Combination of docking, molecular dynamics and quantum mechanical calculations for metabolism prediction of 3,4-methylenedioxybenzoyl-2-thienylhydrazone.

In modern drug discovery process, ADME/Tox properties should be determined as early as possible in the test cascade to allow a timely assessment of their property profiles. To help medicinal chemists in designing new compounds with improved pharmacokinetics, the knowledge of the soft spot position or the site of metabolism (SOM) is needed. In silico methods based on docking, molecular dynamics and quantum chemical calculations can bring us closer to understand drug metabolism and predict drug-drug interactions. We report herein on a combined methodology to explore the site of metabolism prediction of a new cardioactive drug prototype, LASSBio-294 (1), using MetaPrint2D to predict the most likely metabolites, combined with structure-based tools using docking, molecular dynamics and quantum mechanical calculations to predict the binding of the substrate to CYP2C9 enzyme, to estimate the binding free energy and to study the energy profiles for the oxidation of (1). Additionally, the computational study was correlated with a metabolic fingerprint profiling using LC-MS analysis. The results obtained using the computational methods gave valuable information about the probable metabolites of (1) (qualitatively) and also about the important interactions of this lead compound with the amino acid residues of the active site of CYP2C9. Moreover, using a combination of different levels of theory sheds light on the understanding of (1) metabolism by CYP2C9 and its mechanisms. The metabolic fingerprint profiling of (1) has shown that the metabolites founded in highest concentration in different species were metabolites M1, M2 and M3, whereas M8 was found to be a minor metabolite. Therefore, our computational study allowed a qualitative prediction for the metabolism of (1). The approach presented here has afforded new opportunities to improve metabolite identification strategies, mediated by not only CYP2C9 but also other CYP450 family enzymes.

Metabolomic analysis reveals novel isoniazid metabolites and hydrazones in human urine.

Isoniazid (INH) is a first-line drug for tuberculosis control; the side effects of INH are thought to be associated with its metabolism, and this study was designed to globally characterize isoniazid metabolism. Metabolomic strategies were used to profile isoniazid metabolism in humans. Eight known and seven novel INH metabolites and hydrazones were identified in human urine. The novel products included two hydroxylated INH metabolites and five hydrazones. The two novel metabolites were determined as 2-oxo-1,2-dihydro-pyridine-4-carbohydrazide and isoniazid N-oxide. Five novel hydrazones were produced by condensation of isoniazid with keto acids that are intermediates in the metabolism of essential amino acids, namely, leucine and/or isoleucine, lysine, tyrosine, tryptophan, and phenylalanine. This study enhances our knowledge of isoniazid metabolism and disposition and may offer new avenues for investigating INH-induced toxicity.

Spectrophotometric determination of isoxsuprine hydrochloride using 3-methyl-2-benzothiazolinone hydrazone hydrochloride in spiked human urine and pharmaceuticals.

Two selective and sensitive spectrophotometric methods are proposed for the determination of isoxsuprine hydrochloride (ISX) in spiked human urine and in pharmaceuticals. The methods are based on the oxidative-coupling reaction between 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) and ISX in the presence of Ce(SO(4))(2). The novelty of the proposed reaction is the formation of two different colored chromogens at two different pHs. The resulting product at pH<1.5 is a red colored chromogen peaking at 500nm (method A) and that formed between the pH 3.85 and 4.15, is violet colored with an absorption maximum at 580nm (method B). In both the methods, absorbance of the chromogen is found to increase linearly with the concentration of ISX as is corroborated by the correlation coefficients of 0.9989 and 0.9970, and the systems obey Beer's law over the ranges of 1.4-21.0 and 1.0-15.0microgml(-1), for method A and method B, respectively. The calculated molar absorptivities are 1.08 x 10(4) and 1.78 x 10(4)lmol(-1)cm(-1) for method A and method B, respectively with corresponding Sandell sensitivity values of 0.0311 and 0.0190microgcm(-2). The reaction stoichiometry, in both the methods, was evaluated by the limiting logarithmic method and was found to be 1:1 (ISX:MBTH). The methods were successfully applied to the determination of ISX in spiked human urine and pharmaceutical formulation.

Metabolism of OR-1896, a metabolite of levosimendan, in rats and humans.

OR-1896 is a pharmacologically active, long-lived metabolite of levosimendan. In the current study, the metabolism of (14)C-labelled OR-1896 was investigated in six healthy men after intravenous infusion over 10 min and in male rats after an intravenous bolus dose. In human plasma, the only (14)C-compounds detected were (14)C-OR-1896 and its deacetylated form, (14)C-OR-1855, in varying proportions in different subjects. In rat plasma >93% of radioactivity was associated with OR-1896. Radioactivity was mainly excreted to urine in both rats (about 69% of the dose) and humans (about 87% of the dose). OR-1896 was a major urinary compound in both humans and rats. Another major human metabolite was hypothesized as N-conjugated OR-1855. Other human and rat urinary biotransformation products were characterized as N-hydroxylated OR-1896 and N-hydroxylated OR-1855, as well as glucuronide or sulphate conjugates of N-hydroxyl OR-1896. The main difference between rat and human metabolism was a lower amount of OR-1855-related metabolites in the rats. In human faecal homogenates, only OR-1896 and OR-1855 were detected, whereas rat faecal metabolite profile was similar to that in urine.

Pharmacokinetics and excretion balance of OR-1896, a pharmacologically active metabolite of levosimendan, in healthy men.

OBJECTIVE: To investigate the pharmacokinetics and excretion balance of [(14)C]-OR-1896, a pharmacologically active metabolite of levosimendan, in six healthy male subjects. In addition, pharmacokinetic parameters of total radiocarbon and the deacetylated congener, OR-1855, were determined. METHODS: OR-1896 was administered as a single intravenous infusion of 200 microg of [(14)C]-OR-1896 (specific activity 8.6 MBq/mg) over 10 min. The pharmacokinetic parameters were calculated by three-compartmental methods. RESULTS: During the 14-day collection of urine and faeces, excretion (+/-S.D.) averaged 94.2+/-1.4% of the [(14)C]-OR-1896 dose. Mean recovery of radiocarbon in urine was 86.8+/-1.9% and in faeces 7.4+/-1.5%. Mean terminal elimination half-life of OR-1896 (t(1/2)) was 70.0+/-44.9 h. Maximum concentrations of OR-1855 were approximately 30% to that of OR-1896. Total clearance and the volume of distribution of OR-1896 were 2.0+/-0.4 l/h and 175.6+/-74.5l, respectively. Renal clearances of OR-1896 and OR-1855 were 0.9+/-0.4 l/h and (5.4+/-2.3)x10(-4) l/h, respectively. CONCLUSIONS: This study provides data to demonstrate that nearly one half of OR-1896 is eliminated unchanged into urine and that the active metabolites metabolite of levosimendan remain in the body longer than levosimendan. The remaining half of OR-1896 dose is eliminated through other metabolic routes, partially through interconversion back to OR-1855 with further metabolism of OR-1855. Given the fact that the pharmacological activity and potency of OR-1896 is similar to levosimendan, these results emphasize the clinical significance of OR-1896 and its contribution to the long-lasting effects of levosimendan.

High-performance liquid chromatographic determination of urinary 2,5-hexanedione as mono-2,4-dinitrophenylhydrazone using ultraviolet detection.

The good correlation between exposure to n-hexane and 2,5-hexanedione urinary excretion confers on this diketone an important toxicological meaning. this paper proposes a reversed-phase HPLC method which includes, after acid hydrolysis, a derivatization step of 2,5-hexanedione with 2,4-dinitrophenylhydrazine at 70 degrees C for 20 min. The reaction conditions, such as temperature, reagent concentration and time, are optimized so as to allow the condensation of a single carbonyl group. A linear response was obtained in the 0.19-20.0 mg/l range with a detection limit of 0.03 mg/l, corresponding to a signal-to-noise ratio of 3. A phosphate buffer (pH 3.3)-acetonitrile mixture (50:50) as the eluent and UV detection at 334 nm were used.

Pharmacokinetic studies of benzalazine.

The pharmacokinetic properties of benzalazine ((2-hydroxy-5-[(4-carboxyphenyl)azo]benzoic acid, CAS 64896-26-0), a new agent for the treatment of ulcerative colitis and Crohn's disease of the large intestine, were investigated. From jejunal loops of rats in situ no noteworthy absorption of benzalazine was observed. All attempts to demonstrate metabolic conversion of benzalazine in mucosal homogenate of the small intestine of rats were without any success. In faecal suspensions, the half-life of the metabolic conversion of benzalazine was determined as 15 min and the formation of the metabolite 5-aminosalicylic acid (5-ASA) was demonstrated qualitatively. 72 h after single oral administration of 300 mg benzalazine/kg b.w. to rats, an average of 71.83% of the administered dose was recovered in urine and faeces. Only a small amount of unmetabolized benzalazine was excreted with urine and faeces (0.75% and 1.47% of the administered dose, respectively). The benzalazine metabolite 5-ASA and the 5-ASA metabolite acetyl-5-aminosalicylic acid (Ac-5-ASA) were excreted mainly with the faeces (29.22% and 20.66% of the administered dose, respectively) and only in small amounts with the urine (2.54% and 11.06% of the administered dose, respectively).

High-performance liquid chromatographic microassay for methyl ethyl ketone in urine as the 2,4-dinitrophenyl-hydrazone derivative.

We report a high-performance liquid chromatographic procedure for determining methyl ethyl ketone in urine. The method is based on pre-column derivatization with 2,4-dinitrophenylhydrazine and liquid-liquid extraction of the derivative. The analyte is chromatographically separated from other urine constituents in less than 12 min and is detected by UV absorption at 360 nm. Peak height and concentration are linearly related. The relative standard deviation assessed for within-day imprecision was 3.2% at the 2.21 mg/l level. The mean analytical recovery from urines spiked with 1.0 mg/l ketone was 96.0 +/- 6.1%. The simple sample handling, the small volume of urine required and the short amount of time taken for the whole procedure make it suitable for routine biomonitoring of exposure to methyl ethyl ketone in industrial workers. The concentration in urine from nine non-exposed controls was less than 0.1 mg/l. The concentrations measured in urine samples from 60 exposed workers ranged from 0.1 to 1.1 mg/l and from 0.3 to 3.6 mg/l at the before- and the end-shift collections, respectively.

Derivatization and high-performance liquid chromatographic determination of urinary glycolic acid.

A high-performance liquid chromatographic method for the determination of urinary glycolic acid is proposed, based on pre-column derivatization with phenylhydrazine coupled with the enzymatic oxidation of glycolate to glyoxylate. The phenylhydrazone formed is separated by liquid chromatography and detected at 324 nm. The minimum detectable concentration of glycolate was 10.0 mumol/l. The recovery of glycolate added to urine averaged 96.1%. The day-to-day coefficients of variation calculated by analysis of two urine samples with normal and high glycolate contents were 4.6 and 7.5%, respectively. Results of analyses of urine samples from healthy persons, idiopathic calcium stone formers and Type I primary hyperoxaluria patients are reported.