Pharmacodynamic and pharmacokinetic characterisation of RBx 7796: a novel 5-lipoxygenase inhibitor.

OBJECTIVE: RBx 7796, a 5-lipoxygenase inhibitor, was evaluated in in vivo efficacy models, in vitro ADME and in vivo pharmacokinetic models. METHOD: RBx 7796 was evaluated for inhibition of 5-lipoxygenase enzyme and release of LTB4 from isolated rat and human neutrophils. RBx 7796 was tested in allergic bronchoconstriction model in Balb/c mice and LPS induced airway hyperreactivity model in rats. RBx 7796 was evaluated for metabolic stability in liver microsomes and cytochrome P450 inhibition potential. Pharmacokinetic profile of RBx 7796 was also determined in rat and dog. RESULTS: RBx 7796 inhibited 5-lipoxygenase enzyme and inhibited release of LTB4 from neutrophils. RBx 7796 also inhibited early and late airway reactivity following allergen challenge in mouse model. LPS induced increase in airway reactivity was blocked by RBx 7796. Compound was found to be stable in liver microsomes and devoid of major cytochrome P450 inhibition potential. The oral bioavailability of RBx 7796 in rat and dog was 83 % and 47 %, respectively. Following repeated daily administration, compound did not exhibit any sign of accumulation and/or tendency to induce its own metabolism. CONCLUSION: The results suggest that RBx 7796 is an inhibitor of 5-lipoxygenase enzyme that is orally efficacious in two different models of airway reactivity. The molecule also demonstrated acceptable pharmacokinetic profile warranting further development.

Validation of the LC-MS/MS method for the quantification of mevalonic acid in human plasma and determination of the matrix effect.

A simple, specific, and sufficiently sensitive liquid chromatography-tandem mass spectrometry (negative-ion electrospray ionization) methodology to determine mevalonic acid (MVA) in human plasma is described, and its application to the analysis of rat plasma MVA levels after rosuvastatin administration is demonstrated. The method was validated over the linearity range of 0.5-50.0 ng/ml (r(2) > 0.99) using deuterated MVA as an internal standard. The lower limit of quantification was 0.5 ng/ml. The assay procedure involved the isolation of MVA from plasma samples using solid-phase extraction. Chromatographic separation was achieved on a HyPurity Advance column with a mobile phase consisting of ammonium formate buffer (10 mM, pH 8.0) and acetonitrile (70:30, v/v). Excellent precision and accuracy were observed. MVA and deuterated mevalonolactone were stable in water and plasma under different storage and processing conditions. The recovery observed was low, which was attributable to a significant matrix effect. A significant decrease (30-40%; P < 0.05) was observed in rat plasma MVA levels after rosuvastatin administration.

Evaluation of interaction potential of certain concurrently administered drugs with pharmacological and pharmacokinetic profile of centchroman in rats.

Centchroman (Ormeloxifene) is a nonsteroidal, selective estrogen receptor modulator, oral contraceptive and anticancer agent, and is intended for long-term use by women. In view of its vast clinical application and the interaction of steroidal oral contraceptives with certain commonly used therapeutic agents, evaluation of interaction of certain concomitantly administered therapeutic agents (ibuprofen, rifampicin, diazepam, salbutamol, nifedipine, paracetamol, haloperidol, and tetracycline), in terms of both the postcoital contraceptive efficacy and pharmacokinetic profile, with centchroman was undertaken in female Sprague-Dawley rats. Among the representatives from each commonly used therapeutic category, interaction (pharmacokinetic) was observed with ibuprofen (60 mg/kg, twice daily), haloperidol (0.7 mg/kg, twice daily), and tetracycline (140 mg/kg, twice daily) coadministration on Days 1 through 5 postcoitum. Of these three therapeutic agents, only tetracycline interfered with the contraceptive efficacy of centchroman. It reduced the bioavailability of centchroman and its active metabolite by increasing their excretion through bile and feces. Increased metabolite excretion on tetracycline coadministration indicates the enterohepatic recirculation of the metabolite, not the parent drug. However, the effect of tetracycline was negated by the inclusion of lactic acid bacillus spores in the regimen.

Evaluation of absorbability of centpropazine in rats: in-situ and in-vivo appraoches.

Intestinal absorption of centpropazine was studied in rats by both in-situ (closed-loop method) and in-vivo (portal-venous difference) approaches. The drug was found to be well absorbed from solution in in-situ studies. However, the results obtained in-vivo suggested that very low amounts of drug reach the portal circulation after oral dosing. This could imply extensive binding to the mucosa or metabolism in the intestinal wall. The presence of higher amounts of metabolites in the portal vein compared with the inferior vena cava samples signal their formation in the gastrointestinal tract or enterohepatic recirculation. These findings will be useful in incorporating suitable structural and formulation modifications for enhancing the bioavailability of centpropazine and its analogues.

Tissue distribution and excretion of CDRI-81/470 in rats.

Methyl-N[5 [[4-(2-pyridinyl)-1-piperazinyl]carbonyl]- 1H-benzimidazol-2-yl] carbamate (CDRI-81/470) is a broad spectrum anthelmintic agent, effective against both intestinal and systemic parasitism. Tissue distribution and excretion of CDR1-81/470 were studied in rats after a single oral dose of 100 mg kg(-1) CDRI-81/470. One of the metabolites was identified in pilot studies as its N-decarboxylate derivative and characterized by synthesis. HPLC assay methods for the simultaneous estimation of CDRI-81/470 and its N-decarboxylate derivative in tissues, bile, urine, and faeces were developed and validated. The parent compound was quantitated in all major tissues and organs up to 48 h post-dose. Among the tissues other than serum, the highest concentrations of CDRI-81/470 were found in liver, whereas only trace levels were found in brain. Approximately 3% of the administered dose was excreted unchanged in urine at 120 h postdose, whereas approximately 7% was recovered in faeces. The contribution of the biliary route for the excretion of parent compound was less than 0.5%. The N-decarboxylate derivative was quantitated in faeces (1-4%) and bile ( < 0.1%) but was absent in serum, tissues, and urine. An additional metabolite was isolated from bile and characterized as the pyridinyl-5-hydroxy derivative of CDRI-81/470. CDRI-81/470 showed rapid absorption and distribution into all major organs and tissues, and underwent extensive metabolism in rats. Two metabolites in bile were identified and characterized by synthesis.

Binding of centchroman with human serum as determined by charcoal adsorption method.

Protein binding of drugs is an important factor influencing both pharmacokinetic and pharmacodynamic parameters. Thus, knowing the extent of protein binding of drugs is crucial. Centchroman is a non-steroidal once a week oral contraceptive. It has been reported to be useful for the treatment of breast cancer and osteoporosis. Ample data has been generated on pharmacokinetics of centchroman in animals and humans. The extent of protein binding of centchroman has not been established so far. Non-specific adsorption of the drug limits the use of conventional methods like ultrafiltration and equilibrium dialysis. A method of charcoal adsorption as reported by Yuan et al. (method I) was used after modification (method II) to determine its binding to human serum. The extent of protein binding (%) is estimated from decline of percent drug remaining in the supernatant after adding the charcoal. Study was carried out at 1- and 10-microg/ml concentrations in drug free human serum samples and an HPLC assay was used to determine concentration-time data. The percentage of centchroman remaining in serum versus time data was analysed using non-linear fitting programs on WinNonlin software. Method II was found to give higher estimates of protein binding than the former method by preventing the dilution effect. Using this method, the extent of protein binding of centchroman was found to be 101.83+/-1.28 and 94.87+/-3.59% at 1 and 10 microg/ml, respectively. However, it was approximately 90% in the individual serum samples showing intersubject variability in protein binding of centchroman.

Determination of antifilarial compound UMF-078 and its metabolites in plasma by high-performance liquid chromatography.

UMF-078, methyl (+/-)-[5-(alpha-amino-4-fluorobenzyl)benzimidazol-2-yl]carba mate, is a new antifilarial compound being developed by the World Health Organization. In the present study, a HPLC method for the simultaneous estimation of UMF-078 and its metabolites (flubendazole, decarbamoylated flubendazole, UMF060 and decarbamoylated UMF-060) in plasma was developed, validated and applied to pharmacokinetic studies. Linearity was observed between 20 and 1000 ng/ml for decarbamoylated UMF-060 and between 10 and 500 ng/ml for other analytes. Recoveries were consistent over the concentration ranges studied for all the analytes. Variations in intra- and inter-batch accuracy and precision were within acceptable limits of +/-20% at the lowest limit of quantitation, whereas at higher concentrations it was +/-15%. The analytes showed stability up to two freeze-thaw cycles in plasma. No degradation was observed for any of the analytes even after 72 h of storing the dry plasma extracts at -30 degrees C. The assay method was employed to study the pharmacokinetics of hydrochloride salt of UMF-078 in rats. The parent compound and its metabolites viz: decarbamoylated UMF-060, UMF-060 and flubendazole were quantitated in serum and the compounds could be monitored up to 168 h post-dose.

Development and validation of a new high-performance liquid chromatographic assay of centpropazine, a new antidepressant compound, in serum.

Centpropazine is a new anti-depressant compound developed by Central Drug Research Institute, Lucknow (India). We report here the development and validation of a new HPLC assay of a parent drug in the serum of humans, monkeys and rats for pharmacokinetic studies. Centpropazine was eluted on a C18 column with a mobile phase consisting acetonitrile phosphate buffer (60:40) pumped at 1.5 ml min(-1) flow rate and quantitated by UV detector at 270 nm. Considering the sample volume available from different species and to enhance the sensitivity of assay, three sample clean up methods requiring 0.05, 0.5 and 4 ml serum for a linear quantitation range of 312.5 ng ml(-1)-5 microg ml(-1), 0.04-2.5 microg ml(-1) and 2.5-80 ng ml(-1) respectively were developed. The lowest limit of quantitation of the method was 2.5 ng ml(-1) requiring 4 ml serum, 40 ng ml(-1) requiring 0.5 ml serum and 312 ng ml(-1) requiring 50 microl serum sample. All these methods were fully validated in human serum and extended to monkey and rat serum. The recovery of centpropazine at 5, 80, 625, 1280 and 2500 ng ml(-1) ranged between 92 and 105%. The within and between run variability in precision and accuracy were less than 10% and the drug in serum was stable up to three freeze-thaw cycles. Overall the method is simple, quick and robust for biopharmaceutical applications. The method was applied to analyse concentrations of centpropazine in rat serum after administering single 20 mg kg(-1) peroral and 5 mg kg(-1) i.v. dose. The chromatograms of treated rat serum exhibited three well resolved peaks of metabolites and one of them was identified as hydroxy-metabolite of centpropazine.

Choosing the calibration model in assay validation.

Data transformations and weighting schemes are normally used to obtain the best-fit of standard curves in bioanalysis and the calibration model is usually selected during prevalidation. In the present study, a comparison has been made between unweighted and weighted (1/x, 1/x2, and 1/square root of x) regression models with or without an intercept in achieving the best-fit for the standard curve of CDRI compound 81/470, a new anthelmintic agent, in cow milk. Validation samples in milk at the LLOQ, medium, and high concentrations were also analysed by each of the calibration models. An unweighted regression equation with an intercept overestimated the concentrations at the LLOQ. An unweighted equation without intercept and weighted equations with or without an intercept significantly minimized the bias at the LLOQ without distorting the results at higher concentrations. Hence, an unweighted equation for a straight line passing through the origin was found to be the best model for a standard curve of 81/470 in milk. Similar results were obtained for 81/470 and UMF-078 in serum and plasma, respectively. Bioanalysts should routinely test these models to obtain the best fit model for their calibration curves as part of their assay validation not during prevalidation.

Evaluation of metronidazole toxicity: a prospective study.

Metronidazole is an antimicrobial, antiprotozoal agent that has been widely used in the treatment of a variety of infections. Some therapeutic indications necessitate prolonged treatment with metronidazole. Peripheral neuropathy is a potential metronidazole-induced toxicity, which has been reported in only a few isolated retrospective studies. This prospective study was designed to determine the toxic profile of metronidazole in patients undergoing long-term treatment with this drug. In the present study, 17 patients of both sexes, aged between 20 and 50 years, with body weights ranging from 46 to 62 kg and who were suffering from various medical ailments were recruited. The patients received 400 mg t.i.d. oral metronidazole in a total dose of 16.8-39.6 g for 2-4 weeks. It was found that patients usually suffered from some of the toxic symptoms of metallic taste, headache and dry mouth and to a lesser extent nausea, glossitis, urticaria, pruritus, urethral burning and dark colored urine. Symptoms were irrespective of sex and directly proportional to duration of therapy. Deep tendon ankle jerks were maximally reduced in four patients and sense of vibration at the level of olecranon and patella was affected in two patients. Distal latency and velocity of the sural and posterior tibial nerves were significantly affected (p < 0.01) compared with control values. These results indicate possible motor-sensory neurotoxicity involving the lower limbs due to long-term metronidazole therapy.

A rapid and sensitive high performance liquid chromatographic assay of the new antimalarial compound 80/53 in serum with a novel sample clean-up method and its pharmacokinetics in rabbits.

The compound 80/53 (AM) is a new antimalarial agent synthesized by this institute as a safer and less toxic analogue of primaquine. It was found to exhibit fluorescence in acetonitrile solution and this finding was exploited to develop a selective and sensitive high performance liquid chromatographic (HPLC) assay of the AM in rabbit serum. The sample clean-up was done in a single step by simultaneous protein precipitation and extraction with acetonitrile in the presence of sodium sulfate. The lower limit of quantitation of the method was 50 ng ml-1 using 100 microliters of serum sample. The method was fully validated from 50 to 1600 ng ml-1 concentration range with a recovery ranging from 70 to 75%. The within- and between-run variability was less than 10% and the drug in serum was stable over four freeze-thaw cycles and up to 24 h in injection solvent at 4 degrees C. The method was applied to determine the pharmacokinetic parameters of AM in 5 rabbits receiving a single bolus intravenous and peroral dose in a crossover study. The concentration-time data after a 5 mg kg-1 i.v. dose in rabbits was best fitted to the two compartment body model with first order absorption and elimination rate constants. The terminal half-life and MRT of AM were 95.3 +/- 43.5 and 104 +/- 10.6 min respectively. After administering a single 20 mg kg-1 oral dose, the serum levels of AM in all the rabbits declined below the quantitation limit by 90 min and it was not possible to fit the data by the compartmental approach. The MRT and AM after oral dose was 31.1+2-8.3 min. Application of the assay has also been extended to analyze the serum samples of rats, monkeys and humans.

Synthesis and disposition of 14C-labelled 81/470, a new anthelminthic agent in rats.

CDRI compound 81/470 (AH) is a new broad-spectrum anthelminthic agent under development for clinical and veterinary application. We herewith report the synthesis of 14C-labelled AH, and a study of the tissue distribution and excretion of radioactivity after administering a single 1 mg/kg p.o. or i.v. dose in young male rats. After oral administration of the dose, percent radioactivity recovered in 24-h urine, faeces and tissues were 17.9, 59.7 and 22.9 respectively. The levels were below detection limit in brain and gonads up to 24 h. In bile-duct-cannulated rats, the majority (37.8 +/- 2.8 and 43.8 +/- 6.4) of the radioactivity was excreted in the bile within 24 h of p.o. and i.v. administration, respectively. After an oral dose (1 mg/kg), the urinary excretion of radioactivity in rats was found to be approximately one-half (21 +/- 5.7, 18.3 +/- 2.1) of that obtained by i.v. administration of an equal dose (40.2 +/- 3.1 and 35 +/- 1.3), in bile-duct-intact and cannulated rats respectively.

Tissue distribution and pharmacokinetics of centchroman. A new nonsteroidal postcoital contraceptive agent and its 7-desmethyl metabolite in female rats after a single oral dose.

This study reports assay methodology, tissue distribution, and the basic pharmacokinetic behavior of centchroman and its 7-desmethyl metabolite [7-desmethyl centchroman (DMC)] after a single 12.5 mg/kg po dose in young female rats. Plasma, liver, lung, spleen, uterus, and adipose tissue were collected at various time intervals up to 14 days after dose. Reversed-phase HPLC, coupled with fluorescence detector, was used for simultaneous determination of centchroman and DMC in biosamples. The drug and metabolite were quantitated up to 2 and 5 ng/ml in plasma and 10 and 20 ng/g in tissues, respectively. The assay method was validated in terms of accuracy, precision, interassay, and intraassay variability, and was found to be reliable and reproducible. Peak centchroman levels in all of the tissues were found between 8-12 hr, whereas DMC peaks appeared between 8 and 24 hr, except that in liver the first peak of 1.2 micrograms/g appeared in the 1-hr sample. Tissue-to-plasma concentration ratios of centchroman were > 200 times in the lung; > 100 times in the spleen, liver, and adipose tissue; and > 40 times in the uterus at maxima in each tissue. Similarly, tissue concentrations of DMC were > 350 times in the lung, > 100 times in the liver and spleen, and > 25 times in the uterus and adipose tissue than in the plasma. High tissue-to-plasma concentration ratios of metabolites than the parent drug are indicative of its greater affinity for tissues. Terminal half-life of the centchroman and DMC in plasma were 24.1 and 36.6 hr, respectively. The mean residence time of centchroman was highest in the liver (78.4 hr), followed by the uterus (72.7 hr), adipose tissue (47.5 hr), lung (46 hr), spleen (44.1 hr), and plasma (37.7 hr). The mean residence time of DMC was also highest in the liver (133.7 hr), followed by the uterus (122 hr), adipose tissue (85.2 hr), lung (62.6 hr), spleen (62.6 hr), and plasma (48.2 hr).

Centchroman: a new non-steroidal oral contraceptive in human milk.

Centchroman, a non-steroidal oral contraceptive drug, was given to 13 nursing mothers comprising two groups. Each participant in group I (n = 8) received a single 30 mg dose, and in group II (n = 5) each participant received a 30 mg twice a week dose for twelve weeks. Simultaneous blood and milk samples were collected and analyzed for the parent drug by high performance liquid chromatography. In the single dose study (group I), the mean +/- peak centchroman concentrations in milk and serum were 78.7 +/- 28.4 and 63.6 +/- 23.6 ng/ml with milk-to-serum (M/S) ratio of 1.4 +/- 0.9. There was no significant increase in centchroman concentrations in milk after multiple dosing (group II). However, serum concentrations reached up to 112.5 ng/ml at 6 h after the 13th dose. Average M/S ratios were insignificantly different at trough (prior to next dose) and at peak (4-6 h after dose) centchroman levels. Additionally, the breast milk and serum centchroman concentrations showed a significant correlation (r = 0.64, P < 0.01), indicating that the amount of centchroman excreted into breast milk is dependent on serum concentrations. The weekly dose (% of the maternal dose) of centchroman ingested by the breast-fed infant at peak maternal serum and milk levels was in the range of 0.4 to 11.5%, assuming a weekly milk uptake of 1.05 l/kg. There was no significant difference in the dose ingested by the infants between the two dosing groups. These levels of centchroman passing into breast milk and subsequent exposure to the infants are unlikely to be of any physiological consequence.

The effect of competitive and non-linear plasma protein binding on the stereoselective disposition and metabolic inversion of ibuprofen in healthy subjects.

The stereoselective disposition and metabolic inversion of ibuprofen were studied in 12 healthy subjects under conditions of competitive and non-linear plasma protein binding. Each subject received each of four oral treatments according to a Latin-square design: 300 mg R(-)-ibuprofen, 300 mg S(+)-ibuprofen, 300 mg R(-)(-)+300 mg S(+)-ibuprofen, and 300 mg R(-)(-)+600 mg S(+)-ibuprofen. For a given treatment, the partial clearance of S(+)-ibuprofen was greater than that of R(-)-ibuprofen for all stereoisomeric drug species. Likewise, the unbound partial clearances of S(+)-ibuprofen were greater for most stereoisomeric drug species. There was also less difference among treatment groups when partial clearances were referenced to unbound as opposed to total plasma concentrations of enantiomer. The unbound intrinsic clearance and fractional inversion of R(-)-ibuprofen were unchanged across the four treatments, and chiral inversion was systemic, averaging 69%. In conclusion, stereoselective differences exist for the partial and composite clearances of R(-)- and S(+)-ibuprofen even when corrected for differences in plasma protein binding. However, differences among treatment groups for a particular elimination pathway are largely due to ibuprofen's non-linear binding.

Simultaneous liquid chromatographic determination of centchroman and its 7-demethylated metabolite in serum and milk.

A precise and sensitive high-performance liquid chromatographic assay was developed and validated for determination of centchroman (I) and its 7-demethylated metabolite (II) in human serum and milk. The serum, at alkaline pH, was extracted with diethyl ether. In the case of milk, after precipitation of the milk protein with acetonitrile, the supernatant was evaporated to dryness and then extracted with diethyl ether at alkaline pH. After solvent evaporation the residue was reconstituted in mobile phase. Separations were accomplished by reversed-phase liquid chromatography using a Spheri-5 cyano column. Recoveries of I and II were always > 95%. Excellent linear relationships (r > 0.999) were obtained between the measured and added concentration ratios of the corresponding serum and milk concentrations over a range of 1 to 1000 ng/ml and 2.5 to 1000 ng/ml for I and II, respectively.

Quantification of 2,3-dihydro-7-methoxypyrrolo-[2,1-b]-quinazolin-9(1H)-one, a new antiallergic agent, by high-performance liquid chromatography in serum.

The CDRI compound 73/602 (I, 7-methoxydeoxyvasicinone) is an antiallergic agent presently undergoing Phase II clinical trials. A rapid, precise and sensitive high-performance liquid chromatographic assay has been developed and validated for the detection of I in rat serum. The method involves extraction of rat serum samples with diethyl ether after addition of 1 M potassium hydroxide. Separations were accomplished on a RP-18 column using acetonitrile-phosphate buffer (pH 7) (20:80, v/v) as the mobile phase. Recovery of I was always > 90%. An excellent linear relationship (r = 0.9999) was obtained between the quantified and added concentrations over the range of 2.5 to 500 ng/ml. The sensitivity of the assay for 0.5 ml serum was 2.5 ng/ml.

Determination of ampicillin in serum by high-performance liquid chromatography with precolumn derivatization.

A high-performance liquid chromatographic assay method using precolumn derivatization and fluorescence detection has been developed and validated for the determination of ampicillin in serum. The presented method is simple and provides improved selectivity and sensitivity over other existing HPLC methods. It is linear over the concentration range of 100 to 10,000 ng/ml (method 1) and 2 to 1000 ng/ml (method 2) and the extraction recovery is more than 75%. The coefficient of variation is found to be less than 10% over the concentration ranges studied.

Simultaneous determination of a new antimalarial agent, CDRI compound 80/53, and its metabolite primaquine in serum by high-performance liquid chromatography.

Compound 80/53 (I) is a new substance being developed as an antimalarial agent. It is unstable in acidic conditions where it is converted into primaquine. A high-performance liquid chromatographic assay for simultaneous determination in serum of I and primaquine has been developed. Conditions were optimized to minimize the conversion of I into primaquine. The method includes extraction of the unchanged compound and primaquine from serum samples with hexane-2-propanol (pH > 8). Separation was accomplished by reversed-phase chromatography on a C18 column with acetonitrile-tetrahydrofuran-phosphate buffer. The recoveries of I and primaquine were always greater than 70%. No interference was observed in extracts obtained from drug-free serum. The detector response was linear with concentrations of I and the metabolite in the ranges 25-400 and 10-180 ng/ml, respectively, and the within-day precision (coefficient of variation) remained less than 13.7% for I and 12.5% for primaquine. The method is suitable for the determination of concentration-time profiles of I and primaquine in human serum.