In vitro characterization and pharmacokinetics of dapagliflozin (BMS-512148), a potent sodium-glucose cotransporter type II inhibitor, in animals and humans.

(2S,3R,4R,5S,6R)-2-(3-(4-Ethoxybenzyl)-4-chlorophenyl)-6-hydroxymethyl-tetrahydro-2H-pyran-3,4,5-triol (dapagliflozin; BMS-512148) is a potent sodium-glucose cotransporter type II inhibitor in animals and humans and is currently under development for the treatment of type 2 diabetes. The preclinical characterization of dapagliflozin, to allow compound selection and prediction of pharmacological and dispositional behavior in the clinic, involved Caco-2 cell permeability studies, cytochrome P450 (P450) inhibition and induction studies, P450 reaction phenotyping, metabolite identification in hepatocytes, and pharmacokinetics in rats, dogs, and monkeys. Dapagliflozin was found to have good permeability across Caco-2 cell membranes. It was found to be a substrate for P-glycoprotein (P-gp) but not a significant P-gp inhibitor. Dapagliflozin was not found to be an inhibitor or an inducer of human P450 enzymes. The in vitro metabolic profiles of dapagliflozin after incubation with hepatocytes from mice, rats, dogs, monkeys, and humans were qualitatively similar. Rat hepatocyte incubations showed the highest turnover, and dapagliflozin was most stable in human hepatocytes. Prominent in vitro metabolic pathways observed were glucuronidation, hydroxylation, and O-deethylation. Pharmacokinetic parameters for dapagliflozin in preclinical species revealed a compound with adequate oral exposure, clearance, and elimination half-life, consistent with the potential for single daily dosing in humans. The pharmacokinetics in humans after a single dose of 50 mg of [(14)C]dapagliflozin showed good exposure, low clearance, adequate half-life, and no metabolites with significant pharmacological activity or toxicological concern.

Promotion of tubulin assembly by aluminum ion in vitro.

It has been proposed that aluminum ion is a contributing factor in a variety of neurological diseases. In many of these diseases, aberrations in the cytoskeleton have been noted. The effects of aluminum ion on the in vitro assembly of tubulin into microtubules has been examined by determining the association constants for the metal ion-guanosine triphosphate-tubulin ternary complex required for polymerization. The association constant for aluminum ion was approximately 10(7) times that of magnesium ion, the physiological mediator of microtubule assembly. In addition, aluminum ion at 4.0 X 10(-10) mole per liter competed effectively with magnesium ion for support of tubulin polymerization when magnesium ion falls below 1.0 millimole per liter. The microtubules produced by aluminum ion were indistinguishable from those produced by magnesium ion when viewed by electron microscopy, and they showed identical critical tubulin concentrations for assembly and sensitivities to cold-induced depolymerization. However, the rate of guanosine triphosphate hydrolysis and the sensitivity to calcium ion-induced depolymerization, critical regulatory processes of microtubules in vivo, were markedly lower for aluminum ion microtubules than for magnesium ion microtubules.

Integration of Physiologically-Based Pharmacokinetic Modeling into Early Clinical Development: An Investigation of the Pharmacokinetic Nonlinearity.

BMS-911543, a promising anticancer agent, exhibited time-dependent and dose-dependent nonlinear pharmacokinetics (PKs) in its first-in-human (FIH) study. Initial physiologically based pharmacokinetic (PBPK) modeling efforts using CYP1A2-mediated clearance kinetics were unsuccessful; however, further model analysis revealed that CYP1A2 time-dependent inhibition (TDI) and perhaps other factors could be keys to the nonlinearity. Subsequent experiments in human liver microsomes showed that the compound was a time-dependent inhibitor of CYP1A2 and were used to determine the enzyme inactivation parameter values. In addition, a rat tissue distribution study was conducted and human plasma samples were profiled to support the refinement of the PBPK model. It was concluded that the interplay between four BMS-911543 properties, namely, low solubility, saturation of the metabolizing enzyme CYP1A2, CYP1A2 TDI, and CYP1A2 induction likely resulted in the time-dependent and dose-dependent nonlinear PKs. The methodology of PBPK model-guided unmasking of compound properties can serve as a general practice for mechanistic understanding of a new compound's disposition.

The use of radiolabeled compounds for ADME studies in discovery and exploratory development.

Radiolabeled compounds are excellent investigative tools and widely used to carry out ADME studies during drug discovery and development stages. The most commonly used radioisotopes are 14C and 3H. 3H materials are generally easier to synthesize than 14C materials. Therefore, a variety of probes and substrates used in in vitro assays are labeled with 3H. Since synthesis of 14C material requires intensive resources, it is usually not available until after a molecule is considered for potential development or after the molecule enters the development phase. Improvement in the technology in radiochemistry has enabled the use of radiolabeled compounds earlier in pre-clinical and clinical development to address mechanistic issues. For in vitro studies, radiolabeled probes are utilized to test affinity with various transporters, to perform metabolism comparison among species and to assess possible formation of reactive metabolites. For in vivo studies, radiolabeled compounds are employed to identify and elucidate metabolites formed, to investigate the extent of absorption, bioavailability, tissue distribution, mass balance, routes of excretion, and pre-systemic metabolism. Due to the significant impact of radiolabeled studies on drug development, these studies will be performed earlier than have been in the past and will continue to be an integral part of drug discovery and development.

Cardioselective antiischemic ATP-sensitive potassium channel (KATP) openers. 5. Identification of 4-(N-aryl)-substituted benzopyran derivatives with high selectivity.

This paper describes our studies aimed at the discovery of structurally distinct analogs of the cardioprotective KATP opener BMS-180448 (2) with improved selectivity for the ischemic myocardium. The starting compound 6, derived from the indole analog 4. showed good cardioprotective potency and excellent selectivity compared to 2 and the first-generation KATP opener cromakalim (1). The structure-activity studies indicate that increasing the size of the alkyl ester leads to diminished potency as does its replacement with a variety of other groups (nitrile, methyl sulfone). Replacement of the ethyl ester of 6 with an imidazole gave the best compound 3 (BMS-191095) of this series which maintains the potency and selectivity of its predecessor 6. The results described in this publication further support that there is no correlation between vasorelaxant and cardioprotective potencies of KATP openers. Compound 3 is over 20- and 4000-fold more selective for the ischemic myocardium than 2 and cromakalim (1), respectively. The selectivity for the ischemic myocardium is achieved by reduction of vasorelaxant potency rather than enhancement in antiischemic potency. As for cromakalim (1) and 2, the cardioprotective effects of compound 3 are inhibited by cotreatment with the KATP blocker glyburide, indicating that the KATP opening is involved in its mechanism of cardioprotection. With its good oral bioavailability (47%) and plasma elimination half-life (3 h) in rats, compound 3 offers an excellent candidate to investigate the role of residual vasorelaxant potency of 2 toward its cardioprotective activity in vivo.

Thio- and oxoflavopiridols, cyclin-dependent kinase 1-selective inhibitors: synthesis and biological effects.

Flavopiridol analogues, thio- and oxoflavopiridols which contain a sulfur (16) or oxygen (18) atom linker between a chromone ring and the hydrophobic side chain, are selective cyclin-dependent kinase 1 (CDK1) inhibitors with an IC(50) of 110 and 130 nM. These analogues were prepared from key intermediate 7 by substituting the ethyl sulfoxide. Enantio pure intermediate piperidone 10 was obtained from the racemic piperidone 8 via a very efficient "dynamic kinetic resolution" in 76% yield. Hydrophobic side chains such as chlorophenyl or tert-butyl produced potent CDK1 inhibitory activity, while hydrophilic side chains such as pyrimidine or aniline caused a severe reduction in CDK inhibitory activity. These analogues are competitive inhibitors with respect to ATP, and therefore activity was dependent upon the CDK subunit without being affected by the cyclin subunit or protein substrate. Thio- and oxoflavopiridols 16 and 18 are not only selective within the CDK family but also discriminated between unrelated serine/threonine and tyrosine protein kinases. CDK1 selective thio- and oxoflavopiridol analogues inhibit the colony-forming ability of multiple human tumor cell lines and possess a unique antiproliferative profile in comparison to flavopiridol.

Aminodiol HIV protease inhibitors. Synthesis and structure-activity relationships of P1/P1' compounds: correlation between lipophilicity and cytotoxicity.

A series of novel aminodiol inhibitors of HIV protease based on the lead compound 1 with structural modifications at P1' were synthesized in order to reduce the cytotoxicity of 1. We have observed a high degree of correlation between the lipophilicity and cytotoxicity of this series of inhibitors. It was found that appropriate substitution at the para position of the P1' phenyl group of 1 resulted in the identification of equipotent (both against the enzyme and in cell culture) compounds (10l, 10m, 10n, and 15c) which possess significantly decreased cytotoxicity.

Gastric paraganglioma: an immunohistological and ultrastructural case study.

This is a report of the immunohistochemical and ultrastructural features of a case of paraganglioma of the stomach in a 61-year-old woman who presented with melaena. The typical 'Zellballen' arrangement of epithelial cells with granular cytoplasm was present and the tumour was highly vascular. The polygonal epithelial cells stained strongly for neuron specific enolase and the intervening elongated sustentacular or support cells were positive for S100 protein and vimentin. Many dense core granules were present within the epithelial cells while the elongated support cells contained plentiful diffuse intermediate filaments. No hybrid cells or ganglion cells were present in the tumour. This is the sixth recorded case of gastric paraganglioma and the first to be studied in detail by immunostaining and electron microscopy.

Crohn's disease in northern Ireland - A retrospective survey of 159 cases.

A RETROSPECTIVE survey of Crohn's disease in a circumscribed population was carried out to determine the incidence of the disease in Northern Ireland and to compare the findings with previous regional studies in the British Isles. One hundred and fifty-nine cases were selected by strict criteria for detailed examination. Epidemiological data is presented which demonstrates that Crohn's disease has a similar distribution in this population to that seen in other regions. The crude annual incidence rate of 1.3 per 100,000 lies in the lower half of the range of published figures.Remarkable findings were the higher incidence of Crohn's disease with increasing age, particularly in females and the significantly higher incidence in the urban population.

Subungal melanoma.

Subungal melanoma is a rare condition accounting for 1-3% of all melanomas. It has been associated with a poor prognosis, with a 10-30% 5 year survival usually attributed to the delay in diagnosis. Early biopsy of suspicious lesions followed by amputation of the digit in those proving positive is the treatment of choice.

Impact of community violence on the workload of a district general hospital.

Clinical observation suggests that deliberate violence against the person is increasing in both incidence and severity in the community. Over a six-year period there was a trend towards an annual increase in the number of attendances at the Waveney Hospital as the result of assaults. A retrospective study for a six-month period (August 1987 to January 1988) defined the pattern of attendance, injuries and treatment for 284 cases. Most of the victims were young males who presented outside normal working hours with superficial injuries. Admission was required in 12% of patients.

Crohn's disease in Northern Ireland--a retrospective study of 440 cases.

The incidence of Crohn's disease in the community is thought to be changing, with conflicting evidence for increases, decreases or steady state situations being described. A retrospective study, using strict criteria for diagnosis, for a 16 year period in Northern Ireland demonstrated an increasing incidence of Crohn's disease, with a distribution in the population similar to that described in previous studies.

Splenic cysts: a review of 6 cases.

Benign cysts of the spleen are rare. Six cases have been managed in this hospital in the past 25 years. The history, physical findings and investigations are reviewed. It is suggested that splenectomy is the treatment of choice as it carries a low morbidity and mortality.