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Controlled clinical trial of 4-4 diisoamyloxythiocarbanilide in the treatment of pulmonary tuberculosis
192
Tubercle, Lond., (1964), 45, 192
C O N T R O L L E D C L I N I C A L T R I A L OF 4-4 D I I S O A M Y L O X Y T H I O C A R B A N I L I D E IN T H E T R E A T M E N T O F P U L M O N A R Y T U B E R C U L O S I S By A, S. MOODm,* Sister M. AQUINAS, and R. D. FOORD fi'om the Medical and Health Department, Hongkong, Ruttonjee Sanatorium. Hongkong and the Grantham Hospital, Hongkong
SUMMARY A total of l00 Chinese patients suffering from open pulmonary tuberculosis were randomly allocated to treatment with either PAS and isoniazid or with 4-4 diisoamyloxythiocarbanilide ('isoxyl') and isoniazid, Twenty were excluded from further participation because of pretreatment drug resistance. Of the remaining 80 patients, one each from the 39 PH group and 41 IX group were withdrawn at the 16th week, the IX patient being withdrawn because of failure of treatment. There were no noticable toxic effects from treatment. After 24 weeks' treatment 71% ofthe PH group and 40% of the IX group had negative cultures. Isoniazid-resistance emerged in 7 of the 38 PH and in 20 of the 40 IX patients. Pretreatment resistance to 'lsoxyl' was reported in 37 of the 78 patients who completed the trial. This might well have had an effect on the results of treatment with 'lsoxyl' and isoniazid.
Derivatives of thiourea were shown to possess in vitro activity against the tubercle bacillus by Mayer (1941) as well as by Juin & Buu-Hoi (1946). These early compounds were unsuitable for clinical use because of their activity on the thyroid gland, but later compounds were shown to be free of this disadvantage by Mayer, Eisman & Kanopka (1953) and by Buu-Hoi & Xuong (1953), in the treatment of leprosy. Youmans, Youmans, & Doub (1958) and Steenkcn and others (1958) demonstrated in vitro and in vivo anti-tuberculosis activity in the carbanidin group of thiourea derivatives. 'Isoxyl' is a new compound, 4-4 diisoamyloxythiocarbanilide, of the N-N diarylthiourea group synthesised by Buu-Hoi & Xuong and shown by Tacquet and others (1958) to have in vivo and in vitro activity against the tubercle bacillus. Freerksen (1962), Murohashi & Yanagisawa (1963), and Lucchessi (1963), showed that in laboratory animals the anti-tuberculosis activity of 'Isoxyl' was more powerful than that of PAS, comparable to that of ethionamide, but less powerful than streptomycin or isoniazid. Fegiz & Rellini (196i.) showed that the drug was well tolerated in human beings in a dosage of 2 g. daily, and claimed that 'Isoxyl' could with advantage be substituted for PAS in triple drug therapy, and that it produced no evidence of intolerance. Favez, Vulli6moz & Br6aud (1963) reported that in newly diagnosed cases of tuberculosis 'Isoxyl' 6 g. in combination with isoniazid, 600 rag. produced a clinical response comparable with that produced by PAS and isoniazid, streptomycin and isoniazid and that 'Isoxyl' was effective in preventing the emergence of organisms resistant to isoniazid. Thus, the evidence has been accnmulating to suggest that 'Isoxyl' might, with advantage, be considered as a possible substitute for PAS or possibly as an addition to the drugs already available *Present address--Branault, Barrhead, Glasgow.
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for the treatment of tuberculosis. The drug is attractive by virtue of its smaller bulk and greater acceptibility. If the reported good climcal results could be duplicated locally, it might be worth adoption for general use in Hong Kong. It was with this idea in mind that a controlled clinical trial was planned. Plan of the Trial Objectives To measure the therapeutic efficiency of 'Isoxyl' in combination with isoniazid against a combination of PAS and isoniazid in the treatment of newly diagnosed pulmonary tuberculosis. To compare the efficacy of PAS and 'Isoxyl' in their power to prevent the emergence ofisoniazidresistant organisms. To record and compare the toxic effects of the two drug combinations. To make and record observations on the cross resistance pattern between 'lsoxyl' and other antituberculosis drugs. Criteria for Selection of Patients Individuals included conformed to the following: Age 15-50 years. Suffering from untreated pulmonary tuberculosis with or without cavitation. Tubercle bacilli were demonstrated in the sputum by direct smear less than 4 weeks before the date of acceptance. Willing to accept treatment in hospital for a period of at least 24 weeks. Patients showing any of the following were excluded: Moribund state. Excreting tubercle bacilli which showed ht vitro resistance to streptomycin, PAS, or isoniazid. M iliary tuberculosis. Other complicating tuberculosis. Pleural effusion obscuring more than half of one lung field. Diabetes mellitus. Any concurrent disease which might complicate treatment or interfere with results. Prescribed Treatment Patients were allocated to one or other of the following treatment schedules: IX--'lsoxyl' tablets, 4 g. with isoniazid 200 nag. taken in one daily dose. P H - - P A S 10 g. in solution with isoniazid, 200 nag. taken in one daily dose. The Patients A total of 100 Chinese patients was selected on the basis of the acceptance criteria from the public clinics in Hong Kong and admitted to one of the two hospitals taking part in the study. As soon as aceeptibility had been confirmed, application for inclusion in the trial was made to the trial secretary, who was located in a separate institution and did not come into contact with the patients. Allocation was made from a single group of previously prepared and sealed envelopes bearing numbers 501-600 which contained a note bearing one of the two treatment regimes allocated by random numbers, and patients were provisionally included in the trial until the results of the sensitivity tests became available. Exclusions~Of the original 100 patients, 20 were excluded on account of bacterial resistance to one or more of the standard drugs leaving, 39 PAS and isoniazid cases (PH) and 4/ ('Isoxyl' and isoniazid cases (IX). The pretreatment condition of the patients is shown in Table I. The radiographic classification is based on extent of lesions in categories one to six, according to the number of zones involved, and cavitation is ~ivided into categories 0-3 according to the size and number of cavities seen. it will be seen that the two groups corresponded closely in all respects.
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TABLEI.--PRETREATMENTSTATUSOF PATIENTS
PAS/isoniazid (PH)
'Isoxyl'/isoniazid
3"31 1 "54
3.40
(ZX)
First random allocation Resistant to one or more of the standard drugs Remaining in the study General condition
Good Fair Poor
Sex
Males Females
Age (years)
15-24
25-34 35-44 45-50 Sputum status 3 Specimens
Radiographic assessment*
No acid fast bacilliseen 0-I0 per smear 10-20 ,, 20-40 ,, Over 40 ,, No. of zones Cavitation
1.34
*See text for method of assessment.
Routine ExamhTations Pretreatment--Body weight, haemoglobin, fasting blood sugar, blood cholesterol, serum albumen and globulin, S.G.O.T., S.G.P.T., total white and differential count, three sputum specimens for concentration, grading, culture, and sensitivity tests for streptomycin, PAS, isoniazid, thiosemicarbazone, ethionamide and 'Isoxyl'. One 15 in. • 12 in. x-ray film of the chest. Subsequent Exam#~ations--All pretreatment examination procedures to be repeated at the 12th and 24th weeks. At the 4th, 8th, 16th and 20th w e e k s : - Body weight, two sputum specimens for concentration, grading, culture, and sensitivity tests as detailed above in the pre-treatme~t examination. BACTERIOLOGICAL METHODS Specimens were concentrated by Petroff's method, stained by Ziehl-Neelsen and examined by direct microscopy. Smears were classified according to the total number of bacilli present. From the residue two cultures were prepared on L6wenstein-Jensen slopes and incubated for a minimum period of 8 weeks at 37 ~ C. One culture was examined a t the Hoffg K o n g G o v e r n m e n t Institute. of Pathology or at the laboratory of The G r a n t h a m Hospital, H o n g Kong. The resistance ratio method was used, with the following drug concentrations with H37Rv as control organism.
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Micrograms per ml. Streptomycin 0.5 1-0 2.0 4.0 PAS 0.5 1.0 2.0 4.0 lsoniazid 0.12 0.25 0.5 1.0 5.0 Ethionamide 5.0 10.0 20.0 40.0 80.0 100.0 Thiosemicarbazone 0.5 1.0 2.0 4.0 8.0 16 A ratio of 2 or less was recorded as 'sensitive'. A ratio of 4 was recorded as 'probably resistant'. A ratio of 8 or above was recorded as 'definitely resistant'. The second culture was sent by air to the Tuberculose-Forschungsinstitut, Borstel, Hamburg, where a parallel set of sensitivity tests was carried out using the absolute concentration method. This laboratory performed, in addition, a sensitivity test f o r ' I s o x y l ' on each specimen (see Appendix). Thus each culture was tested for drug resistance by two different methods, the resistance ratio and the absolute concentration methods. The measure of agreement obtained in the two different methods in the course of 2,000 consecutive sensitivity tests is shown in Table II. It will be observed that there was complete agreement in over 90 ~o of these tests and that the major part of the disagreement was seen in tests where one laboratory reported partial resistance as against full sensitivity by the other, or where one laboratory reported total resistance in one specimen as against a report of partial resistance by the other. Significant disagreement, where one laboratory reported total sensitivity as against total resistance by the other, occurred in only 8 tests (0.4 ~ ) . hlterpretation o f Bacteriological Findings
Because of the multiplicity of the bacteriological observations a standard procedure was adopted for classification of cases. At each four weeks period there were either two or three graded sputum smears examined and reported. The case was classified according to the highest grading. At each four weeks period there were either four or six cultures, each positive culture being tested for sensitivity to five drugs, streptomycin, PAS, isoniazid, thiosemicarbazone and ethionamide, and in the case of cultures examined at Borstel, to 'lsoxyl' as well. In the pretreatment series a single TABLEII.--THE MEASUREOFAGREEMENTAND DISAGREEMENTOF RESULTSOF SENSITIVITYTESTS CARRIEDOUT ON THE CULTURE FROMTHE SAME SPECIMEN BY TWO DIFFERENT METHODS (HONG KONG--RESISTANCERATIO METHOD" BORSTEL~ABSOLUTECONCENTRATIONMETHOD) Results agree
Results disagree
Hung Kong result
Sens.*
P.R.*
D.R.*
Borstel result
Sens.
P.R.
D.R.
381 367 365 382 224
-m -5 38
1719
43
Ethionamide Streptomycin Thiosemicarbazone PAS Isoniazid Total
Sens. D.R.
16
1
24
I
21
27 3 53
45
100
1807
Grand Total (Percentage)
(90%)
*Sens.=Sensitive.
P.R.
P.R.
P.R.-----Partiallyresistant.
Sens.
D.R. D.R.
Sens.
P.R.
3
5 8 7 12
3
4
42
4
33
3
4
49
3 3
193 (lo%) D.R.=Definitely resistant.
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report of resistance to streptomycin, PAS, or isoniazid was regarded as reasonable ground for excluding the case from the study; while in examinations during treatment a single unsupported report of resistance was disregarded. A case was regarded as having developed resistant cultures only when two or more reports of resistance were made, and where two reports of resistance differed, the higher was the one adopted. WITHDRAWALS One PH case (538) withdrew after the examinations of the 16th week when the sputum was already negative on concentration and culture, probably on account of gastric symptoms, but showing a minor degree of mental instability which appeared to be unassociated with the treatment. One IX case (593) was withdrawn at the end of the 16th week as a treatment failure because of persistant positive sputum and the development of definite resistance to isoniazid which first appeared at ttae 8th week, and partial resistance to 'lsoxyl', which were accompanied by clinical and radiographic deterioration. The remaining 78 patients completed the stated period of treatment of 24 weeks. BACTERIOLOGICALRESULTS Results The principal measure of efficiency of a therapeutic agent in tile treatment of tuberculosis is its ability to render the sputum abacterial and the speed with which it does so. The bacteriological changes brought about by the two treatment regimes are shown in Table III. These results show a clear advantage in favour of the PH regime, 84 ~o showing the sputum to be negative on concentration, an4 71 ~o on culture, at 24 weeks as compared with 55~o, and 4 0 ~ respectively in the IX group. (Tb'." differences are significant at the 2 ~ level.) A further measure of the adequacy of a therapeutic agent is its ability, when used in conjunction with isoniazid, to prevent the emergence of isoniazid-resistant tubercle bacilli. A comparison of the two-drug regimes in this respect is shown in Table IV. In the 38 patients who completed 24 weeks' treatment with PAS and isoniazid, 11 remained sputum positive on culture, of whom 7 (18 ~o) had cultures resistant to isoniazid, while in the 'lsoxyl' and isoniazid group 24 of the 40 patients remained sputum positive on culture, 20 (50 ~o) showing resistance to isoniazid, of whom 4 had also developed resistance to 'lsoxyl'. OTHER CHANGES Radiographically the changes produced by treatment were divided into three grades of improvement, three grades of deterioration and one category which showed no change. Assessments were TABLEIII.--BACTER1OLOGICALCHANGESDURINGTREATMENT 38 patients completed treatment wth PAS/isoniazid
40 patients completed treatment with "lsoxyl'/isoniazid
Negative findings on
Negative findings on
Sputttm conc.
I
Pretreatment 4th week 8th week 12th week 16th week 20th week 24th week
I
Sputum conc.
Culture
Number
%
2 13 15 27 29 31 32
5
1
2
34 40 71 76 81 84
11 17 15 21 22 22
27 42 37 52 55 55
Number
%
Number
%
Culture Nmnber
~
4 - 4 DIISOAMYLOXYTHIOCA RBANILIDE
197
TABLEIV.--EMERGENCEOF ]SONIAZIDRESISTANCEAFTER24 WEEKS TREATMENT I
Treatment with
I Culture results
i PAS]isoniazM 38 patients
I
"Isoxyl'/isoniazid 40 patients
L
Negative Positive and sensitive Positive and resistant to isoniazid Positive and resistant to isoniazid and 'Isoxyl'
t I j i
27 4 7
16 4 16
--
4
TABLEV.--RADIOGRAPHICCHANGESDURINGTREATMENT Weeks 0.12
Degree of change
Worse Unchanged
Weeks 13-24
PH
IX
29 5
25 8
PH
J
Weeks 0-24 I X ..........
PH
i
--
t
--3 --2 --1 0
I
IX
1
!
Improved
+1 +2 +3
23
23
I
1
14 1
1 ,
--
23 12 3
made by one of us (A. S. M.) without knowing the treatment regime used, based on a comparison of the x-rays of pretreatment and 12 weeks, between the film at 12 weeks and 24 weeks, and between the pretreatment and 24 weeks film. The results of these assessments are shown on Table V. One patient in the IX group showed radiographic deterioration and one no change: all in the PH group showed improvement. This slight difference is not statistically significant. The results of the routine examinations are shown in Table VI, which shows the average and range of values found but excludes the results of examinations at 12 weeks, as these showed nothing of note. There was no evidence that either treatment had any adverse effect on the haemopoetic system, the liver, the thyroid gland, the kidneys, nor was there any significant change in the blood sugar levels. The changes produced were common to both groups. T o x I c EFFECTS There were a few complaints of epigastric discomfort, loss of appetite and nausea, but these complaints were of a trivial nature, o f short duration, and appeared to occur with equal frequency in both groups. N o t a single case had to stop treatment because of toxic effects. One IX case (513) also received prednisolone because of fever, deterioration on x-ray and loss of voice. He developed a skin rash three weeks later, the origin of which was not determined, but continued with treatment as before, improving gradually but was still sputum positive at the 24th week. RESISTANCE TO '[SOXYL' BEFORE TREATMENT Routine sensitivity tests to 'Isoxyl' were carried out at the Borstel Institute, but were not dupli-
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TABLEVI.--LABORATORYINVESTIGATIONS(AVERAGEVALUEAND RANGE)
PAS]isoniazid
"Isox),l'/isoniazid
Z-~,e,~eo,- , ~ nlent
~,~,,~o,- I '
[ 24th week % change
nlent
-
24th week % change
i
Average wt. (lbs.) Range Haemoglobin (%) Range Fasting blood sugar (mg./100 nal.) Range Blood cholesterol (rag./100 ml.) Range Serum albumen (g./100 ml.) Range Serum globulin (g./100 ml.) Range S.G.O.T. (Karmen) Range S.G.P.T. (Karmen) Range W.B.C. total Range in tousands
102 112 (68-123) (79-151) 81 84 (60-110) (70-1lO) 77 80 (50-109) (40-120) 153 182 (95-223) (127-270) 3"53 4"24 (2;9.94.5) (3;0.;~.3) (2"7"5"1) I ( 2 " 4 " 4 ' 4 )I11.2 3.4 (7-30)
,
11.6
(3-22) ! 1-0
(4-60) (3-73) 7445 6611 (3.8-13.8) (3.4-10.3)
+10% 100 (67-I24) + 4% i 87 l (60-110) + 4% t 78 !(60-109) +18% i 160 (103-230) +20% 3.73 (3.1-4.6) --22% 3.72 (3.0-4.9) ul % 14.7" (6-38) -% 10.5(3-73) --1% 7805 (4.7-14)
109 (72-139) 93 (70-110) 82 (50-109) 165 (112-240) 4"34 (3 "O-5"3) 3 -32 (2.2-4"2) 9'9"
+ 9% + 7% + 6% + 3% +16% --11% --33% --11%
(2-85) 6430 (3.8-10)
--17%
*One case with values outside the ranges quoted is excluded because of history of previous liver disease. 'lsoxyl' was continued without disturbance of the values. cated in the Hong Kong laboratories because of uncertainty as to their value and doubt as to the most suitable technique. These tests showed that of the 100 patients provisionally selected for inclusion in the study, 41 were excreting organisms with a significant degree of resistance to 'lsoxyl' in the pretreatment examinations by the absolute concentration method. Three 'Isoxyl' resistant cases were included in the 20 cases rejected because of other bacterial resistance, and a further 'lsoxyl' resistant case was lost (538) on withdrawal, leaving 37 'lsoxyl' resistant cases who completed the prescribed period of treatment (20 P H and 17 IX). Sputum cultures results at 24 weeks in the IX group showed that 57 % of the 'lsoxyl'-sensJtive cases were negative as against 18 % in the 'Isoxyl'-resistant cases, while in the PH group, 88 % of the 'Isoxyl'-sensitive as against 55 % of the 'Isoxyl'-resistant cases had become negative (Table VII). The differences in the proportions negative on culture are significant at the 5 level in the IX group but are not significant in the P H group. TABLEVII.--BACTERIOLOGICALRESULTSACCORDINGTO PRETREATMENTSENSITIVITYTO 'ISOXYL'
I Pretreatment set~sitivity to isoxyl
I
Bacteriologicalfinding on sputum culture at 24 weeks
Total
PAS/isoniazid
f l Pos.
'lsoxyl' sensitive 'lsoxyl' resistant
41 37
I
2 9
I I
Neg. no.
%
16 11
88 55
t
'Isoxyl'/isoniazid
E! [
I
Pos. 10 14
Neg. no. [ [
13 3
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With regard to cross resistance, of the 41 patients who showed pretreatment resistance to 'Isoxyl', 12 also had cultures resistant to thiosemicarbazone, and 5 had cultures resistant to thiosemicarbazone but not to "Isoxyl'. Of the four who developed 'Isoxyl' resistance under treatment not a single one developed resistance to thiosemicarbazone. Thus there appears to be some, but not a very well defined association between resistance to the two drugs. Discussion 'lsoxyl' fulfilled all claims as to acceptibility, causing only minor gastric upset and anorexia as well as a few other symptoms of insignificant proportions and duration. Routine tests showed no measurable toxicity, and little difference in toxicity in the two treatment groups. Comparison of the results based on x-ray findings showed, perhaps, a very small advantage to the PH group. Bacteriologically however, the 'Isoxyl' group fared much less favourably: a smaller proportion became culture-negative and a considerably higher proportion developed cultures resistant to isoniazid in the course of treatment. These results do not agree with the findings of Favez, Vulli6moz & Br6aud (1963), but approximate more closely to those obtained by Donohue and others (1959) in their study of carbinidin, and in the East Africa/British Medical Research Council (1960) study of diphenylthi0urea (SU 1906), a related compound. These unsatisfactory results gave rise to the thought that perhaps the drug was not being absorbed from the intestine and was not getting into the blood in satisfactory concentrations. A suggestion was considered that inadequate fat in the diet might have interfered with absorption; but investigation into the diets showed an average daily intake of 3,000 calories, and an average daily fat intake of about 85 g. The next suggestion to be considered was that a reduced gastric acidity might interfere with absorption. Investigations showed normal acidity in the only eleven IX patients remaining in the trial at this point. Final/y, serum 'isoxyl' activity estimations based on the work o f Dye (personal communication) using Kirschner's medium and H37Rv instead of Middlebrook medium 7H9 and the standard tubercle bacillus RvRx, were carried out on the same eleven'remaining IX patients. 'lsoxyl" was administered both in the form of tablets as used in the trial and also in the form of capsules containing "Isoxyl' in corn oil as recommended by Crowle (personal communication). The results of these tests, shown in Table VIII, make it clear that 'lsoxyl' in corn oil as TABLEVIII.~'IsOXYL' SERUMACTIVITYTESTSAFTERADMINISTRATIONOF 'IsOXYL'IN OIL AND IN TABLETS.(SERUM ISOXYLACTIVITY~G./ML. Period after administration (hours)
Number o f patient
572 573 578 582 585 589 590 591 592 596 598
24 w~ sput~ cultt +
Ne~ Ne Ne Ne Ne Ne +
-t+ Ne
PI
e
in ol .,
in tablets
7"2 9'6 9-6 7-2 2"4 7"2 7"2 4"8 4.8 7"2 7-2
3.6 2.4 3.6 4.8 4.8 4.8 3 "6 4.8 4.8 4.8 3.6
!
in oil
in tablets
in oil
,
3 "6 7.2 7.2 7.2 4"8 9"6 4.8 7.2 3.6 7.2 7.2
2-4 2"4 2"4 3-6 2"4 3"6 3"6 3-6 3-6 3-6 3'6
3-6 3.6 3.6 3-6 3.6 3.6 3-6 4.8 3.6 4-8 3.6
]
in tablets
The minimum inhibitory concentration of 'Isoxyl' solution for H37Rv in all three batches of tests was 1.2 ~g./mL
200
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compared to 'Isoxyl' tablets gives much more satisfactory serum levels, but that all results, at 14 18 and 23 hours after administration both for tablets and for 'lsoxyl' in corn oil, indicate absorption levels higher than the minimum inhibitory concentration for H37Rv of 1.2 Fg./ml. which was found in this series of tests and which exactly corresponded with the level found by Dye in his tests, using different medium and standard bacillus. These tests show that 'lsoxyl' gained entry to the circulating blood in concentrations more than adequate to inhibit tubercle bacilli of standard virulence and drug sensitivity, and it seems that non-absorption of 'Isoxyl' was not an important factor in the results. The finding that 41 ~o of the organisms showed pretreatment resistance to "lsoxyl' was most unexpected, as this drug was appearing in the region as an anti-tuberculosis agent for the first time. The very high prevalence in patients who had denied previous anti-tuberculosis therapy of any description makes it extremely unlikely that previous specific drug administration played any part. There appears to be a close association between 'Isoxyi' resistance and response to treatment with this drug, so that clinically the in vitro findings appear to be substantiated, as is shown in Table VII. Thus we have evidence that 'lsoxyl' was absorbed in adequate concentration as measured against a standard tubercle bacillus, but was a less effective supplement to isoniazid than was PAS against pulmonary tuberculosis occurring in Hong Kong. The surprise laboratory finding of widespread primary resistance to 'lsoxyl' appenred to have a considerable effect on the results of treatment. It is tempting to conclude that pr,mary resistance to 'lsoxyl', not previously reported, but found to be widespread in organisms locally prevalent, may be due to innate differences in the local bacillus of either developmental or metabolic origin, and that these differences interfere with results of treatment with this drug.
4-4
DI1SOAMYLOXYTHIOCARBANIL1DE
201
APPENDIX SENSITIVITY TEST FOR 4-4 D I I S O A M Y L O X Y T H I O C A R B A N I L I D E Dr. G. Meissner at the Tuberkulose-Forschungsinstitut, Borstel has supplied the following details: 4-4 diisoamyloxythiocarbanilide ('Isoxyl') is diluted in polyethyleneglyco1200* and added to the LOwenstein-Jensen medium so that the final concentrations before inspissation are 1000, 500, 200, 100 and 50 ~g./ml. Method 1000 nag. 'lsoxyl' are added while heating to 10 ml. of polyethylenglycol 200 and warming continued until the solution is brown. The 'lsoxyl' remains in solution for I-2 hours, even after cooling. This solution contains 100,000 ~.g./ml. From this, other dilutions in polyethyleneglycol are made. 1:2, 1:5,1:10 and 1:20. One part each of the standard solution and of the 4 dilutions are added to 99 parts of liquid L0wenstein-Jensen medium before inspissation. It is important to add the medium very gently and to mix carefully after each addition. 5 ml. of the drug-containing medium is then put into the culture tubes. For the sensitivity test the inocula must/rot be large. We inoculate only 0.1 ml. of suspension, containing at most 10 -a nag. per ml. Strains of normal sensitivity grow in 50-100 ~.g./ml., those growing in higher concentrations are resistant. It is recommended that the results should be expressed in 'resistance ratios' or according to the proportion method, the control strain being N37Rv, titrated at the same time. In this way there is a control titration for each of the strains tested. A control with medium containing 1% of the diluent without any drug is also done. *Polyethyleneglycol is obtainable under the trade name of'Polydiol' from Hiils A. G., 437 Marl, bei Recklinghausen, Grateful acknowledgment is made to Messrs. Continental Pharma of Brussels the manufacturers of 'lsoxyl' who financed this trial and in particular, to Dr. Lambotte for his helpful attitude at all times. We also express gratitude to the staff of Ruttonjee Sanatorium and The Grantham Hospital, to Dr. Chan Hip-shing, who for some time was in charge of one section of the project, and to Mrs. O. Sheldon for secretarial assistance. Acknowledgment is made also in respect of laboratory work carried out at the Tuberkulose-Forschungsinstitut, Borstel (Professor E. Freerksen) by arrangement with Messrs. Continental Pharma, and to the Hong Kong Government Institute of Pathology, particularly to Mr. Lee King-see, and to the laboratory at the Grantham Hospital. We are grateful to Dr. G. Meissner for carrying out the bacteriological work at Borstel and for the report of the sensitivity test to "lsoxyl' in the appendix. Finally, acknowledgment is made to the Director of Medical & Health Services, Hong Kong, for permission to publish. REFERENCES Buu-Hol, N. P., & XUONG, N. D. (1953). C. R. ,4cad. Sci. (Paris), 237, 498. DONOHUE, R. F., DUKE, C. J., KATZ, S., & ROMANSKY, M. J. (1959). ,4mer. Rev. resp. Dis., 80, 590. EAST AVR~CA/BRITmH MEDICAL RESEARCH COUNCIL Thiocetazone/Diphenylthiourea Investigation (1960). Tubercle, Lond., 41,399. FAVEZ, G., VULU~MOZ, P., BR~AUD (1963). Acta tuberc, pneltmol, belg., 54, 103. FEGIZ, G., & RELLINL G. (1961). Gazz. #ltittel. Med. Chir. 66, 24, 1. FREERKSEN, E. (1962), Arzneim-Forsch., 12, 280. JU1N, J. P., Buu-Hol, N. P. (1946). Ann. Inst. Pasteur, 72, 580. LUCCHESSL M. (1963). Acta tuberc, pneumol, belg., 54, 42. MAYER, R. L. (I~A1). Rev. reed. fi'anc., 9, 393. MAYER, R. L., EISMAN, P. C., KANOPKA, E. A. (1953). Proc. Soc. exp. Biol., N.Y., 82, 769. MUROHASHI & YANAGlSAWA(1963). ,4cta tuberc., pneumol, belg., 54, 35. STEENKEN, W., MONTALmNE, V., SMITH, M. M., & WOUNSKL R. (1958). Amer. Rev. Tuberc., 78, 570. TACQUET, A., GERNEZ-RIEUX, CJ-L, MACQUET, W., Buu-HoL N. P., XUONG, N. D. (1959). Ann. Inst. Pasteur, Lille, 10, 43. YOUMANS, G. P., YOUMANS, A. S., & DOUB, L. (1958), Amer. Rev. Tuberc., 77, 301.
Tubercle, Lond., (1964), 45, 192
C O N T R O L L E D C L I N I C A L T R I A L OF 4-4 D I I S O A M Y L O X Y T H I O C A R B A N I L I D E IN T H E T R E A T M E N T O F P U L M O N A R Y T U B E R C U L O S I S By A, S. MOODm,* Sister M. AQUINAS, and R. D. FOORD fi'om the Medical and Health Department, Hongkong, Ruttonjee Sanatorium. Hongkong and the Grantham Hospital, Hongkong
SUMMARY A total of l00 Chinese patients suffering from open pulmonary tuberculosis were randomly allocated to treatment with either PAS and isoniazid or with 4-4 diisoamyloxythiocarbanilide ('isoxyl') and isoniazid, Twenty were excluded from further participation because of pretreatment drug resistance. Of the remaining 80 patients, one each from the 39 PH group and 41 IX group were withdrawn at the 16th week, the IX patient being withdrawn because of failure of treatment. There were no noticable toxic effects from treatment. After 24 weeks' treatment 71% ofthe PH group and 40% of the IX group had negative cultures. Isoniazid-resistance emerged in 7 of the 38 PH and in 20 of the 40 IX patients. Pretreatment resistance to 'lsoxyl' was reported in 37 of the 78 patients who completed the trial. This might well have had an effect on the results of treatment with 'lsoxyl' and isoniazid.
Derivatives of thiourea were shown to possess in vitro activity against the tubercle bacillus by Mayer (1941) as well as by Juin & Buu-Hoi (1946). These early compounds were unsuitable for clinical use because of their activity on the thyroid gland, but later compounds were shown to be free of this disadvantage by Mayer, Eisman & Kanopka (1953) and by Buu-Hoi & Xuong (1953), in the treatment of leprosy. Youmans, Youmans, & Doub (1958) and Steenkcn and others (1958) demonstrated in vitro and in vivo anti-tuberculosis activity in the carbanidin group of thiourea derivatives. 'Isoxyl' is a new compound, 4-4 diisoamyloxythiocarbanilide, of the N-N diarylthiourea group synthesised by Buu-Hoi & Xuong and shown by Tacquet and others (1958) to have in vivo and in vitro activity against the tubercle bacillus. Freerksen (1962), Murohashi & Yanagisawa (1963), and Lucchessi (1963), showed that in laboratory animals the anti-tuberculosis activity of 'Isoxyl' was more powerful than that of PAS, comparable to that of ethionamide, but less powerful than streptomycin or isoniazid. Fegiz & Rellini (196i.) showed that the drug was well tolerated in human beings in a dosage of 2 g. daily, and claimed that 'Isoxyl' could with advantage be substituted for PAS in triple drug therapy, and that it produced no evidence of intolerance. Favez, Vulli6moz & Br6aud (1963) reported that in newly diagnosed cases of tuberculosis 'Isoxyl' 6 g. in combination with isoniazid, 600 rag. produced a clinical response comparable with that produced by PAS and isoniazid, streptomycin and isoniazid and that 'Isoxyl' was effective in preventing the emergence of organisms resistant to isoniazid. Thus, the evidence has been accnmulating to suggest that 'Isoxyl' might, with advantage, be considered as a possible substitute for PAS or possibly as an addition to the drugs already available *Present address--Branault, Barrhead, Glasgow.
4-4 DIISOAMYLOXYTHIOCARBANILIDE
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for the treatment of tuberculosis. The drug is attractive by virtue of its smaller bulk and greater acceptibility. If the reported good climcal results could be duplicated locally, it might be worth adoption for general use in Hong Kong. It was with this idea in mind that a controlled clinical trial was planned. Plan of the Trial Objectives To measure the therapeutic efficiency of 'Isoxyl' in combination with isoniazid against a combination of PAS and isoniazid in the treatment of newly diagnosed pulmonary tuberculosis. To compare the efficacy of PAS and 'Isoxyl' in their power to prevent the emergence ofisoniazidresistant organisms. To record and compare the toxic effects of the two drug combinations. To make and record observations on the cross resistance pattern between 'lsoxyl' and other antituberculosis drugs. Criteria for Selection of Patients Individuals included conformed to the following: Age 15-50 years. Suffering from untreated pulmonary tuberculosis with or without cavitation. Tubercle bacilli were demonstrated in the sputum by direct smear less than 4 weeks before the date of acceptance. Willing to accept treatment in hospital for a period of at least 24 weeks. Patients showing any of the following were excluded: Moribund state. Excreting tubercle bacilli which showed ht vitro resistance to streptomycin, PAS, or isoniazid. M iliary tuberculosis. Other complicating tuberculosis. Pleural effusion obscuring more than half of one lung field. Diabetes mellitus. Any concurrent disease which might complicate treatment or interfere with results. Prescribed Treatment Patients were allocated to one or other of the following treatment schedules: IX--'lsoxyl' tablets, 4 g. with isoniazid 200 nag. taken in one daily dose. P H - - P A S 10 g. in solution with isoniazid, 200 nag. taken in one daily dose. The Patients A total of 100 Chinese patients was selected on the basis of the acceptance criteria from the public clinics in Hong Kong and admitted to one of the two hospitals taking part in the study. As soon as aceeptibility had been confirmed, application for inclusion in the trial was made to the trial secretary, who was located in a separate institution and did not come into contact with the patients. Allocation was made from a single group of previously prepared and sealed envelopes bearing numbers 501-600 which contained a note bearing one of the two treatment regimes allocated by random numbers, and patients were provisionally included in the trial until the results of the sensitivity tests became available. Exclusions~Of the original 100 patients, 20 were excluded on account of bacterial resistance to one or more of the standard drugs leaving, 39 PAS and isoniazid cases (PH) and 4/ ('Isoxyl' and isoniazid cases (IX). The pretreatment condition of the patients is shown in Table I. The radiographic classification is based on extent of lesions in categories one to six, according to the number of zones involved, and cavitation is ~ivided into categories 0-3 according to the size and number of cavities seen. it will be seen that the two groups corresponded closely in all respects.
TUBERCLE
194
TABLEI.--PRETREATMENTSTATUSOF PATIENTS
PAS/isoniazid (PH)
'Isoxyl'/isoniazid
3"31 1 "54
3.40
(ZX)
First random allocation Resistant to one or more of the standard drugs Remaining in the study General condition
Good Fair Poor
Sex
Males Females
Age (years)
15-24
25-34 35-44 45-50 Sputum status 3 Specimens
Radiographic assessment*
No acid fast bacilliseen 0-I0 per smear 10-20 ,, 20-40 ,, Over 40 ,, No. of zones Cavitation
1.34
*See text for method of assessment.
Routine ExamhTations Pretreatment--Body weight, haemoglobin, fasting blood sugar, blood cholesterol, serum albumen and globulin, S.G.O.T., S.G.P.T., total white and differential count, three sputum specimens for concentration, grading, culture, and sensitivity tests for streptomycin, PAS, isoniazid, thiosemicarbazone, ethionamide and 'Isoxyl'. One 15 in. • 12 in. x-ray film of the chest. Subsequent Exam#~ations--All pretreatment examination procedures to be repeated at the 12th and 24th weeks. At the 4th, 8th, 16th and 20th w e e k s : - Body weight, two sputum specimens for concentration, grading, culture, and sensitivity tests as detailed above in the pre-treatme~t examination. BACTERIOLOGICAL METHODS Specimens were concentrated by Petroff's method, stained by Ziehl-Neelsen and examined by direct microscopy. Smears were classified according to the total number of bacilli present. From the residue two cultures were prepared on L6wenstein-Jensen slopes and incubated for a minimum period of 8 weeks at 37 ~ C. One culture was examined a t the Hoffg K o n g G o v e r n m e n t Institute. of Pathology or at the laboratory of The G r a n t h a m Hospital, H o n g Kong. The resistance ratio method was used, with the following drug concentrations with H37Rv as control organism.
4 - 4 DIISOAMYLOXYTHIOCARBANILIDE
195
Micrograms per ml. Streptomycin 0.5 1-0 2.0 4.0 PAS 0.5 1.0 2.0 4.0 lsoniazid 0.12 0.25 0.5 1.0 5.0 Ethionamide 5.0 10.0 20.0 40.0 80.0 100.0 Thiosemicarbazone 0.5 1.0 2.0 4.0 8.0 16 A ratio of 2 or less was recorded as 'sensitive'. A ratio of 4 was recorded as 'probably resistant'. A ratio of 8 or above was recorded as 'definitely resistant'. The second culture was sent by air to the Tuberculose-Forschungsinstitut, Borstel, Hamburg, where a parallel set of sensitivity tests was carried out using the absolute concentration method. This laboratory performed, in addition, a sensitivity test f o r ' I s o x y l ' on each specimen (see Appendix). Thus each culture was tested for drug resistance by two different methods, the resistance ratio and the absolute concentration methods. The measure of agreement obtained in the two different methods in the course of 2,000 consecutive sensitivity tests is shown in Table II. It will be observed that there was complete agreement in over 90 ~o of these tests and that the major part of the disagreement was seen in tests where one laboratory reported partial resistance as against full sensitivity by the other, or where one laboratory reported total resistance in one specimen as against a report of partial resistance by the other. Significant disagreement, where one laboratory reported total sensitivity as against total resistance by the other, occurred in only 8 tests (0.4 ~ ) . hlterpretation o f Bacteriological Findings
Because of the multiplicity of the bacteriological observations a standard procedure was adopted for classification of cases. At each four weeks period there were either two or three graded sputum smears examined and reported. The case was classified according to the highest grading. At each four weeks period there were either four or six cultures, each positive culture being tested for sensitivity to five drugs, streptomycin, PAS, isoniazid, thiosemicarbazone and ethionamide, and in the case of cultures examined at Borstel, to 'lsoxyl' as well. In the pretreatment series a single TABLEII.--THE MEASUREOFAGREEMENTAND DISAGREEMENTOF RESULTSOF SENSITIVITYTESTS CARRIEDOUT ON THE CULTURE FROMTHE SAME SPECIMEN BY TWO DIFFERENT METHODS (HONG KONG--RESISTANCERATIO METHOD" BORSTEL~ABSOLUTECONCENTRATIONMETHOD) Results agree
Results disagree
Hung Kong result
Sens.*
P.R.*
D.R.*
Borstel result
Sens.
P.R.
D.R.
381 367 365 382 224
-m -5 38
1719
43
Ethionamide Streptomycin Thiosemicarbazone PAS Isoniazid Total
Sens. D.R.
16
1
24
I
21
27 3 53
45
100
1807
Grand Total (Percentage)
(90%)
*Sens.=Sensitive.
P.R.
P.R.
P.R.-----Partiallyresistant.
Sens.
D.R. D.R.
Sens.
P.R.
3
5 8 7 12
3
4
42
4
33
3
4
49
3 3
193 (lo%) D.R.=Definitely resistant.
196
TUBERCLE
report of resistance to streptomycin, PAS, or isoniazid was regarded as reasonable ground for excluding the case from the study; while in examinations during treatment a single unsupported report of resistance was disregarded. A case was regarded as having developed resistant cultures only when two or more reports of resistance were made, and where two reports of resistance differed, the higher was the one adopted. WITHDRAWALS One PH case (538) withdrew after the examinations of the 16th week when the sputum was already negative on concentration and culture, probably on account of gastric symptoms, but showing a minor degree of mental instability which appeared to be unassociated with the treatment. One IX case (593) was withdrawn at the end of the 16th week as a treatment failure because of persistant positive sputum and the development of definite resistance to isoniazid which first appeared at ttae 8th week, and partial resistance to 'lsoxyl', which were accompanied by clinical and radiographic deterioration. The remaining 78 patients completed the stated period of treatment of 24 weeks. BACTERIOLOGICALRESULTS Results The principal measure of efficiency of a therapeutic agent in tile treatment of tuberculosis is its ability to render the sputum abacterial and the speed with which it does so. The bacteriological changes brought about by the two treatment regimes are shown in Table III. These results show a clear advantage in favour of the PH regime, 84 ~o showing the sputum to be negative on concentration, an4 71 ~o on culture, at 24 weeks as compared with 55~o, and 4 0 ~ respectively in the IX group. (Tb'." differences are significant at the 2 ~ level.) A further measure of the adequacy of a therapeutic agent is its ability, when used in conjunction with isoniazid, to prevent the emergence of isoniazid-resistant tubercle bacilli. A comparison of the two-drug regimes in this respect is shown in Table IV. In the 38 patients who completed 24 weeks' treatment with PAS and isoniazid, 11 remained sputum positive on culture, of whom 7 (18 ~o) had cultures resistant to isoniazid, while in the 'lsoxyl' and isoniazid group 24 of the 40 patients remained sputum positive on culture, 20 (50 ~o) showing resistance to isoniazid, of whom 4 had also developed resistance to 'lsoxyl'. OTHER CHANGES Radiographically the changes produced by treatment were divided into three grades of improvement, three grades of deterioration and one category which showed no change. Assessments were TABLEIII.--BACTER1OLOGICALCHANGESDURINGTREATMENT 38 patients completed treatment wth PAS/isoniazid
40 patients completed treatment with "lsoxyl'/isoniazid
Negative findings on
Negative findings on
Sputttm conc.
I
Pretreatment 4th week 8th week 12th week 16th week 20th week 24th week
I
Sputum conc.
Culture
Number
%
2 13 15 27 29 31 32
5
1
2
34 40 71 76 81 84
11 17 15 21 22 22
27 42 37 52 55 55
Number
%
Number
%
Culture Nmnber
~
4 - 4 DIISOAMYLOXYTHIOCA RBANILIDE
197
TABLEIV.--EMERGENCEOF ]SONIAZIDRESISTANCEAFTER24 WEEKS TREATMENT I
Treatment with
I Culture results
i PAS]isoniazM 38 patients
I
"Isoxyl'/isoniazid 40 patients
L
Negative Positive and sensitive Positive and resistant to isoniazid Positive and resistant to isoniazid and 'Isoxyl'
t I j i
27 4 7
16 4 16
--
4
TABLEV.--RADIOGRAPHICCHANGESDURINGTREATMENT Weeks 0.12
Degree of change
Worse Unchanged
Weeks 13-24
PH
IX
29 5
25 8
PH
J
Weeks 0-24 I X ..........
PH
i
--
t
--3 --2 --1 0
I
IX
1
!
Improved
+1 +2 +3
23
23
I
1
14 1
1 ,
--
23 12 3
made by one of us (A. S. M.) without knowing the treatment regime used, based on a comparison of the x-rays of pretreatment and 12 weeks, between the film at 12 weeks and 24 weeks, and between the pretreatment and 24 weeks film. The results of these assessments are shown on Table V. One patient in the IX group showed radiographic deterioration and one no change: all in the PH group showed improvement. This slight difference is not statistically significant. The results of the routine examinations are shown in Table VI, which shows the average and range of values found but excludes the results of examinations at 12 weeks, as these showed nothing of note. There was no evidence that either treatment had any adverse effect on the haemopoetic system, the liver, the thyroid gland, the kidneys, nor was there any significant change in the blood sugar levels. The changes produced were common to both groups. T o x I c EFFECTS There were a few complaints of epigastric discomfort, loss of appetite and nausea, but these complaints were of a trivial nature, o f short duration, and appeared to occur with equal frequency in both groups. N o t a single case had to stop treatment because of toxic effects. One IX case (513) also received prednisolone because of fever, deterioration on x-ray and loss of voice. He developed a skin rash three weeks later, the origin of which was not determined, but continued with treatment as before, improving gradually but was still sputum positive at the 24th week. RESISTANCE TO '[SOXYL' BEFORE TREATMENT Routine sensitivity tests to 'Isoxyl' were carried out at the Borstel Institute, but were not dupli-
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198
TABLEVI.--LABORATORYINVESTIGATIONS(AVERAGEVALUEAND RANGE)
PAS]isoniazid
"Isox),l'/isoniazid
Z-~,e,~eo,- , ~ nlent
~,~,,~o,- I '
[ 24th week % change
nlent
-
24th week % change
i
Average wt. (lbs.) Range Haemoglobin (%) Range Fasting blood sugar (mg./100 nal.) Range Blood cholesterol (rag./100 ml.) Range Serum albumen (g./100 ml.) Range Serum globulin (g./100 ml.) Range S.G.O.T. (Karmen) Range S.G.P.T. (Karmen) Range W.B.C. total Range in tousands
102 112 (68-123) (79-151) 81 84 (60-110) (70-1lO) 77 80 (50-109) (40-120) 153 182 (95-223) (127-270) 3"53 4"24 (2;9.94.5) (3;0.;~.3) (2"7"5"1) I ( 2 " 4 " 4 ' 4 )I11.2 3.4 (7-30)
,
11.6
(3-22) ! 1-0
(4-60) (3-73) 7445 6611 (3.8-13.8) (3.4-10.3)
+10% 100 (67-I24) + 4% i 87 l (60-110) + 4% t 78 !(60-109) +18% i 160 (103-230) +20% 3.73 (3.1-4.6) --22% 3.72 (3.0-4.9) ul % 14.7" (6-38) -% 10.5(3-73) --1% 7805 (4.7-14)
109 (72-139) 93 (70-110) 82 (50-109) 165 (112-240) 4"34 (3 "O-5"3) 3 -32 (2.2-4"2) 9'9"
+ 9% + 7% + 6% + 3% +16% --11% --33% --11%
(2-85) 6430 (3.8-10)
--17%
*One case with values outside the ranges quoted is excluded because of history of previous liver disease. 'lsoxyl' was continued without disturbance of the values. cated in the Hong Kong laboratories because of uncertainty as to their value and doubt as to the most suitable technique. These tests showed that of the 100 patients provisionally selected for inclusion in the study, 41 were excreting organisms with a significant degree of resistance to 'lsoxyl' in the pretreatment examinations by the absolute concentration method. Three 'Isoxyl' resistant cases were included in the 20 cases rejected because of other bacterial resistance, and a further 'lsoxyl' resistant case was lost (538) on withdrawal, leaving 37 'lsoxyl' resistant cases who completed the prescribed period of treatment (20 P H and 17 IX). Sputum cultures results at 24 weeks in the IX group showed that 57 % of the 'lsoxyl'-sensJtive cases were negative as against 18 % in the 'Isoxyl'-resistant cases, while in the PH group, 88 % of the 'Isoxyl'-sensitive as against 55 % of the 'Isoxyl'-resistant cases had become negative (Table VII). The differences in the proportions negative on culture are significant at the 5 level in the IX group but are not significant in the P H group. TABLEVII.--BACTERIOLOGICALRESULTSACCORDINGTO PRETREATMENTSENSITIVITYTO 'ISOXYL'
I Pretreatment set~sitivity to isoxyl
I
Bacteriologicalfinding on sputum culture at 24 weeks
Total
PAS/isoniazid
f l Pos.
'lsoxyl' sensitive 'lsoxyl' resistant
41 37
I
2 9
I I
Neg. no.
%
16 11
88 55
t
'Isoxyl'/isoniazid
E! [
I
Pos. 10 14
Neg. no. [ [
13 3
4-4 DIISOAMYLOXYTHIOCARBANILIDE
199
With regard to cross resistance, of the 41 patients who showed pretreatment resistance to 'Isoxyl', 12 also had cultures resistant to thiosemicarbazone, and 5 had cultures resistant to thiosemicarbazone but not to "Isoxyl'. Of the four who developed 'Isoxyl' resistance under treatment not a single one developed resistance to thiosemicarbazone. Thus there appears to be some, but not a very well defined association between resistance to the two drugs. Discussion 'lsoxyl' fulfilled all claims as to acceptibility, causing only minor gastric upset and anorexia as well as a few other symptoms of insignificant proportions and duration. Routine tests showed no measurable toxicity, and little difference in toxicity in the two treatment groups. Comparison of the results based on x-ray findings showed, perhaps, a very small advantage to the PH group. Bacteriologically however, the 'Isoxyl' group fared much less favourably: a smaller proportion became culture-negative and a considerably higher proportion developed cultures resistant to isoniazid in the course of treatment. These results do not agree with the findings of Favez, Vulli6moz & Br6aud (1963), but approximate more closely to those obtained by Donohue and others (1959) in their study of carbinidin, and in the East Africa/British Medical Research Council (1960) study of diphenylthi0urea (SU 1906), a related compound. These unsatisfactory results gave rise to the thought that perhaps the drug was not being absorbed from the intestine and was not getting into the blood in satisfactory concentrations. A suggestion was considered that inadequate fat in the diet might have interfered with absorption; but investigation into the diets showed an average daily intake of 3,000 calories, and an average daily fat intake of about 85 g. The next suggestion to be considered was that a reduced gastric acidity might interfere with absorption. Investigations showed normal acidity in the only eleven IX patients remaining in the trial at this point. Final/y, serum 'isoxyl' activity estimations based on the work o f Dye (personal communication) using Kirschner's medium and H37Rv instead of Middlebrook medium 7H9 and the standard tubercle bacillus RvRx, were carried out on the same eleven'remaining IX patients. 'lsoxyl" was administered both in the form of tablets as used in the trial and also in the form of capsules containing "Isoxyl' in corn oil as recommended by Crowle (personal communication). The results of these tests, shown in Table VIII, make it clear that 'lsoxyl' in corn oil as TABLEVIII.~'IsOXYL' SERUMACTIVITYTESTSAFTERADMINISTRATIONOF 'IsOXYL'IN OIL AND IN TABLETS.(SERUM ISOXYLACTIVITY~G./ML. Period after administration (hours)
Number o f patient
572 573 578 582 585 589 590 591 592 596 598
24 w~ sput~ cultt +
Ne~ Ne Ne Ne Ne Ne +
-t+ Ne
PI
e
in ol .,
in tablets
7"2 9'6 9-6 7-2 2"4 7"2 7"2 4"8 4.8 7"2 7-2
3.6 2.4 3.6 4.8 4.8 4.8 3 "6 4.8 4.8 4.8 3.6
!
in oil
in tablets
in oil
,
3 "6 7.2 7.2 7.2 4"8 9"6 4.8 7.2 3.6 7.2 7.2
2-4 2"4 2"4 3-6 2"4 3"6 3"6 3-6 3-6 3-6 3'6
3-6 3.6 3.6 3-6 3.6 3.6 3-6 4.8 3.6 4-8 3.6
]
in tablets
The minimum inhibitory concentration of 'Isoxyl' solution for H37Rv in all three batches of tests was 1.2 ~g./mL
200
TUBERCLE
compared to 'Isoxyl' tablets gives much more satisfactory serum levels, but that all results, at 14 18 and 23 hours after administration both for tablets and for 'lsoxyl' in corn oil, indicate absorption levels higher than the minimum inhibitory concentration for H37Rv of 1.2 Fg./ml. which was found in this series of tests and which exactly corresponded with the level found by Dye in his tests, using different medium and standard bacillus. These tests show that 'lsoxyl' gained entry to the circulating blood in concentrations more than adequate to inhibit tubercle bacilli of standard virulence and drug sensitivity, and it seems that non-absorption of 'Isoxyl' was not an important factor in the results. The finding that 41 ~o of the organisms showed pretreatment resistance to "lsoxyl' was most unexpected, as this drug was appearing in the region as an anti-tuberculosis agent for the first time. The very high prevalence in patients who had denied previous anti-tuberculosis therapy of any description makes it extremely unlikely that previous specific drug administration played any part. There appears to be a close association between 'Isoxyi' resistance and response to treatment with this drug, so that clinically the in vitro findings appear to be substantiated, as is shown in Table VII. Thus we have evidence that 'lsoxyl' was absorbed in adequate concentration as measured against a standard tubercle bacillus, but was a less effective supplement to isoniazid than was PAS against pulmonary tuberculosis occurring in Hong Kong. The surprise laboratory finding of widespread primary resistance to 'lsoxyl' appenred to have a considerable effect on the results of treatment. It is tempting to conclude that pr,mary resistance to 'lsoxyl', not previously reported, but found to be widespread in organisms locally prevalent, may be due to innate differences in the local bacillus of either developmental or metabolic origin, and that these differences interfere with results of treatment with this drug.
4-4
DI1SOAMYLOXYTHIOCARBANIL1DE
201
APPENDIX SENSITIVITY TEST FOR 4-4 D I I S O A M Y L O X Y T H I O C A R B A N I L I D E Dr. G. Meissner at the Tuberkulose-Forschungsinstitut, Borstel has supplied the following details: 4-4 diisoamyloxythiocarbanilide ('Isoxyl') is diluted in polyethyleneglyco1200* and added to the LOwenstein-Jensen medium so that the final concentrations before inspissation are 1000, 500, 200, 100 and 50 ~g./ml. Method 1000 nag. 'lsoxyl' are added while heating to 10 ml. of polyethylenglycol 200 and warming continued until the solution is brown. The 'lsoxyl' remains in solution for I-2 hours, even after cooling. This solution contains 100,000 ~.g./ml. From this, other dilutions in polyethyleneglycol are made. 1:2, 1:5,1:10 and 1:20. One part each of the standard solution and of the 4 dilutions are added to 99 parts of liquid L0wenstein-Jensen medium before inspissation. It is important to add the medium very gently and to mix carefully after each addition. 5 ml. of the drug-containing medium is then put into the culture tubes. For the sensitivity test the inocula must/rot be large. We inoculate only 0.1 ml. of suspension, containing at most 10 -a nag. per ml. Strains of normal sensitivity grow in 50-100 ~.g./ml., those growing in higher concentrations are resistant. It is recommended that the results should be expressed in 'resistance ratios' or according to the proportion method, the control strain being N37Rv, titrated at the same time. In this way there is a control titration for each of the strains tested. A control with medium containing 1% of the diluent without any drug is also done. *Polyethyleneglycol is obtainable under the trade name of'Polydiol' from Hiils A. G., 437 Marl, bei Recklinghausen, Grateful acknowledgment is made to Messrs. Continental Pharma of Brussels the manufacturers of 'lsoxyl' who financed this trial and in particular, to Dr. Lambotte for his helpful attitude at all times. We also express gratitude to the staff of Ruttonjee Sanatorium and The Grantham Hospital, to Dr. Chan Hip-shing, who for some time was in charge of one section of the project, and to Mrs. O. Sheldon for secretarial assistance. Acknowledgment is made also in respect of laboratory work carried out at the Tuberkulose-Forschungsinstitut, Borstel (Professor E. Freerksen) by arrangement with Messrs. Continental Pharma, and to the Hong Kong Government Institute of Pathology, particularly to Mr. Lee King-see, and to the laboratory at the Grantham Hospital. We are grateful to Dr. G. Meissner for carrying out the bacteriological work at Borstel and for the report of the sensitivity test to "lsoxyl' in the appendix. Finally, acknowledgment is made to the Director of Medical & Health Services, Hong Kong, for permission to publish. REFERENCES Buu-Hol, N. P., & XUONG, N. D. (1953). C. R. ,4cad. Sci. (Paris), 237, 498. DONOHUE, R. F., DUKE, C. J., KATZ, S., & ROMANSKY, M. J. (1959). ,4mer. Rev. resp. Dis., 80, 590. EAST AVR~CA/BRITmH MEDICAL RESEARCH COUNCIL Thiocetazone/Diphenylthiourea Investigation (1960). Tubercle, Lond., 41,399. FAVEZ, G., VULU~MOZ, P., BR~AUD (1963). Acta tuberc, pneltmol, belg., 54, 103. FEGIZ, G., & RELLINL G. (1961). Gazz. #ltittel. Med. Chir. 66, 24, 1. FREERKSEN, E. (1962), Arzneim-Forsch., 12, 280. JU1N, J. P., Buu-Hol, N. P. (1946). Ann. Inst. Pasteur, 72, 580. LUCCHESSL M. (1963). Acta tuberc, pneumol, belg., 54, 42. MAYER, R. L. (I~A1). Rev. reed. fi'anc., 9, 393. MAYER, R. L., EISMAN, P. C., KANOPKA, E. A. (1953). Proc. Soc. exp. Biol., N.Y., 82, 769. MUROHASHI & YANAGlSAWA(1963). ,4cta tuberc., pneumol, belg., 54, 35. STEENKEN, W., MONTALmNE, V., SMITH, M. M., & WOUNSKL R. (1958). Amer. Rev. Tuberc., 78, 570. TACQUET, A., GERNEZ-RIEUX, CJ-L, MACQUET, W., Buu-HoL N. P., XUONG, N. D. (1959). Ann. Inst. Pasteur, Lille, 10, 43. YOUMANS, G. P., YOUMANS, A. S., & DOUB, L. (1958), Amer. Rev. Tuberc., 77, 301.