Concentration-Dependent Antagonism and Culture Conversion in Pulmonary Tuberculosis.

BACKGROUND: There is scant evidence to support target drug exposures for optimal tuberculosis outcomes. We therefore assessed whether pharmacokinetic/pharmacodynamic (PK/PD) parameters could predict 2-month culture conversion. METHODS: One hundred patients with pulmonary tuberculosis (65% human immunodeficiency virus coinfected) were intensively sampled to determine rifampicin, isoniazid, and pyrazinamide plasma concentrations after 7-8 weeks of therapy, and PK parameters determined using nonlinear mixed-effects models. Detailed clinical data and sputum for culture were collected at baseline, 2 months, and 5-6 months. Minimum inhibitory concentrations (MICs) were determined on baseline isolates. Multivariate logistic regression and the assumption-free multivariate adaptive regression splines (MARS) were used to identify clinical and PK/PD predictors of 2-month culture conversion. Potential PK/PD predictors included 0- to 24-hour area under the curve (AUC0-24), maximum concentration (Cmax), AUC0-24/MIC, Cmax/MIC, and percentage of time that concentrations persisted above the MIC (%TMIC). RESULTS: Twenty-six percent of patients had Cmax of rifampicin <8 mg/L, pyrazinamide <35 mg/L, and isoniazid <3 mg/L. No relationship was found between PK exposures and 2-month culture conversion using multivariate logistic regression after adjusting for MIC. However, MARS identified negative interactions between isoniazid Cmax and rifampicin Cmax/MIC ratio on 2-month culture conversion. If isoniazid Cmax was <4.6 mg/L and rifampicin Cmax/MIC <28, the isoniazid concentration had an antagonistic effect on culture conversion. For patients with isoniazid Cmax >4.6 mg/L, higher isoniazid exposures were associated with improved rates of culture conversion. CONCLUSIONS: PK/PD analyses using MARS identified isoniazid Cmax and rifampicin Cmax/MIC thresholds below which there is concentration-dependent antagonism that reduces 2-month sputum culture conversion.

Attenuation of anti-tuberculosis therapy induced hepatotoxicity by Spirulina fusiformis, a candidate food supplement.

Therapy using Isoniazid (INH) and Rifampicin (RIF) leads to induction of hepatotoxicity in some individuals undergoing anti-tuberculosis treatment. In this study, we assessed the effect of Spirulina fusiformis on INH and RIF induced hepatotoxicity in rats compared with hepatoprotective drug Silymarin. Induction of hepatotoxicity was measured by changes in the liver marker enzymes (aspartate transaminase, alanine transaminase, and alkaline phosphatase). The antioxidant status was also analyzed in liver tissue homogenate and plasma by measurement of superoxide dismutase, catalase, glutathione-S-transferase, glutathione reductase, and lipid peroxidation levels. We also aimed to study the binding and interactions of the transcription factors Pregnane X Receptor (PXR) and Farnesoid X Receptor (FXR) with INH, RIF, and representative active compounds of Spirulina fusiformis by in silico methods. The administration of INH and RIF resulted in significant (p < 0.05) decrease in the antioxidant levels and total protein levels. There was also a significant (p < 0.05) increase in the levels of liver marker enzymes. Spirulina fusiformis was seen to protect the parameters from significant changes upon challenge with INH and RIF in a dose-dependent manner. This was corroborated by histological examination of the liver. The results of the in silico analyses further support the wet lab results.

Isoniazid-induced hepatotoxicity in rat hepatocytes of gel entrapment culture.

Gel entrapment culture of rat hepatocytes in hollow fibers were evaluated as a potential in vitro model for studies on isoniazid-induced hepatotoxicity. After exposure to isoniazid (0.11 mM and 1.1 mM) for 24-96 h, gel entrapped hepatocytes were more severely damaged than hepatocyte monolayers according to the assays on methyl thiazolyl tetrazolium (MTT) reduction, intracellular glutathione (GSH) content, reactive oxygen species (ROS) levels, and albumin secretion. Furthermore, CYP 2E1 activity detected by 4-nitrocatechol (4-NC) formation maintained at least 7 days in gel entrapped hepatocytes but decreased to an undetectable level within 2 days in hepatocyte monolayer. And the addition of CYP 2E1 inhibitor, diethyl-dithiocarbamate (DDC), significantly reduced isoniazid-induced GSH depletion in gel entrapped hepatocytes. In addition, the protective effects of N-acetylcysteine (NAC), GSH, liquorice extract and glycyrrhizic acid (GA), a purified compound from liquorice extract, against isoniazid hepatotoxicity were clearly observed in gel entrapped hepatocytes at 72 h incubation. Overall, gel entrapped hepatocytes were more susceptible to isoniazid-induced hepatotoxicity than hepatocyte monolayers by a possible mechanism that higher CYP 2E1 activity in gel entrapped hepatocytes could enhance isoniazid toxicity. This indicates that gel entrapped hepatocytes in hollow fibers could be a more effective model than hepatocyte monolayer for hepatotoxicity research in vitro.

Terminalia chebula (fruit) prevents liver toxicity caused by sub-chronic administration of rifampicin, isoniazid and pyrazinamide in combination.

Terminalia chebula Gertn. (Combetraceae) is an important herbal drug in Ayurvedic pharmacopea. In the present study, a 95% ethanolic extract of T. chebula (fruit) (TC extract), which was chemically characterized on the basis of chebuloside II as a marker, was investigated for hepatoprotective activity against anti-tuberculosis (anti-TB) drug-induced toxicity. TC extract was found to prevent the hepatotoxicity caused by the administration of rifampicin (RIF), isoniazid (INH) and pyrazinamide (PZA) (in combination) in a sub-chronic mode (12 weeks). The hepatoprotective effect of TC extract could be attributed to its prominent anti-oxidative and membrane stabilizing activities. The changes in biochemical observations were supported by histological profile.

INH induced status epilepticus: response to pyridoxine.

Isoniazid is an effective and widely used drug in tuberculosis treatment. The administration of toxic amounts of INH causes recurrent seizures, profound metabolic acidosis, coma and even death but therapeutic dose of isoniazid is a very rare cause of seizures. We present a case of 44-year-old HIV positive African-American female who was recently started on a preventive dose of INH after being found purified protein derivative (PPD) positive. She developed status-epilepticus that did not respond to most of the antiepileptics. As soon as she received intravenous pyridoxine, the seizures terminated abruptly.

Reversal of prolonged isoniazid-induced coma by pyridoxine.

Isoniazid overdose is known to result in the rapid onset of seizures, metabolic acidosis, and prolonged obtundation. Pyridoxine has been reported to be effective in treating isoniazid-induced seizures. We report three cases of obtundation secondary to isoniazid overdose that was immediately reversed by intravenous pyridoxine. In two of these cases, status seizures were stopped by intravenous pyridoxine administration, but the patients remained comatose for prolonged periods. The comas were immediately reversed by the administration of additional pyridoxine. In the third case, the patient's lethargy was treated by intravenous pyridoxine on presentation and was followed by immediate awakening. Pyridoxine is effective in treating not only isoniazid-induced seizures, but also the mental status changes associated with this overdose. The dose required to induce awakening may be higher than that required to control seizures.

Hepatoprotective effect of isonicotinoylhydrazone SH7 against chronic isoniazid toxicity.

This study was carried out to investigate whether 3,5-dichloro-salicylaldehyde isonicotinoyl hydrazone (SH7), an analogue of the antituberculosis drug isoniazid (INH), recently synthesized in our laboratory, could prevent isoniazid-induced liver damage. Forty-two white healthy mice were treated, once daily for 30 days, with solutions of INH, SH7 and combinations of them at doses of 7.5 mg/kg p.o. and 15 mg/kg p.o. At the end of this period, livers were harvested for histopathological analysis. The levels of lipid peroxidation products (MDA) and the activities of antioxidant defense enzymes, superoxide dismutase (SOD) and catalase (CAT), were also measured in the liver homogenates. Treatments with combinations of INH and SH7 decreased the levels of MDA, normalized the previously depressed levels of SOD and CAT and prevented isoniazid-induced liver damages. A previously demonstrated tuberculostatic and superoxide scavenger activity (SSA) of the isonicotinoylhydrazone SH7 and results from the present study thus set out a proposal for a new isoniazid/SH7 combination therapy designed to provide hepatoprotection.

Prevention of isoniazid tumorigenicity by antitoxicants of isoniazid in Swiss mice.

Effect of isoniazid on blood ammonia levels and protein biosynthesis in tissues of Swiss mice was studied. Isoniazid treatment resulted in an elevation of the blood ammonia. It inhibited the protein biosynthesis in lung and kidney tissues of Swiss mice after 24 h of treatment. Simultaneous administration of L-arginine or L-sodium glutamate or pyridoxine hydrochloride or folic acid prevented the inhibition. Further, effect of these antitoxicants of isoniazid was studied on its tumorigenicity. Mice were fed isoniazid (1.1 mg/mouse/day) and the antitoxicant (1.1 mg/mouse/day) and killed when they appeared moribund. Incidence of tumor in mice receiving L-arginine or L-sodium glutamate along with isoniazid did not show any significant difference when compared with the mice treated with isoniazid alone. On the other hand, observations with folic acid gave full protection against tumorigenic action of isoniazid while pyridoxine hydrochloride displayed partial protection.

L-alpha-amino-beta-chloropropionic acid hydroxamide: an inhibitor of GABA-lapha-oxoglutarate aminotrarsferase.

The intramuscular administration of L-alpha-amino-beta-chloropropinonic acid hydroxamide (2 mmol/kg) to mice strongly inhibited the activity of GABA-T, but not GAD, in the brain of the animals. Adminstration of the compound 3 h prior to isonicotinic acid hydrazide treatment significantly delayed the onset of seizures induced by the hydrazide.

Pharmacological profile of a novel class of muscarinic acetylcholine receptor agonists.

Some in vivo pharmacological effects of a number of muscarinic acetylcholine receptor agonists containing the bicyclic 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO) skeleton were studied in rats and mice. The key compounds O,N-dimethyl-THPO (2) and O-methyl-THPO (4) are bioisosteres of arecoline and norarecoline, respectively. The vasodepressor effects of arecoline and the title compounds in anaesthetized rats gave parallel log dose-response curves. The order of potency of the compounds in this test system was identical with that measured earlier using a guinea-pig ileum preparation, arecoline and 2 being the most active compounds. This order of potency was different from those for the antinociceptive and anticonvulsant effects of the compounds using grid shock and isoniazid antagonism tests, respectively, where O-propargyl-THPO (3) proved to be the most active. The pA2 values for the atropine or scopolamine antagonism of these effects of arecoline and 4 were calculated. The partition coefficients (log P values) of the compounds were measured and shown to conform with their ability to penetrate the blood-brain barrier.

Felbamate antagonizes isoniazid- and FG 7142-induced reduction of GABAA receptor function in mouse brain.

Injection of the antiepileptic drug, felbamate (2-phenyl-1,3-propanediol dicarbamate), into mice reduced in a dose-dependent manner (150-300 mg/kg i.p.) the isoniazid (200 mg/kg s.c.)-induced increase in ex vivo binding of t-[35S]butylbicyclophosphorothionate ([35S]TBPS) to cerebral cortical and hippocampal membranes. The same doses of felbamate reduced significantly the number of mice exhibiting isoniazid-induced seizures. A dose of felbamate (50 mg/kg) ineffective in isoniazid-treated mice completely antagonized the increase of [35S]TBPS binding elicited by FG 7142 (N-methyl-beta-carboline-3-carboxamide), a benzodiazepine receptor inverse agonist. The above effects of felbamate resembled those of diazepam. Accordingly, the combination of ineffective doses of felbamate (50 mg/kg) and diazepam (0.2 mg/kg) elicited a marked decrease of [35S]TBPS binding. The results indicate that facilitation of gamma-aminobutyric acid type A (GABAA) receptor function may play a role in the anticonvulsant action of felbamate.

Translational activation of ethanol-inducible cytochrome P450 (CYP2E1) by isoniazid.

The molecular mechanism of ethanol-inducible cytochrome P450(CYP2E1) induction by isoniazid was studied and compared to that of pyridine, an inducer of CYP2E1. Aniline hydroxylase and immunoreactive CYP2E1 protein were significantly induced by isoniazid without or with only slight activation of other cytochromes P450. In contrast, pyridine increased the activities of a broad range of P450s. The effects of two structural analogs of isoniazid, isonicotinamide and isonicotinic acid were also tested and found to have a markedly decreased ability to induce CYP2E1. The induction of CYP2E1 by isoniazid was not accompanied by an increased level of CYP2E1 mRNA, and was completely blocked by pretreatment with cycloheximide or sodium fluoride, inhibitors of mRNA translation. These data thus suggest that CYP2E1 induction by isoniazid is due to activation of CYP2E1 mRNA translation and that the hydrazide group on the pyridine ring of isoniazid is important both in the selective induction of CYP2E1 and for magnitude of effect.

Effect of convulsant and anticonvulsant agents on level and metabolism of gamma-aminobutyric acid in mouse brain.

1. The effect of the convulsant agents pentetrazole, picrotoxin, bicuculline, strychnine and isoniazid on the central level of gamma-aminobutyric acid (GABA) and the activity of the enzymes glutamate decarboxylase (GAD) and GABA-alpha-oxoglutarate aminotransferase (GABA-T) from mice brain was studied in vivo and vitro. In vivo, convulsant doses of picrotoxin and isoniazid lowered the level of GABA and the activity of GAD, whereas strychnine and bicuculline had no such effect. Pentetrazole inhibited GAD, but did not alter the GABA content. In vitro, all convulsants, except bicuculline, inhibited the activity of GAD; however, the concentrations of strychnine were far beyond the range that is reached in vivo by convulsant doses. Only isoniazid inhibited the activity of GABA-T in vivo as well as in vitro. 2. Phenobarbital, ethosuximide and trimethadione were about equally active in preventing convulsions induced by strychnine and picrotoxin, whereas diazepam was 9 times, and sodium valproate 3.5 times more active against convulsions elicited by picrotoxin. Phenytoin up to 100 mg/kg was ineffective against all chemoconvulsants. 3. Diazepam, sodium valproate, ethosuximide and trimethadione antagonized the inhibition of GAD and the decrease in GABA concentrations caused by isoniazid. Phenobarbital and phenytoin prevented the decrease of GABA but did not reverse the inhibition of GAD. 4. The results suggest a role played by the transmitter pool of GABA in the convulsant action of chemoconvulsants and in the anticonvulsant effect of antiepileptics clinically used in petit mal epilepsy.

Convulsions induced by hyperbaric oxygen: inhibition by phenobarbital, diazepam and baclofen.

1. The anticonvulsive potencies of diazepam, phenobarbital and baclofen against convulsions induced by oxygen under high pressure (OHP), by isoniazid and by strychnine were investigated in mice and rats. 2. The anticonvulsive potency of diazepam was much higher than that of phenobarbital and baclofen against all three types of convulsions. 3. The selective activity of diazepam against isoniazid induced convulsions in rats (dose ratio ED50 phenobarbital/ED50 diazepam: 400) could not be confirmed in mice (dose ratio ED50 phenobarbital/ED50 diazepam: 20-40), where diazepam was equipotent against all three types of convulsions. 4. Baclofen which does not inhibit strychnine induced convulsions was equipotent in inhibiting convulsions evoked by isoniazid and OHP in mice. 5. The results are in agreement with the postulated GABA-inhibitory mechanism of OHP induced convulsions, whereas they make a glycine inhibitory mechanism very unlikely. Although the results do not allow further conclusions about the mode of action of diazepam, a clinical trial of diazepam in OHP-induced convulsions should be considered.

Antagonism of isoniazid-induced convulsions by abecarnil in mice tolerant to diazepam.

The ability of the benzodiazepine receptor full agonist diazepam, the selective agonist abecarnil, and the partial agonist imidazenil to antagonize convulsions induced by isoniazid (200 mg/kg, S.C.) was studied in mice chronically treated with diazepam (3 mg/kg, i.p., three times daily) or abecarnil (0.1 or 1 mg/kg, i.p., three times daily or 6 mg/kg, S.C., daily). Diazepam induced tolerance to its own anticonvulsant effect. In contrast, chronic treatment with abecarnil failed to induce tolerance to its own anticonvulsant activity. Animals treated with abecarnil at 0.1 mg/kg developed cross-tolerance to imidazenil, whereas those treated with 1 mg/kg became less sensitive to diazepam. Mice chronically treated with abecarnil at 6 mg/kg showed almost complete tolerance to diazepam. Abecarnil was able to antagonize the convulsions elicited by isoniazid in diazepam-tolerant mice. These data indicate that chronic administration of abecarnil, unlike that of classical benzodiazepines, does not induce tolerance to its anticonvulsant effect, and that abecarnil overcomes tolerance induced by long-term treatment with the full agonist diazepam.

Isonicotinic acid hydrazide inhibits cell population growth during teratogenesis of chick embryo.

In chick embryos treated with a 4 hr pulse of 7.2 X 10(-5) M isonicotinic acid hydrazide (INH) the cell population growth is inhibited with an increased population doubling time. Teratogenised blastoderm cells complete their ongoing cell cycle and arrest in G1 phase. A chase with an equimolar concentration of pyridoxal-5-phosphate restores the growth rate after a lag of 4 hr equivalent to the duration of treatment with INH. Presumptive mesoblast cells invaginated through the primitive streak and neuroectoblast cells induced prior to the application of INH differentiate, while the teratogen inhibits morphogenesis and organization of organ primordia.

Protective effect of N-acetylcysteine in isoniazid induced hepatic injury in growing rats.

Status of oxidative/antioxidative profile was the mechanistic approach to inumerate the nature of protection by N-acetylcysteine (NAC) in isoniazid (INH) exposed experimental animals. Analysis of lipid peroxidation, thiol levels, cytochrome P450, superoxide dismutase (SOD), catalase, glutathione peroxidase, reductase and transferase were estimated in liver along with the body and liver weight of animals and histological observations. Isoniazid exposure to animals resulted in no change in body and liver weights. Thiols, lipid peroxidation, catalase, SOD glutathione peroxidase, reductase, transferase and cytochrome P450 levels were altered with INH exposure. Supplementation of NAC with INH protected the animals against hepatotoxic reactions by minimizing the free radical induced tissue injury and overall maintenance of the endogenous scavengers of free radicals.

Therapeutic potential of thymoquinone against anti-tuberculosis drugs induced liver damage.

Hepatotoxicity is the most serious adverse effect related to tuberculosis treatment which interrupts the successful completion of tuberculosis treatment. The purpose of this study was to assess therapeutic effect of thymoquinone (TQ) against anti-tuberculosis drugs (ATD) induced liver damage. Rats were treated with ATD for 8 weeks (3 days/week) as given for the treatment of TB. This was followed by therapy of TQ for 8 weeks (3 days/week). Administration of combined ATD induced hepatotoxicity was evident from a significant elevation in the AST, ALT, ALP, bilirubin, albumin, cholesterol, urea, uric acid, creatinine, LPO and decreased activities of enzymes. These altered variables were significantly reversed toward control after treatment with TQ. Histological studies also supported biochemical findings. Results of this study strongly indicated protective effect of TQ and thus, can be expected as promising protective agent in maintenance of normal hepatic function during treatment with ATD.

Protective effects of kaempferol on isoniazid- and rifampicin-induced hepatotoxicity.

Isoniazid (INH) and rifampicin (RIF) are the first-line drugs for antituberculosis (anti-TB) chemotherapy. The levels of serum transaminases [aspartate aminotransferase (AST) and alanine aminotransferase (ALT)] are abnormal in 27% of patients undergoing INH and RIF treatments and in 19% of patients undergoing treatment with INH alone. Cytochrome P450 2E1 (CYP2E1) metabolizes many toxic substrates, including ethanol, carbon tetrachloride, and INH, which ultimately results in liver injury. The objective of this study was to screen for CYP2E1 inhibitors in vitro and investigate whether the selected compound could prevent INH/RIF-induced hepatotoxicity in vivo. We screened 83 known compounds from food and herbal medicines as inhibitors of CYP2E1. The hepatotoxic dose of INH/RIF was 50/100 mg kg(-1) day(-1). Hepatotoxicity was assessed using galactose single-point (GSP) method (a quantitative measurement of liver function), histopathological examination of the liver, malondialdehyde (MDA) assay, and measurement of AST and ALT activities. Kaempferol inhibited CYP2E1 activity in mice by 0.31- to 0.48-fold (p < 0.005). Mice with INH/RIF-induced hepatotoxicity showed significantly abnormal serum levels of AST and ALT, and GSP value, and these values could be decreased by the administration of kaempferol (p < 0.005). Kaempferol significantly reduced the depletion of hepatic glutathione and prevented the increase in MDA formation in mice. Furthermore, kaempferol did not affect the anti-TB effects of INH/RIF. To our knowledge, this is the first report of kaempferol's utility as an adjuvant for preventing CYP2E1-mediated hepatotoxicity induced by drugs such as INH and RIF.

Role of oxidative stress and nitric oxide in the protective effects of alpha-lipoic acid and aminoguanidine against isoniazid-rifampicin-induced hepatotoxicity in rats.

Despite the great efficacy of isoniazid (INH) and rifampicin (RIF) combination, in the treatment of tuberculosis, hepatotoxicity is the most common serious complication. The potential protective effect of alpha-lipoic acid and aminoguanidine; against combination-induced hepatotoxicity was investigated in the present study. Administration of INH-RIF combination (50 mg/kg each for 14 days) resulted in an elevation of serum hepatic marker enzymes and a significant increase in lipid profile parameters. Combinations treatment increased lipid peroxidation products, decreased glutathione content, superoxide dismutase, catalase and myeloperoxidase activities. Furthermore, liver total nitrite level was significantly increased in INH-RIF treated rats. Co-administration of either alpha-lipoic acid or aminoguanidine significantly ameliorate combination-induced alterations in hepatic marker enzymes. These effects were directly linked to a greater decrease in the combination-induced elevation in lipid peroxidation products and total nitrite levels. Furthermore, co-administration of alpha-lipoic acid and aminoguanidine restore superoxide dismutase, catalase and myeloperoxidase activities and maintained the imbalance in the glutathione level. Additionally, such beneficial effect of alpha-lipoic acid was linked to a marked lipid-lowering effect. Histopathological examination revealed preservation of liver integrity of the protected groups compared to combination-treated rats alone.