mean serum blanks of the calorimetric and fluorimetric methods determined in the third study were equivalent to 0.2 kO.2 pg/ml and 0.1 20.1 pg/mI thiacetazone respectively. Variance analyses of the duplicate sets of results obtained from each patient in the first study and the 0 and 24 hour serum samples obtained from the Singapore patients receiving 150 mg thiacetazone daily in the second study, also suggested that the error of Method 1 in actual practice was to about + O-3 kg/ml thiacetazone. The results obtained when the thiacetazone concentrations of the 216 serum samples obtained in the third study were determined by the ultraviolet method 2. the calorimetric method 3 and the fluorimetric method 4, did not differ significantly and the residual term from a variance analysis of the data was equivalent to an overall error of + 0.27 pg/ml thiacetazone for the three methods. The correlation coefficients of the results obtained by the calorimetric and fluorimetric methods against those obtained by the ultraviolet method were 0.974 and 0.980, respectively. When normal urines from the second and third studies were assayed by the ultraviolet method I and 2, values equivalent to a daily urinary excretion of about l-2 mg thiacetazone were obtained. When the calorimetric and fluorimetric methods were employed the blanks were negligible. .SpeciJicit)-

qf the

methods used to determine

thiaceta-_one

The possible interference caused by other commonly used antituberculosis drugs and by potential metabolities of thiacetazone to the determination of the drug by the 4 methods described in this study was evaluated. On a molar basis p-acetylamino-benzaldehyde gave about 2& times as much fluorescence as thiacetazone in Method 4 but none of the other potential metabohties interfered significantly. Three potential metabolites (p-amino-benzaldehyde-thiosemicarbazone, pacetylamino-benzaldehyde and y-amino-benzaldehyde) gave identical amounts of colour to that given by thiacetazone in Method 3, but neither p-acetylamino-benzoic acid or p-amino-benzoic acid interfered. The ultraviolet method suffered interference from p-amino-benzaldehyde-thiosemicarbazone and p-amino-benzaldehyde. None of the methods was subject to interference from isoniazid, streptomycin, p-amino-salicylic acid, ethambutol or cycloserine, but rifampicin, pyrazinamide and ethionamide interfered with the determination of thiacetazone by the ultraviolet methods. Minimal

inhibitor)?

concentrations

qf‘thiacetazone

Several conclusions can be drawn from the results of the slide culture thiacetazone sensitivity tests (Table IV). First, strains from Kenya were considerably more sensitive than those from Hong Kong. Thus, 0.5 pg/ml thiacetazone completely inhibited all 12 Kenyan strains over a 7-day period, but only 11 per cent of 107 Hong Kong strains. Secondly, the concentration that completely inhibited growth of the Hong Kong strains was 8-16 times higher than the concentration causing just detectable inhibition. Nevertheless 77 per cent of the Hong Kong strains were partially inhibited by 0.5 pg/ml thiacetazone. A large difference between the end-points for the two types of inhibition also existed for the Kenyan strains. Thirdly, a comparison of the readings of the Hong Kong tests at 7 and 14 days suggested that thiacetazone deteriorated by less than 50 per cent during a 7-day period. Hence the concentration causing complete inhibition of all of the Kenyan strains and detectable inhibition of most of the Hong Kong strains would be about 0.4 Fg/ml at day 0 (as in a tuberculous lesion). Application of estimates of deterioration from the Hong Kong data to the Kenyan results is supported by an experiment in which 6 strains of M. tuberculosis with natural or acquired resistance

50

ELLARD TABLE IV.-RESULTS

AND

OTHERS

OF SLIDE CULTURE SENSITIVITY TESTS TO THIACETAZONE

Minimal inhibitory concentration of thiacetozone (~g/ml) Origin of sputum

Incubation period

Type of‘ inhibition

(days)

-_

Kenya

I

(12 patients)

-------m+ ~----~ 0.12 0.25 or Iexss

complete detectable

~~~ 0.5

~~~~--~~-~ -~ I.0

2.0

7

14

4.0

I

6

5

0

0

0

12

0

0

0

0

0

~__ Hong Kong (107 patients) -_ Hong Kong (107 patients)

-~ ~~~~~~-~

complete detectable

--

7* 54*

6 28

23 15

20 9

-__20 I

complete detectable

.._

4* 50*

2 30

4 II

19 13

25 3

8.0 or more 0

--..

0 ~-31 0 53 0

to thiacetazone, from patients in Hong Kong or Madras, and strain H37Rv were incubated at 37°C in 7H9 Tween-albumin medium containing 20 Kg/ml thiacetazone initially. Samples were removed at intervals of up to 14 days, passed through a cellulose membrane filter, and formolized. Chemical estimation (method 2) showed no difference in the thiacetazone concentration between any of the media containing organisms and the uninoculated medium; thiacetazone concentrations decreased on average by 15 per cent/week of incubation. Thus, there was no evidence that tubercle bacilli, and especially naturally resistant Hong Kong strains, appear resistant in vitro because they destroy thiacetazone. Discussion Serum concentrations and urinary excretion

qf thiacetazone

The studies on the stability of thiacetazone in serum suggested that storage of the samples from the first 3 studies prior to their analysis may have resulted in some decomposition, but that this was likely to have been less than 20 per cent in any of the samples. The fact that the serum concentrations and urinary excretion of thiacetazone by the Chinese, Indian and Malay patients investigated in Singapore did not differ significantly is of particular interest since the frequency of gastric, cutaneous and vestibular side-effects due to thiacetazone in Singapore was also shown to be unaffected by the racial origin of the patients (Ferguson and others, 1971; Miller and others, 1972; Singapore Tuberculosis Services/Brompton Hospital/British Medical Research Council Investigation, 1971). It may also be noted that the vitamin and antihistamine supplement studied was without effect both on the overall incidence of side-effects (Miller and others 1970) and, as shown in the present study, on the pharmacology of thiacetazone. The similarity between the urinary excretion of unchanged thiacetazone by patients in Kenya and Singapore (Table III) also suggests that pharmacology of thiacetazone in patients in the two centres does not differ significantly. Peak thiacetazone serum concentrations occurred about 4-5 hours after giving 150 mg doses of the drug (Table I and II). When the size of the dose was increased to 600 mg the time of the peak shifted to 6 hours or later, possibly because absorption of the larger doses was prolonged owing to the low aqueous solubility of thiacetazone. Peak thiacetazone concentrations were nevertheless approximately proportional to dosage. These findings, together with the similarity in the percentages of thiacetazone excreted in the urine after giving single doses ranging from 150 to 600 mg in the third study, suggest that all the doses of thiacetazone examined were well absorbed. From a consideration of the fall in the urinary excretion of unchanged thiacetazone over a 3-day period after giving single doses of the drug in the third study, together with the falls in the serum concentra-

SERUM

CONCENTRATIONS

OF

THIACETAZONE

51

tions found in the first three studies after peak concentrations had been achieved, it was concluded that the half-life of thiacetazone in man probably averages a little under 12 hours. Metabolism of thiacetazone

The failure to detect significant amounts of p-amino-benzaldehyde-thiosemicarbazone or p-acetylamino-benzoic acid in the urine after giving thiacetazone indicates that it is not extensively deacetylated or cleaved to form p-acetylamino-benzaldehyde and thiosemicarbazone. The excretion of thiacetazone, as estimated by the ultraviolet methods, averaged about 20 per cent of the dose. When the calorimetric and fluorimetric methods were employed (third study) the proportion of the dose accounted for rose to 30 per cent and 36 per cent respectively. These results suggest the formation of extractable metabolites of thiacetazone containing the acetylamino group intact but having undergone metabolism in the thiosemicarbazone portion of the molecule. Comparison of methods for the determination of thiacetazone serum concentrations

The two ultraviolet methods were considerably simpler than the calorimetric and fluorometric methods devised for the determination of thiacetazone. The chief limitation of the ultraviolet method resulted from the magnitude of normal serum blanks. In the first and second studies these blanks were equivalent to about O-6pg/ml thiacetazone, a value of the same order as the minimum concentrations after daily dosage with 150 mg of the drug. In these circumstances it was essential to incorporate into the analyses precisely equivalent samples of normal blank serum. Sera from healthy controls might be employed, or from tuberculous patients treated with other drugs, provided they did not include rifampicin, pyrazinamide or ethionamide. In circumstances where this is not possible it would be preferable to employ the more time-consuming calorimetric or fluorometric methods. Of the two ultraviolet methods, the second is to be preferred, since if other ultravioletabsorbing materials were demonstrated in the chloroform/propan-2-01 extract, an aliquot can then be evaporated to dryness and thiacetazone determined calorimetrically or fluorimetrically. Despite their fundamentally different specificities, the 3 methods employed to determine the thiacetazone serum concentrations in the third study yielded results that did not differ significantly. The ultraviolet methods depend primarily on thiacetazone’s benzaldehyde-thiosemicarbazone grouping, the calorimetric method on its conversion by acid-hydrolysis to a diazotisable-amine, and the fluorimetric method on its oxidation by alkaline permangamate to p-acetylamino-benzoic acid. It may therefore be concluded that serum concentrations of any metabolites of thiacetazone are much less than those of the unchanged drug. Variance analysis of the)results obtained in the first 2 studies, as well as a consideration of the determinations on the blank (0-hr) sera analysed in the third study, indicated that the error of the ultraviolet methods for determining thiacetazone in the serum was equivalent to about 5 0.3 Erg/ml of the drug. Since the maximum serum concentrations achieved by daily dosage with 150 mg thiacetazone were less than 3 kg/ml, it is apparent that more sensitive methods are needed. The results obtained in the third study indicated that the calorimetric and f’luorometric methods were slightly more sensitive than the ultraviolet methods. Thus the accuracy of the fluorometric method was to about + 0.15 [*g/ml thiacetazone. The sensitivity of this method might be improved if means could be found for quantitatively converting thiacetazone to p-acetylamino-benzoic acid. The method described in this paper employing alkaline permanganate yielded only about 40 per cent of the theoretical amount of p-acetylamino-benzoic acid perhaps due to cyclisation reactions to thiadiazole and triazole derivatives similar to those described by Hoggarth (1951). Antituberculosis activity of thiacetazone

A number of problems arise in attempting to estimate what concentrations of thiacetazone must be present in tuberculous lesions to prevent the emergence of isoniazid resistance during chemotherapy with isoniazid plus thiacetazone (the minimal effective concentration or MEC). As first shown by Rist, Gals & Jullien (1951) many European strains of tubercle bacilli contain a small

proportion of physiologically resistant organisms which can grow slowly in the preaencc 01‘high concentrations of thiacetazone. but retain their original sensitivity to the drug OII subculti\ation after growth. The heterogeneity in response is even more marked in 21small proportion OI‘\tr;lin\ from E. Africa and in the majority or strains from Madras and Hong Kong. These strains appeat. resistant in conventional sensitivity tests. because the resistant organisms grow more rapidly in the presence of thiacetazone and are often present in larger proportions than the corresponding organisms from European strains (see review by Grosset & Benhassine. 1970). Lest & Marks (19701. while recognising the phenomenon. concluded that the sensitivity to thiacetazone of the ma.jtrrit! of the bacterial population (the sensitive moiety) of British and Hong Kong strains was similar. and that the appearance of colonies of physiologically resistant organisms could be pre\,entcd in sensitivity tests by using a small inoculum, filtered to remove large clumps, and a very short incubation period. III the present study, slide culture sensitivity tests were used, which avoided complications due to clumping and were read after only 7 days incubation. In these tests the concentration of thiacetazone required to inhibit growth completely was much higher than the concentration producing just detectable inhibition, indicating heterogeneity of response in both the Kenyan and the Hong Kong strains. However, using either end-point. the Hong Kong strains appeared considerably more resistant and this difference in sensitivity did not seem to be due to destruction of the drug by the bacilli. There was, nevertheless, a limited range of thiacetazone concentrations of about 0.4 !Jg/ml which on the one hand completely prevented multiplication of the Kenyan strains and on the other hand partially inhibited most of the Hong Kong strains. The results set out in Tables I and III indicate that the pharmacology of thiacetazone with very similar in African patients from Kenya and Chinese patients from Singapore. In view of the greater natural resistance in IYIN) of most of the Hong Kong strains, it is therefore remarkable that the daily regimen of 300 mg isoniazid and 150 mg thiacetazone was of similar efficacy in patients of about the same mean weight in E. Africa and in Ho11g Kong (East African/British Medical Research Council, 1963a: 1966; Hong Kong Antituberculosis Association and Government Tuberculosis Service/British Medical Research Council. 1968). High efficacy in both places co~tld be explained on the assumptions that the concentrations of thiacetazone maintained in the lesions were just above the MEC in the Hong Kong patients and were considerably above a lower MEC in the the E. African patients. The tatter assumption cannot however be correct, since reduction of the daily dose of thiacetazone frotn 150 mg to 100 mg resulted in a considerable loss of efficacy of the thiacetazone/isoniazid regimen in E. African patients (East African/British Medical Research Council, 1963a). The MEC therefore appears to be similar for E. African and Hong Kong patients. Furthermore, the slide culture sensitivity test data indicates that the only common value of ~hc MEC is about 0.4 pg/ml. We therefore suggest that there must be a difference in the ability of the host defences of E. African and Hong Kong patients to prevent the growth of small number ot isoniazid resistant mutant bacilli. The hypothesis proposes that the immune defences of Africans, who usually have a very acute form of pulmonary tuberculosis, are incapable of preventing such organisms growing in the lesions. even when they are partially inhibited by thiacetazone: for thiacetazone to be effective the concentration required should be suficient to inhibit all growth. All of the I2 Kenyan strains were completely inhibited by 0.4 pg/ml, a concentration continuously exceeded when patients were treated with 150 mg thiacetazone daily (Table I). Although the penetration of thiacetazone into lesions has not been studied, its lipid solubility, lack of charge at body pH and relatively long half-life indicate that the concentrations achieved in the lesions are likely to be similar to those in the serum. Thus an estimated value for the MEC of 0.4 pg/ml is consistent with the pharmacological data. Furthermore, the hypothesis accounts for the associations between the initial sensitivity of tubercle bacilli to thiacetazone and the response to treatment with thiacetazone/isoniazid regimens (E. African/ British Medical Research Council, 1963b; International Union Against Tuberculosis, 1970). On the other hand, the hypothesis suggests that the defense mechanism of the Hong

SERUM

CONCENTRATIONS

OF

THIACETAZONE

53

Kong patients, who usually have a chronic type of disease, are capable of arresting the growth of isoniazid-resistant mutant bacilli, providing the bacilli are partially inhibited by thiacetazone. It would account for the efficacy of daily treatment with 150 mg thiacetazone and 300 mg isoniazid in Hong Kong, since thiacetazone concentrations of 0.4 pg/ml or more would partially inhibit most of the strains isolated from the Hong Kong patients (Table IV). Furthermore, as inhibition by 0.4 pg/ml was only just detectable in the majority of the strains, the proportion of the organisms capable of growth at higher concentrations would be expected to have little influence on their ability to grow during treatment. Thus the hypothesis expects little or no association between the results of initial sensitivity tests and the response of the patients. Despitee vidence of real variation from patient to patient in the sensitivity of their pretreatment strains as measured in conventional tests, these estimates of sensitivity were not associated with response (Hong Kong Antituberculosis Association and Government Tuberculosis Service/British Medical Research Council, 1968), nor were associations found between the slide culture results reported here and response. However, associations between the results of conventional tests and of the slide culture tests were poor or absent, suggesting that much of the apparent variation between strains is technical in origin. The efficacy of thiacetazone-containing regimens depends on the concentrations of thiacetazone achieved in the lesions and on the MEC. Our findings suggest that whereas the pharmacology of the drug and hence the concentrations in lesions are not influenced by the race of the patient, the MEC is dependent on characteristics of the tubercle bacilli and on the immune response of the patient, which can vary greatly from one area of the world to another. Although the MEC was probably about 0.4 pg/ml both for E. African and Hong Kong patients, there is no certainty that similar values would be obtained elsewhere, as for instance in W. Africa where thiacetazone resistant strains of ‘M. qfricanum’ are frequently isolated. It is therefore necessary to re-emphasise that before a thiacetazone-containing regimen is introduced for wide-scale use in a new area. its efficacy should first be established by means of a controlled clinical trial. We should like to thank Drs. S. Devi, S. Doraisingham and J. M. J. Supramaniam from the Tan TockSengHospital, Singapore; and Mr. E. A. Edwards and Drs. G. C. Gould, D. M. Macfadyen and A. Jan Mohamed from the East African Tuberculosis Investigation Centre and Infectious Diseases Hospital, Nairobi for their co-operation in obtaining the serum samples without which this investigation could not have been undertaken. We should also like to thank Mr. A. J. Nunn and Miss Ruth Tall for statistical help and Dr. Wallace Fox for his keen interest throughout the whole

investigation.

REFERENCES BURKE, J. C. & TITUS, 0. E. (1950). The pharmacology of p-formylacetanilide thiosemicarbazone (Myvizone) in animals. Transactions of the 46th Annual Meeting of the National Tuberculosis Association, p.l. BRATTON, A. C. & MARSHALL, E. K. Jr. (I 939). A new coupling component for sulfanilamide determination. Journal of Biological Chemistry, 128, 537. CHATTERJEE, K. R. &BOSE, R. (1953). A simple method of estimation of thiosemicarbazone in tissue and body fluids. Leprosy in India, 25, 199. EAST AFRICAN/BRITISH MEDICAL RESEARCH COUNCIL SECOND THIACE~AZONE INVESTIGATION(1963~). Isoniazid with thiacetazone in the treatment of pulmonary tuberculosis in East Africa - Second investigation. Tubercle, 44, 301. EAST AFRICAN/BRITISH MEDICAL RESEARCH COUNCILPRETREATMENT DRUG RESISTANCE REPORT( 1963b). Influence of pretreatment bacterial resistance to isoniazid, thiacetazone or PAS on the response to chemotherapy of African patients with pulmonary tuberculosis Tubercle, 44, 393. EAST AFRICAN/BRITISH MEDICAL RESEARCH COUNCIL THIRD THIACE~AZONE INVESTIGATION (1966). Isoniazid with thiacetazone (thioacetazone) in the treatment of pulmonary tuberculosis in East Africa-Third investigation. The effect of an initial streptomycin supplement. Tubercle, 47, I. FERGIJSON, G. C., NUNN, A. J., Fox, W., MILLER, A. B., ROBINSON, D. K. & TALL, R. (1971). A second international co-operative investigation into thiacetazone side-effects. Rashes on two thiacetazone-containing regimens. Tubercle, 52, 166. Fox, W., STARK, A. J., TALL, R., BHATIA, J. L., CLARKE, J. H. C., DONIA, T. O., KRISHNASWAMI, K. V. & OUSSEDIK, N. (I 974). A study of adverse reactions to high dosage intermittent thiacetazone. Tubercle, 55, 27.

54

ELLARD

AND

OTHERS

GROSSET, J. & RENHASSINE, M. (1970). La thiacetazone (Tbl): donneesexperimentales et cliniques recentes. Advctnce.\ in Tuberculosis Research, 17, 107. HEILMEYER, I. & HEILMEYER,L. (1950). Quantitive BestimmungvonBenzaldehyde-thiosemicarbazonderivaten (TBl698) in Serum, Exsudat und Harn bei oraler und parenteraler Belastung sowie bei therapeutischen Gaben. Archit fur experimentelle Pathologie und Pharmakologie, 211, 3 13. HENDRICKS, F. D.. WELCH, H., ARNAULT, L. T., LEHMAN, A. J., CANNON, M. L., FISCHBACH, H. & LEVINE, J. (1950). Studies of the pharmacology of tibione and a method for its assay in body fluids. Transactions 9th Streptomycin Conference (Veterans Administration) p. 160. HOGC;ARTH, E. (1951). Compounds related to thiosemicarbazide. Part 8. The oxidation of thiosemicarbazones. Journal of’the Chemical Society, 2202. HONG KONGANTI-TUBERCULOSIS ASSOCIATION AND GOVERNMENT TUBERCULOSIS SERVICE/BRITISH MEDICAL RESEARCH COUNCIL INVESTIGATION (1968). A controlled comparison of thiacetazone (thioacetazone) plus isoniazid with PAS plus isoniazid in Hong Kong. Tubercle. 49, 243. HONK; KONG TUBERCXLOSIS TREATMENT SERVICES/BRITISH MEDICAL RESEARCH COUNCIL INVESTIGATION (1972). A study in Hong Kong to evaluate the role of pretreatment susceptibility tests in the selection of regimens of Amer.can Review of Respiratory Disease, 106, 1. chemotherapy for pulmonary tuberculosis. INTERNATIONAL UNION AGAINST TUBERCULOSIS (1970). A controlled trial of three regimens of self-administered and supervised chemotherapy for pulmonary tuberculosis. Bulletin International Union against Tuberculosis, 44, 8. KING, E. J. & W~OTON, 1. D. P. (1956). Microanalysis in Medical Biochemistry, 3rd edition, p. 161, J. &A. Churchill. London. LEAT, J. L. & MARKS, J. (1970). Improvement of drug-sensitivity tests on tubercle bacilli. Tubercfe, 51, 68. MILLER. A. B., Fox, W. & TALL, R. (1966). An international co-operative investigation into thiacetazone (thioacetazone) side-effects. Tubercle, 47, 33. MILLER, A. H., NUNN, A. J., ROBINSON, D. K., FERC~USON,G. C., Fox, W. & TALL, R. (1970). A second international co-operative investigation into thioacetazone side-effects. 1. The influence of a vitamin and antihistamine supplement. Bulletin of the World Health Organisation, 43, 107. MILLER, A. B., NUNN, A. J., ROBINSON, D. K., Fox, W., SOMASUNDRAM. P. R. & TALL, R. (1972). A second international co-operative investigation into thioacetazone side-effects. 2. Frequency and geographical distribution of side-effects. Bulletin of the World Health Organisation, 47, 21 I. RIST, N., CALS, S. & JULLIEN, W. (1951). Aspects varies de la sensibilite de bacille tuberculeux a la paraacetylbenzaldehyde-thiosemicarbazone (Tbl) en milieu de Youmans. Annales de I’lnstitut Pasteur, 81, 324. SHORT, E. 1. (1961). The detection of thiacetazone in the urine. Tubercle, 42, 524. SINGAPORE TUBERCULOSISSERVICES/BROMPTON HOSPITAL/BRITISH MEDICAL RESEARCH COUNCIL INVESTIGATION(1971). A controlled clinical trial of the role of thiacetazone-containing regimens in the treatment of pulmonary tuberculosis in Singapore. Tuber&, 52, 88. SPINKS, A. (1949). The estimation of some thiosemicarbazones and their blood concentrations in experimental animals. British Journal of Pharmacology and Chemotherapy, 4, 254. SPINKS, A. (1951). Estimation and photolability of some thiosemicarbazones. British Journal of Pharmacology and Chemotherapy, 6, 35. TUBERCULOSIS CHEMOTHERAPY CENTRE, MADRAS(1966). Isoniazid plus thioacetazone compared with two regimens Bulletin of isoniazid plus PAS in the domiciliary treatment of pulmonary tuberculosis in South Indian patients. of the World Health Organisation, 34, 483. WERNITZ. W. & TORNUSS,H. (1952). Quantitative Contebenstudien. 4 Mitteilung. Contebenblutspiegel beim Menschen. Zeitsehrift fur klinische Medizin, 150, 170. WOLLENBERC;, 0. (1950). Uber die kolorimetrische Bestimmung von Thiosemicarbozonen im Harn. Deutsche medizinische Wochenschrift, 75, 899.