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Spectrophotofluorometric assay for isoniazid and acetyl isoniazid in plasma adapted to pediatric studies.

We modified the micro-scale spectrophotofluorometric method of Miceli et al. [Biochem. Med. 12, 348 (1975)] for the assay of "apparent" isoniazid (isoniazid acid-lable hydrazones) to improve its clinical application. We also adapted the method for determination of acetyl isoniazid. Data are presented showing how long plasma containing isoniazid may validly be stored. The applicability of the method was demonstrated in studies on children and small animals.

Adolescent↗

ACONCURRENT COMPARISON OF INTERMITTENT (TWICE-WEEKLY) ISONIAZID PLUS STREPTOMYCIN AND DAILY ISONIAZID PLUS PAS IN THE DOMICILIARY TREATMENT OF PULMONARY TUBERCULOSIS; TUBERCULOSIS CHEMOTHERAPY CENTRE, MADRAS.

Previous reports from the Tuberculosis Chemotherapy Centre, Madras, have established that ambulatory treatment of pulmonary tuberculosis with the combination of isoniazid and PAS, administered daily, yields satisfactory results. However, in the usage of any unsupervised regimen, reliance must be placed on the co-operation of patients in self-administering their drugs. Irregularities in drug-taking, which are not uncommon, may lead to unfavourable therapeutic results; this might be avoided by supervised administration of the drugs. Daily supervision is clearly impracticable in developing countries but regimens in which the drug is administered intermittently-say, twice a week or less frequently-are, if effective, more likely to gain general application.This paper presents the results of a controlled study of a fully supervised intermittent regimen of isoniazid (12.5-16.1 mg/kg body-weight, orally) plus streptomycin (injected in a uniform dose of 1 g), given together twice weekly, compared with a standard, unsupervised, daily, oral regimen of isoniazid (3.7-6.3 mg/kg body-weight) plus sodium PAS (0.2-0.3 g/kg body-weight), given in two doses. The intermittent regimen was at least as effective as the standard oral regimen, and although the incidence of temporary giddiness in patients receiving this regimen was rather high, this did not appear to have any long-term importance nor did it appear unduly to affect the co-operation of the patients. These encouraging findings suggest a possible change in the orientation of drug-administration for tuberculosis in developing countries.

Aminosalicylic Acid↗

Ethambutol plus isoniazid compared with rifampicin plus isoniazid in antituberculosis continuation treatment.

Patients with bacteriologically positive pulmonary tuberculosis were treated initially for an average of three and a half months with isoniazid, rifampicin, and ethambutol and then a total of one year's treatment was completed with either rifampicin plus isoniazid (R+I) or with ethambutol plus isoniazid (E+I). 63 patients in each continuation group were followed up for at least one year, and no relapses occurred. Continuation treatment with E+I was as effective and acceptable as that with R+I and was much less costly.

Drug Therapy, Combination↗

Bioequivalence assessment of rifampicin, isoniazid and pyrazinamide in a fixed dose combination of rifampicin, isoniazid, pyrazinamide and ethambutol vs. separate formulations.

Depending on the patient category, tuberculosis requires treatment with 3 to 5 drugs which means that patient's compliance to therapy may not be optimal. To increase patient's adherence to treatment schedules, these drugs can be given as single drug preparations or fixed dose combinations (FDCs) of 2 or more drugs in a single formulation. However, an important issue associated with a rifampicin-containing FDC is its quality. Hence, to avoid spurious formulations entering the market, the World Health Organization and the International Union Against Tuberculosis and Lung Disease have recommended FDCs only of proven bioavailability. In this study, the relative bioavailability of rifampicin, isoniazid and pyrazinamide was assessed in a group of 14 healthy male subjects using the FDC tablet containing 4 drugs versus separate formulations at the same dose levels. The study was designed as an open, crossover trial. A total of 9 blood samples were collected over a period of 24 h. The concentration of rifampicin, its main metabolite desacetyl rifampicin, isoniazid and pyrazinamide in plasma were assessed using HPLC analysis. The pharmacokinetic parameters AUC(0-24) and Cmax were subjected to parametric and non-parametric statistical tests at 90% confidence interval. In addition, time to reach peak concentration (tmax), elimination rate constant (Kel) and terminal elimination half-life (t1/2) for each drug were also calculated. It was concluded that the FDC tablet containing 4 drugs is bioequivalent to separate rifampicin, isoniazid and pyrazinamide formulations at the same dose levels.

Analysis of Variance↗

[A high pressure liquid chromatography method for detecting the isoniazid derivative isonicotinoyl-hydrazine sodium glucuronide, free isoniazid and acetylisoniazid and its use in pharmacologic studies].

A specific direct method for determining the isoniazid derivative isonicotinoyl-hydrazine sodium glucuronide (INHG-Na) with the aid of high-pressure liquid chromatography has been developed, which permits the analysis of this derivative of isoniazid in addition to isoniazid itself and acetylisoniazid in the plasma of patients. In initial pharmacokinetic applications of the method, it has been shown that plasma INHG-Na is a stable substance that liberates only negligible concentrations of INH.

Acetylation↗

A 5-year study of patients with pulmonary tuberculosis treated at home in a controlled comparison of isoniazid plus PAS with 3 regimens of isoniazid alone.

This report from the Tuberculosis Chemotherapy Centre, Madras, describes the progress, over a 5-year period, of 341 patients with newly diagnosed, sputum-positive tuberculosis. All the patients were treated on a domiciliary basis. In the first year, the patients received, on the basis of random allocation, a standard regimen of isoniazid plus PAS or 1 of 3 regimens of isoniazid alone. Previous reports have shown that the response in the first year was substantially superior with the standard regimen, and that the bacteriological relapse rates in the second year were fairly similar for the 4 regimens. The findings in the present report extend the latter conclusion to the end of 5 years. Further, when considered together with the findings in an earlier study, they have shown that isoniazid, given as maintenance chemotherapy in the second year, was highly effective in preventing bacteriological relapse in patients who, at 1 year, had bacteriologically quiescent disease and no residual cavitation; the effect was, however, less marked in patients with residual cavitation at 1 year.Patients who were clear-cut failures of the allocated chemotherapy and those who had a bacteriological relapse in the second or subsequent years were usually re-treated with streptomycin plus PAS or streptomycin plus pyrazinamide, and if this was ineffective, with cycloserine plus thioacetazone or cycloserine plus ethionamide.Considering the findings over the 5-year period for all patients, 16 died from non-tuberculous causes and 1 took his discharge prematurely. Of the remainder, 86% had bacteriologically quiescent disease at 5 years, 6% had bacteriologically active disease and 8% had died of tuberculosis. These findings confirm the value of well-organized domiciliary chemotherapy, which was established by an earlier report from the Centre, and are particularly encouraging for developing countries such as India, where tuberculosis is a major problem and resources are limited.

Aminosalicylic Acids↗

Attack rate of tuberculosis in a 5-year period among close family contacts of tuberculous patients under domiciliary treatment with isoniazid plus PAS or isoniazid alone.

This report from the Tuberculosis Chemotherapy Centre, Madras, considers the risk, over a 5-year period, to close family contacts of sputum-positive patients treated at home for 1 year with a standard regimen of isoniazid plus PAS or one of 3 regimens of isoniazid alone. The attack rate of tuberculosis in the contacts did not appear to be influenced by the treatment received by the patients in the first year or by the duration in the 5-year period for which the patients had (1) positive sputum smears, (2) positive cultures, or (3) isoniazid-resistant cultures. Further, over half the cases of tuberculosis developed in the first year, many of these being in the first 3 months. These findings confirm the conclusions reached from an earlier study, namely, that the major risk to the contacts is from exposure to the infectious patient before diagnosis, and that the risks from the other possible sources of infection (the patient during treatment and the urban environment of Madras) are, in comparison, small.

Ambulatory Care↗

Comparative trial of thiacetazone with isoniazid and paraaminosalicyclic acid (PAS) with isoniazid in the treatment of pulmonary tuberculosis in Nigerians.

A comparative trial of a combination of thiacetazone 150 mg with isoniazid 300 mg in a once daily dose and that paraaminosalicyclic acid 12 gm uith isoniazid 300 mg daily in divided doses was carried out on 72 specially selected patients with pulmonary tuberculosis over a period of 12 months. The result of the trial showed that patients treated with thiacetazone/isoniazid combination responded equally favourably to therapy as the patients treated with PAS/isoniazid combination. The result of sputum conversion was similar in both groups. Radiological changes after treatment showed a significant similarity in both groups. It was observed that the thiacetazone/isoniazid combination is no more toxic than the PAS/isoniazid combination. There was no case of blood dyscrasia during the trial. Of the 72 patients who started the trial, a total of 19 (26 per cent) patients defaulted before the end of the third month. However, the remaining 53 patients (74 per cent) completed the trial, and the final analysis was based on this number. It was concluded that thiacetazone (150 mg/day) was of similar efficacy when compared with PAS (12 gm/day) as a companion drug for isoniazid (300 mg/day) in producing sputum conversion, and radiological changes in Nigerian tuberculosis patients and is therefore to be preferred because of its relative cheapness and once-a-day administration.

Adolescent↗

Failure of commercially prepared isoniazid diet to produce isoniazid serum concentrations in rhesus monkeys.

A comparison was made of serum isoniazid concentrations and the time course of isoniazid elimination in rhesus monkeys given the drug im, orally on sugar cubes, or in a commercially medicated diet. When the drug was administered orally on sugar cubes or im, peak serum concentrations were usually reached within 2 hr. A threefold individual variation in the rate of elimination was noted, and in most animals the serum concentration at 6 hr approached zero. Isoniazid was not detected in the serum of any animal receiving the commercially medicated diet. Others using this medicated food in their tuberculosis prophylaxis programs should be aware that the diet probably does not provide therapeutically effective dosage to the animals.

Administration, Oral↗

Mechanisms of isoniazid resistance in Mycobacterium tuberculosis: enzymatic characterization of enoyl reductase mutants identified in isoniazid-resistant clinical isolates.

Mutants in the structural gene of the inhA-encoded NADH-dependent 2-trans enoyl-acyl carrier protein reductase were identified from isoniazid-resistant clinical isolates of Mycobacterium tuberculosis. Recombinant InhA proteins with defined single amino acid replacements were expressed in Escherichia coli and purified to homogeneity. Steady-state kinetic parameters for wild type (WT) and I16T, I21V, I47T, and I95P mutants of the enoyl reductase were measured spectrophotometrically. NADH binding to WT and I16T, I21V, I47T, S94A, and I95P mutant reductases were determined by fluorescence spectroscopy and demonstrated that all mutant enzymes had reduced NADH affinity and that NADH binding to all mutants was cooperative as compared with the hyperbolic binding of NADH to the WT enzyme. Since KatG-produced electrophilic derivatives of isoniazid have been suggested to inactivate the enoyl reductase-NADH complex, the kinetics of inactivation for the WT and I21V and I95P mutants was determined. Both mutations resulted in significantly increased values for the apparent first-order rate constant of inactivation.

Antitubercular Agents↗

Fibrinogen and antithrombin III blood levels fluctuations during isoniazid or isoniazid plus rifampicin administration.

Nineteen randomized patients, treated with isoniazid (INH, CAS 54-85-3) for tuberculosis chemoprophylaxis or isoniazid plus rifampicin (RMP, CAS 13292-46-1) combination for tuberculosis therapy, were studied in order to explore the effects of these drugs on fibrinogen and antithrombin III blood levels. Other hepatic biology indices were also measured (aminotransferases, alkaline phosphatase, prothrombin time etc). The results suggested a relationship between INH or INH + RMP administration and fibrinogen as well as antithrombin III blood levels. The data indicate a protective effect of the RMP synchronous administration (by enzyme induction mechanisms) in the preservation of fibrinogen blood levels.

Adult↗

Studies on immunomodulatory properties of isoniazid. II. Effect of isoniazid on interleukin 2 production and interleukin 2-receptor expression.

The effect of isoniazid on interleukin 2 production and interleukin 2-receptor expression by phytohemagglutinin-stimulated human T cells was investigated. High concentrations of the drug decreased interleukin 2 production while low doses (10(-5)-10(-6) M) produced a slight increase in interleukin 2 production. Isoniazid did not affect the expression of interleukin 2-receptors on the surface of T cells, except a slight decrease in cells exposed to high levels of the drug.

Cell Division↗

[A case of tuberculous pleurisy associated with myoclonus and Quincke's edema due to isoniazid and isoniazid sodium methanesulfonate].

A case of tuberculous pleurisy associated with myoclonus and Quincke's edema due to isoniazid (INH) and isoniazid sodium methanesulfonate (IHMS) was reported. A 75-year-old man was admitted to our division because of chest discomfort and the left chest pain of one month's duration. A conventional chest roentgenogram revealed pleural effusion in the left thoracic cavity. The pleural specimen obtained from the left parietal pleura revealed caseating granuloma. Myoclonus suddenly appeared two months after the administration of antituberculous drugs for tuberculous pleurisy. Therefore, INH was discontinued. Three days later the patient's myoclonus disappeared and nine days later IHMS was newly administered. The patient abruptly developed myoclonus and Quincke's edema. IHMS was discontinued and 30 mg of prednisolone was simultaneously given. Two days later myoclonus disappeared and two days more later Quincke's edema was improved. The lymphocyte stimulation test using IHMS was positive. At that time, levels of serum vitamin B6 were within normal levels. These results suggest that myoclonus may result from epileptogenic action caused by INH or IHMS, and Quincke's edema may result from hypersensitive reaction associated with IHMS.

Aged↗

Studies on immunomodulatory properties of isoniazid. Influence of isoniazid on responsiveness of peripheral blood mononuclear cells to interleukin-2.

Peripheral blood mononuclear cells which were "resting" i.e. interleukin-2-receptor-negative were stimulated to proliferate by interleukin-2 in vitro. It was found that isoniazid at concentration of 10(-5)-10(-6) M increased the peak proliferation of "resting" cells but had no influence on the kinetics of proliferation. The mechanism of this effect of isoniazid remains unclear.

Cell Division↗

A fast and efficient metal-mediated oxidation of isoniazid and identification of isoniazid-NAD(H) adducts.

It is currently believed that isoniazid (INH) is oxidised inside Mycobacterium tuberculosis to generate, by covalent attachment to the nicotinamide ring of NAD(H) (beta-nicotinamide adenine dinucleotide), a strong inhibitor of InhA, an enzyme essential for mycolic acid biosynthesis. This work was carried out to characterise the InhA inhibitors (named INH-NAD(H) adducts) which are generated, in the presence of the nicotinamide coenzyme NAD+, by oxidation of INH with manganese(III) pyrophosphate, a nonenzymatic and efficient oxidant used to mimic INH activation by the catalase-peroxidase KatG inside M. tuberculosis. The oxidation process is almost complete in less than 15 minutes (in comparison to the slow activation obtained in the KatG-dependent process (2.5 hours) or in the nonenzymatic O2/Mn(II)-dependent activation (5 hours)). The alkylation of NAD+ by the postulated isonicotinoyl radical generates, in solution, a family of INH-NAD(H) adducts. Analyses with liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS) and experiments performed with 18O- and 2H-labelled substrates allowed us to propose two open and four hemiamidal cyclised dihydropyridine structures as the main forms present in solution; these result from the combination of the isonicotinoyl radical and the nicotinamide part of NAD+. A small amount of a secondary oxidation product was also detected. Structural data on the forms present in solution should help in the design of inhibitors of enzymes involved in the biosynthesis of mycolic acids to act as potential antituberculosis drugs.

Antitubercular Agents↗

Effect of antioxidants and antitoxicants of isoniazid on the formation of lung tumours in mice by isoniazid and hydrazine sulphate.

The present study reports the effects of antioxidants and antitoxicants on the formation of lung tumours in Swiss mice induced by isoniazid (INH) and hydrazine sulphate (HS). Dietary administration of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) or simultaneous oral administration of antitoxicants (L-arginine, L-sodium glutamate and pyridoxine hydrochloride) failed to prevent HS-induced lung tumours. On the other hand BHA and BHT inhibited the formation of lung tumours in groups of mice receiving INH. Folic acid supplementation had marginal effect on the formation of lung tumours in groups receiving HS (P less than 0.1). Higher lung tumour incidence was observed in groups maintained only on BHT diet as compared to animals maintained on standard diet.

Animals↗

Determination of isonicotinic acid in the presence of isoniazid and acetylisoniazid. Studies on isonicotinic acid formation from isoniazid in isolated rat hepatocytes.

In comparison with the hepatocytes obtained from intact rats and rats pretreated with phenobarbital or 3-methylchoranthrene, the amount of isonicotinic acid (INA) formed from isoniazid (INH) increased substantially after incubation at 37 degrees C using the pretreated hepatocytes. This suggests an oxidative pathway for INA formation from INH, apart from hydrolysis. In order to explore the exact mechanism of INA formation in the hepatocytes, an HPLC assay for INA in the presence of INH and acetylisoniazid was developed. In this assay, INA was extracted after the preparation of an ion pair with tetra-n-butylammonium hydroxide, and analysed using an ODS column and a mobile phase consisting of 0.067 M potassium dihydrogenphosphate solution-methanol (96:4, v/v). The method is simple, accurate and especially suitable for INA determination after incubation of INH in isolated rat hepatocytes.

Animals↗