PHARMACOLOGY OF SOME SLOW-RELEASE PREPARATIONS OF ISONIAZID OF POTENTIAL USE IN INTERMITTENT TREATMENT OF TUBERCULOSIS

340

PHARMACOLOGY OF SOME SLOWRELEASE PREPARATIONS OF ISONIAZID OF POTENTIAL USE IN INTERMITTENT TREATMENT OF TUBERCULOSIS V. R. ABER G. A. ELLARD D. A. MITCHISON PATRICIA T. GAMMON S. LAKSHMINARAYAN K. M. CITRON WALLACE Fox RUTH TALL Medical Research Council Unit for Laboratory Studies of Tuberculosis, Royal Postgraduate Medical School, Brompton Hospital; and Medical Research Council Tuberculosis and Chest Diseases Unit, Brompton Hospital, London

Sum ary been

The pharmacology of three slow-release preparations of isoniazid (I.N.H.) have studied for their potential use in the once-weekly

treatment

of tuberculosis.

The

I.N.H.

in the matrix

preparation was almost completely absorbed, yet the rate of absorption should allow a dose of at least 30 mg. per kg. body-weight to be given to slow and rapid inactivators of I.N.H. without acute toxicity. Such a dose should be therapeutically effective in once-weekly regimens. Effective and safe once-weekly chemotherapy might also be achieved in rapid inactivators by giving 15 mg. per kg. ordinary I.N.H. with 15 mg. per kg. of the enteric-coated preparation studied, but this regimen would not be safe in slow

I.N.H. cannot be increased further because of the risk of acute toxicity-especially vasomotor collapse and

convulsions-at about the time that the peak serum concentrations are attained. Other attempts to increase the half-life of I.N.H. by inhibiting its acetylation using sulphamethazine or acetylisoniazid have been

disappointing. 1.2,13 We hoped that an effective exposure could be achieved without drug toxicity by giving a dose of at least 30 mg. per kg. I.N.H. in a slow-release formulation, resulting in peak serum-i.N.H. levels not much above 16 g. per ml. (the average peak level obtained after 15 mg. per kg. ordinary I.N.H. in slow inactivators 8). Two approaches were envisaged. In the first, a dose of 15 mg. per kg. ordinary soluble I.N.H. might be given at the same time as a 15 mg. per kg. dose of enteric-coated per

kg.

I.N.H.

or more I.N.H.

matrix

preparation. approached, and we studies of the three

In the

dose of 30 mg. in might given a slow-release Two drug manufacturers were describe here pharmacological

second,

a

be

l.N.H.

preparations provided.

Methods

Preparations of LN.H. in 100 mg. sugar-coated tablets (" ordinary Roche-Rimifon 625) and the following slowrelease preparations were investigated: 100 mg. tablets of enteric-coated I.N.H. (" enteric-i.N.H. "; Roche-Rimifon 646), 100 mg. tablets of an enteric-coated I.N.H. matrix (" enteric-matrix "; Roche-Rimifon 647), and 300 mg. uncoated tablets of an i.N.H. matrix (" matrix"; Smith and Nephew HS 82). I.N.H. I.N.H. ";

inactivators. Introduction

of pulmonary tuberculosis with of 15 mg. isoniazid (I.N.H.) per doses twice-weekly THE

increased

1’4-fold, either by giving 6 g. sodium p-aminosalicylate concomitantly or by raising the dose from 13 to 17 mg. per kg. l.N.H., the response to treatment was only slightly improved.3.4 The dose of was

treatment

kg. body-weight and 0’75-1 g. streptomycin is effective in both slow and rapid inactivators of I.N.H.1,2 However, when the same doses were given once a week, the results were less satisfactory, especially in rapid inactivators.2 When the basic once-weekly regimen was strengthened by giving an initial month of daily treatment with I.N.H. and streptomycin, a bacterioSogically quiescent disease state was attained in 95% of the slow inactivators, but in only 75% of the rapid inactivators.2,3 The deficiency of I.N.H. in the rapid inactivators resulted in the multiplication of sensitive organisms between the once-weekly doses and the 4 emergence of drug resistance.2, Studies of the action of l.N.H. against Mycobacterium tuberculosis in vitro have indicated that the period for which growth is inhibited after a pulse of the drug is related to the " exposure " to l.N.H., where exposure is defined as the product of the concentration of l.N.H. used and the duration of the exposure. 5-7 The exposure obtained in vivo is proportional to the half-life of I.N.1-I. in the body and to the total dose of I.N.H. administered, whether it be given in single or multiple doses, and in ordinary or slow-release formulations. The exposure achieved was 2-4 times greater in slow than in rapid inactivators following a dose of 13-17 mg. per kg. I.N.H., 4,8 and a similar result was obtained after a dose of 10 mg. per kg. I.N.H.9 Experimental work in the guineapig suggested that the maximum therapeutic effect would be obtained if the exposure of rapid inactivators given 15 mg. per kg. I.N.H. could be doubled. 10, 11 When the exposure in rapid inactivators

Design of the Investigation The investigation was divided into three parts. In the first study, I.N.H. serum concentrations were determined in twelve adult patient-volunteers from the Brompton Hospital who had previously been classified as six slow and six rapid inactivators of I.N.H. Eight of these patients Each patient received a had pulmonary tuberculosis. 300 mg. dose of one of the three slow-release I.N.H. preparations or ordinary I.N.H. followed, after at least a week, by a 300 mg. dose of a second of these four preparations,

according to a randomised incomplete block design. The results suggested that the matrix was the most promising preparation, and its pharmacology was further investigated in the second and third studies. The second study was carried out on volunteers from staff of the Royal Postgraduate Medical School. The kinetics of the elimination of I.N.H. and its metabolites in the urine were determined in one slow and one rapid inactivator over a period of 48 hours after dosage with 600 mg. matrix and also after 250 mg. ordinary I.N.H. The absorption of I.N.H. from 600 mg. matrix was then assessed in a further five slow and six rapid inactivators by determining the urinary excretion of l.N.H. and its metabolites during the periods 0-23 hours, 23-24 hours, and 24-48 hours. In the third study, plasma-i.N.H. concentrations were determined after giving 600 mg. matrix to twenty adult patient-volunteers at the Brompton Hospital. Most of these patients had chronic respiratory diseases such as chronic bronchitis or asthma. All doses of l.N.H. were swallowed, without chewing,

341 empty stomach, and if patients in the first and third studies were being treated with I.N.H., P.A.S., or pyrazinamide, these drugs were stopped for at least 24 hours before the start of any investigation. All blood specimens were collected within 10 minutes of the stated times. Samples of plasma or serum were stored at -70°C, and samples of urine at -20°C until analysis up to 2 months later. on an

Analytical Methods and Determination of LN.H. Inactivator Status of Subjects The concentrations of I.N.H. and of acetyl-i.N.H. in the serum or plasma were determined fluorimetrically.14 The same methods were used to determine the concentrations of I.N.H. together with its acid-labile hydrazones (" acidlabile I.N.H.") and of acetyl-i.N.H. in the urine. These results were confirmed by minor modifications of colorimetric methods for the determination of these compounds.15,16 Isonicotinic acid and isonicotinylglycine were determined colorimetrically.14 The completeness of the urine collections in the second study was confirmed by determining their creatinine contents by the alkalinepicrate method.17 In the first study, patients were classified as slow or rapid inactivators of I.N.H. according to the ratio of the excretion in the urine of acetyl-I.N.H. to that of acidlabile I.N.H. from 0 to 2 hours after an oral dose of 300 mg. ordinary I.N.H.13 Those with ratios of 0-20-0-49 were classified as slow inactivators and those with ratios of 1-10-4-40 as rapid inactivators. This classification was confirmed by determining the ratios of acetyl-i.N.H. to I.N.H. in the serum after administration of the various preparations of I.N.H.13,lS In the second and third studies, subjects were classified according to the ratio of the urinary excretion of acetyl-i.N.H. to that of acid-labile I.N.H. 23-24 hours after dosage with 600 mg. matrix, and in the third study this classification was confirmed by determining the ratios of acetyl-I.N.H. to I.N.H. in the plasma after giving the matrix.18

Results

No symptom likely to be associated with the ingestion of any of the preparations was noted in any subject. 1 st

Study

The serum-l.N.H. concentrations determined in the groups of three slow and three rapid inactivators after they had been given 300 mg. ordinary I.N.H. or

the slow-release preparations are summarised in table i. After ordinary I.N.H., peak serum concentrations were attained at about 1 hour, and averaged about 6 {.Lg. per ml. in the slow inactivators and 4 {.Lg. per ml. in the rapid inactivators. Absorption was considerably delayed when enteric-i.N.H. was given, and in only

the I.N.H. concentration at 2 hours exceed 0-2 g. per ml. 4 hours after dosage, peak serum concentrations similar to those found with ordinary I.N.H. occurred in five of the six subjects. In the sixth subject the absorption of I.N.H. was further retarded and the peak serum concentration was probably attained between 5 and 7 hours. The results obtained with the enteric-matrix were one

patient did

very variable. The highest serum concentrations were attained in four of the patients at about 6 hours. In the other two, absorption was grossly delayed and the maximum serum concentrations probably occurred between 12 and 24 hours. By contrast, remarkably uniform results were obtained with the matrix between patients within each inactivator group. The highest I.N.H. concentrations were found at 2 hours. In the slow inactivators the highest I.N.H. serum concentrations were less than a half of those achieved with ordinary a.N.l3., although similar concentrations were found 12 hours after dosage. In the rapid inactivators, the highest I.N.H. concentrations, after giving the matrix, were less than a fifth of those obtained after giving ordinary I.N.H., but at 12 hours the concentrations were approximately three times those from

ordinary 2nd

I.N.H.

Study

The kinetics of the urinary elimination of acid-labile I.N.H. by the slow and the rapid inactivator after giving either ordinary I.N.H. or the matrix are illustrated in figs. 1 and 2. The total elimination of I.N.H. and its metabolites (0-48 hours) was similar whether ordinary l.N.H. or the matrix had been given (87 % and 85% in the slow inactivator, and 96% and 91% in the rapid inactivator, respectively). The rate of elimination of I.N.H. was, however, much slower when the matrix was given. The total 48-hour urinary excretion of acid-labile I.N.H., together with acetyl-i.N.H., isonicotinic acid, and isonicotinylglycine after 600 mg. matrix, accounted for 89% (range 76-98%) of the dose in the six slow inactivators and 95 % (range 90-103 %) in the seven rapid inactivators. 3rd

Study The plasma-i.N.H. concentrations found after giving 600 mg. matrix to ten slow and ten rapid inactivators are summarised in table 11. Peak plasma concentrations occurred at about 2 hours and were considerably lower than those that would have been anticipated after giving ordinary I.N.H., especially in the rapid

TABLE I-MEAN SERUM CONCENTRATIONS OF I.N.H. AFTER ORAL DOSAGE WITH SLOW-RELEASE I.N.H. PREPARATIONS

300 mg. ORDINARY AND

THREE

342

Fig. 1-Urinary excretion of acid-labile

I.N.H.

by

slow inacti-

vator.

inactivators. The range of the concentrations found was partly due to the fact that the same dose was given to all the patients irrespective of their weights, which varied from 48 to 82 kg. To make some allowance for this, and to facilitate comparison with results obtained in other pharmacological studies with ordinary I.N.H., the plasma concentrations obtained per mg. per kg. of I.N.H. administered in the matrix formulation were calculated for each patient. These calculations are summarised in table ill. Discussion

Enteric-LN.H. The results obtained in the first study indicated that the coating used in the enteric-I.N.H. preparation had delayed the absorption of I.N.H. by about 3 hours, and that thereafter absorption was as rapid and probably as complete as with ordinary I.N.H. Since the half-life of I.N.H. in slow inactivators is about 3 hours, combining a 15 mg. per kg. dose of the enteric-I.N.H. with a 15 mg. per kg. dose of ordinary I.N.H. would be expected to increase the peak l.N.H. concentrations by

Fig. 2-Urinary excretion of acid-labile I.N.H. by rapid inactivator.

and result in a potentially toxic combinaHowever, if patients could be accurately and simply classified, then such a combination might be given to rapid inactivators without serious risk of acute toxicity, while slow inactivators could receive 15 mg. per kg. ordinary I.N.H. It would, however, be essential to preserve the integrity of the enteric coating of the preparation from its manufacture until it was taken by the patient, otherwise rapid absorption could occur and this would lead to the patient consuming the equivalent of 30 mg. per kg. ordinary I.N.H., a potentially toxic dose. about

50%

tion.

Enteric-matrix The results obtained in the first study with this preparation were too variable in the small number of patients studied to make an assessment of its potential use in the intermittent treatment of tuberculosis, and for this reason it was not investigated further.

TABLE II-MEAN PLASMA CONCENTRATIONS OF I.N.H. AFTER ORAL DOSAGE WITH

600 mg. MATRIX

I

TABLE III-MEAN PLASMA CONCENTRATIONS OF I.N.H. AFTER ORAL DOSAGE WITH

10

Based

on

isoniazid half-lives of 3 hours for slow inactivators and 1-25 hours for

600 mg. MATRIX

rapid inactivators (see text).

343

Matrix The in-vitro release properties of the matrix suggested that it could be considered as being made up of 85% of a matrix releasing I.N.H. with a half-life of 5-25 hours and 15% of ordinary I.N.H.19 The results obtained in the first study demonstrated that the matrix was acting as a slow-release preparation in vivo and suggested that it might be possible to give doses of at least 30 mg. per kg. to both slow and rapid inactivators without causing acute toxicity. In the second study, the kinetics of the elimination of I.N.H. in the urine confirmed the in vivo slow-release properties of the matrix. Furthermore, the elimination of I.N.H. and its metabolites in the urine was similar to that obtained after giving ordinary I.N.H.,20 indicating that the absorption of I.N.H. from the matrix was virtually complete. If the release properties of the matrix in vivo paralleled those in vitro, it would suggest that I.N.H. is probably absorbed throughout the whole length of the intestine and probably even from the colon and rectum-a conclusion supported by earlier evidence of absorption of I.N.H. from rectal suppositories.21 The third study enabled the magnitude of peak I.N.H. plasma concentrations to be established with greater precision. The peak l.N.H. plasma concentrations after 600 mg. matrix averaged about 0-45 Vg. per ml. per mg. per kg. in slow inactivators and about 0-2 jjLg. per ml. per mg. per kg. in rapid inactivators (table ill). These results may be compared with an average peak concentration of 1-07 g. per ml. per mg. per kg. after giving 15 mg. per kg. ordinary I.N.H. to slow inactivators.8This would suggest that the matrix could be safely given to both slow and rapid inactivators at doses of up to 35 mg. per kg. Such a dose of the matrix should result in a sufficient exposure for effective once-weekly chemotherapy in both inactivator groups.

plasma concentrations that might be after giving the matrix were calculated using expected a one-compartment model, assuming that the I.N.H.release properties of the matrix in vivo were identical to those in vitro and that I.N.H. was absorbed from the gastrointestinal tract by a process with first-order kinetics (rate constant equivalent to 50% absorption every 0-4 hours, calculated from the data illustrated in fig. 3 of Gelber et al. 9), distributed throughout the total body water 18 (volume equivalent to 68% of the body-weight) and eliminated from the body with an average half-life of 3 hours in slow inactivators and 1-25 hours in rapid inactivators. This model accurately predicted the peak serum I.N.H. concentrations obtained in slow and rapid inactivators after giving an oral dose of 15 mg. per kg. ordinary I.N.H.8 It also correctly predicted the plasma l.N.H. concentrations found in the slow inactivators after giving the matrix, but gave rather higher concentrations for the rapid inactivators than those found in the third study (table ill). Similar results to those in the third study were obtained when the 24-hour plasma-i.N.H. concentrations were determined in fifty healthy student volunteers (thirty slow and twenty rapid inactivators) who were given 600 mg. of the matrix. 18e Further studies on the pharmacology of the matrix The

in patients with pulmonary tuberculosis are in progress. Providing that suitable plasma concentrations of I.N.H. can be achieved in rapid inactivators at a dose of the matrix that is free from important adverse reactions in both slow and rapid inactivators, these investigations will lead to clinical trials of the efficacy of the matrix in once-weekly combined regimens for the treatment of tuberculosis. We thank the following consultant physicians at the Brompton Hospital for allowing their patients to participate in this study: Dr. J. C. Batten, Dr. J. R. Bignall, Dr. E. E. Keal, Dr. H. Nicholson, Dr. N. C. Oswald, Sir Kenneth Robson, Prof. J. G. Scadding, Dr. J. Smart, and Dr. P. A. Zorab. Requests for reprints should be addressed to D. A. M., M.R.C. Unit for Laboratory Studies of Tuberculosis, Royal Postgraduate Medical School, Ducane Road, London W12 OHS. REFERENCES 1. Tuberculosis Chemotherapy Centre, Madras. Bull. Wld Hlth Org. 1964, 31, 247. 2. Tuberculosis Chemotherapy Centre, Madras. ibid. 1970, 43, 143. 3. Menon, N. K. Bull. int. Un. Tuberc. 1970, 43, 271. 4. Tripathy, S. P. ibid. p. 276. 5. Armstrong, A. R. Am. Rev. resp. Dis. 1960, 81, 498. 6. Beggs, W. H., Jenne, J. W. Tubercle, Lond. 1969, 50, 377. 7. Bourgeois, P., Dubois-Verlière, M., Maël, M. Revue Tuberc. 1958,

22, 108. 8. Tuberculosis

Chemotherapy Centre, Madras. Personal communi-

cation. 9. Gelber, R., Jacobsen, P., Levy, L. Clin. Pharmac. Ther. 1969, 10, 841. 10. Dickinson, J. M., Ellard, G. A., Mitchison, D. A. Tubercle, Lond. 1968, 49, 351. 11. Mitchison, D. A., Dickinson, J. M. Postgrad. med. J. 1971, 47, 737. 12. Tiitinen, H. Scand. J. resp. Dis. 1969, 50, 281. 13. Ellard, G. A., Gammon, P. T. Unpublished. 14. Ellard, G. A., Gammon, P. T., Wallace, S. M. Biochem. J. 1972, 126 (in the press). 15. Dymond, L. C., Russell, D. W. Clinica chim. Acta, 1970, 27, 513. 16. Venkataraman, P., Eidus, L., Tripathy, S. P. Tubercle, Lond. 1968, 49, 210. 17. Wootton, I. D. P. Micro-analysis in Medical Biochemistry, p. 174.

London, 1964. 18. Ellard, G. A., Gammon, P. T., Tiitinen, H., Evans, D. A. P.

Unpublished. 19. Smith and Nephew Research Ltd. Personal communication. 20. Peters, J. H., Miller, K. S., Brown, P. J. Pharmac. exp. Ther. 1965, 150, 298. 21. Laplane, R., Sarel, J., Joly, J., Couinard, Presse méd. 1955, 63, 723.

I.N.H.

INSULIN ANTIBODIES IN ÆTIOLOGY OF LABILE DIABETES

P. D. EXON

K. DIXON

H. R. HUGHES*

Department of Clinical Chemistry and Diabetic Clinic, General

Summary

Hospital, Birmingham

The maximum

affinity

binding capacity and of insulin-binding

constant

antibodies have been measured in sera of insulintreated patients attending a diabetic outpatient clinic. The patients were classified as stable (thirteen) or labile (six) on the basis of clinical criteria. The sera of stable patients had moderate amounts of insulinbinding antibodies of low affinity: patients in the labile group had very small amounts of antibody or antibodies which were avid binders of insulin. The avid-binding antibodies were less effective in buffering changes in free-insulin concentration which occur when therapeutic insulin enters the vascular space. *

Present address: Group

Pathology Laboratory, Warwick.