Metabolic profiling of a novel antithrombotic compound, S002-333 and enantiomers: metabolic stability, species comparison and in vitro-in vivo extrapolation.

OBJECTIVE: The aim of this research work was to characterize the metabolism of S002-333, (2-(4'-methoxy-benzenesulfonyl)-2,3,4,9-tetrahydro-1H-pyrido (3,4-b) indole-3-carboxylic acid amide) and its enantiomers, S004-1032 (R-form) and S007-1558 (S-form) in pooled human liver microsomes (PHLM) and pooled liver microsomes (LM) of rat (RLM), rabbit (RABLM), dog (DLM) and monkey (MLM). Another objective of this study was to identify suitable surrogate species to humans for further development of lead candidates. METHOD: In vitro metabolic stability and metabolite identification of S002-333 and enantiomers were carried out in PHLM and LM of various species. The prediction of surrogate species and in vitro in vivo extrapolation were performed based upon the calculated in vitro intrinsic clearance (CLint ). RESULTS/CONCLUSION: The in vitro CLint values for S002-333, S004-1032 and S007-1558 were 0.027 ± 0.005, 0.025 ± 0.004 and 0.036 ± 0.005 ml/min/mg, respectively, in PHLM, indicating that S007-1558 was the most metabolically unstable of the three. The LM of other species showed similar results. A common surrogate species to humans for S002-333 and enantiomers was predicted as rabbit where the extrapolated hepatic clearance (CLH ) did not show a significant difference to the in vivo CLH values. However, none of the species closely mimic humans with respect to the proportion of major metabolites (M-1-M-4) formed in vitro. Likewise, the CLH values were also predicted in humans for S002-333 and enantiomers using various mathematical models. During analysis, there was no chiral inversion evident among the individual isomers throughout in vitro and in vivo experiments. In conclusion, the in vitro results indicate a prominent role of phase I metabolism in the degradation of S002-333 and enantiomers and predict rabbit as an alternative species to conduct further safety and efficacy studies. Copyright © 2016 John Wiley & Sons, Ltd.

Pharmacokinetics, dose proportionality and permeability of S002-333 and its enantiomers, a potent antithrombotic agent, in rabbits.

1. S002-333 [(2-(4'-methoxy-benzenesulfonyl)-2,3,4,9-tetrahydro-1H-pyrido (3,4-b) indole-3-carboxylic acid amide)] is a novel and potent antithrombotic active agent. The present work investigates the pharmacokinetics, bioavailability, dose proportionality and permeability of the racemate, S002-333 in male New Zealand White (NZW) rabbits. 2. Rabbits were administered single intravenous (i.v.) (2 mg/kg) and three oral doses of 10, 20 and 40 mg/kg of S002-333, respectively, at different occasions to evaluate dose proportionality. Serial blood samples were collected and analyzed by a liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Since S002-333 is a racemate consisting of S004-1032 (R) and S007-1558 (S), same samples were analyzed using a chiralcel column so as to evaluate the respective enantiomers. 3. The peak plasma concentration, after oral administration, occurred at ∼10 h post-dose. The clearance (CL) and volume of distribution (Vd) after i.v. dose were found to be 3.05 ± 0.09 l/h/kg and 6.73 ± 1.16 l/kg, respectively. The absolute oral bioavailability of S002-333 was 16.32%, whereas it was 6.62 and 5.90% for R- and S-enantiomers, respectively. The absolute bioavailability of 10, 20 and 40 mg/kg doses were found to be 27.91, 14.39 and 16.91%, respectively. The PAMPA (parallel artificial membrane permeability assay) assay shows that S002-333 has a low-passive permeability at gastric and intestinal environment. 4. In conclusion, S002-333 has low-passive permeability, low CL and large Vd. The R-enantiomer has a "slightly" greater bioavailability than the S-enantiomer.

In vitro metabolism of a novel antithrombotic compound, S002-333, and its enantiomers: quantitative cytochrome P450 phenotyping, metabolic profiling and enzyme kinetic studies.

1. S002-333, (2-(4'-methoxy-benzenesulfonyl)-2,3,4,9-tetrahydro-1H-pyrido (3,4-b) indole-3-carboxylic acid amide) is a novel potent antithrombotic molecule currently under development phase. It is the racemic mixture of two enantiomers, namely S004-1032 (R-form) and S007-1558 (S-form). 2. The contribution of five major isoenzymes, namely CYP2B6, 2C9, 2C19, 2D6 and 3A4 was quantified using recombinant P450s in the phase-I metabolism through relative activity factor approach. CYP2C19 was found to be the major contributor for S002-333 and S007-1558, while CYP3A4 showed greater involvement in S004-1032 metabolism. Chemical inhibition and immunoinhibition studies reconfirmed the results in human liver microsomes (HLM). 3. Four major phase-I metabolites of S002-333; M-1 and M-3 (oxidative), M-2 (O-demethylated) and M-4 (dehydrogenated) were characterized in HLM. These metabolites constituted 11.2, 11.3 and 21.5% of the parent in comparison with the net phase-I metabolism of 29.9, 31.4 and 38.3% of S002-333, S004-1032 and S007-1558, respectively. 4. Among CYP2C9, 2C19 and 3A4, the relative contribution of CYP2C9 was found to be maximum during M-1 through M-4 formation. Enzyme kinetic analysis for detected metabolites indicated that M-1 to M-3 followed classical hyperbolic kinetics, whereas M-4 showed evidence of autoactivation. In conclusion, the results suggest prominent role of CYP2C9, 2C19 and 3A4 isoforms for enantioselective disposition of S002-333 in vitro.

A standardized phytopreparation from an Indian medicinal plant (Dalbergia sissoo) has antiresorptive and bone-forming effects on a postmenopausal osteoporosis model of rat.

OBJECTIVE: The aim of this study was to evaluate the skeletal effects of an extract made from the leaves and pods of Dalbergia sissoo (butanol-soluble standardized fraction [BSSF]) on ovariectomized rats, a model for postmenopausal osteopenia. METHODS: Adult Sprague-Dawley rats were ovariectomized and administered BSSF (50 and 100 mg/kg per day) or 17ß-estradiol orally for 12 weeks. The sham-operated group and the ovariectomy + vehicle group served as controls. Bone microarchitecture, bone turnover markers (serum osteocalcin and C-telopeptide fragment of collagen type I), biomechanical strength, new bone formation (based on mineral apposition rate and bone formation rate), and skeletal expressions of osteogenic and resorptive gene markers were studied. Uterine histomorphometry was used to assess estrogenicity. Bioactive marker compounds in BSSF were analyzed by high-performance liquid chromatography. One-way analysis of variance was used to test the significance of effects. RESULTS: In comparison with ovariectomized rats treated with vehicle, BSSF treatment in ovariectomized rats resulted in an improved trabecular microarchitecture of the long bones, increased biomechanical strength parameters of the vertebra and femur, decreased bone turnover markers (osteocalcin and type I collagen) and expression of skeletal osteoclastogenic genes, and increased new bone formation and expression of osteogenic genes in the femur. Overall, the osteoprotective effects of BSSF were comparable to those of 17ß-estradiol. BSSF did not exhibit uterine estrogenicity. Analysis of marker compounds revealed the presence of osteogenic methoxyisoflavones, including caviunin 7-O-[ß-D-apiofuranosyl-(1→6)-ß-D-glucopyranoside] (a novel compound), biochanin A, and pratensin. CONCLUSIONS: Oral doses of BSSF in the preclinical setting are effective in preventing estrogen deficiency-induced bone loss by dual action: inhibition of bone resorption and stimulation of new bone formation.

Medicarpin, a legume phytoalexin, stimulates osteoblast differentiation and promotes peak bone mass achievement in rats: evidence for estrogen receptor ß-mediated osteogenic action of medicarpin.

Dietary isoflavones including genistein and daidzein have been shown to have favorable bone conserving effects during estrogen deficiency in experimental animals and humans. We have evaluated osteogenic effect of medicarpin (Med); a phytoalexin that is structurally related to isoflavones and is found in dietary legumes. Med stimulated osteoblast differentiation and mineralization at as low as 10⁻¹° M. Studies with signal transduction inhibitors demonstrated involvement of a p38 mitogen activated protein kinase-ER-bone morphogenic protein-2 pathway in mediating Med action in osteoblasts. Co-activator interaction studies demonstrated that Med acted as an estrogen receptor (ER) agonist; however, in contrast to 17ß-estradiol, Med had no uterine estrogenicity and blocked proliferation of MCF-7 cells. Med increased protein levels of ERß in osteoblasts. Selective knockdown of ERα and ERß in osteoblasts established that osteogenic action of Med is ERß-dependent. Female Sprague-Dawley (weaning) rats were administered Med at 1.0- and 10.0 mg.kg⁻¹ doses by gavage for 30 days along with vehicle control. Med treatment resulted in increased formation of osteoporgenitor cells in the bone marrow and osteoid formation (mineralization surface, mineral apposition/bone formation rates) compared with vehicle group. In addition, Med increased cortical thickness and bone biomechanical strength. In pharmacokinetic studies, Med exhibited oral bioavailability of 22.34% and did not produce equol. Together, our results demonstrate Med stimulates osteoblast differentiation likely via ERß, promotes achievement of peak bone mass, and is devoid of uterine estrogenicity. In addition, given its excellent oral bioavailability, Med can be potential osteogenic agent.

Intravenous pharmacokinetics, oral bioavailability, dose proportionality and in situ permeability of anti-malarial lumefantrine in rats.

BACKGROUND: Despite the wide spread use of lumefantrine, there is no study reporting the detailed preclinical pharmacokinetics of lumefantrine. For the development of newer anti-malarial combination(s) and selection of better partner drugs, it is long felt need to understand the detailed preclinical pharmacokinetics of lumefantrine in preclinical experimental animal species. The focus of present study is to report bioavailability, pharmacokinetics, dose linearity and permeability of lumefantrine in rats. METHODS: A single dose of 10, 20 or 40 mg/kg of lumefantrine was given orally to male rats (N = 5 per dose level) to evaluate dose proportionality. In another study, a single intravenous bolus dose of lumefantrine was given to rats (N = 4) at 0.5 mg/kg dose following administration through the lateral tail vein in order to obtain the absolute oral bioavailability and clearance parameters. Blood samples were drawn at predetermined intervals and the concentration of lumefantrine and its metabolite desbutyl-lumefantrine in plasma were determined by partially validated LC-MS/MS method. In-situ permeability study was carried in anaesthetized rats. The concentration of lumefantrine in permeability samples was determined using RP-HPLC. RESULTS: For nominal doses increasing in a 1:2:4 proportion, the C(max) and AUC(0-∞) values increased in the proportions of 1:0.6:1.5 and 1:0.8:1.8, respectively. For lumefantrine nominal doses increasing in a 1:2:4 proportion, the C(max) and the AUC(0-t) values for desbutyl-lumefantrine increased in the proportions of 1:1.45:2.57 and 1:1.08:1.87, respectively. After intravenous administration the clearance (Cl) and volume of distribution (Vd) of lumefantrine in rats were 0.03 (± 0.02) L/h/kg and 2.40 (± 0.67) L/kg, respectively. Absolute oral bioavailability of lumefantrine across the tested doses ranged between 4.97% and 11.98%. Lumefantrine showed high permeability (4.37 × 10(-5) cm/s) in permeability study. CONCLUSIONS: The pharmacokinetic parameters of lumefantrine and its metabolite desbutyl-lumefantrine were successfully determined in rats for the first time. Lumefantrine displayed similar pharmacokinetics in the rat as in humans, with multiphasic disposition, low clearance, and a large volume of distribution resulting in a long terminal elimination half-life. The absolute oral bioavailability of lumefantrine was found to be dose dependent. Lumefantrine displayed high permeability in the in-situ permeability study.

2,3-dideoxy hex-2-enopyranosid-4-uloses as promising new anti-tubercular agents: design, synthesis, biological evaluation and SAR studies.

The alarming resurgence of tuberculosis (TB) underlines the urgent need for development of new and potent anti-TB drugs. Towards this goal we herein report the design and synthesis of 2,3-dideoxy hex-2-enopyranosid-4-uloses as promising new anti-tubercular agents. These easily accessible, small molecules were found to exhibit in vitro activity against Mycobacterium tuberculosis H37Rv in a MIC range of 0.78 µg/mL to 25 µg/mL. A detailed SAR study on these hex-2-enopyranosid-4-uloses led to the identification of compound 5g (S007-724) which on the basis of low MIC (0.78 µg/mL-M. tuberculosis H37Rv; 1.56 µg/mL-MDR, SDR strains of M. tuberculosis; 0.78 µg/mL-inhibition of intracellular replication of M. tuberculosis) and SI value of 13.5 has been identified as a promising lead molecule.

Differential effects of formononetin and cladrin on osteoblast function, peak bone mass achievement and bioavailability in rats.

Dietary soy isoflavones including genistein and daidzein have been shown to have favorable effects during estrogen deficiency in experimental animals and humans. We have evaluated osteogenic effect of cladrin and formononetin, two structurally related methoxydaidzeins found in soy food and other natural sources. Cladrin, at as low as 10 nM, maximally stimulated both osteoblast proliferation and differentiation by activating MEK-Erk pathway. On the other hand, formononetin maximally stimulated osteoblast differentiation at 100 nM that involved p38 MAPK pathway but had no effect on osteoblast proliferation. Unlike daidzein, these two compounds neither activated estrogen receptor in osteoblast nor had any effect on osteoclast differentiation. Daily oral administration of each of these compounds at 10.0 mg kg(-1) day(-1) dose to recently weaned female Sprague-Dawley rats for 30 consecutive days, increased bone mineral density at various anatomic positions studied. By dynamic histomorphometry of bone, we observed that rats treated with cladrin exhibited increased mineral apposition and bone formation rates compared with control, while formononetin had no effect. Cladrin had much better plasma bioavailability compared with formononetin. None of these compounds exhibited estrogen agonistic effect in uteri. Our data suggest that cladrin is more potent among the two in promoting parameters of peak bone mass achievement, which could be attributed to its stimulatory effect on osteoblast proliferation and better bioavailability. To the best of our knowledge, this is the first attempt to elucidate structure-activity relationship between the methoxylated forms of daidzein and their osteogenic effects.

Effect of centchroman coadministration on the pharmacokinetics of metformin in rats.

OBJECTIVES: To study the effect of centchroman, a non-steroidal oral contraceptive, coadministration on the pharmacokinetics of metformin in rats. MATERIALS AND METHODS: The pharmacokinetic interaction of metformin was studied in normal Sprague-Dawley female rats with and without centchroman coadministration. Blood samples were analyzed using a validated high-performance liquid chromatography method to generate the pharmacokinetic profile of metformin. The C(max) and t(max) were directly read from the concentration-time data. Other pharmacokinetic parameters were estimated using non-compartmental analyses. RESULTS: Metformin was monitored up to 10 h, and it exhibited a double-peak phenomenon. The C(max 1), 2.62 ± 0.32 µg/ml, and C(max 2,) 2.96 ± 0.65 µg/ml, occurred after 0.75 and 3 h post-dose, respectively. The mean residence time (MRT), AUC(0-4 h) and volume of distribution (Vd/F) were 4.20 ± 0.30 h, 8.53 ± 1.89 µg.h/ml and 14.24 ± 5.42 L/kg, respectively. Following centchroman coadministration, metformin showed significantly (P < 0.05) higher C(max) (C(max 1,) 3.96 ± 0.55 µg/ml and C(max 2,) 5.21 ± 0.59 µg/ml), AUC(0-4 h) (12.28 ± 0.73 µg.h/ml) and Vd/F (18.29 ± 1.19 L/kg), but lower MRT (3.19 ± 0.36 h) than the values obtained after metformin dosing alone. However, AUC(0-t) (17.74 ± 5.58 µg.h/ml) and clearance (3.76 ± 0.80 L/h/kg) remained unchanged. CONCLUSIONS: The results indicate that centchroman coadministration increases the rate but not the extent of absorption of metformin in rats. However, it does not seem to alter the pharmacokinetics of metformin to clinically significant levels.

Total extract and standardized fraction from the stem bark of Butea monosperma have osteoprotective action: evidence for the nonestrogenic osteogenic effect of the standardized fraction.

OBJECTIVE: The aim of this study was to determine the skeletal effects of Butea total extract (BTE) and its acetone soluble fraction (ASF) from Butea monosperma, which is rich in methoxyisoflavones, in ovariectomized (OVx) rats, a model for postmenopausal bone loss. METHODS: BTE (1.0 g kg d) and ASF (100 mg kg d) were given orally for 12 weeks to adult OVx rats. The sham-operated and ovariectomy + vehicle groups served as controls. Bone mineral density, osteoid formation (mineral apposition rate and bone formation rate), bone microarchitecture, and bone turnover/resorption markers were studied. Phytoestrogens in rats given BTE and ASF were analyzed by high-performance liquid chromatography. One-way analysis of variance was used to test significance of effects. RESULTS: OVx rats treated with either BTE or ASF exhibited increased bone mineral density in trabecular bones and improved trabecular microarchitecture compared with the ovariectomy + vehicle group. ASF treatment was more efficient than BTE treatment in maintaining trabecular microarchitecture. Serum osteocalcin and urinary type 1 collagen levels in OVx rats treated with either BTE or ASF were significantly lower than those of the ovariectomy + vehicle group. ASF treatment led to increased mineral apposition rate and bone formation rate compared with ovariectomy + vehicle, whereas BTE had no such effect. In the uterotropic assay, BTE was mildly estrogenic in adult OVx rats. In immature rats, BTE exhibited both estrogenicity and antiestrogenicity. ASF had neither uterine estrogenicity nor antiestrogenicity. Analysis of phytoestrogens revealed significant enrichment of cladrin, isoformononetin, and medicarpin in ASF over BTE. CONCLUSIONS: Derived from B monosperma, ASF at a 10-fold lower dose than that of BTE was effective in preventing OVx-induced bone loss and stimulated new-bone formation.

A novel flavonoid, 6-C-beta-d-glucopyranosyl-(2S,3S)-(+)-3',4',5,7-tetrahydroxyflavanone, isolated from Ulmus wallichiana Planchon mitigates ovariectomy-induced osteoporosis in rats.

OBJECTIVE: The aim of this study was to determine the skeletal effect of 6-C-beta-d-glucopyranosyl-(2S,3S)-(+)-3',4',5,7-tetrahydroxyflavanone (GTDF)/Ulmoside A, a new compound isolated from the extract of Ulmus wallichiana in a rat model of postmenopausal bone loss. METHODS: GTDF (1.0 and 5.0 mg kg d) was given orally to ovariectomized (OVx) rats (180-200 g) for 12 weeks. Sham operated + vehicle, ovariectomy + 17beta-estradiol (2.5 microg kg d), and ovariectomy + vehicle groups served as various controls. Bone mineral density (BMD), trabecular microarchitecture, bone biomechanical strength, levels of bone turnover/resorption markers, uterotropic effect, and plasma pharmacokinetics were studied. One-way analysis of variance was used to test significance of effects. RESULTS: OVx rats treated with both doses of GTDF exhibited significantly higher BMD in the trabecular (distal femur, proximal tibia, and vertebrae) and cortical (femur shaft) regions compared with the ovariectomy + vehicle group. Micro-CT demonstrated that OVx rats treated with 5.0 mg kg day of GTDF had better bone microarchitectural parameters compared with the ovariectomy + vehicle group. Serum osteocalcin and urinary C-terminal teleopeptide of Type I collagen levels in OVx rats treated with GTDF (at both doses) were significantly lower than those in the ovariectomy + vehicle group. At neither of the two doses did GTDF exhibit uterine estrogenicity. A pharmacokinetic study revealed that GTDF achieved maximum plasma concentration (40.67 ng mL) at approximately 1 hour, indicating its slow absorption. Its absolute bioavailability was found to be 1.04% with a plasma elimination half-life of approximately 5 hours. CONCLUSIONS: GTDF, a novel compound isolated from U wallichiana extract, improves bone biomechanical quality through positive modifications of BMD and trabecular microarchitecture without a hyperplastic effect on the uterus.

Extract and fraction from Ulmus wallichiana Planchon promote peak bone achievement and have a nonestrogenic osteoprotective effect.

OBJECTIVE: This study aimed to determine the skeletal effects of total ethanolic extract (TEE) and its butanolic fraction (BF) from the stem-bark of Ulmus wallichiana, which is rich in C-glycosylated flavonoids, in growing rats (for peak bone [PB] achievement) and in ovariectomized (OVx) rats (for menopausal bone loss). METHODS: TEE (750 mg kg(-1) d(-1)) and BF (50 mg kg(-1) d(-1)) were given orally for 10 weeks to weaning female Sprague-Dawley rats and for 12 weeks to adult OVx rats of the same strain, respectively. In studies with OVx rats, sham operated + vehicle, OVx + 17beta-estradiol, and OVx + vehicle groups served as various controls. Bone mineral density (BMD), biomechanical strength, bone histology, formations of osteoprogenitor cells, osteoid formation, and bone turnover/resorption markers were studied. Bioactive marker compounds in TEE and BF were analyzed by high-performance liquid chromatography. One-way analysis of variance was used to test significance of effects. RESULTS: In growing rats, both TEE and BF increased BMD, bone strength, and bone formation rate, suggesting higher PB achievement. OVx rats treated with either TEE or BF exhibited increased BMD at various anatomical positions and improved bone strength and trabecular architecture compared with the OVx + vehicle group. Serum osteocalcin and urinary type 1 collagen degradation product levels in OVx rats treated with either TEE or BF were significantly lower than those of the OVx + vehicle group. Neither TEE nor BF exhibited uterine estrogenicity. Analysis of marker compounds revealed significant enrichment of two bioactive markers in BF over TEE. CONCLUSIONS: Derived from U wallichiana, BF at much a lower dose than TEE was effective in PB achievement and prevention of OVx-induced bone loss.