Using a single tablet daily to treat latent tuberculosis infection in Brazil: bioequivalence of two different isoniazid formulations (300 mg and 100 mg) demonstrated by a sensitive and rapid high-performance liquid chromatography-tandem mass spectrometry method in a randomised, crossover study

The recommended treatment for latent tuberculosis (TB) infection in adults is a daily dose of isoniazid (INH) 300 mg for six months. In Brazil, INH was formulated as 100 mg tablets. The treatment duration and the high pill burden compromised patient adherence to the treatment. The Brazilian National Programme for Tuberculosis requested a new 300 mg INH formulation. The aim of our study was to compare the bioavailability of the new INH 300 mg formulation and three 100 mg tablets of the reference formulation. We conducted a randomised, single dose, open label, two-phase crossover bioequivalence study in 28 healthy human volunteers. The 90% confidence interval for the INH maximum concentration of drug observed in plasma and area under the plasma concentration vs. time curve from time zero to the last measurable concentration “time t” was 89.61-115.92 and 94.82-119.44, respectively. The main limitation of our study was that neither adherence nor the safety profile of multiple doses was evaluated. To determine the level of INH in human plasma, we developed and validated a sensitive, simple and rapid high-performance liquid chromatography-tandem mass spectrometry method. Our results showed that the new formulation was bioequivalent to the 100 mg reference product. This finding supports the use of a single 300 mg tablet daily strategy to treat latent TB. This new formulation may increase patients’ adherence to the treatment and quality of life.

ELISA protocol for rapid screening of potential antitubercular drugs based on antigenic reactivity of mycobacterial ES-31 serine protease - a drug target supported by axenic culture of Mycobacterium tuberculosis H37 Ra strain in the presence of inhibitor.

Background: Mycobacterial ES-31 serine protease has been reported to be a drug target using protease and lipase inhibitors in axenic and macrophage cultures. Simple screening techniques are needed for rapid testing of anti-tubercular drugs. Aim: To demonstrate the usefulness of ELISA protocol based on antigenic reactivity of mycobacterial serine protease by indirect ELISA for detecting anti-tubercular activity. Material and Methods: Indirect ELISA for assessment of antigenic reactivity of mycobacterial ES-31 serine protease was standardized using ES-31Ag and anti-DSS-goat-serum and assessed the inhibition of the antigenic reactivity by isoniazid, an anti-tubercular drug and serine protease inhibitor and orlistat, a lipase inhibitor. Results: Optimal antigenic reactivity of mycobacterial ES-31 serine protease was observed at 5μg/well of ES-31 antigen and at 1:25 dilution of anti-DSS-goat-serum. Isoniazid showed 42% inhibition of ES-31 serine protease at 0.4μg/well, while orlistat showed inhibition of 60% at 0.5μg/well. Inhibition of Mtb H37Ra bacilli is further confirmed in axenic culture. 35% and 29% inhibition by isoniazid at 0.4μg/well and orlistat at 0.5μg/well were observed respectively on bacterial growth. Conclusion: Simple ELISA protocol based on assay of antigenic reactivity of mycobacterial ES-31 serine protease, a drug target, has been standardized for rapid screening of potential anti-tubercular drugs.

Desarrollo de nuevos sistemas de liberación modificada y administración oral para el tratamiento de la tuberculosis / Development of new oral drug delivery systems for tuberculosis treatment

INTRODUCCIÓN: Rifampicina es inestable en medio ácido, y su descomposición es acelerada por isoniazida. El desarrollo de una formulación que permita la iberación secuencial de rifampicina (en estómago) e isoniazida (en intestino) podría superar este inconveniente. OBJETIVO: Obtener materiales portadores de rifampicina e isoniazida mediante acomplejamiento con polielectrolitos y caracterizarlos para determinar su utilidad en el desarrollo de sistemas de liberación sitio-específica, en combinación a dosisfija. MÉTODOS: Se utilizaron carboximetilcelulosa (CMC) y ácidoalgínico (AA) como polielectrolitos modelo. Se obtuvieron series de complejos CMC-rifampicina y AA-isoniazida. Se caracterizó el tipo de interacción, la capacidad de carga y las características reológicas de los materiales sólidos que, tras ser compactados bajo la forma de matrices simples o mixtas, fueron sometidos a ensayos de liberación en medios biorrelevantes. RESULTADOS: La interacción entre los grupos involucrados fue iónica, con una capacidad de carga del 100%. Los materiales presentaron propiedades de flujo desfavorables, con mejora por granulación. Las matrices liberaron rápidamente rifampicina en medio ácido con mínimos niveles concomitantes de isoniazida. La matriz seleccionada presentó liberación modulada de isoniazida, completada al cabo de 3 horas en un medio que simulaba el contenido intestinal. CONCLUSIONES: Los nuevos materiales pueden ser utilizados en el desarrollo de una formulación oral de liberación sitio-específica, capaz de mejorar la efectividad, reducir efectos adversos e incrementar la estabilidad de rifampicina.

INTRODUCTION: Rifampicin is unstable in acidic medium and its decomposition is accelerated by isoniazid. The development of a formulation to allow the sequential release of rifampicin (in stomach) and isoniazid (in gut) could overcome this problem. OBJECTIVE: To obtain materials with rifampicin and isoniazid, loaded in polyelectrolyte polymers and characterize them in order to determine their utility in the development of oral delivery systems for site-specific fixeddose combination. METHODS: Carboxymethyl cellulose (CMC) and alginic acid (AA) were used as polyelectrolytes. Series of complexes CMC-rifampicin and AA-isoniazid were obtained. The type of interaction, loading capacity and rheological properties were characterized in solid materials, which after compaction under simple or combined matrixes were testedin biorrelevant media. RESULTS: The interaction between components was ionic, and loading capacity was 100%.The powders showed unfavorable flow properties, improving by granulation. Release of rifampicin in acidic medium was fast, with minimal concomitant levels of isoniazid. The selected matrix showed a controlled release of isoniazid, which was completed after 3 hours in simulated intestinal media. CONCLUSIONS: The new materials can be used for the development of site specific oral formulations of rifampicin and isoniazid. They may lead to improved effectiveness, reduced side effects and higher rifampicin stability.

Intestinal barrier function and serum concentrations of rifampin, isoniazid and pyrazinamide in patients with pulmonary tuberculosis

Intestinal barrier function and serum concentrations of rifampin, isoniazid and pyrazinamide were studied in healthy controls and patients with active pulmonary tuberculosis. A case-control study of 29 controls and 30 cases attending at the Health Center, July, 2004 to December, 2005 was conducted. The body mass index was significantly reduced in cases compared to controls (p < 0.001). The intestinal paracellular transport of lactulose was significantly (p = 0.019) reduced in cases compared to controls. The transcellular transport of mannitol and the lactulose:mannitol ratio were not significantly (p = 0.0698) reduced in cases compared to controls. Low serum concentrations of rifampin, isoniazid and pyrazinamide were observed in 81 percent (48/59), 92 percent (54/59) and 28 percent (12/59), respectively, in all individuals. The results demonstrated a marked decrease on intestinal paracellular transport in patients with active pulmonary tuberculosis and reduced serum concentrations of rifampin and isoniazid in both groups.

Intestinal permeability and malabsorption of rifampin and isoniazid in active pulmonary tuberculosis

Low antimycobacterial drug concentrations have been observed in tuberculosis (TB) patients under treatment. The lactulose/mannitol urinary excretion test (L/M), normally used to measure intestinal permeability, may be useful to assess drug absorption. The objective of this research was to study intestinal absorptive function and bioavailability of rifampin and isoniazid in TB patients. A cross sectional study was done with 41 patients and 28 healthy controls, using the L/M test. The bioavailabilities of rifampin (R) and isoniazid (H) were evaluated in 18 patients receiving full doses. Urinary excretion of mannitol and lactulose, measured by HPLC, was significantly lower in TB patients. The serum concentrations of the drugs were below the expected range for R (8-24 mcg/mL) or H (3-6 mcg/mL) in 16/18 patients. Analyzing the drugs individually, 12/18 patients had low serum concentrations of R, 13/18 for H and 8/18 for both drugs. We suggest that there is a decrease in the functional absorptive area of the intestine in TB patients, which would explain the reduced serum concentrations of antituberculosis drugs. There is a need for new approaches to improve drug bioavailability in TB patients.

In virto and in vivo effects of isoniazid on stabilityand pharmacokinetics of rifampicin

The stability of rifampicin in the presence of isoniazid was studied using several in vitro examinations including differential scanning calorimetry, infrared spectroscopy and thin layer chromatography. The results suggested the probability of the interaction between rifampicin and isoniazid. Regarding in vivo studies, the effect of isoniazid 5 mg kg-1 on the pharmacokinetics of rifampicin 10 mg kg-1 was investigated in dogs after single oral administration. Plasma rifampicin concentrations were determined using high performance liquid chromatographic assay for the determination of the drug. The effect of repeated oral administration of rifampicin-isoniazid [R-I] combination, for a period of five days in dogs, on hepatic enzymatic activity and renal function tests was studied. The present study showed a good agreement between both in vitro and in vivo results. In addition, the pharmacokinetic of rifampicin was found to be influenced by the presence of isoniazid

Consideraciones generales sobre el uso de agentes antimicrobianos en pacientes hepáticos / General remarks: antimicrobial agents use in hepatic patients

El hígado juega un papel fundamental en el metabolismo de las drogas, incluyendo los antimicrobianos. En los pacientes con enfermedad hepática, se deben monitorizar cuidadosamente los efectos adversos y toxicidad de estos medicamentos. En este artículo se analizan los aspectos del metabolismo de los antimicrobianos, particularmente relacionados a los cambios farmacocinéticos en los pacientes hepáticos. Además, se dan algunas recomendaciones prácticas sobre su uso en estos pacientes

CYP1A2 activity as a risk factor for bladder cancer

CYP1A2, CYP2D6 and N-acetyltransferase activities were estimated in 100 patients with bladder cancer and 84 control subjects from measurements of theophylline, metoprolol and isoniazid and their metabolites in urine, respectively. The frequency of occurrence of slow acetylators of isoniazid and poor metabolizers of metoprolol were 16.7% and 1.2% in the control group and 16.3% and 2.0% in the cancer patient group. These differences were not significant. The recovery ratio of 1-methyluric acid(1-MU) from theophylline was significantly higher in patients with bladder cancer than in control subjects(0.340 +/- 0.016 versus 0.260 +/- 0.020, p< 0.05). The 1-MU recovery ratio was a significant, independent risk factor among the metabolic capacities tested as shown by logistic regression analysis, controlling for N-acetylation of isoniazid, hydroxylation of metoprolol, age, sex, and smoking. We concluded that the capacity for 3-demethylation of theophylline, as a reflection of CYP1A2 activity, is significantly associated with increased risk of nonoccupational urinary bladder cancer.

Inhibition of paracetamol induced reduction in hepatic glutathione level by INH of rifampin coadministration in mice

Repated administration of paracetamol induces hepatotoxicity due to depletion of the liver tissue from its glutathione contents. As paracetamol may be currently used in combination with INH and rifampin it was notworthy to explore the effect of these drugs on hepatic glutathione contents. Paracetamol, INH and rifampin were given either alone or in combination. After certain period of drug treatment, the animals were sacrificed and their livers were homogenized to determine its glutathione contents. It was observed that paracetamol significantly decreased while either INH or rifampin significantly increased the glutathione contents of the liver. When given in combination, both INH and rifampin could prevent the decrease in glutathione level when paracetamol was given with either of them for 2 days and only partially when it was given for 7 days. How much this effect can be benificial ? this needs thorough clinical investigation

Food reduces isoniazid bioavailability in normal volunteers.

Food and drug interaction is a major variable in bioavailability of drugs. Isoniazid is the most common antitubercular drug used in India. We studied the effect of standard Indian breakfast and lunch on the bioavailability of isoniazid in a single dose crossover study in normal male volunteers. The standard breakfast and lunch significantly reduced plasma AUC, Cmax and Kabs values of isoniazid. Isoniazid, thus, should not be administered with food.

Effect of subacute DDT on pharmacokinetics of isoniazid and liver function in rabbits.

DDT administration (30 mg/kg per day, po, for 21 consecutive days) to rabbits showed an increase in peak plasma concentration and a decrease in time to reach peak plasma concentration of isoniazid whereas no change was observed in elimination rate constant and area under the plasma concentration-time curve. DDT treatment caused increased absorption of isoniazid. Early signs of hepatic damage were also observed. Since there was no change in the levels of serum glutamate oxaloacetate transaminase and serum glutamate pyruvate transaminase, it can be concluded that DDT does not significantly affect liver function at the dosage used. The observed elevated levels of alkaline phosphatase could be due to direct activation of the enzyme. Leukopaenia and neutropaenia with relative lymphocytosis indicated that DDT might have suppressant effect on granulocyte cell line of WBCs.