In vitro and in vivo studies on the metabolism and pharmacokinetics of the selective gut microbial ß-glucuronidase targeting compound Inh 1.

1. In vitro studies using rat, mouse and human microsomes and hepatocytes on the bacterial ß-glucuronidase inhibitor (1-((6,8-dimethyl-2-oxo-1,2-dihydroquinolin-3-yl)methyl)-3-(4-ethoxyphenyl)-1-(2-hydroxyethyl)thiourea) (Inh 1) revealed extensive metabolism in all species.2. The intrinsic clearances of Inh 1 in human, mouse and rat hepatic microsomes were 30.9, 67.8 and 201 µL/min/mg, respectively. For intact hepatocytes intrinsic clearances of 21.6, 96.0 and 129 µL/min/106 cells were seen for human, mouse and rat, respectively.3. The metabolism of Inh 1 involved an uncommon desulphurisation reaction in addition to oxidation, deethylation and conjugation reactions at multiple sites. Six metabolites were detected in microsomal incubations in human and rat, and seven for the mouse. With hepatocytes, eighteen metabolites were characterised, nine for human, and eleven for mouse and rat.4. Following IV administration to mice (3 mg/kg), plasma concentrations of Inh 1 declined bi-exponentially with a terminal elimination half-life of 0.91 h and low systemic clearance (11.8% of liver blood flow). After PO dosing to mice (3 mg/kg), peak observed Inh 1 concentrations of 495 ng/ml were measured 0.5 hr post dose, declining to under 10 ng/ml at 8 hr post dose. The absolute oral bioavailability of Inh 1in the mouse was ca. 26%.

The in vitro metabolism and in vivo pharmacokinetics of the bacterial ß-glucuronidase inhibitor UNC10201652.

In vitro incubation of the bacterial ß-glucuronidase inhibitor UNC10201652 (4-(8-(piperazin-1-yl)-1,2,3,4-tetrahydro-[1,2,3]triazino[4',5':4,5]thieno[2,3-c]isoquinolin-5-yl)morpholine) with mouse, rat, and human liver microsomes and hepatocytes generated metabolites at multiple sites via deethylations, oxidations and glucuronidation.Two UNC10201652 metabolites were detected in human, and four in mouse and rat liver microsomal incubations. Intrinsic clearances of UNC10201652 in human, mouse, and rat liver microsomes were 48.1, 115, and 194 µL/min/mg respectively.Intrinsic clearances for human, mouse, and rat hepatocytes were 20.9, 116, and 140 µL/min/106 cells respectively and 24 metabolites were characterised: 9 for human and 11 for both rodent species.Plasma clearance was 324.8 mL/min/kg with an elimination half-life of 0.66 h following IV administration of UNC10201652 to Swiss Albino mice (3 mg/kg). Pre-treatment with 1-aminobenzotriazole (ABT) decreased clearance to 127.43 mL/min/kg, increasing the t1/2 to 3.66 h.Comparison of profiles after oral administration of UNC10201652 to control and pre-treated mice demonstrated a large increase in Cmax (from 15.2 ng/mL to 184.0 ng/mL), a delay in Tmax from 0.25 to 1 h and increased AUC from 20.1 to 253 h ng/ml. ABT pre-treatment increased oral bioavailability from 15% to >100% suggesting that CYP450's contributed significantly to UNC10201652 clearance in mice.

Discovery and evaluation of novel FAAH inhibitors in neuropathic pain model.

Conceptual design and modification of urea moiety in chemotype PF-3845/04457845, the bench marking irreversible inhibitor of fatty acid amide hydrolase (FAAH), led to discovery of a novel nicotinamide-based lead 12a having reversible mechanism of action. Focused SAR around the pyridine heterocycle (Ar) in 12a (Tables 1 and 2) resulted into four shortlisted compounds, (-)-12a, (-)-12i, (-)-12l-m. The required (-)-enantiomers were obtained via diastereomeric resolution of a novel chiral dissymmetric intermediate 15. Based on comparative profile of FAAH potency, metabolic stability in liver microsome, liability of inhibiting major hCYP450 isoforms, rat PK, and brain penetration ability, two SAR optimized compounds, (-)-12l and (-)-12m, were selected for efficacy study in rat model of chemotherapy-induced peripheral neuropathy (CIPN). Both the compounds exhibited dose related antihyperalgesic effects, when treated with 3-30 mg/kg po for 7 days. The effects at 30 mg/kg are comparable to that of PF-04457845 (10 mg/kg) and Tramadol (40 mg/kg).

Evaluation of separate role of intestine and liver in first pass metabolism of budesonide in rat.

1. Budesonide, a potent topical corticosteroid, reported to have low oral bioavailability in mice, rat, dog and human due to rapid first pass metabolism. However, there is insufficient information available in literature regarding the role of intestine and or liver responsible for the first pass metabolism of budesonide. 2. Current study in rats investigates the role of intestine and liver in first pass metabolism of budesonide using two in vivo models. Additionally, budesonide was also evaluated in in vitro assays such as thermodynamic solubility, permeability in Caco-2 cells and stability in simulated gastric (SGF), intestinal fluids (SIF) to understand the underlaying cause for low oral bioavailability. 3. Budesonide showed low oral, intra-duodenal and high intra-portal bioavailability in rat. In a dual vein cannulated rat model, intestinal and hepatic extraction ratios calculated based upon intestinal availability (Fa·Fg) and hepatic availability (Fh), suggests hepatic extraction of budesonide is minimal compared to intestinal. 4. In vitro results suggest, solubility and permeability may not be a barrier for the observed low oral bioavailability in rats. 5. Correlating the in vitro and in vivo data together, it can be concluded that, intestine might be playing major role in first pass metabolism of budesonide.

Discovery of Potent and Selective A2A Antagonists with Efficacy in Animal Models of Parkinson's Disease and Depression.

Adenosine A2A receptor (A2AAdoR) antagonism is a nondopaminergic approach to Parkinson's disease treatment that is under development. Earlier we had reported the therapeutic potential of 7-methoxy-4-morpholino-benzothiazole derivatives as A2AAdoR antagonists. We herein described a novel series of [1,2,4]triazolo[5,1-f]purin-2-one derivatives that displays functional antagonism of the A2A receptor with a high degree of selectivity over A1, A2B, and A3 receptors. Compounds from this new scaffold resulted in the discovery of highly potent, selective, stable, and moderate brain penetrating compound 33. Compound 33 endowed with satisfactory in vitro and in vivo pharmacokinetics properties. Compound 33 demonstrated robust oral efficacies in two commonly used models of Parkinson's disease (haloperidol-induced catalepsy and 6-OHDA lesioned rat models) and depression (TST and FST mice models).

Discovery of liver-directed glucokinase activator having anti-hyperglycemic effect without hypoglycemia.

Glucokinase activators (GKAs) are among the emerging drug candidates for the treatment of type 2 diabetes (T2D). Despite effective blood glucose lowering in clinical trials, many pan-GKAs "acting both in pancreas and liver" have been discontinued from clinical development mainly because of their potential to cause hypoglycemia. Pan-GKAs over sensitize pancreatic GK, resulting in insulin secretion even at sub-normoglycemic level which might be a possible explanation for hypoglycemia. An alternative approach to minimize the risk of hypoglycemia is to use liver-directed GKAs, which are reported to be advancing well in clinical development. Here, we report the discovery and structure-activity relationship (SAR) studies on a novel 2-phenoxy-acetamide series with the aim of identifying a liver-directed GKA. Incorporation of a carboxylic acid moiety as an active hepatocyte uptake recognizing element at appropriate position of 2-phenoxy-acetamide core led to the identification of 26, a potent GKA with predominant liver-directed pharmacokinetics in mice. Compound 26 on oral administration significantly reduced blood glucose levels during an oral glucose tolerance test (oGTT) performed in diet-induced obese (DIO) mice, while showing no sign of hypoglycemia in normal C57 mice over a 10-fold dose range, even when dosed at fasted condition. Together, these data demonstrate a liver-directed GKA has beneficial effect on glucose homeostasis with reduced risk of hypoglycemia.

Discovery and evaluation of 1H-pyrrolo[2,3-b]pyridine based selective and reversible small molecule BTK inhibitors for the treatment of rheumatoid arthritis.

In a pursuit to identify reversible and selective BTK inhibitors, two series based on 7H-pyrrolo[2,3-d]pyrimidine and 1H-pyrrolo[2,3-b]pyridine as the hinge binding core, have been identified. Structure activity relationship (SAR) exploration led to identification of two advanced lead molecules, 11 and 13, which demonstrated desired BTK inhibitory potency in different cellular assays, excellent selectivity in a panel of 50 diverse kinases, favorable in vivo PK properties in mice and anti-arthritic effect in a mouse model of CIA.

Liquid chromatography-tandem mass spectrometry analysis of urine specimens for K2 (JWH-018) metabolites.

Marijuana is the most widely used drug of abuse all over the world. The major active constituent of the drug is Δ9- tetrahydrocannabinol (Δ9-THC). Δ9-THC exerts its psychological activities by interacting with the cannabinoid receptors (CB1 and CB2) in the brain. JWH-018, HU-210, and CP-47497, with CB1 agonist activity (similar to Δ9-THC), have been used by the drug culture to spike smokable herbal products to attain psychological effects similar to those obtained by smoking marijuana. The products spiked with these CB1 agonists are commonly referred to as "Spice" or "K2". The most common compound used in these products is JWH-018 and related compounds (JWH-073 and JWH-250). Little work has been done on the detection of these synthetic cannabimimetic compounds in biological specimens. This report investigated the metabolism of JWH-018 by human liver microsomes, identification of the metabolites of JWH-018 in urine specimen of an individual who admitted use of the drug, and reports on the quantitation of three of its urinary metabolites, namely the 6-OH-, the N-alkyl OH (terminal hydroxyl)-, and the N-alkyl terminal carboxy metabolites using liquid chromatography-tandem mass spectrometry. The concentrations of these metabolites are determined in several forensic urine specimens.

Bioavailability, pharmacokinetics, and tissue distribution of the oxypregnane steroidal glycoside P57AS3 (P57) from Hoodia gordonii in mouse model.

P57AS3 (P57), an oxypregnane steroidal glycoside, is known to be responsible for the appetite suppressing activity of HOODIA GORDONII, a dietary supplement used for weight loss. In this study, bioavailability, pharmacokinetics, and tissue distribution of P57 were determined in CD1 female mice after administration of a single dose of enriched methanolic extract of HOODIA GORDONII (equivalent to a dose of 25 mg of P57/kg) by oral gavage or a single dose of purified P57 (25 mg/kg) intravenously. The level of P57 in plasma and tissues (brain, liver, kidney, and intestine) was determined by UPLC-MS. After oral administration of HOODIA extract, the peak plasma level of P57 was achieved in 0.6 h. Upon intravenous administration, the plasma clearance rate of P57 was 1.09 L/h/kg. P57 was rapidly distributed and eliminated from the tissues within 4 hours. The level of tissue distribution was highest in the kidney followed by liver and brain. Upon oral administration, P57 was not detected in the brain and a very low concentration was seen in the intestine, kidney, and liver. Tissue/plasma ratio was 0.33 for brain, 0.57 for liver, and 0.75 for kidney with IV route and 0.11 for intestine, 0.02 for liver, and 0.04 for kidney with oral route. The half-life of the elimination phase was similar with both routes. The oral bioavailability was 47.5 % and the half-life of the absorption phase was 0.13 h. In conclusion, P57 showed moderate bioavailability and was eliminated rapidly.

Structure-activity relationship and mechanism of action studies of manzamine analogues for the control of neuroinflammation and cerebral infections.

Structure-activity relationship studies were carried out by chemical modification of manzamine A (1), 8-hydroxymanzamine A (2), manzamine F (14), and ircinal isolated from the sponge Acanthostrongylophora. The derived analogues were evaluated for antimalarial, antimicrobial, and antineuroinflammatory activities. Several modified products exhibited potent and improved in vitro antineuroinflammatory, antimicrobial, and antimalarial activity. 1 showed improved activity against malaria compared to chloroquine in both multi- and single-dose in vivo experiments. The significant antimalarial potential was revealed by a 100% cure rate of malaria in mice with one administration of 100 mg/kg of 1. The potent antineuroinflammatory activity of the manzamines will provide great benefit for the prevention and treatment of cerebral infections (e.g., Cryptococcus and Plasmodium). In addition, 1 was shown to permeate across the blood-brain barrier (BBB) in an in vitro model using a MDR-MDCK monolayer. Docking studies support that 2 binds to the ATP-noncompetitive pocket of glycogen synthesis kinase-3beta (GSK-3beta), which is a putative target of manzamines. On the basis of the results presented here, it will be possible to initiate rational drug design efforts around this natural product scaffold for the treatment of several different diseases.

Characterization of in vitro pharmacokinetic properties of hoodigogenin A from Hoodia gordonii.

This study was aimed to predict the pharmacokinetic properties of hoodigogenin A, which is the aglycone of the oxypregnane steroidal glycoside P57AS3 (P57) isolated from Hoodia gordonii. A series of in vitro assays was used to predict its gastric, intestinal and metabolic stability, intestinal and blood brain barrier (BBB) transport, protein binding and interaction with major drug metabolising enzymes. In the simulated gastric fluid, hoodigogenin A was stable (2 % degradation in 60 minutes) whereas P57 was unstable (45 % degradation in 30 minutes). In simulated intestinal fluid, P57 was degraded to an extent of 8 % in 180 minutes, while hoodigogenin A was stable. Hoodigogenin A was efficiently transported by passive diffusion across Caco-2 and MDR1-MDCK monolayers with P(app) values in the range of 32 x 10(-6) cm/sec and 22 x 10(-6) cm/sec, respectively. The compound was metabolically unstable in human liver microsomes and S9 fractions with a CL' (int) of 71 and 120 mL/min/kg, respectively and was bound to the plasma proteins to an extent of 92 %. The compound strongly inhibited CYP3A4 activity (IC(50) 3 microM), indicating a possibility of drug-herb/botanical interactions when products containing H. gordonii are used simultaneously with other botanicals/herbs/drugs.

Intestinal and blood-brain barrier permeability of ginkgolides and bilobalide: in vitro and in vivo approaches.

In this study intestinal and blood-brain barrier (BBB) permeability of ginkgolides A, B, C, J and bilobalide, isolated from Ginkgo biloba (Ginkgoaceae), was evaluated in Caco-2 and MDR1-MDCK cell monolayer models. The transport was examined for 2 hours in both absorptive and secretory directions. Quantitation was performed by UPLC-MS. In the Caco-2 model, each compound (100 microM) displayed a pH-dependent transport in the absorptive direction. A low permeability of ginkgolides was observed across the MDR1-MDCK model in the absorptive direction. An efflux was observed for all compounds in both the models. The efflux ratio was much higher in the MDR1-MDCK cell model (> 10) compared to the Caco-2 cell model (1.5-3.6). In comparison to ginkgolides, the permeability of bilobalide was much higher across the Caco-2 monolayer in both directions. However, a poor transport of bilobalide was observed in the MDR1-MDCK model in the absorptive direction. A high efflux was observed for all compounds in the mixture form as compared to their isolated forms. In rats, a single dose of bilobalide (8 mg/kg) administered intravenously resulted in a significant level of bilobalide in both plasma and brain. A brain-to-plasma partition coefficient of 0.56 at 120 min indicated its possibility of brain uptake.

Alkaloids and saponins as cytochrome P450 inhibitors from blue cohosh (Caulophyllum thalictroides) in an in vitro assay.

Blue cohosh, Caulophyllum thalictroides (L.) Michx. is a popular herb, roots and rhizomes of which have been extensively used for women's health. Alkaloids and saponins are considered to be responsible for its pharmacological effects. In this investigation the methanolic extract of the roots of blue cohosh, the alkaloidal fraction and isolated constituents were evaluated for their inhibition of major drug metabolizing cytochrome P450 (CYP450) enzymes. The methanolic extract did not show any effect but the alkaloidal fraction showed a strong inhibition of CYP 2C19, 3A4, 2D6, and 1A2 (>80% inhibition at 100 microg/mL) with IC50 values in the range of 2-20 microg/mL. Among the isolated alkaloids, a piperidine-type alkaloid (caulophyllumine B) and three lysine-derived alkaloids (O-acetlybaptifolin, anagyrine, and lupanine) inhibited these enzymes to various extents (IC50:2.5-50 microM). N-Methylcytisine weakly inhibited CYP3A4 only (32% inhibition at 100 microM). An equimolar mixture of alkaloids exhibited a more pronounced inhibitory effect on all four enzymes as compared to the isolated alkaloids. Among the saponins, caulosides C and D inhibited CYP3A4 at the highest test concentration of 100 microM (43% and 35% inhibition, respectively). Other enzymes were not affected. This in vitro study indicates that dietary supplements containing blue cohosh may pose a risk of drug-drug interactions if taken with other drugs or herbs, metabolism of which involves CYP450 enzymes.

In vitro metabolic stability and intestinal transport of P57AS3 (P57) from Hoodia gordonii and its interaction with drug metabolizing enzymes.

Hoodia gordonii, a succulent cactus-like plant growing in South Africa, has been used in traditional medicine for its appetite suppressant properties. Its use as a dietary supplement to promote weight loss has recently gained popularity. An oxypregnane steroidal glycoside P57AS3 (P57) is reported to be the active constituent of the sap extract responsible for anorexigenic activity. No information is available about its metabolic stability, intestinal transport and interaction with drug metabolizing enzymes. In the present investigation, the metabolic stability of P57 in human liver microsomes and its interaction with drug metabolizing enzymes (CYP1A2, 2C9, 3A4 and 2D6) were determined. Intestinal transport of P57 was studied in the Caco-2 cell model of intestinal transport and absorption. P57 was metabolically stable in the presence of human liver microsomes. The compound inhibited CYP3A4 activity with an IC50 value of 45 microM, whereas the activity of CYP 1A2, 2C9 and 2D6 was not inhibited. In the Caco-2 model, P57 exhibited a higher transport in the secretory direction than in the absorptive direction with efflux ratios of 3.1 and 3.8 at 100 and 200 microM, respectively. The efflux was inhibited by selective inhibitors of multidrug resistance associated proteins MRP1/MRP2 (MK-571) and P-gp (verapamil). In conclusion, intestinal transport of P57 was mediated by P-gp and MRP transporters. The compound was metabolically stable and showed weak inhibition of CYP 3A4.

Transport of Schisandra chinensis extract and its biologically-active constituents across Caco-2 cell monolayers - an in-vitro model of intestinal transport.

We have determined the intestinal transport of Schisandra chinensis extract and its lignans (gomisin A, gomisin N and schisandrin C) in the Caco-2 cell monolayer model. The transport across monolayers was examined for 2 h in absorptive and secretory directions. Quantitation of lignans was performed by HPLC. Out of the three lignans, gomisin A exhibited bi-directional transport, with P(app) values in the range of 25-29 x 10(-6) cm s(-1), indicating a passive diffusion. Gomisin N, mixture and Schisandra extract displayed a higher transport in the secretory direction with efflux ratios in the range of 2.2-5.2. The efflux was decreased in the presence of inhibitors of multidrug resistance protein (MRP) transporter (MK-571) and P-glycoprotein (verapamil) indicating a possible involvement of an efflux pump and MRP in the transport of Schisandra lignans. Poor transport of schisandrin C was observed which could not be quantitated. The permeability of gomisin A in the isolated form was significantly different compared with the mixture or extract.

Transport of decursin and decursinol angelate across Caco-2 and MDR-MDCK cell monolayers: in vitro models for intestinal and blood-brain barrier permeability.

Decursin (DE) and decursinol angelate (DA) were isolated from the roots of Angelica gigas (Apiaceae) and purified by HPLC. DE and DA have been reported to exhibit significant neuropharmacological activities, but their intestinal transport and permeability in terms of CNS penetration across the blood-brain barrier (BBB) are unknown. This study was undertaken to evaluate the IN VITRO intestinal and BBB transport of DE and DA using Caco-2 and MDR-MDCK cell monolayer models, respectively. The bidirectional transport of DE and DA across Caco-2 and MDR-MDCK monolayers was examined for 2 hours. Integrity of the monolayer was determined by TEER value and by monitoring the transport of Lucifer yellow (Ly) across the monolayers. Quantitation of DE and DA was performed by HPLC. DE and DA exhibited bidirectional transport with a Papp value in the range of 9.0-12.0x10(-6) cm/sec and 7.2-11.7x10(-6) cm/sec in Caco-2 and MDR-MDCK monolayers, respectively. The TEER values were in the range of 410-440 and 1170-1230 ohm cm2 for Caco-2 and MDR-MDCK monolayers, respectively. Ly measurement, the fluorescent marker of passive paracellular diffusion, resulted in Papp values of 2.5-5.0x10(-6) in Caco-2 and 6.0-8.0x10(-6) cm/sec in MDR-MDCK monolayers, confirming that the monolayer integrity was intact at the end of the experiment. Caco-2:human colonic adenocarcinoma DA:decursinol angelate DE:decursin Ly:Lucifer yellow MDCK:Madin-Darby canine kidney MDR:multidrug resistant Papp:apparent permeability TEER:transepithelial electrical resistance.

Triplet repeats in human genome: distribution and their association with genes and other genomic regions.

MOTIVATION: Simple sequence repeats (SSRs) or microsatellite repeats are found abundantly in many prokaryotic and eukaryotic genomes. Among SSRs, triplet repeats are of special significance because some of them have been linked to various genetic disorders. The objective of the study is to analyze the triplet repeats of complete human genome and to identify the genes that contain the triplet repeats in their coding region. The analysis will help us to identify the candidate genes that have potential for repeat expansion. RESULTS: We have analyzed triplet repeats in the complete human genome from the publicly available sequences. Our analysis revealed that AGC and CCG repeat were predominantly present in the coding regions of the genome while UTRs and the upstream sequences contained CCG repeats in relative abundance. Analysis of density of triplet repeats (bp/Mb) revealed that AAT and AAC were the abundant repeats whereas ACT and ACG were the rare repeats found in human genome. We could identify about 2135 known or predicted genes that were associated with at least one of the triplet repeat types. A large proportion of putative transcripts that were identified by gene finding programs were found to be associated with triplet repeats. These transcripts will be the candidate genes for analysis of triplet repeat expansion and a possible association with disease phenotypes. Identification of 171 genes which contain a minimum of ten repeat units will be of particular interest in future in correlating their association with any disease phenotype due to the expansion potential of repeats present in them. The list of genes and other details of analysis are given in the online supplementary data (http://www.ingenovis.com/tripletrepeats).

SSRD: simple sequence repeats database of the human genome.

Simple sequence repeats are predominantly found in most organisms. They play a major role in studies of genetic diversity, and are useful as diagnostic markers for many diseases. The simple sequence repeats database (SSRD) for the human genome was created for easy access to such repeats, for analysis, and to be used to understand their biological significance. The data includes the abundance and distribution of SSRs in the coding and non-coding regions of the genome, as well as their association with the UTRs of genes. The exact locations of repeats with respect to genomic regions (such as UTRs, exons, introns or intergenic regions) and their association with STS markers are also highlighted. The resource will facilitate repeat sequence analysis in the human genome and the understanding of the functional and evolutionary significance of simple sequence repeats. SSRD is available through two websites, http://www.ccmb.res.in/ssr and http://www.ingenovis.com/ssr.

MRD: a microsatellite repeats database for prokaryotic and eukaryotic genomes.

MRD is a database system to access the microsatellite repeats information of genomes such as archea, eubacteria, and other eukaryotic genomes whose sequence information is available in public domains. MRD stores information about simple tandemly repeated k-mer sequences where k= 1 to 6, i.e. monomer to hexamer. The web interface allows the users to search for the repeat of their interest and to know about the association of the repeat with genes and genomic regions in the specific organism. The data contains the abundance and distribution of microsatellites in the coding and non-coding regions of the genome. The exact location of repeats with respect to genomic regions of interest (such as UTR, exon, intron or intergenic regions) whichever is applicable to organism is highlighted. MRD is available on the World Wide Web at http://www.ccmb.res.in/mrd and/or http://www.ingenovis.com/mrd. The database is designed as an open-ended system to accommodate the microsatellite repeats information of other genomes whose complete sequences will be available in future through public domain.