The syntheses of [(14) C]BMS-823778 for use in a human ADME clinical study and of [(13) CD3 (13) CD2 ]BMT-094817, a stable-isotope labeled standard of a newly detected human metabolite.

Type 2 diabetes is a significant worldwide health problem. To support the development of BMS-823778 as an inhibitor of 11ß-hydroxysteroid dehydrogenase type 1 for type 2 diabetes, the synthesis of carbon-14-labeled material was required for use in a human adsorption, distribution, metabolism, and excretion (ADME) study. The HCl salt form of [(14) C]BMS-823778 was synthesized in two steps from commercially available [2-(14) C]acetone. The radiochemical purity of the synthesized [(14) C]BMS-823778 after dilution with unlabeled clinical-grade BMS-823778 was 99.5% having a specific activity of 7.379 µCi/mg. One result of the human ADME study was the detection of a new human metabolite, BMT-094817. To support the quantification of BMT-094817 in clinical samples, it was necessary to synthesize [(13) CD3 (13) CD2 ]BMT-094817 for use as a liquid chromatography/mass spectrometry standard. [(13) CD3 (13) CD2 ]BMT-094817 was prepared in five labeled steps from [(13) CD3 ]iodomethane.

A validated LC-MS/MS assay for the simultaneous determination of the anti-leukemic agent dasatinib and two pharmacologically active metabolites in human plasma: application to a clinical pharmacokinetic study.

Dasatinib (Sprycel) is a potent antitumor agent prescribed for patients with chronic myeloid leukemia (CML). To enable reliable quantification of dasatinib and its pharmacologically active metabolites in human plasma during clinical testing, a sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated. Samples were prepared using solid phase extraction on Oasis HLB 96-well plates. Chromatographic separation was achieved isocratically on a Luna phenyl-hexyl analytical column. Analytes and the stable labeled internal standards were detected by positive ion electrospray tandem mass spectrometry. The assay was validated over a concentration range of 1.00-1000 ng/mL for dasatinib and its two active metabolites. Intra- and inter-assay precision values for replicate QC control samples were within 5.3% for all analytes during the assay validation. Mean QC control accuracy values were within ± 9.0% of nominal values for all analytes. Assay recoveries were high (>79%) and internal standard normalized matrix effects were minimal. The three analytes were stable in human plasma for at least 22 h at room temperature, for at least 123 days at -20°C, and following at least six freeze-thaw cycles. The validated method was successfully applied to the quantification of dasatinib and two active metabolites in a human pharmacokinetic study.

Spinal cord injury triggers an intrinsic growth-promoting state in nociceptors.

Although most investigations of the mechanisms underlying chronic pain after spinal cord injury (SCI) have examined the central nervous system (CNS), recent studies have shown that nociceptive primary afferent neurons display persistent hyperexcitability and spontaneous activity in their peripheral branches and somata in dorsal root ganglia (DRG) after SCI. This suggests that SCI-induced alterations of primary nociceptors contribute to central sensitization and chronic pain after SCI. Does SCI also promote growth of these neurons' fibers, as has been suggested in some reports? The present study tests the hypothesis that SCI induces an intrinsic growth-promoting state in DRG neurons. This was tested by dissociating DRG neurons 3 days or 1 month after spinal contusion injury at thoracic level T10 and measuring neuritic growth 1 day later. Neurons cultured 3 days after SCI exhibited longer neurites without increases in branching ("elongating growth"), compared to neurons from sham-treated or untreated (naïve) rats. Robust promotion of elongating growth was found in small and medium-sized neurons (but not large neurons) from lumbar (L3-L5) and thoracic ganglia immediately above (T9) and below (T10-T11) the contusion site, but not from cervical DRG. Elongating growth was also found in neurons immunoreactive to calcitonin gene-related peptide (CGRP), suggesting that some of the neurons exhibiting enhanced neuritic growth were nociceptors. The same measurements made on neurons dissociated 1 month after SCI revealed no evidence of elongating growth, although evidence for accelerated initiation of neurite outgrowth was found. Under certain conditions this transient growth-promoting state in nociceptors might be important for the development of chronic pain and hyperreflexia after SCI.

Metabolism and disposition of [14C]brivanib alaninate after oral administration to rats, monkeys, and humans.

Brivanib [(R)-1-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[1,2,4]triazin-6-yloxy)propan-2-ol, BMS-540215] is a potent and selective dual inhibitor of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) signaling pathways. Its alanine prodrug, brivanib alaninate [(1R,2S)-2-aminopropionic acid 2-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy]-1-methylethyl ester, BMS-582664], is currently under development as an oral agent for the treatment of cancer. This study describes the in vivo biotransformation of brivanib after a single oral dose of [(14)C]brivanib alaninate to intact rats, bile duct-cannulated (BDC) rats, intact monkeys, BDC monkeys, and humans. Fecal excretion was the primary route of elimination of drug-derived radioactivity in animals and humans. In BDC rats and monkeys, the majority of radioactivity was excreted in bile. Brivanib alaninate was rapidly and completely converted via hydrolysis to brivanib in vivo. The area under the curve from zero to infinity of brivanib accounted for 14.2 to 54.3% of circulating radioactivity in plasma in animals and humans, suggesting that metabolites contributed significantly to the total drug-related radioactivity. In plasma from animals and humans, brivanib was a prominent circulating component. All the metabolites that humans were exposed to were also present in toxicological species. On the basis of metabolite exposure and activity against VEGF and FGF receptors of the prominent human circulating metabolites, only brivanib is expected to contribute to the pharmacological effects in humans. Unchanged brivanib was not detected in urine or bile samples, suggesting that metabolic clearance was the primary route of elimination. The primary metabolic pathways were oxidative and conjugative metabolism of brivanib.

CYP3A4-mediated ester cleavage as the major metabolic pathway of the oral taxane 3'-tert-butyl-3'-N-tert-butyloxycarbonyl-4-deacetyl-3'-dephenyl-3'-N-debenzoyl-4-O-methoxycarbonyl-paclitaxel (BMS-275183).

3'-tert-Butyl-3'-N-tert-butyloxycarbonyl-4-deacetyl-3'-dephenyl-3'-N-debenzoyl-4-O-methoxycarbonyl-paclitaxel (BMS-275183) is an orally available taxane analog that has the potential to be used as an oral agent to treat cancers. The compound is similar to the two clinically intravenously administered taxanes, paclitaxel and docetaxel, in that it contains a baccatin ring linked to a side chain through an ester bond. Unlike the other taxanes, the hydrolysis of this ester bond leads to formation of a free baccatin core (M13) that was the major metabolism pathway in incubations of [(14)C]BMS-275183 in human liver microsomes (HLMs) in the presence of NADPH, but it was not formed in incubations with human liver cytosol or HLM in the absence of NADPH. The other prominent metabolites formed in HLM incubations resulted from oxidation of t-butyl groups on the side chain (M20, M20B, M21, M22, and M23). All these metabolites were formed by cDNA-expressed CYP3A and not by other cytochrome P450 (P450) enzymes tested. Formation of these metabolites was selectively inhibited by ketoconazole and troleandomycin. The formation of M13 followed Michaelis-Menten kinetics with the K(m) values of 1.3 to 2.4 muM in HLM or CYP3A4; the V(max) value for the formation of M13 and M23 in the cDNA-expressed CYP3A4 matched well (within 2-fold difference) with that determined in HLM when expressed in units of per picomole of P450. These results showed that BMS-275183 is metabolized by CYP3A4 to yield baccatin through oxidation of side-chain t-butyl groups. An intramolecular cyclization of a side-chain hydroxylation metabolite is proposed to be responsible for the formation of M13, the side-chain hydrolysis metabolite.

Lacteal secretion, fetal and maternal tissue distribution of dasatinib in rats.

Dasatinib [N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl]-2-methyl-4-pyrimidinyl]amino]-5-thiazolecarboxamide; BMS-354825] is a potent and broad-spectrum kinase inhibitor used for the treatment of chronic myeloid leukemia and Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia. Dasatinib exhibited extensive lacteal secretion in Sprague-Dawley rats following a single p.o. dose of [14C]dasatinib (10 mg/kg, 300 microCi/kg). Radioactivity was detected through 72 h postdose, with a milk/plasma area under concentration-time curve from 0 to infinity (AUC(0-inf)) ratio of approximately 25. The majority of the total radioactivity in milk was attributed to unchanged dasatinib. After a single dose of [14C]dasatinib to pregnant Sprague-Dawley rats at gestation day 18, radioactivity was extensively distributed in maternal tissues. The radioactivity detected by tissue excision or quantitative whole-body autoradiography was highest in adrenal gland, mammary tissue, lungs, kidneys, liver, and placenta. Compared with maternal tissues, a relatively low level of radioactivity was detected in fetal tissues. The concentrations of dasatinib-equivalents in fetal liver and kidneys were <13% of the respective maternal organs. The C(max) of dasatinib-equivalents in fetal blood was approximately 39% of that in maternal blood; however, the AUC values were comparable. Fetal brain/blood ratios of C(max) and AUC(0-inf) were approximately 1.58 and 1.48, respectively, which were much greater than the maternal ratios of 0.12 and 0.13. In summary, dasatinib was extensively distributed in maternal tissues and secreted into milk, but its penetration into the adult brain was limited. Transporters may be involved in mediating dasatinib distribution in the adult rat, whereas in the fetus, tissue and blood exposures were similar, suggesting that distribution in the fetus is predominantly mediated by diffusion.

Metabolism and disposition of dasatinib after oral administration to humans.

SPRYCEL (dasatinib, BMS-354825; Bristol-Myers Squibb, Princeton, NJ), a multiple kinase inhibitor, is currently approved to treat chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia tumors in patients who are resistant or intolerant to imatinib mesylate (Gleevec; Novartis, Basel, Switzerland). After a 100-mg single p.o. dose of [(14)C]dasatinib to healthy volunteers, the radioactivity was rapidly absorbed (T(max) approximately 0.5 h). Both dasatinib and total radioactivity (TRA) plasma concentrations decreased rapidly with elimination half-life values of <4 h. Dasatinib was the major drug-related component in human plasma. At 2 h, dasatinib accounted for 25% of the TRA in plasma, suggesting that metabolites contributed significantly to the total drug-related component. There were many circulating metabolites detected that included hydroxylated metabolites (M20 and M24), an N-dealkylated metabolite (M4), an N-oxide (M5), an acid metabolite (M6), glucuronide conjugates (M8a,b), and products of further metabolism of these primary metabolites. Most of the administered radioactivity was eliminated in the feces (85%). Urine recovery accounted for <4% of the dose. Dasatinib accounted for <1 and 19% of the dose in urine and feces, respectively, suggesting that dasatinib was well absorbed after p.o. administration and extensively metabolized before being eliminated from the body. The exposures of pharmacologically active metabolites M4, M5, M6, M20, and M24 in patients, along with their cell-based IC(50) for Src and Bcr-Abl kinase inhibition, suggested that these metabolites were not expected to contribute significantly toward in vivo activity.

Biotransformation of [14C]dasatinib: in vitro studies in rat, monkey, and human and disposition after administration to rats and monkeys.

This study describes the in vitro metabolism of [(14)C]dasatinib in liver tissue incubations from rat, monkey, and human and the in vivo metabolism in rat and monkey. Across species, dasatinib underwent in vitro oxidative metabolism to form five primary oxidative metabolites. In addition to the primary metabolites, secondary metabolites formed from combinations of the oxidative pathways and conjugated metabolites of dasatinib and its oxidative metabolites were also observed in hepatocytes incubations. In in vivo studies in rats and monkeys, the majority of the radioactive dose was excreted in the bile and feces. In bile duct-cannulated monkeys after an i.v. dose, 13.7% of the radioactive dose was excreted in the feces through direct secretion. Dasatinib comprised 56 and 26% of the area under the curve (AUC) (0-8 h) of total radioactivity (TRA) in plasma, whereas multiple metabolites accounted for the remaining 44 and 74% of the AUC (0-8 h) of TRA for rats and monkeys, respectively. In rat and monkey bile, dasatinib accounted for < 12% of the excreted dose, suggesting that dasatinib was extensively metabolized before elimination. The metabolic profiles in bile were similar to the hepatocyte profiles. In both species, a large portion of the radioactivity excreted in bile (> or = 29% of the dose) was attributed to N-oxides and conjugated metabolites. In rat and monkey feces, only the oxidative metabolites and their further oxidation products were identified. The absence of conjugative or N-oxide metabolites in the feces suggests hydrolysis or reduction, respectively, in the gastrointestinal tract before elimination.