Novel tricyclic small molecule inhibitors of Nicotinamide N-methyltransferase for the treatment of metabolic disorders.

Nicotinamide N-methyltransferase (NNMT) is a metabolic regulator that catalyzes the methylation of nicotinamide (Nam) using the co-factor S-adenosyl-L-methionine to form 1-methyl-nicotinamide (MNA). Overexpression of NNMT and the presence of the active metabolite MNA is associated with a number of diseases including metabolic disorders. We conducted a high-throughput screening campaign that led to the identification of a tricyclic core as a potential NNMT small molecule inhibitor series. Elaborate medicinal chemistry efforts were undertaken and hundreds of analogs were synthesized to understand the structure activity relationship and structure property relationship of this tricyclic series. A lead molecule, JBSNF-000028, was identified that inhibits human and mouse NNMT activity, reduces MNA levels in mouse plasma, liver and adipose tissue, and drives insulin sensitization, glucose modulation and body weight reduction in a diet-induced obese mouse model of diabetes. The co-crystal structure showed that JBSNF-000028 binds below a hairpin structural motif at the nicotinamide pocket and stacks between Tyr-204 (from Hairpin) and Leu-164 (from central domain). JBSNF-000028 was inactive against a broad panel of targets related to metabolism and safety. Interestingly, the improvement in glucose tolerance upon treatment with JBSNF-000028 was also observed in NNMT knockout mice with diet-induced obesity, pointing towards the glucose-normalizing effect that may go beyond NNMT inhibition. JBSNF-000028 can be a potential therapeutic option for metabolic disorders and developmental studies are warranted.

Highly sensitive LC-MS/MS-ESI method for determination of phenelzine in human plasma and its application to a human pharmacokinetic study.

A selective, sensitive and rapid LC-MS/MS method has been developed and validated for quantification of the phenelzine (PZ) in 200µL of human plasma using hydroxyzine (HZ) as an internal standard (IS) as per regulatory guidelines. The sample preparation involved the derivatization of PZ using pentaflurobenzaldehyde followed by solid phase extraction process to extract PZ and HZ from human plasma. LC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electro spray ionization technique in positive ion mode and the transitions of m/z 305.1→105.1 and m/z 375.3→201.1 were used to measure the derivative of PZ and IS, respectively. The total run time was 3.5min and the elution of PZ and HZ occurred at 2.53, and 1.92min, respectively; this was achieved with a mobile phase consisting of 10mM ammonium acetate: acetonitrile (20:80, v/v) at a flow rate of 1.0mL/min on an Ace C18 column with a split ratio of 70:30. The developed method was validated in human plasma with a lower limit of quantitation 0.51ng/mL. A linear response function was established for the range of concentrations 0.51-25.2ng/mL (r>0.995) for PZ. The intra- and inter-day precision values met the acceptance criteria. PZ was stable in the battery of stability studies viz., stock solution, bench-top, auto-sampler, long-term and freeze/thaw cycles. The developed assay method was applied to an oral bioequivalence study in humans.

Lymphatic transport of α-linolenic acid and its conversion to long chain n-3 fatty acids in rats fed microemulsions of linseed oil.

In the present study we evaluated the uptake of ALA and its conversion to EPA + DHA in rats given linseed oil (LSO) in native form or as a microemulsion in whey protein or in lipoid. In a single oral dose study in which rats maintained on rodent pellets deficient in ω-3 fatty acids were intubated with 0.35 g LSO in lipoid, the amount of ALA present in lymph was increased reaching a maximum concentration of 16.23 mg/ml at 2.5 h. The amount of ALA present in lymph was increased to a maximum level of 10.95 mg/ml at 4 h in rats given LSO as a microemulsion in whey protein. When LSO was given without emulsification, the amount of ALA present in lymph was found to reach a maximum level of 7.08 mg/ml at 6 h. A similar result was observed when weaning rats were intubated with 0.15 g of LSO per day for a period of 60 days. Higher levels of ALA by 41 and 103 % were observed in lymph lipids of rats given microemulsions of LSO in whey protein and in lipoid respectively as compared to rats given LSO without pre-emulsification. Very little conversion of ALA to EPA and DHA was observed in lymph lipids but higher amounts of EPA + DHA was observed in liver and serum of rats given LSO in microemulsion form. This study indicated that ALA concentration in lymph lipids was increased when LSO was given in microemulsion form in lipoid and further conversion to EPA and DHA was facilitated in liver and serum.

A sensitive LC-MS/MS method for the quantification of febuxostat in human plasma and its pharmacokinetic application.

An improved, simple and highly sensitive LC-MS/MS method has been developed and validated for quantification of febuxostat with 100 µL human plasma using febuxostat-d7 as an internal standard (IS) according to regulatory guidelines. The analyte and IS were extracted from human plasma via liquid-liquid extraction using diethyl ether. The chromatographic separation was achieved on a Zorbax C18 column using a mixture of acetonitrile and 5 mm ammonium formate (60:40, v/v) as the mobile phase at a flow rate of 0.5 mL/min. The total run time was 5.0 min and the elution of febuxostat and IS occurred at 1.0 and 1.5 min, respectively. A linear response function was established for the range of concentrations 1-6000 ng/mL (r > 0.99). The precursor to product ion transitions monitored for febuxostat and IS were m/z 317.1 → 261.1 and 324.2 → 262.1, respectively. The intra- and inter-day precisions (%RSD) were within 1.29-9.19 and 2.85-7.69%, respectively. The proposed method was successfully applied to pharmacokinetic studies in humans.

Validated LC-ESI-MS/MS method for simultaneous quantitation of felodipine and metoprolol in rat plasma: application to a pharmacokinetic study in rats.

A highly sensitive and specific LC-ESI-MS/MS method has been developed and validated for simultaneous quantification of felodipine (FDP) and metoprolol (MPL) in rat plasma (50 µL) using phenacetin as an internal standard (IS) as per the FDA guidelines. Liquid-liquid extraction method was used to extract the analytes and IS from rat plasma. The chromatographic resolution of FDP, MPL and IS was achieved with a mobile phase consisting of 0.2% formic acid in water-acetonitrile (25:75, v/v) with a time program flow gradient on a C18 column. The total chromatographic run time was 4.0 min and the elution of FDP, MPL and IS occurred at 1.05, 2.59 and 1.65 min, respectively. A linear response function was established for the range of concentrations 0.59-1148 and 0.53-991 ng/mL for FDP and MPL, respectively, in rat plasma. The intra- and inter-day accuracy and precision values for FDP and MPL met the acceptance as per FDA guidelines. FDP and MPL were stable in battery of stability studies viz., bench-top, auto-sampler and freeze-thaw cycles. The validated assay was applied to a pharmacokinetic study in rats.

Sensitive LC-MS/MS-ESI method for simultaneous determination of nifedipine and atenolol in human plasma and its application to a human pharmacokinetic study.

A rapid, simple, sensitive and selective LC-MS/MS method has been developed and validated for quantification of nifedipine (NF) and atenolol (AT) in human plasma (250 µL). The analytical procedure involves a one-step liquid-liquid extraction method using carbamazepine as an internal standard (IS). The chromatographic resolution was achieved on a Hypurity Advance C(18) column using an isocratic mobile phase consisting of 5 mm ammonium acetate-acetonitrile (15:85, v/v) at flow rate of 1.0 mL/min. The LC-MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. The total run time of analysis was 2 min and elution of NF, AT and IS occurred at 0.79, 1.04 and 0.76 min, respectively. A detailed method validation was performed as per the FDA guidelines and the standard curves found to be linear in the range of 1.02-101 ng/mL for NF and 5.05-503 ng/mL for AT, with a coefficient of correlation of ≥ 0.99 for both the drugs. NF and AT were found to be stable in a battery of stability studies, viz. bench-top, auto-sampler and repeated freeze-thaw cycles. The validated assay method was successfully applied to a pharmacokinetic study in humans.

Simultaneous determination of carisoprodol and aspirin in human plasma using liquid chromatography-tandem mass spectrometry in polarity switch mode: application to a human pharmacokinetic study.

A simple, sensitive and rapid LC-MS/MS-ESI method has been developed and validated for simultaneous quantification of the carisoprodol and aspirin in human plasma. Carisoprodol was detected in positive ion mode, whereas aspirin was detected in negative ion mode. Carbamazepine and furosemide were used as internal standards (IS) for quantification of carisoprodol and aspirin, respectively. The extraction procedure involves a liquid-liquid extraction method with ter-butyl methyl ether. Chromatographic separation was achieved on a Zorbax XDB-Phenyl (4.6 × 75 mm, 3.5 µm) column using an isocratic mobile phase (5 mm ammonium acetate:methanol, 20:80, v/v) at a flow rate of 0.8 mL/min with a total run time of 2.2 min. A detailed method validation was performed as per the FDA guidelines. The standard curves found to be linear in the range of 25.5-4900 and 15.3-3000 ng/mL for carisoprodol and aspirin, respectively. The results met the acceptance criteria. Carisoprodol and aspirin were found to be stable in various stability studies. The validated method was successfully applied to a pharmacokinetic study following co-administration of carisoprodol (250 mg) and aspirin (75 mg) tablets by oral route to human volunteers.

Irinotecan and its active metabolite, SN-38: review of bioanalytical methods and recent update from clinical pharmacology perspectives.

The introduction of irinotecan has revolutionized the applicability of camptothecins as predominant topoisomerase I inhibitor for anti-cancer therapy. The potent anti-tumor activity of irinotecan is due to rapid formation of an in vivo active metabolite, SN-38. Therefore, irinotecan is considered as a pro-drug to generate SN-38. Over the past decade, side-by-side with the clinical advancement of the use of irinotecan in the oncology field, a plethora of bioanalytical methods have been published to quantify irinotecan, SN-38 and other metabolites. Because of the availability of HPLC, LC-MS and LC-MS/MS methods, the pharmacokinetic profiling of irinotecan and its metabolites has been accomplished in multiple species, including cancer patients. The developed assays continue to find use in the optimization of newly designed delivery systems with regard to pharmacokinetics to promote safe and effective use of either irinotecan or SN-38. This review intends to: firstly, provide an exhaustive compilation of the published assays for irinotecan, SN-38 and other metabolite(s) of irinotecan, as applicable; secondly, to enumerate the validation parameters and applicable conclusions; and thirdly, provide some recent perspectives in the clinical pharmacology arena pertaining to efflux transporters, pediatric profiling, role of kidney function in defining toxicity, drug-drug interaction potential of irinotecan, etc.

Torcetrapib for animal and human pharmacokinetic studies: applicability of chiral and achiral methodologies.

The emergence of bioanalysis as a key tool in the drug-discovery and -development process has enabled the development of sensitive, precise and specific bioanalytical methods in recent years. These methods have enabled the progress of novel chemical entities through the life cycle of drug discovery and development. The focus of this review article is on a well-known cholesteryl ester transfer protein (CETP) inhibitor known as torcetrapib. Although torcetrapib was withdrawn from clinical development, it is important to understand the various bioanalytical methodologies (chiral and achiral) that are readily available for the pharmacokinetic/pharmacodynamic characterization of the drug. Additionally, these methodologies may be applicable to the bioanalysis of the next-generation CETP inhibitors. This review covers the development and validation of assay methods that were used to obtain preclinical and clinical pharmacokinetic parameters of torcetrapib. Accordingly, methods are available for the determination of torcetrapib in various species, namely dogs, hamsters, rats, mice, monkeys and humans. Since torcetrapib is a chiral compound, methods have been developed for stereoselective bioanalysis to evaluate in vivo chiral inversion phenomena. Interestingly, torcetrapib can be analyzed by various bioanalytical options (e.g., HPLC-UV, LC-MS, LC-MS/MS and GC-MS assays) depending on the type of species under consideration with the associated sensitivity requirements. This review covers all the available methodologies for torcetrapib, providing both assay-development and -optimization strategies. It also tabulates validation parameters and enumerates the difficulties, challenges and nuances of the various published assays for torcetrapib.

Digoxin - a therapeutic agent and mechanistic probe: review of liquid chromatographic mass spectrometric methods and recent nuances in the clinical pharmacology attributes of digoxin.

Digoxin is an important therapeutic agent for the treatment of congestive cardiac failure. In spite of its narrow therapeutic index, digoxin has been used extensively by the medical community and, lately, the use of digoxin as a mechanistic probe for p-glycoprotein transporter activity has increased. This review describes recent trends in the bioanalysis of digoxin, where scores of liquid chromatographic-mass spectrometric assays have been successfully employed to measure digoxin in preclinical, clinical and mechanistic studies. It provides various considerations such as internal standard selection, extraction schemes, matrix effect, selectivity evaluation and optimization of mass spectral conditions, for example, to enable the development of sound bioanalytical methods for digoxin. Some recent updates with regard to clinical pharmacology, absorption and disposition aspects of digoxin have been included. Overall, liquid chromatographic-mass spectrometric assays represent an important tool for many future preclinical, clinical and mechanistic probe studies that would probe digoxin with or without other coadministered substrates.

Bioavailability enhancement of poorly water soluble and weakly acidic new chemical entity with 2-hydroxy propyl-beta-cyclodextrin: selection of meglumine, a polyhydroxy base, as a novel ternary component.

The purpose of the present study was to investigate the influence of a polyhydroxy base, N-acetyl glucamine (also know as Meglumine), as a ternary component on the complexation of DRF-4367, a poorly water-soluble and weakly acidic anti-inflammatory molecule, with 2-hydroxypropyl-beta-cyclodextrin (HPbetaCD). The molecular inclusion of DRF-4367 with HPbetaCD alone and in combination with ternary component was aimed at improvement in solubility and, subsequently, dissolution rate-limited oral bioavailability. The solid complexes of DRF-4367 and HPbetaCD with or without meglumine (binary and ternary systems, respectively) were prepared as coevaporated product in different stoichiometric ratios and compared against physical mixture. The formation of inclusion complexes was confirmed by using classical instrumental techniques. Phase solubility studies suggested that meglumine was responsible for solubility improvement via multiple factors rather than just providing a favorable pH. Mechanisms and factors governing solubility enhancement were investigated by using phase solubility and thermodynamic parameters. The complexation of DRF-4367 with HPbetaCD is thermodynamically favored because the Gibbs free energies of transfer of the drug to the cyclodextrin cavity are negative. The solubilization efficiency and stability were further improved while retaining the favorable Gibbs free energies of transfer with the addition of meglumine. Inclusion ternary complex of DRF-4367 with HPbetaCD and meglumine showed significant improvement in dissolution compared with uncomplexed drug and binary system. Moreover, the phenomena of reprecipitation observed with binary system during dissolution could be avoided with meglumine as an enabling ternary component. This improved physicochemical behavior of ternary complex with the novel inclusion of a polyhydroxy base translated into an enhanced oral bioavailability of DRF-4367 compared with either uncomplexed drug or nanosuspension.

Evaluation of glycolamide esters of indomethacin as potential cyclooxygenase-2 (COX-2) inhibitors.

A number of novel indomethacin glycolamide esters were synthesized and tested for their cyclooxygenase (COX-1 and COX-2) inhibition properties in vitro. Many of these compounds proved to be selective COX-2 inhibitors, and subtle structural changes in the substituents on the glycolamide ester moiety altered the inhibitory properties as well as potencies significantly. Their in vitro data were rationalized through molecular modeling studies. Few of them displayed anti-inflammatory activity in vivo. Compound 32, [1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetic acid 2-morpholin-4-yl-2-oxo ethyl ester, was identified as a promising compound in this class and its good anti-inflammatory activity was demonstrated in the in vivo model.

Simple method for the determination of rosiglitazone in human plasma using a commercially available internal standard.

To the best of our knowledge, bioanalytical methods to determine rosiglitazone in human plasma reported in literature use internal standards that are not commercially available. Our purpose was to develop a simple method for the determination of rosiglitazone in plasma employing a commercially available internal standard (IS). After the addition of celecoxib (IS), plasma (0.25 mL) samples were extracted into ethyl acetate. The residue after evaporation of the organic layer was dissolved in 750 microL of mobile phase and 50 microL was injected on to HPLC. The separation was achieved using a Hichrom KR 100, 250 x 4.6 mm C(18) with a mobile phase composition potassium dihydrogen phosphate buffer (0.01 m, pH 6.5):acetonitrile:methanol (40:50:10, v/v/v). The flow-rate of the mobile phase was set at 1 mL/min. The column eluate was monitored by fluorescence detector set at an excitation wavelength of 247 nm and emission wavelength of 367 nm. Linear relationships (r(2) > 0.99) were observed between the peak area ratio rosiglitazone to IS vs rosiglitazone concentrations across the concentration range 5-1000 ng/mL. The intra-run precision (%RSD) and accuracy (%Dev) in the measurement of rosiglitazone were <+/-10.69 and <-12.35%, respectively across the QC levels (50-1000 ng/mL). The extraction efficiency was >80% for both rosiglitazone and IS from human plasma. The lower limit of quantitation of the assay was 5 ng/mL. In summary, the methodology for rosiglitazone measurement in plasma was simple, sensitive and employed a commercially available IS.

Oral bioavailability and pharmacokinetics of DRF-4367, a new COX-2 inhibitor in rats.

The pharmacokinetic characterization of DRF-4367 (a new diaryl pyrazole derivative), a potent selective COX-2 inhibitor was performed in Wistar rats. In the first study, a single dose of 2, 5, 10, 30 or 100 mg/kg DRF-4367 was given orally to rats for investigating the dose proportionality and/or linearity in the pharmacokinetics. In the second study, a single intravenous bolus dose of DRF-4367 was given at a dose of 10 mg/kg to calculate the absolute oral bioavailability, clearance and volume of distribution parameters. Blood samples were drawn at predetermined intervals up to 24 h post-dose. The concentrations of DRF-4367 in various plasma samples were determined by a validated HPLC method. Plasma concentration versus time data was generated following oral and i.v dosing and subjected to a noncompartmental pharmacokinetic analysis. Following oral administration, maximum concentrations of DRF-4367 were achieved at about 3 h and were unchanged with incremental doses. Both Cmax and AUC0-infinity appeared to increases less than proportional to the administered oral doses. While the doses increased in the ratio of 1.0 : 2.5 : 5.0 : 15.0 : 50.0, the mean AUC0-infinity and Cmax increased in the ratios of 1.0 : 2.8 : 4.5 : 8.6 : 14.5 and 1 : 2.4 : 4.1 : 6.2 : 8.3, respectively. Following i.v. administration, the concentration of DRF4367 declined in a monoexponential fashion with terminal elimination half-life of 5.7 h. The systemic clearance and volume of distribution of DRF-4367 in rats were 0.36 L/h/Kg and 2.2 L/Kg respectively after i.v administration. Elimination half-life was unchanged with route of administration and with increase in oral doses. Absolute oral bioavailability of DRF-4367 in the efficacy dose range was 70-80%.

HPLC method for the determination of rosiglitazone in human plasma and its application in a clinical pharmacokinetic study.

Rosiglitazone (CAS 155141-29-0, Avandia) is a novel insulin sensitizer used in the treatment of type 2 diabetes. A sensitive high performance liquid chromatography (HPLC) method for its determination in human plasma using fluorescence detection (excitation: 247 nm, emission: 367 nm) with a suitable internal standard (I. S.) is described. Ethyl acetate was used as extraction solvent. A mobile phase consisting of phosphate buffer, acetonitrile and methanol was used at a flow rate of 1.0 ml/min on a C18 column. The absolute recovery was > 90% and the lower limit of quantitation was 5 ng/ml. The intra- and inter-day relative standard deviations ranged from 0.58-6.69% and 0.82-6.63%, respectively. The method described is simple, economical, precise and accurate and has been successfully applied in a pharmacokinetic study conducted in healthy human volunteers.