Spin trapping of free radical intermediates produced during the metabolism of isoniazid and iproniazid in isolated hepatocytes

By the use of spin trapping agents phenyl-t-butyl nitrone (PBN) and 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) free radical species were detected in isolated hepatocytes incubated with either isoniazid, iproniazid and their respective metabolites acetyl-hydrazine and isopropyl-hydrazine. The addition of bis-nitrophenyl phosphate, an inhibitor of the acylamidase enzymes, to isolated hepatocytes decreased the free radical activation of isoniazid and iproniazid, but not that of acetyl- and isopropyl-hydrazine, confirming that the radical species were originating from the biotransformation of these latter compounds. The ESR spectra were ascribed to the trapping of, respectively, acetyl and isopropyl free radicals on the basis of the analogies of the spectral feature with those of chemically-prepared spin adducts. Comparable ESR spectra were also observed during the metabolism of acetyl- and isopropyl-hydrazines by liver microsomes and their formation was inhibited by the omission of NADP+, anaerobic incubation and enzyme denaturation. In the microsomal preparations inhibitors of the mixed function oxidase system decreased to various extents the free radical formation and a similar effect was also observed following the destruction of cytochrome P-450 induced by pretreating the rats with CoCl2. The addition of reduced glutathione also decreased the radical trapping indicating that GSH can effectively compete with the spin traps for the reaction with the free radicals. The incubation of isolated hepatocytes with isoniazid or acetyl-hydrazine reduced by 20-25% the intracellular GSH content, while a 50% decrease in GSH was present in the cells exposed to iproniazid and isopropyl-hydrazine. In the same hepatocyte preparations stimulation of lipid peroxidation and leakage of LDH were also observed during cell incubation with iproniazid and isopropyl-hydrazine, but not with isoniazid or acetyl-hydrazine and the extent of both phenomena correlated with the susceptibility of the above compounds to the free radical activation.     

Reductive destruction of dacarbazine, procarbazine hydrochloride, isoniazid, and iproniazid

Reductive destruction of dacarbazine, procarbazine hydrochloride, isoniazid, and iproniazid using nickel-aluminum alloy in basic solution is described. Solutions of dacarbazine 10 mg/mL were prepared by adding dacarbazine 100 mg, citric acid 100 mg, and mannitol 50 mg to 10 mL of water. Aqueous solutions of procarbazine hydrochloride 10 mg/mL were prepared from commercially available capsules, and aqueous solutions of isoniazid 10 mg/mL and iproniazid 5 mg/mL were prepared from powdered drug. Reductive destruction of drugs was accomplished by mixing each solution with an equal volume of 1 M potassium hydroxide solution and adding 1 g of nickel-aluminum alloy for each 20 mL of basified solution. The resulting mixtures were stirred for 20 hours (96 hours for iproniazid) and analyzed by high-performance liquid chromatography and gas chromatography for the presence of residual drug and degradation products. Dacarbazine solutions were also subjected to destruction by photolysis and by oxidation using potassium permanganate in sulfuric acid, and the results were compared with those obtained by reductive destruction. All reaction mixtures were tested for mutagenicity in Salmonella strains. All drugs subjected to reductive destruction were completely degraded to the limits of detection of the assay and produced only nonmutagenic reaction mixtures. The only acceptable results for dacarbazine were obtained by the reductive destruction method. Reduction of dacarbazine, procarbazine hydrochloride, isoniazid, and iproniazid with nickel-aluminum alloy in dilute base appears to be a good method for the destruction of these toxic compounds.     

Spectrophotometric determination of some MAO inhibitors using 7,7,8,8-tetracyanoquinodimethane and iodine monochloride

Two simple and sensitive spectrophotometric methods are described for the assay of three MAO inhibitors: isocarboxazid, tranylcypromine sulphate and iproniazid phosphate. The first method is based on the formation of a highly coloured stable radical anion between the drug as an n-donor and 7,7,8,8-tetracyanoquinodimethane (TCNQ) as a pi-electron acceptor. Beer's law is obeyed in the concentration range 0.2-3, 0.2-4 and 0.5-4 micrograms ml-1 for isocarboxazid, tranylcypromine sulphate and iproniazid phosphate, respectively. The second method involves the use of iodine monochloride (ICl) as an sigma-acceptor. It was found that ICl reacts quantitatively only with isocarboxazid and tranylcypromine sulphate; with iproniazid phosphate results were very poor. Beer's law is obeyed in the concentration range 2-20 micrograms ml-1 for both drugs. The optimum experimental parameters for colour production in each case were determined. The percentage recoveries obtained were in accordance with those obtained by the official methods. The proposed methods are characterized by high sensitivity.     

Fast-flow EPR spectroscopic observation of the isoniazid, iproniazid, and phenylhydrazine hydrazyl radicals

Hydrazyl radical intermediates have been suggested as important intermediates in the biochemistry of hydrazides and hydrazines. Although spin-trapping studies have intercepted those species previously, there has been no report of the direct observation of the unstable hydrazyl radicals of isoniazid and iproniazid. We have employed the fast-flow technique in electron paramagnetic resonance (EPR) spectroscopy to measure spectra for the short-lived hydrazyl radicals of a family of hydrazides, including the pharmacologically important compounds isoniazid and iproniazid, as well as for a series of phenylhydrazines. Our investigations of the phenylhydrazine radical and the related chloro-substituted analogues have allowed definitive assignments of the hyperfine coupling constants of that toxicologically important free radical. Theoretical values of hyperfine coupling constants, calculated by density functional formalism, provided a guide to assignments for the hydrazyl species and confirmed the experimentally based assignments for phenylhydrazyl radical.     

The effect of antidepressive drugs and some related compounds on the levels of adenine nucleotides, inorganic phosphate and phosphocreatine in the rat brain

The effects upon levels of adenine nucleotides, phosphocreatine and inorganic phosphate of iproniazid, isoniazid, phenelzine, pheniprazine, tranylcypromine, harmine, imipramine, amitriptyline, orphenadrine, diphenhydramine and cocaine have been studied. With the exception of harmine and diphenhydramine, each of these compounds increased the brain level of adenosine triphosphate and, with the exception of imipramine and cocaine, the level of adenosine diphosphate decreased. Harmine had no effect on levels of adenine nucleotides and, in the case of diphenhydramine, the level of adenosine diphosphate increased and the level of adenosine triphosphate tended to decrease. There appears to be a relationship between the ability of the drugs to cause behavioural signs of central nervous stimulation and to produce an increase in the adenosine triphosphate/diphosphate ratio. This effect may be a factor in the action of antidepressive drugs.     

In vivo rat hemoglobin thiyl free radical formation following administration of phenylhydrazine and hydrazine-based drugs

We have employed the ESR spin trapping technique in vivo to detect the formation of the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/hemoglobin thiyl free radical adduct in the blood of rats following administration of hydrazine-based drugs. The drugs examined were isoniazid, iproniazid, phenelzine, and hydralazine. In addition, phenylhydrazine and acetylhydrazine were also studied in a like manner. Of the four drugs, only phenelzine and iproniazid were able to induce the formation of the DMPO/hemoglobin thiyl free radical adduct in vivo, whereas only phenelzine and hydralazine were able to form this adduct in vitro. We were able to decrease the in vivo iproniazid-induced adduct formation by pretreating the rats with bis-para-nitrophenylphosphate, an arylamidase inhibitor. Our results support the idea that iproniazid is hydrolyzed in the liver to a more reactive metabolite, most likely isopropylhydrazine, which is subsequently released into the blood stream. In addition to the drug studies, experiments were performed to provide additional evidence that the radical adduct we detected was indeed of a hemoglobin thiyl free radical. Studies employing alpha-phenyl-N-t-butylnitrone (PBN) as the spin trap in place of DMPO also showed the formation of the PBN/hemoglobin thiyl free radical adduct.     

Uniform procedure of (1)H NMR analysis of rat urine and toxicometabonomics Part II: comparison of NMR profiles for classification of hepatotoxicity.

A procedure of nuclear magnetic resonance (NMR) urinalysis using pattern recognition is proposed for early detection of toxicity of investigational compounds in rats. The method is applied to detect toxicity upon administration of 13 toxic reference compounds and one nontoxic control compound (mianserine) in rats. The toxic compounds are expected to induce necrosis (bromobenzene, paracetamol, carbon tetrachloride, iproniazid, isoniazid, thioacetamide), cholestasis (alpha-naphthylisothiocyanate (ANIT), chlorpromazine, ethinylestradiol, methyltestosterone, ibuprofen), or steatosis (phenobarbital, tetracycline). Animals were treated daily for 2 or 4 days except for paracetamol and bromobenzene (1 and 2 days) and carbon tetrachloride (1 day only). Urine was collected 24 h after the first and second treatment. The animals were sacrificed 24 h after the last treatment, and NMR data were compared with liver histopathology as well as blood and urine biochemistry. Pathology and biochemistry showed marked toxicity in the liver at high doses of bromobenzene, paracetamol, carbon tetrachloride, ANIT, and ibuprofen. Thioacetamide and chlorpromazine showed less extensive changes, while the influences of iproniazid, isoniazid, phenobarbital, ethinylestradiol, and tetracycline on the toxic parameters were marginal or for methyltestosterone and mianserine negligible. NMR spectroscopy revealed significant changes upon dosing in 88 NMR biomarker signals preselected with the Procrustus Rotation method on principal component discriminant analysis (PCDA) plots. Further evaluation of the specific changes led to the identification of biomarker patterns for the specific types of liver toxicity. Comparison of our rat NMR PCDA data with histopathological changes reported in humans and/or rats suggests that rat NMR urinalysis can be used to predict hepatotoxicity.     

Metabolism of the hallucinogen N,N-dimethyltryptamine in rat brain homogenates

The metabolism of the hallucinogen N,N-dimethyltryptamine (DMT) in whole rat brain homogenate is reported. Studies were conducted using tritiated DMT and DMT-N-oxide (DMT-NO), and metabolites were identified and quantified using thin-layer chromatography and liquid scintillation counting techniques. Metabolite confirmation was obtained by incubation of α,α,β,β-tetradeutero-DMT (DDMT) with whole brain homogenate followed by combined gas chromatographic/mass spectrometric analyses. The metabolites of DMT were identified as indoleacetic acid (IAA), DMT-NO, N-methyltryptamine (NMT), 2-methyl-1,2,3,4-tetrahydro-β-carboline (2-MTHBC), tryptamine (TA) and 1,2,3,4- tetrahydro-β-carboline (THBC). DMT-NO was metabolized to give DMT, NMT, IAA and 2-MTHBC. Formation of these metabolites from DMT-NO was stimulated by anaerobic incubation. Mechanisms for the formation of β-carbolines from DMT and DMT-NO are discussed. The effects of the monamine oxidase inhibitor iproniazid phosphate on DMT metabolism were also studied. Iproniazid inhibited the formation of IAA from DMT by 83 per cent. However, the formation of NMT and DMT-NO was inhibited by 90 per cent under these conditions. Thus, the reported extension of half-life and potentiation of DMT behavioral effects by iproniazid may be due to inhibition of NMT and DMT-NO formation rather than inhibition of monoamine oxidase. A cyclic pathway for the synthesis and metabolism of DMT in brain tissue is proposed.     

Exploration of Cold Extrusion for the Preparation of Enteric Minitablets of Isoniazid

The objective of the present work was to formulate the enteric minitablets of isoniazid by cold extrusion method. The minitablets were prepared using isoniazid, hydroxylpropylmethylcellulose phthalate and dibasic calcium phosphate. The minitablets were coated using hydroxypropylmethylcellulose phthalate. Full factorial design was adopted to optimize the formulation. The minitablets showed good flow and acceptable friability. The drug release was resisted in 0.1 N HCl for 2 h from the optimized batch. The optimized batch showed more than 90% of drug release in phosphate buffer in 15 min. Capsules containing rifampicin powder and enteric isoniazid minitablets showed complete drug release in acidic and alkaline media respectively. The process of cold extrusion appears to be an attractive alternative to by-pass the existing patents.     

高效液相色谱法测定异烟肼的血药浓度

目的 :建立测定血浆中异烟肼浓度的高效液相色谱法。方法 :色谱柱 :EclipseXDBC18(4 6mm×150mm ,5μm ) ;流动相 :0 05mol/L磷酸二氢铵 -乙腈 (65∶35)。0 5ml血浆用10 %三氯醋酸沉淀蛋白后 ,加桂皮醛衍生化 ,再用乙醚提取分离后进样。结果 :在0 10～12 0μg/ml范围内线性关系良好 ;日内误差小于7 % ,日间误差小于14 % ;方法平均回收率95 %～105 %。结论 :本方法具有快捷、灵敏度较高、干扰少等特点 ,可用于异烟肼的药代动力学研究     

Neurochemical correlates of behavior: levels of amino acids in four areas of the brain of the rat during drug-induced behavioral excitation.

The levels of GABA, aspartate, glutamate, glycine and alanine were determined in 4 specific brain areas (telencephalon, diencephalon-mesencephalon cerebellum and pons-medulla oblongata) of rats killed during a period of drug-induced behavioral excitation. Behavioral excitation was obtained in adult, male Wistar rats working on a Sidman shock-avoidance schedule following administration of 2 mg/kg tetrabenazine (TBZ) 18 hr after iproniazid (50 mg/kg) pretreatment. When compared to trained animals (working on the avoidance schedule but receiving no drugs), the excited rats had increased levels of GABA in the telencephalon and diencephalon-mesencephalon, decreased levels of aspartate in all 4 brain areas, and a lower content of glycine in the pons-medulla region. The changes in the levels of aspartate in all areas of the brain, GABA in the diencephalon-mesencephalon, and glycine in the pons-medulla were significantly correlated (p less than 0.01) with the degree of excitation. It was observed that avoidance training alone produced increases in the levels of four amino acids: aspartate in telencephalon and cerebellum, GABA in cerebellum, and glycine and glutamate in the pons-medulla. The injection of iproniazid alone or iproniazid followed by TBZ into naive animals had little effect on the levels of the five amino acids. The data are discussed in terms of aspartate and GABA interacting as neurotransmitters with cholinergic and catecholaminergic and/or serotonergic neurons to produced the behavioral excitation.     

The effect of enzyme inhibitors on histamine catabolism in man

The catabolism of histamine was studied in three female psychiatric patients by analysis of the [¹⁴C]-labelled metabolic products occurring in the urine after a subcutaneous injection of [¹⁴C]histamine. Each patient was studied before and during treatment with aminoguanidine or iproniazid.

Without treatment the patients had a normal histamine catabolism. Aminoguanidine and iproniazid inhibited the oxidation of histamine to imidazoleacetic acid; iproniazid produed a 50% inhibition of the oxidation of methylhistamine to methylimidazoleacetic acid. After iproniazid a large proportion of the injected [C¹⁴]histamine was excreted as methyl [¹⁴C]histamine.

     

The action of dopamine on the arterial blood pressure of the guinea-pig

The depressor action of dopamine (β-3:4-dihydroxyphenylethylamine) upon the arterial blood pressure of the guinea-pig has been studied. This effect begins without a latent period. It is often enhanced after the intravenous injection of iproniazid (Marsilid). The depressor response is sufficiently sensitive to serve as a method of bioassay of dopamine in microgram quantities. Observations on the depressor action of L-dopa have also been made. This effect is also enhanced by iproniazid; it begins after a latent period. Epinine (β-3:4-dihydroxyphenylethylmethylamine) caused a pressor response, followed by a fall of arterial blood pressure. No evidence was obtained in support of the suggestion that the two amines, which are oxidized at similar rates by amine oxidase, cause a fall of blood pressure after their conversion to an aldehyde by the action of amine oxidase.     

Dissolution test for site-specific release isoniazid pellets in USP apparatus 3 (reciprocating cylinder): Optimization using response surface methodology

The present work aims to predict drug release from novel site-specific release isoniazid pellets, in USP dissolution test apparatus 3, using the response surface methodology (RSM). Site-specific release isoniazid pellets were prepared by extrusion-spheronization followed by aqueous coating of Acryl-EZE. RSM was employed for designing of the experiment, generation of mathematical models and optimization study. A 3(2) full factorial design was used to study the effect of two factors (at three levels), namely volume of dissolution medium (150, 200, 250 ml) and reciprocation rate (5, 15, 25 dips per min). Amount of drug released in 0.1N hydrochloric acid at 2h and in pH 6.8 phosphate buffer at 45 min were selected as responses. Results revealed that both, the volume of medium and reciprocation rate, are significant factors affecting isoniazid release. A second order polynomial equation fitted to the data was used to predict the responses in the optimal region. The optimized conditions resulted in dissolution data that were close to the predicted values. The proposed mathematical model is found to be robust and accurate for optimization of dissolution test conditions for site-specific release isoniazid pellets.     

Colorimetric and titrimetric assay of isoniazid.

Two methods are proposed for the determination of isoniazid in pure form or in tablets. In the first method chlorpromazine hydrochloride, when treated with 2-iodoxybenzoic acid as an oxidant in 50% w/v o-phosphoric acid solution, is oxidized to chlorpromazine free radical which absorbs at 530 nm. The red free radical is readily reduced quantitatively by isoniazid to the colourless chloropromazine. The addition of isoniazid to a red solution of chlorpromazine free radical results in a decrease in absorbance in direct proportion to the quantity of isoniazid. This forms the basis for the quantitative determination of micro-quantities of isoniazid (3-18 micrograms ml-1). The second method involves the titrimetric determination of isoniazid using N-bromophthalimide as a titrant. The end-point is determined either directly using methyl red or amaranth as indicator, or by a back titration method in which a known excess of N-bromophthalimide solution is added to isoniazid solution and then the residual unreacted reagent is determined iodometrically. The results by the proposed procedures were in good agreement with those obtained by the official methods.     

采用多条溶出曲线评价异烟肼片的质量

目的：研究国内外异烟肼片在4种溶出介质中的溶出曲线,以评价该制剂质量。方法：浆法,50 r·min^-1,分别在pH1.2盐酸溶液、pH4.5醋酸盐缓冲液、pH6.8磷酸盐缓冲液和水中测定溶出曲线,比较了各企业样品的溶出均一性,并用单点法或f₂因子法进行了比较分析。结果：美国山德士样品批内批间溶出均一性均较好,国内企业溶出均一性参差不齐。以山德士样品作为参比制剂,A、B、C、F、G企业样品在4种溶出介质中均与参比制剂溶出过程相似;D、E、H企业样品在部分介质中与参比制剂溶出过程相似。结论：国内部分厂家异烟肼片生产工艺不稳定,各企业产品质量存在差异,亟待提高。     

Restoration of the chronotropic effect of tyramine on rat atria after reserpine

1. The restoration by various sympathomimetic amines of the chronotropic response to tyramine was studied on the isolated atria of rats pretreated with reserpine. The atria were exposed to the "restorative" sympathomimetic amine for 10 min, washed over a period of 1 hr and then tested with 10 muM tyramine. The effect of noradrenaline, dopamine and norphenylephrine before and after inhibition of monoamine oxidase by 0.5 mM iproniazid were compared with their alpha-methyl and N-alkyl analogues in their ability to restore the chronotropic response to tyramine.2. Noradrenaline and adrenaline restored the chronotropic response to tyramine, the degree of restoration depending on the concentration of the restorative amine used. Noradrenaline after iproniazid and alpha-methylnoradrenaline were equipotent and were about 1,000 times more active than noradrenaline where monoamine oxidase was not inhibited. Dopamine, epinine, norphenylephrine, phenylephrine, octopamine, synephrine and isoprenaline in the absence of monoamine oxidase inhibition had no effect. Dopamine after iproniazid and alpha-methyldopamine were equipotent and were about 1/10 as active as alpha-methylnoradrenaline. Norphenylephrine after iproniazid and metaraminol were equipotent and were about 1/500 as active as alpha-methylnoradrenaline. Octopamine after iproniazid was even less active. The N-methylated analogues were about 1/10 as active as their nor-compounds but the N-isopropyl analogue, isoprenaline, was devoid of activity.3. Dopamine after iproniazid and alpha-methyldopamine were inactive if a dopamine-beta-hydroxylase inhibitor, disulphiram or sodium diethyldithiocarbamate, was present.4. It is concluded that, in atria of reserpinized rats, (a) protection from monoamine oxidase increases; (b) N-substitution decreases; and (c) hydroxyl groups at the beta-carbon and ring positions 3 and 4 increase the capabilities of a sympathomimetic amine to restore the chronotropic response to tyramine.     

The 5-hydroxytryptamine content of mouse brain and whole mice after treatment with some drugs affecting the central nervous system

A number of drugs were examined for their ability to change the concentration of 5-hydroxytryptamine in mouse brain and in whole mice treated with 5-hydroxytryptophan. After β-phenylisopropylhydrazine or iproniazid, two inhibitors of monoamine oxidase, the brain 5-hydroxytryptamine rose to a maximum value in 8 hr., after which it declined, although a slight rise remained for as long as 6 days. Dose-effect relationships, determined 6 hr. after administration, showed β-phenyl-isopropylhydrazine to be approximately 60 times as effective as iproniazid in raising the brain 5-hydroxytryptamine. When mice were given 5-hydroxytryptophan and the amine content of the whole mice estimated, pretreatment with β-phenylisopropylhydrazine increased their 5-hydroxytryptamine content whereas pretreatment with iproniazid did not change it. The concentration of the amine in mouse brain and in whole mice was lower after reserpine, but was raised when reserpine and β-phenylisopropylhydrazine were given together. A small rise in brain 5-hydroxytryptamine was found after chlorpromazine; when chlorpromazine was given with iproniazid, however, the resulting increase was less than that found after iproniazid alone. Brain 5-hydroxytryptamine was unaltered after prolonged treatment with morphine.     

Microspheres of alginate-chitosan containing isoniazid

Isoniazid was encapsulated into microspheres of alginate-chitosan by means of a complex coacervation method in an emulsion system. Since the encapsulation of isoniazid tends to be limited by its hydrophilic characteristics, this study proposes its microencapsulation by adsorption. The particles were prepared in three steps: (1) preparation of a W/O emulsion; (2) phase separation; and (3) adsorption of the drug. The isolated particles were placed in a solution of the drug under stirring to allow adsorption. The morphology and particle size were analysed by scanning electron microscopy (SEM). The isoniazid content was determined by extraction in 1 M phosphate buffer pH 7.5 under stirring for 4 h. Finally, the samples were filtered and analysed in an UV/VIS spectrophotometer at 260 nm. In vitro release tests were carried out in 0.05 M phosphate buffer pH 7.5. The results showed that microspheres of alginate-chitosan obtained were of spherical shape. The emulsion used for microparticle formation allows the preparation of particles with a narrow size distribution. The adsorption observed is probably of chemical nature, i.e. there is an ionic interaction between the drug and the surface of the particles.     

Über die Hemmung der Dopa-Decarboxylase durch Isonicotinsäurehydrazid

Summary 1. Isonicotinyl hydrazide inhibits the activity of dopa-decarboxylase by blocking the co-enzyme pyridoxal-5-phosphate.2. Pyridoxal-5-phosphate isonicotinyl hydrazone is obtained on incubation of pyridoxal-5-phosphate with isoniazid; in this form the co-enzyme is protected against inactivation by dopa and dopamine, but will be released by slow hydrolysis. Protection and slow release of pyridoxal phosphate may explain why in the presence of pyridoxal phosphate isonicotinyl hydrazone the decarboxylation is slower than in the presence of pure pyridoxal phosphate. On the other hand, there is a practically complete decarboxylation of the substrate which is not found with pyridoxal phosphate alone.

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Ausgeführt mit Unterstützung der Deutschen Forschungsgemeinschaft.