Lisa J. Christopher

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 [14C]dasatinib to healthy volunteers, the radioactivity was rapidly absorbed (Tmax ∼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 IC50 for Src and Bcr-Abl kinase inhibition, suggested that these metabolites were not expected to contribute significantly toward in vivo activity.

Pharmacokinetics of the Dipeptidyl Peptidase 4 Inhibitor Saxagliptin in Rats, Dogs, and Monkeys and Clinical Projections

Saxagliptin is a potent, selective, reversible dipeptidyl peptidase 4 (DPP4) inhibitor specifically designed for extended inhibition of the DPP4 enzyme and is currently under development for the treatment of type-2 diabetes. The pharmacokinetics of saxagliptin were evaluated in rats, dogs, and monkeys and used to predict its human pharmacokinetics. Saxagliptin was rapidly absorbed and had good bioavailability (50–75%) in the species tested. The plasma clearance of saxagliptin was higher in rats (115 ml/min/kg) than in dogs (9.3 ml/min/kg) and monkeys (14.5 ml/min/kg) and was predicted to be low to moderate in humans. The plasma elimination half-life was between 2.1 and 4.4 h in rats, dogs, and monkeys, and both metabolism and renal excretion contributed to the overall elimination. The primary metabolic clearance pathway involved the formation of a significant circulating, pharmacologically active hydroxylated metabolite, M2. The volume of distribution values observed in rats, dogs, and monkeys (1.3–5.2 l/kg) and predicted for humans (2.7 l/kg) were greater than those for total body water, indicating extravascular distribution. The in vitro serum protein binding was low (≤30%) in rats, dogs, monkeys, and humans. After intra-arterial administration of saxagliptin to Sprague-Dawley and Zucker diabetic fatty rats, higher levels of saxagliptin and M2 were observed in the intestine (a proposed major site of drug action) relative to that in plasma. Saxagliptin has prolonged pharmacodynamic properties relative to its plasma pharmacokinetic profile, presumably due to additional contributions from M2, distribution of saxagliptin and M2 to the intestinal tissue, and prolonged dissociation of both saxagliptin and M2 from DPP4.

Identification of the Human Enzymes Involved in the Oxidative Metabolism of Dasatinib: An Effective Approach for Determining Metabolite Formation Kinetics

N-(2-Chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl]-2-methyl-4-pyrimidinyl]amino]-5-thiazolecarboxamide (dasatinib, Sprycel, BMS-354825; Bristol-Myers Squibb, Princeton, NJ) is a potent protein kinase inhibitor to treat chronic myeloid leukemia. In vivo studies have shown that the primary oxidative metabolites of dasatinib are M4 (N-dealkylation), M5 (N-oxidation), M6 (carboxylic acid formation), M20, and M24 (hydroxylation). To identify the enzymes responsible for the formation of these metabolites, [14C]-dasatinib and nonradiolabeled dasatinib were incubated with human cDNA-expressed enzymes [cytochromes P450 (P450s) and flavin-containing monooxygenase (FMO) 3] or human liver microsome (HLM) in the presence of selective P450 inhibitors (antibodies and chemical inhibitors). The results of these experiments showed that metabolites M4, M20, and M24 were mainly generated by CYP3A4; M5 was primarily formed by FMO3; and M6 was formed by a cytosolic oxidoreductase. The enzyme kinetic analysis showed that the formation of M4 and M5 in HLM followed the Michaelis-Menten kinetics, and the formation data of M20 and M24 fitted well to a partial substrate inhibition kinetic model. The Km values were determined by the kinetic analysis of the substrate-dependent metabolite formation plots from a large number of incubations with the nonlabeled dasatinib; the Vmax values were calculated with the predetermined Km values and the metabolite formation rates from a limited number of incubations with [14C]dasatinib. The intrinsic formation clearance values (Vmax/Km) of 52, 14, 274, and 20 μl/mg protein/min for the formation of M4, M5, M20, and M24, respectively, suggested that the formation of M20 was more efficient than other metabolites. Collectively, multiple in vitro experiments showed that dasatinib was predominately metabolized by CYP3A4.

Simultaneous oral therapeutic and intravenous 14C‐microdoses to determine the absolute oral bioavailability of saxagliptin and dapagliflozin

AIM To determine the absolute oral bioavailability (F(p.o.) ) of saxagliptin and dapagliflozin using simultaneous intravenous ¹⁴C-microdose/therapeutic oral dosing (i.v.micro + oraltherap). METHODS The F(p.o.) values of saxagliptin and dapagliflozin were determined in healthy subjects (n = 7 and 8, respectively) following the concomitant administration of single i.v. micro doses with unlabelled oraltherap doses. Accelerator mass spectrometry and liquid chromatography-tandem mass spectrometry were used to quantify the labelled and unlabelled drug, respectively. RESULTS The geometric mean point estimates (90% confidence interval) F(p.o) . values for saxagliptin and dapagliflozin were 50% (48, 53%) and 78% (73, 83%), respectively. The i.v.micro had similar pharmacokinetics to oraltherap. CONCLUSIONS Simultaneous i.v.micro + oraltherap dosing is a valuable tool to assess human absolute bioavailability.

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 μCi/kg). Radioactivity was detected through 72 h postdose, with a milk/plasma area under concentration-time curve from 0 to infinity (AUC0-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 Cmax 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 Cmax and AUC0-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.

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 [14C]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 (≥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.

Characterization of Organic Anion Transporter 2 (SLC22A7): A Highly Efficient Transporter for Creatinine and Species-Dependent Renal Tubular Expression

The contribution of organic anion transporter OAT2 (SLC22A7) to the renal tubular secretion of creatinine and its exact localization in the kidney are reportedly controversial. In the present investigation, the transport of creatinine was assessed in human embryonic kidney (HEK) cells that stably expressed human OAT2 (OAT2-HEK) and isolated human renal proximal tubule cells (HRPTCs). The tubular localization of OAT2 in human, monkey, and rat kidney was characterized. The overexpression of OAT2 significantly enhanced the uptake of creatinine in OAT2-HEK cells. Under physiologic conditions (creatinine concentrations of 41.2 and 123.5 µM), the initial rate of OAT2-mediated creatinine transport was approximately 11-, 80-, and 80-fold higher than OCT2, multidrug and toxin extrusion protein (MATE)1, and MATE2K, respectively, resulting in approximately 37-, 1850-, and 80-fold increase of the intrinsic transport clearance when normalized to the transporter protein concentrations. Creatinine intracellular uptake and transcellular transport in HRPTCs were decreased in the presence of 50 µM bromosulfophthalein and 100 µM indomethacin, which inhibited OAT2 more potently than other known creatinine transporters, OCT2 and multidrug and toxin extrusion proteins MATE1 and MATE2K (IC50: 1.3 µM vs. > 100 µM and 2.1 µM vs. > 200 µM for bromosulfophthalein and indomethacin, respectively) Immunohistochemistry analysis showed that OAT2 protein was localized to both basolateral and apical membranes of human and cynomolgus monkey renal proximal tubules, but appeared only on the apical membrane of rat proximal tubules. Collectively, the findings revealed the important role of OAT2 in renal secretion and possible reabsorption of creatinine and suggested a molecular basis for potential species difference in the transporter handling of creatinine.

Characterization of the in vitro and in vivo metabolism and disposition and cytochrome P450 inhibition/induction profile of saxagliptin in human.

Saxagliptin is a potent dipeptidyl peptidase-4 inhibitor approved for the treatment of type 2 diabetes mellitus. The pharmacokinetics and disposition of [14C]saxagliptin were investigated in healthy male subjects after a single 50-mg (91.5 μCi) oral dose. Saxagliptin was rapidly absorbed (Tmax, 0.5 h). Unchanged saxagliptin and 5-hydroxy saxagliptin (M2), a major, active metabolite, were the prominent drug-related components in the plasma, together accounting for most of the circulating radioactivity. Approximately 97% of the administered radioactivity was recovered in the excreta within 7 days postdose, of which 74.9% was eliminated in the urine and 22.1% was excreted in the feces. The parent compound and M2 represented 24.0 and 44.1%, respectively, of the radioactivity recovered in the urine and feces combined. Taken together, the excretion data suggest that saxagliptin was well absorbed and was subsequently cleared by both urinary excretion and metabolism; the formation of M2 was the major metabolic pathway. Additional minor metabolic pathways included hydroxylation at other positions and glucuronide or sulfate conjugation. Cytochrome P450 (P450) enzymes CYP3A4 and CYP3A5 metabolized saxagliptin and formed M2. Kinetic experiments indicated that the catalytic efficiency (Vmax/Km) for CYP3A4 was approximately 4-fold higher than that for CYP3A5. Therefore, it is unlikely that variability in expression levels of CYP3A5 due to genetic polymorphism will impact clearance of saxagliptin. Saxagliptin and M2 each showed little potential to inhibit or induce important P450 enzymes, suggesting that saxagliptin is unlikely to affect the metabolic clearance of coadministered drugs that are substrates for these enzymes.

Liquid chromatography and tandem mass spectrometry method for the quantitative determination of saxagliptin and its major pharmacologically active 5-monohydroxy metabolite in human plasma: Method validation and overcoming specific and non-specific binding at low concentrations

A liquid chromatography and tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously determine the concentrations of saxagliptin (Onglyza™, BMS-477118) and its major active metabolite, 5-hydroxy saxagliptin to support pharmacokinetic analyses in clinical studies. The dynamic range of the assay was 0.1-50 ng/mL for saxagliptin and 0.2-100 ng/mL for 5-hydroxy saxagliptin. Protein precipitation (PPT) with acetonitrile was used to extract the analytes from plasma matrix before injecting on an Atlantis(®) dC18 column (50 mm × 2.1 mm, 5 μm) for LC-MS/MS analysis. The sample pre-treatment process was carefully controlled to disrupt DPP4-specific binding and non-specific binding observed at lower concentrations. The recoveries for both analytes were >90%. The assay was selective, rugged and reproducible; storage stability of at least 401 days at -20°C was demonstrated. Under these chromatographic conditions, the isomers of saxagliptin and 5-hydroxy saxagliptin were chromatographically separated from saxagliptin and 5-hydroxy saxagliptin. The assay has been used to support multiple clinical studies and regulatory approvals.

Preclinical Pharmacokinetics and In Vitro Metabolism of BMS-690514, a Potent Inhibitor of EGFR and VEGFR2

BMS-690514, a potent inhibitor of human epidermal growth factor receptor (HER) 1 (EGFR), 2, and 4, and vascular endothelial growth factor receptors (VEGFR) 1-3, is currently under investigation as an oral agent for the treatment of solid tumors. In vitro and in vivo studies were conducted to characterize the pharmacokinetics and metabolism. Through integration of in vitro and in vivo pharmacokinetic data and antitumor efficacy in nude mice, human pharmacokinetics and efficacious doses were projected for BMS-690514. The oral bioavailability of BMS-690514 was 78% in mice, approximately 100% in rats, 8% in monkeys, and 29% in dogs. The low oral bioavailability in monkeys could be attributed to high systemic clearance in that species, which was also consistent with predicted clearance using in vitro data from monkey liver microsomes. Permeability of BMS-690514 in Caco-2 cells was in the intermediate range with a moderate potential to be a P-gp substrate. Experiments using recombinant human CYP enzymes and human liver microsomes suggested that CYP2D6 and CYP3A4 are likely to play a key role in the metabolic clearance of BMS-690514; in addition, direct glucuronidation of BMS-690514 was also observed in human hepatocytes. BMS-690514 was able to cross the blood-brain barrier with a brain-to-plasma ratio of approximately 1. The preclinical ADME properties of BMS-690514 suggest good oral bioavailability in humans and metabolism by multiple pathways including oxidation and glucuronidation. Based on the efficacious AUC in nude mice and predicted human pharmacokinetics, the human efficacious QD dose is predicted to be in the range of 100-200 mg.