Search

The teratogenicity of the chemotherapeutic drug thalidomide is species-specific and affects humans, non-human primates, and rabbits. The primary oxidation of thalidomide in previously investigated rodents predominantly resulted in the formation of deactivated 5'-hydroxythalidomide. In the current study, similar in vivo biotransformations to 5-hydroxythalidomide and 5'-hydroxythalidomide were confirmed by the analysis of blood plasma from male rabbits, a thalidomide-sensitive species, after oral administration of thalidomide (2.0 mg/kg). Similar levels of thalidomide in seminal plasma and in blood plasma were detected using liquid chromatography-tandem mass spectrometry at 4 hr and 7 hr after oral doses in male rabbits. Seminal plasma concentrations of 5-hydroxythalidomide and 5'-hydroxythalidomide were also seen in male rabbits in a roughly similar time-dependent manner to those in the blood plasma after oral doses of thalidomide (2.0 mg/kg). Furthermore, the values generated by a simplified physiologically based pharmacokinetic rabbit model were in agreement with the measured in vivo blood plasma data under metabolic ratios of 0.01 for the hepatic intrinsic clearance of thalidomide to both unconjugated 5-hydroxythalidomide and 5'-hydroxythalidomide. These results suggest that metabolic activation of thalidomide may be dependent on rabbit liver enzymes just it was for cytochrome P450 enzymes in humanized-liver mice; in contrast, rodent livers predominantly mediate biotransformation of thalidomide to 5'-hydroxythalidomide. A developmental toxicity test system with experimental animals that involves intravaginal exposures to the chemotherapeutic drug thalidomide via semen should be considered in the future.

3-(6-Methylpyridine-3-yl-sulfanyl)-6-(4H-[1,2,4]triazole-3-yl-sulfanyl)-N-(1,3-thiazole-2-yl)-2-pyridine carboxamide (Cpd-D) is a novel glucokinase activator that is being developed for the treatment of type 2 diabetes. Large interindividual variations were observed in the pharmacokinetics of Cpd-D in male Sprague-Dawley (SD) rats, which were subsequently divided into two phenotypes; >6-fold longer terminal-phase half-life and ∼10-fold larger AUC0-∞ values were observed in slow metabolizers (SM) than in fast metabolizers (FM) after the oral administration of Cpd-D. The thiohydantoic acid analog (M2) was the predominant metabolite detected in the urine, bile, and plasma after the oral administration of [(14)C]Cpd-D to the FM phenotypes of bile-duct cannulated SD rats. The liver microsomes prepared from FM phenotyped rats extensively formed M2 with the highest affinity (Km = 0.09 μM) and largest Vmax/Km value in primary metabolism, whereas those from SM phenotypes had little capacity to form M2. Of the rat cytochrome P450 isoforms tested, the formation of M2 was only catalyzed by recombinant CYP2D1. Sequence substitutions (418A/421C and 418G/421T) were detected in the CYP2D1 gene and were designated F and S alleles, respectively. The genotype-phenotype correlation analysis indicated that two S alleles were homozygous (S/S) in the SM phenotypes, whereas the FM phenotypes were either homozygous for the F-alleles (F/F) or heterozygous (F/S). These results indicated that the CYP2D1 polymorphism caused by nucleotide substitutions (418A/421C versus 418G/421T) was responsible for interindividual variations leading to the polymorphism in the major metabolism and pharmacokinetics of Cpd-D in male SD rats.

The optimization of our lead GK activator 2a to 3-[(1S)-2-hydroxy-1-methylethoxy]-5-[4-(methylsulfonyl)phenoxy]-N-1,3-thiazol-2-ylbenzamide (6g), a potent GK activator with good oral availability, is described, including to uncouple the relationship between potency and hydrophobicity. Following oral administration, this compound exhibited robust glucose lowering in diabetic model rodents.

Identification and synthesis of novel 3-alkoxy-5-phenoxy-N-thiazolyl benzamides as glucokinase activators are described. Removal of an aniline structure of the prototype lead (2a) and incorporation of an alkoxy or phenoxy substituent led to the identification of 3-Isopropoxy-5-[4-(methylsulfonyl)phenoxy]-N-(4-methyl-1,3-thiazol-2-yl)benzamide (27e) as a novel, potent, and orally bioavailable GK activator. Rat oral glucose tolerance test indicated that 27e exhibited a glucose-lowering effect after 10 mg/kg oral administration.

The osteoinductive activity induced by recombinant human BMP-2 (rhBMP-2) blunts proportionately as the recipient ages. In order to compensate for this bluntness administration of fibroblast growth factor-2 (FGF-2) has been considered. The aim of this study was to determine whether FGF-2 administration augments osteoinductive activity caused by rhBMP-2 and to evaluate the effect of aging on bone formation induced by coadministration of rhBMP-2 and FGF-2. Sixty-four Wistar strain male rats of 8-week-old (prepubertal) and 16-week-old (postpubertal) received bone defects bilaterally in the parietal bone and the defects were filled by a polylactic acid polyglycolic acid copolymer/gelatin sponge (PGS) impregnated with rhBMP-2 plus 0 ng, 25 ng, and 250 ng FGF-2 (n=10 in each). At 2 weeks after grafting, the new bone volume seemed to be larger in the rhBMP-2+FGF-2 groups than in the rhBMP-2 alone group. At 4 weeks, the new bone formation was linked to the adjacent original bone. In the prepubertal rats, all newly formed bone was similarly calcified. In the postpubertal rats, only the rhBMP-2+25 ng FGF-2 group showed this higher degree of calcification. At 2 weeks, alkaline phosphatase (ALP) activity in the rhBMP-2+25 ng FGF-2 group was significantly (p

A novel series of (2R)-2-[(1R)-3, 3-difluorocyclopentyl]-2-hydroxy-2-phenylacetamides was designed and synthesized based on the structure and biological profiles of an active metabolite 2 of our prototype muscarinic M(3) receptor selective antagonist 1, to develop a potent, long-acting, orally active M(3) antagonist for the treatment of urinary tract disorders, irritable bowel syndrome, and respiratory disorders. Investigation of (2R)-2-[(1R)-3, 3-difluorocyclopentyl]-2-hydroxy-2-phenylacetamides containing a phenyl or heterocyclic ring as the piperidinyl side chain in place of the 4-methyl-3-pentenyl moiety of 15a revealed that this acid moiety was a versatile template for improving the selectivity for M(3) over M(2) receptors in comparison with the corresponding cyclopentylphenylacetic acid group. However, since the in vitro metabolic stability of these analogues was insufficient compared with that of 2, further derivatization was performed by introducing an appropriate hydrophilic group into the phenyl or 2-pyridyl ring. Thus, the 1-(6-aminopyridin-2-ylmethyl)piperidine analogue 15y exhibiting 190-fold selectivity for M(3) receptors (K(i) = 2.8 nM) over M(2) receptors (K(i) = 530 nM) in a human binding assay and good in vitro metabolic stability in dog and human hepatic microsomes was identified. This compound has excellent oral activity at 4 h after oral dosing (1 mg/kg), inhibiting methacholine-induced bronchoconstriction in dogs, and may be useful in clinical situations in which M(3) over M(2) selectivity is desirable.

In the course of developing a metabolically stable M3 receptor antagonist from the prototype antagonist, J-104129 (1), introduction of certain substituents into the cyclopentane ring of 1 was found to be effective not only in improving metabolic stability but also in greatly enhancing the subtype selectivity. Among the cyclopentane analogues, sulfonamide derivatives (10f) and (10g) displayed 160- and 310-fold selectivity for M3 over M2 receptors, and both were significantly more selective than the prototype antagonist (120-fold). Subsequent derivatization of the sulfonamide series led to the highly selective M3 receptor antagonists (10h, 10i and 10j) with >490-fold selectivity for M3 over M2 receptors. Among them, p-nitrophenylsulfonamide (J-107320, 10h) exhibited 1100-fold selectivity for M3 receptors (Ki = 2.5 nM) over M2 receptors (Ki = 2800 nM) in the human muscarinic receptor binding assay using [3H]-NMS as a radio ligand.

Dorzolamide hydrochloride (MK-507), (-)-(4S, 6S)-4-ethylamino-5, 6-dihydro-6-methyl-4H-thieno-[2, 3-b]-thiopyran-2-sulfonamide-7, 7-dioxide monohydrochloride, is a new topical carbonic anhydrase inhibitor. The binding of dorzolamide and its N-deethylated metabolite (L-706, 803) to rat erythrocytes was investigated, and the pharmacokinetics of dorzolamide in rats was studied after a single intravenous administration.1. Dorzolamide was incorporated into rat erythrocytes rapidly, while the uptake of L-706, 803 was slower.2. There were two types of binding site for dorzolamide and L-706, 803 in rat erythrocytes. For both dorzolamide and L-706, 803, the maximum binding capacity was about 60 μM for the high affinity isozyme and 30 μM for the low affinity isozyme.3. The Kd values of dorzolamide and L-706, 803 for low affinity binding were 0.387 and 0.497 μM, respectively. The Kd values of dorzolamide and L-706, 803 for high affinity binding were about 1/100 and 1/250 of those for low affinity binding, respectively.4. After administration of dorzolamide hydrochloride to rats at the dose of 0.5 mg/kg, dorzolamide and L-706, 803 were not detected in plasma. At the dose of 5 mg/kg, the plasma half-lives of dorzolamide and L-706, 803 were 1.0 and 2.6 hr, respectively.5. At the dose of 0.5 mg/kg, the half-lives of dorzolamide and L-706, 803 in blood were 157 and 116 hr, respectively, and decreased to respective values of 101 and 99 hr after administration of 5 mg/kg.6. After administration of dorzolamide hydrochloride at the dose of 0.5 mg/ kg, the blood AUC values of dorzolamide and L-706, 803 were 1093 and 115 μg hr/ml, respectively. The AUC value of dorzolamide at the dose of 5 mg/kg decreased by 50% and that of L-706, 803 increased about five-fold.7. The Vdss and CLtot of dorzolamide were 99.9 ml/kg and 0.46 ml/hr/kg after administration of 0.5 mg/kg and increased 15 and 25-fold after 5 mg/kg, respectively.

The absorption, distribution, metabolism and excretion of [14C]dorzolamide hydrochloride (MK-507) were studied in rats after single administration. In vitro plasma protein binding was also investigated.1. Following intravenous administration of [14C]dorzolamide hydrochloride to male rats at doses ranging from 0.1 to 5 mg/kg, blood AUC values increased proportionally with doses up to 0.5 mg/kg, but no increase in the values was observed for the dose range of 1 to 5 mg/kg. Most of the radioactivity in blood was present in erythrocytes. The elimination of radioactivity from the blood was very slow and the estimated t1/2 was 10.5-11.3 days.2. No sex differences were observed in blood concentration profiles regardless route of administration, oral or intravenous. After oral administration, almost all [14C]dorzolamide was absorbed.3. After intravenous administration at the dose of 0.5 mg/kg, most of the radioactivity was distributed in blood. At 30 min after dosing, the highest radioactivity was found in the kidney, followed by lung, spleen, bone marrow, stomach, liver, caecum and large intestine. The concentration-time profiles of most tissues were similar to that of blood.4. Within 31 days after intravenous administration at the dose of 0.5 mg/kg, the excretion of radioactivity accounted for 74.7% in urine and 11.8% in feces. Within 24 hr after oral administration at the dose of 0.5 and 5 mg/kg, 24.7 and 73.8% of administered radioactivity were excreted in urine, respectively.5. N-deethylated dorzolamide was found in the blood, urine and tissues. The ratio of the radioactive N-deethylated metabolite to the total radioactivity in blood or in tissues after administration at the dose of 5 mg/kg was larger than those after dosing at the dose of 0.5 mg/kg.6. The in vitro binding of dorzolamide and N-deethylated dorzolamide to plasma proteins accounted for less than 30% in all species studied, and these compounds were mainly bound to albumin.

Glucokinase activators (GKAs) are currently under investigation as potential antidiabetic agents by many pharmaceutical companies. Most of GKAs reported previously possess N-aminothiazol-2-yl amide moiety in their structures because the aminothiazole moiety interacts with glucokinase (GK) and shows strong GK activation. During the development of N-aminothiazol-2-yl amide derivatives, we identified a bioactivation and metabolic liability of 2-aminothizole substructure of GKA 3 by assessing covalent binding, metabolites in liver microsomes and glutathione (GSH) trap assay.

Optimization of the amine part of our original muscarinic M(3) receptor antagonist 1 was performed to identify M(3) receptor antagonists that are superior to 1. Compounds carrying a variety of diamine moieties without hydrophobic substituent on the nitrogen atom were screened against the binding affinity for the M(3) receptor and the selectivity for M(3) over the M(1) and M(2) receptors. This process led to a 4-aminopiperidinamide (2l) with a K(i) value of 5.1 nM and with a selectivity of the M(3) receptor that was 46-fold greater than that of the M(2) receptor. Further derivatization of 2l by inserting a spacer group or by incorporating alkyl group(s) into the amine part resulted in the identification of an 4-(aminoethyl)piperidinamide 2l-b with a K(i) value of 3.7 nM for the M(3) receptor and a selectivity for the M(3) receptor that was 170-fold greater than that of the M(2) receptor.

Background: Understanding fate choice and fate switching between the osteoblast lineage (ObL) and adipocyte lineage (AdL) is important to understand both the developmental inter-relationships between osteoblasts and adipocytes and the impact of changes in fate allocation between the two lineages in normal aging and certain diseases. The goal of this study was to determine when during lineage progression ObL cells are susceptible to an AdL fate switch by activation of endogenous peroxisome proliferator-activated receptor (PPAR)c. Methodology/Principal Findings: Multiple rat calvaria cells within the ObL developmental hierarchy were isolated by either fractionation on the basis of expression of alkaline phosphatase or retrospective identification of single cell-derived colonies, and treated with BRL-49653 (BRL), a synthetic ligand for PPARc. About 30% of the total single cell-derived colonies expressed adipogenic potential (defined cytochemically) when BRL was present. Profiling of ObL and AdL markers by qRTPCR on amplified cRNA from over 160 colonies revealed that BRL-dependent adipogenic potential correlated with endogenous PPARc mRNA levels. Unexpectedly, a significant subset of relatively mature ObL cells exhibited osteoadipogenic bipotentiality. Western blotting and immunocytochemistry confirmed that ObL cells co-expressed multiple mesenchymal lineage determinants (runt-related transcription factor 2 (Runx2), PPARc, Sox9 and MyoD which localized in the cytoplasm initially, and only Runx2 translocated to the nucleus during ObL progression. Notably, however, some cells exhibited both PPARc and Runx2 nuclear labeling with concomitant upregulation of expression of their target genes with BRL treatment. Conclusions/Significance: We conclude that not only immature but a subset of relatively mature ObL cells characterized by relatively high levels of endogenous PPARc expression can be switched to the AdL. The fact that some ObL cells maintain capacity for adipogenic fate selection even at relatively mature developmental stages implies an unexpected plasticity with important implications in normal and pathological bone development., This work was supported by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan (13771074 to YY) and the Canadian Institutes of Health Research (FRN 83704 to JEA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.