Application of response surface method to evaluate the cytotoxic potency of Ulva fasciata Delile, a marine macro alga.

Bioprospecting of marine natural products has recently produced a substantial number of drug candidates. Ulva fasciata Delile, belonging to the family Ulvaceae, is a green marine macro alga that grows profusely on the coastal seashore of South India. In the present study, we investigated the in vitro cytotoxic potential of a methanolic extract of U . fasciata Delile (MEUF) using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay against human colon carcinoma (HT-29), human hepatocyte carcinoma (Hep-G2), and human breast carcinoma (MCF-7) cell lines. Response surface methodology (RSM) was applied using central-composite experimental design (CCD) to obtain optimum combined effect of concentration and cancer cells with highest cytotoxicity. The effect of concentration, cancer cell lines as independent variables on absorbance (OD), percent cell survival and percent cell inhibition as dependent variables was investigated. Maximum cytotoxic activity of MEUF was established for Hep-G2 with lowest OD or percent cell survival; highest percent cell inhibition with significant difference (p > 0.05) was compared to HT-29 and MCF-7.

Estimation of enantiomeric impurity in piperidin-3-amine by chiral HPLC with precolumn derivatization.

A simple, precise, accurate, robust chiral high-performance liquid chromatographic (chiral HPLC) method was developed for estimation of (S)-piperidin-3-amine (S-isomer) in (R)-piperidin-3-amine dihydrochloride (R-AMP). As AMP is a high-melting solid and nonchromophoric compound, development of a suitable chiral method is a challenging task. The proposed chiral HPLC-UV method involves a precolumn derivatization technique with para toluene sulphonyl chloride (PTSC) in the presence of a base to introduce chromophore into analytes. It utilizes chiralpak AD-H column with a simple mobile phase of 0.1% diethyl amine in ethanol with a 0.5 mL/min flow rate. Analytes were monitored by using a UV detector at 228 nm. The resolution between the two enantiomers was more than 4.0. The developed method was validated as per current International Conference on Harmonization (ICH) guidelines.

A facile synthesis of deuterium labeled 2,2-dimethyl-[2H6]-succinic acid and its anhydride.

Deuterium labeled 2,2-dimethyl-[(2)H(6)]-succinic anhydride by a sequence of reactions involving Knoevenagel condensation of [(2)H(6)]-acetone with ethyl cyanoacetate in the presence of piperidine, Michael addition of cyanide, HCl hydrolysis, simultaneous decarboxylation, and subsequent dehydration using acetic anhydride in an overall yield of 34.23% based on [(2)H(6)]-acetone utilized in the reaction is reported. The title compounds were characterized and confirmed spectroscopically by Fourier transform infrared, (1) H-NMR, and Mass. The chemical purity as determined by HPLC was 99%. To the best of our knowledge, the synthesis of these specifically deuterium labeled compounds has not been reported so far.

A novel no-carrier-added submicromolar scale radiosynthesis of [S-methyl-14C]-florfenicol.

In this paper is reported a novel reaction scheme for the no-carrier-added submicromolar scale radiosynthesis of [S-methyl-(14)C]-florfenicol that has been newly designed, developed and employed by us successfully. The [(14)C]-product was obtained in an overall radiochemical yield of 30% based on [(14)C]-methyl iodide taken for the reaction with a radiochemical purity of more than 96%. The specific activity of the product was ~50 mCi (1.85 GBq)/mmol. Chlorosulfonation of compound I was followed by sodium salt formation in situ and it was succeeded by the introduction of [(14)C]-methyl group by coupling with [(14)C]-CH3 I. Subsequently, the oxazolidin-2-one protecting group was opened up by a reaction with sulfuric acid in dioxane and later, the amino group was dichloroacetylated with methyl-2,2-dichloroacetate in triethylamine to obtain [S-methyl-(14)C]-florfenicol.

Ligand/PTC-free intramolecular Heck reaction: synthesis of pyrroloquinoxalines and their evaluation against PDE4/luciferase/oral cancer cell growth in vitro and zebrafish in vivo.

A series of 1,3-disubstituted pyrrolo[2,3-b]quinoxalines has been designed for the potential inhibition of PDE4 without inhibiting luciferase. A ligand/PTC (phase transfer catalyst) free intramolecular Heck cyclization strategy was used to prepare these compounds, some of which showed significant inhibition of PDE4B (IC50≈ 5-14 µM) and growth inhibition of oral cancer cells (CAL 27) but not inhibition of luciferase in vitro. They also showed acceptable safety profiles but no apoptosis in zebrafish embryos.

Novel N-indolylmethyl substituted olanzapine derivatives: their design, synthesis and evaluation as PDE4B inhibitors.

A new strategy for converting antipsychotic drug olanzapine into PDE4 inhibitors is described via the design and Pd/C mediated synthesis of novel N-indolylmethyl olanzapine derivatives. One compound showed good inhibition (IC50 1.1 µM) and >10 fold selectivity towards PDE4B over D that was supported by docking studies. This compound also showed significant inhibition of TNF-α and no major toxicities in cell lines and a zebrafish embryo model except the teratogenic effects to be re-assessed in rodents.

A facile no-carrier-added one pot micro-molar scale radiosynthesis of 2-[¹4C]-uracil.

A facile no-carrier-added one pot micro-molar scale radiolabelled synthesis of 2-[(14)C]-uracil from [(14)C]-urea and propiolic acid in the presence of polyphosphoric acid (PPA) with an yield of 48.33% and with radiochemical purity of 98% is reported in this paper.

A convenient photosynthesis of uniformly [¹4C]-labelled D-glucose, D-fructose and sucrose, and chemical synthesis of methyl-α-D-glucopyranoside ([U-¹4C]-glucose).

This paper describes a convenient procedure for the radiochemical preparation of d-[U-(14)C]-glucose, d-[U-(14)C]-fructose and [U-(14)C]-sucrose with high specific activity by photosynthesis using 'canna indica' leaf, [(14)C]-carbon dioxide and water in presence of light in a closed system. The [(14)C]-sugars formed were extracted, separated and then purified by paper chromatography. Further, the pure d-[U-(14)C]-glucose obtained was converted to methyl-α-d-glucopyranoside ([U-(14)C]-glucose) by glycosidation with methanol using (i) HCl, the conventional Fischer method (ii) heterogeneous organic cation exchange resin (Amberlite IR-120 (H(+))) and (iii) heterogeneous inorganic cation exchanged montmorillonites called metal M(+n)-monts. The results indicated that the latter in the form of Fe(+3)-montmorillonite gave a better yield ( 65%) as compared to others (40-56%). The radiochemical purity of the no-carrier added product was more than 98%. The product retained its specific activity as that of the starting material which is in the range of 250-300 mCi/mmole (9.25-11.1 GBq/mmole), suitable for use as a radiotracer in biochemical investigations.

Development and validation of RP-UPLC method for the determination of darifenacin hydrobromide, its related compounds and its degradation products using design of experiments.

A selective stability-indicating ultra-performance liquid chromatographic (UPLC) method was developed for the quantitative determination of darifenacin hydrobromide (DFN) and its related compounds in API and pharmaceutical dosages. The chromatographic separation was achieved on an Acquity UPLC BEH C18 column (100, 2.1 mm and 1.7 µm) at a flow rate of 0.3 mL/min, and detection was performed at 210 nm. The typical retention behaviors of impurities at various pH values were depicted graphically. The LC conditions were optimized by design of experiments (DOE) to obtain optimal separation in the shortest possible run time. A central composite design (CCD) was employed to study the main effects and interactions of the independent variables. The drug and its thirteen impurities were eluted within 13 min. The method exhibited consistent, high-quality recoveries (93.8 ± 2.1 to 99.8 ± 1.5 (mean ± RSD)) with a high precision for the drug and impurities. Linear regression analysis revealed an excellent correlation between peak responses and concentrations (R(2) values of 0.9991-0.9999) for the drug and impurities. The stability-indicating capability of the method was verified by forced degradation experiments and mass balance study. LC-MS revealed protonated molecular ion peaks [M+H](+) at m/z 428.20, m/z 425.20 and m/z 281.30 for the acid (Imp-4), oxidized (Imp-6) and N-dealkylated (Imp-1) forms of DFN, respectively. Possible degradation pathways were established based on the known reactivity of the drug through hydrolysis, oxidation, N-dealkylation, phenyl hydroxylation, dihydrobenzofuran ring hydroxylation and ring opening. The m/z values of unknown degradation products were matched with the proposed structures and reported DFN metabolites.

A novel reverse phase stability indicating RP-UPLC method for the quantitative determination of fifteen related substances in Ranolazine drug substance and drug product.

A gradient reverse-phase ultra performance liquid chromatographic (RP-UPLC) method was developed for the quantitative determination of Ranolazine and potential process-related impurities (starting materials, positional isomers, degradants and byproducts) at the level of 0.1 µg mL(-1) to 0.3 µg mL(-1). Fifteen potential impurities were identified in the crude samples during the process development. Tentative structures for all the impurities were assigned based on m/z values from LC-MS/MS analysis. This method can be used for the quality control of both drug substance and drug product. All these impurities were separated with a gradient UPLC method by using a polar embedded Waters Acquity BEH RP18 100 mm × 2.1 mm,1.7 µm column, monobasic sodium buffer, a basic organic modifier and acetonitrile in the mobile phase. Further, this method is also capable of separating a major oxidative degradant Di-N-oxide. Impurities having electron donating groups(+I effect) on the phenyl ring increased the retention by improved п-п interactions. The drug was subjected to the International Conference on Harmonization (ICH)-prescribed hydrolytic, oxidative, photolytic and thermal stress conditions. The performance of the method was validated according to the present ICH guidelines for specificity, limit of detection, limit of quantification, linearity, accuracy, precision, ruggedness and robustness.

Liquid chromatography - tandem mass spectrometry for the simultaneous quantitation of glipizide, cilostazol and its active metabolite 3, 4-dehydro-cilostazol in rat plasma: application for a pharmacokinetic study.

A liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method for the simultaneous quantitation of glipizide, cilostazol and 3, 4-dehydro-cilostazol in rat plasma was developed and validated. Glimepride was used as an internal standard (IS). The analytes were extracted by using liquid-liquid extraction procedure and separated on a reverse phase C18 column (50 mm×4.6 mm i. d., 5 µ) using acetonitrile: 2 mM ammonium acetate buffer, pH 3.2 (90:10, v/v) as mobile phase at a flow rate 0.4 mL/min in an isocratic mode. Selective reaction monitoring was performed using the transitions m/z 446.4>321.1, 370.2>288.3, 368.3>286.2, and 491.4>352.2 to quantify glipizide, cilostazol, 3, 4-dehydro-cilostazol and glimepride, respectively. Calibration curves were constructed over the range of 25-2 000 ng/mL for glipizide, cilostazol and 3, 4-dehydro-cilostazol. The lower limit of quantitation was 25 ng/mL for all the analytes. The recoveries from spiked control samples were>76% for all analytes and internal standard. Intra and inter day accuracy and precision of validated method were within the acceptable limits of at all concentration. The quantitation method was successfully applied for simultaneous estimation of glipizide, cilostazol and 3, 4-dehydro-cilostazol in a pharmacokinetic drug-drug interaction study in wistar rats.

Pharmacokinetic profiling of efavirenz-emtricitabine-tenofovir fixed dose combination in pregnant and non-pregnant rats.

During pregnancy, the disposition of various drugs is altered due to changes in physiological condition, maternal gastrointestinal absorption, gastric secretion and motility. A fixed dose combination of antiretrovirals is commonly prescribed for the treatment of HIV infection. There is a need to understand the pharmacokinetics and placental transfer of efavirenz-emtricitabine-tenofovir in fixed dose combination during pregnancy. The pharmacokinetics and placental transfer of efavirenz-emtricitabine-tenofovir fixed dose combination was evaluated in timed pregnant and non-pregnant Sprague-Dawley rats at 30, 10, 15 mg/kg p.o., respectively. The plasma, placental tissue, amniotic fluid and fetal tissue concentrations were measured using high performance liquid chromatography combined with tandem mass spectrometric detector (LC-MS/MS). To summarize, the pharmacokinetic profile of efavirenz remained similar in the pregnant and non-pregnant rats. However, a considerable difference in the pharmacokinetics of emtricitabine and tenofovir was observed in pregnant and non-pregnant rats. Efavirenz and emtricitabine showed appreciable placental, amniotic fluid and fetal exposure compared with tenofovir. The present study suggests that a profound impact on antiretroviral pharmacokinetics was observed during pregnancy and there is a need to monitor the exposure levels of each drug when administered as a fixed dose combination during pregnancy. Further studies to explore the pharmacokinetic parameters of fixed dose antiretrovirals during the preclinical stage in a timed-pregnancy rat model are required. Such studies can help in the development of safe and effective medications with a reduced risk of perinatal transmission of HIV-1 infection.

Stability-indicating UPLC method for determination of Imatinib Mesylate and their degradation products in active pharmaceutical ingredient and pharmaceutical dosage forms.

A simple, precise, accurate stability-indicating gradient reverse phase ultra-performance liquid chromatographic (RP-UPLC) method was developed for quantitative determination of purity of Imatinib Mesylate (IMM) drug substance and drug products in the presence of its process related impurities, and degradation products. The proposed RP-UPLC method utilizes Acquity UPLC BEH 50-mm, 2.1mm and 1.7 µm C-18 column at 30 °C, with a gradient program of 9.0 min at a flow rate of 0.3 mL/min. The compounds of interest were monitored at 237 nm. Resolution for Imatinib and eight related components was found to be greater than 1.5 for any pair of components. The correlation coefficients (r(2)>0.9990) obtained indicate clear correlations between the concentrations and their peak areas for the investigated compounds. RSD obtained for the repeatability and intermediate precision experiments, was less than 5.0%. Accuracy of the method was further ascertained by performing recovery studies through spiking experiments. The drug substance was subjected to hydrolytic, oxidative, photolytic and thermal stress conditions as per ICH. The developed method was validated according to the current ICH guidelines for specificity, limit of detection, limit of quantitation, linearity, accuracy, precision, ruggedness and robustness. The method is also suitable for the assay determination of IMM in pharmaceutical dosage forms.

Solvent effect on copper-catalyzed azide-alkyne cycloaddition (CuAAC): synthesis of novel triazolyl substituted quinolines as potential anticancer agents.

A regioselective route to novel mono triazolyl substituted quinolines has been developed via copper-catalyzed azide-alkyne cycloaddition (CuAAC) of 2,4-diazidoquinoline with terminal alkynes in DMF. The reaction provided bis triazolyl substituted quinolines when performed in water in the presence of Et(3)N. A number of the compounds synthesized showed promising anti-proliferative properties when tested in vitro especially against breast cancer cells.

Design and synthesis of 4-alkynyl pyrazoles as inhibitors of PDE4: a practical access via Pd/C-Cu catalysis.

The design and synthesis of 4-alkynyl pyrazole derivatives has led to the identification of new class of PDE4 inhibitors. All these compounds were accessed for the first time via a facile Pd/C-CuI-PPh(3) mediated C-C bond forming reaction between an appropriate pyrazole iodide and various terminal alkynes. In vitro PDE4B inhibitory properties and molecular modeling studies of some of the compounds synthesized indicated that 4-alkynyl pyrazole could be a promising template for the discovery of novel PDE4 inhibitors.

Concurrent administration of atypical antipsychotics and donepezil: drug interaction study in rats.

Psychotic and behavioral symptoms are common in patients with dementia. Thus, it is rational to assume that patients with dementia would gain benefit from combination therapy of an antipsychotic agent and a cognitive enhancer. Antipsychotics are not approved by the US FDA in elderly patients with dementia but their use is still prevalent in other population. In the current study, we investigate the effect of atypical antipsychotics on acetylcholine modulation by donepezil. In addition, the plasma pharmacokinetics on concurrent administration of these drugs was studied. Acetylcholine modulation was carried out in the ventral hippocampus of Sprague-Dawley rats using brain microdialysis technique. In a parallel group of animals, pharmacokinetic parameters were evaluated on administration of donepezil (5.0 mg kg(-1), ip) alone and in combination with olanzapine, clozapine, or quetiapine. Donepezil produced 348% increase in hippocampal acetylcholine levels. Coadministration of olanzapine and donepezil produced 393% increase in extracellular acetylcholine, and the effect was supported by a significantly (p < 0.05) decreased clearance of donepezil in plasma. Whereas, other plasma pharmacokinetic parameters of donepezil "AUC(0-24h), T (1/2) and T (max)" were moderately altered after this combination treatment. Concurrent administrations of clozapine or quetiapine with donepezil produced a non-significant change in acetylcholine levels in comparison to donepezil alone. The plasma pharmacokinetics of donepezil was unaltered. Results from this preclinical investigation indicate that extrapyramidal side effects may precipitate upon coadministration of donepezil with olanzapine. Care must be exercised by physicians and caregivers while administering these two drugs together.

Simultaneous quantitation of IC87114, roflumilast and its active metabolite roflumilast N-oxide in plasma by LC-MS/MS: application for a pharmacokinetic study.

A sensitive and reliable high-performance liquid chromatography-mass spectrometry (LC-MS/MS) was developed and validated for simultaneous quantification IC87114, roflumilast (RFM), and its active metabolite roflumilast N-oxide (RFN) using tolbutamide as an internal standard. The analytes were extracted by using liquid-liquid extraction and separated on a reverse phase C(18) column (50 mm × 3 mm i.d., 4.6 µ) using methanol: 2 mM ammonium acetate buffer, pH 4.0 as mobile phase at a flow rate 1 mL/min in gradient mode. Selective reaction monitoring was performed using the transitions m/z 398.3 > 145.9, 403.1 >186.9, 419.1 > 187.0 and 271.1 > 155.0 to quantify quantification IC87114, RFM, RFN and tolbutamide, respectively. The method was validated over the concentration range of 0.1-60 ng.mL(-1) for RFM and RFN and 6 to 2980 ng.mL(-1) for IC87114. Intra- and inter-day accuracy and precision of validated method were within the acceptable limits of <15% at all concentrations. Coefficients of correlation (r(2) ) for the calibration curves were >0.99 for all analytes. The quantitation method was successfully applied for simultaneous estimation of IC87114, RFM and RFN in a pharmacokinetic drug-drug interaction study in Wistar rats.

RP-UPLC method development and validation for the simultaneous estimation of ibuprofen and famotidine in pharmaceutical dosage form.

AIM AND BACKGROUND: A stability-indicating LC method was developed for the simultaneous determination of Ibuprofen and Famotidine in pharmaceutical dosage forms. MATERIALS AND METHODS: The chromatographic separation was achieved on Acquity UPLC BEH C-18,50 mm × 2.1 mm and 1.7 µm column with gradient elution. The mobile phase A contains a mixture of 50 mM sodium acetate buffer (pH 5.5): methanol (85:15, v/v), and the mobile phase B contains a mixture of 50 mM sodium acetate buffer (pH 5.5): methanol (25:75, v/v). The flow rate was 0.3 mL min(-1), and the detection wavelength was 260 nm. RESULTS: The limit of detection for Ibuprofen and Famotidine was 1.6 and 1.2 µg mL(-1), respectively. The limit of quantification (LOQ) for Ibuprofen and Famotidine was 5.1 and 4.3 µg mL(-1), respectively. CONCLUSION: This method was validated for accuracy, precision, and linearity. The method was also found to be stability indicating.

Construction of a six-membered fused N-heterocyclic ring via a new 3-component reaction: synthesis of (pyrazolo)pyrimidines/pyridines.

A conceptually new three-component reaction was developed to construct a six-membered fused N-heterocyclic ring affording (pyrazolo)pyrimidines/pyridines as potential inhibitors of PDE4. The reaction is catalyzed by triflic acid in acetic acid in the presence of aerial oxygen.

Identification of oxidative degradation impurities of olanzapine drug substance as well as drug product.

Impurities found in stressed and stability studies of olanzapine (polymorphic form-I) [1-7] in both drug substance and drug product are described. These impurities are identified as 4-(4-methyl-1-piperazinyl)-3-hydroxymethylidene-1H-benzo[b][1,4]diazepine-2(3H)-thione (hydroxymethylidene thione) and (Z)-4-(4-methyl-1-piperazinyl)-3-acetoxymethylidene-1H-benzo[b][1,4]diazapine-2(3H)-thione (acetoxymethylidene thione). An oxidative degradation pathway of olanzapine, for the formation of these impurities, has been proposed.