Metabolomic Profiling and Drug Interaction Characterization Reveal Riboflavin As a Breast Cancer Resistance Protein-Specific Endogenous Biomarker That Demonstrates Prediction of Transporter Activity In Vivo.

Advancement of endogenous biomarkers for drug transporters as a tool for assessing drug-drug interactions (DDIs) depends on initial identification of biomarker candidates and relies heavily on biomarker validation and its response to reference inhibitors in vivo. To identify endogenous biomarkers of breast cancer resistance protein (BCRP), we applied metabolomic approaches to profile plasma from Bcrp-/-, multidrug resistance protein (Mdr)1a/1b-/-, and Bcrp/Mdr1a/1b-/- mice. Approximately 130 metabolites were significantly altered in Bcrp and P-glycoprotein (P-gp) knockout mice, indicating numerous metabolite-transporter interactions. We focused on BCRP-specific substrates and identified riboflavin, which was significantly elevated in the plasma of Bcrp single- and Bcrp/P-gp double- but not P-gp single-knockout mice. Dual BCRP/P-gp inhibitor elacridar caused a dose-dependent increase of the area under the plasma concentration-time curve (AUC) of riboflavin in mice (1.51- and 1.93-fold increases by 30 and 150 mg/kg elacridar, respectively). In three cynomolgus monkeys, we observed approximately 1.7-fold increases in the riboflavin concentrations caused by ML753286 (10 mg/kg), which correlated well with the increase of sulfasalazine, a known BCRP probe in monkeys. However, the BCRP inhibitor had no effect on isobutyryl carnitine, arginine, or 2-arachidonoyl glycerol levels. Additionally, clinical studies on healthy volunteers indicated low intrasubject and intermeal variability of plasma riboflavin concentrations. In vitro experiments using membrane vesicles demonstrated riboflavin as a select substrate of monkey and human BCRP over P-gp. Collectively, this proof-of-principle study indicates that riboflavin is a suitable endogenous probe for BCRP activity in mice and monkeys and that future investigation of riboflavin as a blood-based biomarker of human BCRP is warranted. SIGNIFICANCE STATEMENT: Our results identified riboflavin as an endogenous biomarker candidate of BCRP. Its selectivity, sensitivity, and predictivity regarding BCRP inhibition have been explored. The findings of this study highlight riboflavin as an informative BCRP plasma biomarker in animal models. The utility of this biomarker requires further validation by evaluating the effects of BCRP inhibitors of different potencies on riboflavin plasma concentrations in humans. Ultimately, riboflavin may shed light on the risk assessment of BCRP DDIs in early clinical trials.

Interindividual Variability and Differential Tissue Abundance of Mitochondrial Amidoxime Reducing Component Enzymes in Humans.

Mitochondrial amidoxime-reducing component (mARC) enzymes are molybdenum-containing proteins that metabolize a number of endobiotics and xenobiotics. The interindividual variability and differential tissue abundance of mARC1 and mARC2 were quantified using targeted proteomics in three types of tissue fractions: 1) pediatric liver tissue homogenates, 2) total membrane fraction of the paired liver and kidney samples from pediatric and adult donors, and 3) pooled S9 fractions of the liver, intestine, kidney, lung, and heart. The absolute levels of mARC1 and mARC2 in the pediatric liver homogenate were 40.08 ± 4.26 and 24.58 ± 4.02 pmol/mg homogenate protein, respectively, and were independent of age and sex. In the total membrane fraction of the paired liver and kidney samples, the abundance of hepatic mARC1 and mARC2 was comparable, whereas mARC2 abundance in the kidney was approximately 9-fold higher in comparison with mARC1. The analysis of the third set of samples (i.e., S9 fraction) revealed that mARC1 abundance in the kidney, intestine, and lung was 5- to 13-fold lower than the liver S9 abundance, whereas mARC2 abundance was approximately 3- and 16-fold lower in the intestine and lung than the liver S9, respectively. In contrast, the kidney mARC2 abundance in the S9 fraction was approximately 2.5-fold higher as compared with the hepatic mARC2 abundance. The abundance of mARC enzymes in the heart was below the limit of quantification (∼0.6 pmol/mg protein). The mARC enzyme abundance data presented here can be used to develop physiologically based pharmacokinetic models for the prediction of in vivo pharmacokinetics of mARC substrates. SIGNIFICANCE STATEMENT: A precise targeted quantitative proteomics method was developed and applied to quantify newly discovered drug-metabolizing enzymes, mARC1 and mARC2, in pediatric and adult tissue samples. The data suggest that mARC enzymes are ubiquitously expressed in an isoform-specific manner in the human liver, kidney, intestine, and lung, and the enzyme abundance is not associated with age and sex. These data are important for developing physiologically based pharmacokinetic models for the prediction of in vivo pharmacokinetics of mARC substrates.

Preclinical metabolism and disposition of an orally bioavailable macrocyclic FXIa inhibitor.

FXIa-6f is a high affinity, orally bioavailable macrocyclic FXIa inhibitor with antithrombotic activity in preclinical species.The objectives of this study were to characterize the in vitro metabolism, determine circulating metabolites in pre-clinical species, and examine the disposition of the compound in a bile duct-cannulated rat study (BDC) study to inform clinical development of the compound and the medicinal chemistry approach to identify molecules with improved properties.Across species, metabolic pathways included several oxidative metabolites, including hydroxylated metabolites on the macrocycle or P1 region, descarbamoylation of the methyl carbamate side chain, and a glutathione conjugate on the 2,6-difluoro-3-chlorophenyl ring.In BDC rat, the absorbed dose of [3H]FXIa-6f was cleared mainly by metabolism, with excretion of drug-related material in the bile, mostly as metabolites.In all preclinical species, the parent drug was the primary drug-related component in circulation, but the species differences in the metabolic pathways observed in vitro were reflected in the plasma, where M6, a descarbamoylated metabolite, was more prominent in rat plasma, and M9, a hydroxylated metabolite, was more prominent in monkey plasma. Based on the available data, the human metabolism appears to be most similar to monkey.

Hepatocyte spheroids as a viable in vitro model for recapitulation of complex in vivo metabolism pathways of loratadine in humans.

Accurate prediction of in vivo metabolic pathways in humans can be challenging because in vitro liver matrices may fail to produce certain in vivo metabolites.Rat and human spheroids, generated from cryopreserved hepatocytes in media that contained minimal amount of serum, maintained morphology, viability and cytochrome P450 (CYP) activities for at least a week without media exchange.With spheroid cultures, multiple Phase I and Phase II metabolites were observed in rat and human spheroid cultures that were incubated with loratadine (LOR) for multiple days. Consistent with in vivo observations, 3-hydroxydesloratadine, (3-OH-DL), along with its glucuronide, were observed in human spheroids, but not in rat spheroids. Interestingly, the putative intermediate metabolite leading to 3-OH-DL, DL-N-glucuronide, was observed in incubations with both rat and human spheroids. In conclusion, hepatocyte spheroid were capable of recapitulating the inter-species differences in metabolism between human and rat for LOR, therefore, it may represent a viable model for studying complex metabolic pathways.

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.

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.

The synthesis of a carbon-14 labeled pegylated Adnectin™ for placental transfer studies in guinea pigs.

Adnectins™ are novel fibronectin-based proteins containing domains engineered to bind to targets of therapeutic interest. The molecular weights of adnectins are less than conventional monoclonal antibodies but larger than traditional small molecules. Until now, there has been no information on the placental transfer of adnectins. To assess placental permeability to adnectins in pregnant guinea pigs, a radiolabeled adnectin, ATI-1072, bound to polyethylene glycol through a [(14) C]Maleimide linker, was synthesized from [1,4-(14) C]Maleic acid. This publication describes the synthesis and analysis of PEG-[(14) C]Maleimide-adnectin ([(14) C]ATI-1072).

The Importance of Tracking "Missing" Metabolites: How and Why?

Technologies currently employed to find and identify drug metabolites in complex biological matrices generally yield results that offer a comprehensive picture of the drug metabolite profile. However, drug metabolites can be missed or are captured only late in the drug development process. This could be due to a variety of factors, such as metabolism that results in partial loss of the molecule, covalent bonding to macromolecules, the drug being metabolized in specific human tissues, or poor ionization in a mass spectrometer. These scenarios often draw a great deal of attention from chemistry, safety assessment, and pharmacology. This review will summarize scenarios of missing metabolites, why they are missing, and associated uncovering strategies from deeper investigations. Uncovering previously missed metabolites can have ramifications in drug development with toxicological and pharmacological consequences, and knowledge of these can help in the design of new drugs.

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 [(14)C]saxagliptin were investigated in healthy male subjects after a single 50-mg (91.5 µCi) oral dose. Saxagliptin was rapidly absorbed (T(max), 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 (V(max)/K(m)) 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.

Prediction of drug-drug interaction potential mediated by transporters between dasatinib and metformin, pravastatin, and rosuvastatin using physiologically based pharmacokinetic modeling.

PURPOSE: Recent in vitro studies demonstrated that dasatinib inhibits organic cation transporter 2 (OCT2), multidrug and toxin extrusion proteins (MATEs), and organic anion transporting polypeptide 1B1/1B3 (OATP1B1/1B3). We developed a physiologically based pharmacokinetic (PBPK) model to assess drug-drug interaction (DDI) potential between dasatinib and known substrates for these transporters in a virtual population. METHODS: The dasatinib PBPK model was constructed using Simcyp® Simulator by combining its physicochemical properties, in vitro data, in silico predictions, and pharmacokinetic (PK) results from clinical studies. Model validation against three independent clinical trials not used for model development included dasatinib DDI studies with ketoconazole, rifampin, and simvastatin. The validated model was used to simulate DDIs of dasatinib and known substrates for OCT2 and MATEs (metformin) and OATP1B1/1B3 (pravastatin and rosuvastatin). RESULTS: Simulations of metformin PK in the presence and absence of dasatinib, using inhibitor constant (Ki) values measured in vitro, produced estimated geometric mean ratios (GMRs) of the maximum observed concentration (Cmax) and area under the concentration-time curve (AUC) of 1.05 and 1.06, respectively. Sensitivity analysis showed metformin exposure increased < 30% in both AUC and Cmax when dasatinib Ki was reduced by tenfold for OCT2 and MATEs simultaneously, and < 40% with a 20-fold Ki reduction. The estimated GMRs of Cmax and AUC for pravastatin and rosuvastatin with co-administration of dasatinib were unity (1.00). CONCLUSIONS: This PBPK model accurately described the observed PK profiles of dasatinib. The validated PBPK model predicts low risk of clinically significant DDIs between dasatinib and metformin, pravastatin, or rosuvastatin.

Effects of Itraconazole and Diltiazem on the Pharmacokinetics and Pharmacodynamics of Milvexian, A Factor XIa Inhibitor.

INTRODUCTION: Modulation of Factor XIa (FXIa) may provide a novel mechanism for systemic anticoagulation with the potential to improve the risk-benefit profile observed with existing anticoagulants through greater efficacy or a safer bleeding profile. This study assessed the effects of co-administration with strong and moderate CYP3A inhibitors itraconazole and diltiazem, respectively, on the pharmacokinetic and pharmacodynamic properties of milvexian, a Factor XIa inhibitor. METHODS: This was an open-label, non-randomized, two-period crossover study in healthy participants. In period 1, participants received a single oral dose of milvexian (30 mg) on day 1, followed by a washout on days 2 and 3. In period 2, participants received multiple oral doses of itraconazole (200 mg) or diltiazem (240 mg) with a single dose of milvexian. RESULTS: A total of 28 participants entered the treatment period. Following itraconazole co-administration, milvexian exposure was increased; AUC(0-T), AUC(INF), and C24 were 2.5-, 2.5-, and 3.8-fold higher, while mean Cmax was 28% higher versus milvexian alone. Diltiazem co-administration also increased milvexian exposure; AUC(0-T), AUC(INF), and C24 were 38, 38, and 64% higher, and mean Cmax was 9.6% higher versus milvexian alone. Prolongation of activated partial thromboplastin time was observed with milvexian in a concentration-dependent fashion irrespective of co-administration with itraconazole or diltiazem. Administration of a single dose of milvexian, alone or in combination with itraconazole or diltiazem, was generally safe and well tolerated; there were no deaths or serious adverse events. CONCLUSIONS: A moderate increase in milvexian exposure was observed following co-administration of itraconazole while a minimal increase was seen with diltiazem, consistent with the involvement of CYP3A metabolism and P-glycoprotein in drug absorption/elimination. Milvexian was generally safe and well tolerated in healthy participants. TRIAL REGISTRATION: The study was registered with ClinicalTrials.gov (NCT02807909; submitted June 17, 2016).

Effects of rifampin on the pharmacokinetics and pharmacodynamics of milvexian, a potent, selective, oral small molecule factor XIa inhibitor.

Milvexian (BMS-986177/JNJ-70033093) is a potent, oral small molecule that inhibits the active form of factor XI with high affinity and selectivity. This study assessed the single-dose pharmacokinetic and pharmacodynamic properties of milvexian co-administered with rifampin, an organic anion transport protein (OATP) inhibitor and potent cytochrome P450 (CYP) 3A and P-glycoprotein (P-gp) inducer. In this open-label, nonrandomized, single-sequence study, healthy participants (N = 16) received single doses of milvexian on Day 1 (100 mg), milvexian and rifampin (600 mg) on Day 4, rifampin on Days 5-11, milvexian and rifampin on Day 12, and rifampin on Days 13-14. Pharmacokinetic data were summarized using descriptive statistics. Administration of milvexian, alone or in combination with rifampin, was generally safe and well tolerated. Single-dose co-administration of rifampin and milvexian demonstrated no meaningful changes in milvexian exposure versus milvexian alone (Cmax, 110%; AUC[0-T], 102%; AUC[INF], 101%). After multiple doses of rifampin and milvexian, peak and total milvexian exposure substantially decreased versus milvexian alone (Cmax, 22%; AUC[0-T], 15%; AUC[INF], 15%). Results were consistent with preclinical data, indicating that milvexian is a substrate for CYP3A4/5 and P-gp but not OATP. The implications of these results on the need for dose adjustment of milvexian will be further elucidated following the completion of phase 2 and 3 trials.Trial registration The study was registered with ClinicalTrials.gov (NCT02959060; submitted 7/11/2016, first posted 8/11/2016).

In vitro characterization of the metabolic pathways and cytochrome P450 inhibition and induction potential of BMS-690514, an ErbB/vascular endothelial growth factor receptor inhibitor.

(3R,4R)-4-Amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)-3-piperidinol (BMS-690514) is a potent inhibitor of ErbB human epidermal growth factor receptors (HER1, 2, and 4) and vascular endothelial growth factor receptors 1 to 3 that has been under clinical development for solid tumor malignancies. BMS-690514 is primarily cleared by metabolism with the primary metabolic pathways being direct glucuronidation (M6), hydroxylation (M1, M2, and M37), and O-demethylation (M3). In the current investigation, the metabolic drug-drug interaction potential of BMS-690514 was evaluated in a series of in vitro studies. Reaction phenotyping experiments with cDNA-expressed human cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) enzymes and human liver microsomes (HLM) in the presence of P450 or UGT inhibitors suggested that CYP3A4, CYP2D6, and CYP2C9 were the major enzymes responsible for the oxidative metabolism of BMS-690514, whereas both UGT2B4 and UGT2B7 were responsible for the formation of M6. BMS-690514 did not cause direct or time-dependent inhibition of P450 enzymes (IC(50) values ≥40 µM) in incubations with HLM and probe substrates of CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, or 3A4. The compound also did not substantially induce CYP1A1, CYP1A2, CYP2B6, CYP3A4, or UGT1A1 at concentrations up to 10 µM in cultured human hepatocytes. Considering the submicromolar plasma C(max) concentration at the anticipated clinical dose of 200 mg, BMS-690514 is unlikely to cause clinically relevant drug-drug interactions when coadministered with other medications. In addition, because multiple enzymatic clearance pathways are available for the compound, inhibition of an individual metabolic pathway either via coadministered drugs or gene polymorphisms is not expected to cause pronounced (>2-fold) increases in BMS-690514 exposure.

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.

Mechanistic studies on a P450-mediated rearrangement of BMS-690514: conversion of a pyrrolotriazine to a hydroxypyridotriazine.

BMS-690514 ((3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4] triazin-5-yl)methyl)-3-piperidinol) is an oral oncologic agent being developed for the treatment of patients with advanced nonsmall cell lung cancer and breast cancer. The compound is metabolized via multiple metabolic pathways, including P450-mediated oxidation at one of the carbons of its pyrrolotriazine group. Oxidation at this site results in the formation of two metabolites, M1 and M37. Mass spectrometric and NMR analysis revealed that M1 underwent an unusual structural change, where the pyrrolotriazine moiety rearranged to yield a hydroxypyridotriazine group. In contrast, the structure of the pyrrolotriazine moiety remained intact in M37. In vitro experiments with liver microsomes and deuterated or tritiated BMS-690514 containing the isotopic label on the carbon that underwent oxidation indicated that during the formation of M1, the isotope label was retained at the site of hydroxylation, while the label was lost during the formation of M37. On the basis of these results, a mechanism for the formation of M1 was proposed as follows: BMS-690514 was first oxidized by P450 enzymes either via epoxidation or an iron-oxo addition pathway to form a zwitterionic intermediate. This was followed by opening of the pyrrolotriazine ring to form an aldehyde intermediate, which could be partially trapped with methoxyamine. The aldehyde intermediate then reacted with the secondary amine of the methoxyaniline group in the molecule to form the pyridotriazine moiety of M1. This mechanism is consistent with the observed retention of the isotope label in M1. Metabolite M37 may be formed either via a common zwitterionic intermediate, shared with M1, or through a direct insertion pathway. In in vitro human liver microsome incubations, the abundance of M1 was higher than M37, suggesting that breaking of the carbon-nitrogen bond to generate the aldehyde intermediate, a process similar to N-dealkylation, was a preferred pathway.

Metabolism and disposition of [14C]BMS-690514 after oral administration to rats, rabbits, and dogs.

(3R,4R)-4-Amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f] [1,2,4]triazin-5-yl)methyl)-3-piperidinol (BMS-690514) is a potent inhibitor of human epidermal growth factor receptors 1, 2, and 4 and vascular endothelial growth factor receptors 1 through 3. BMS-690514 is an oral oncologic agent currently being developed for the treatment of patients with advanced non-small cell lung cancer and breast cancer. In this investigation, a series of studies was conducted to determine the biotransformation of [(14)C]BMS-690514 after oral administration to rats, rabbits, and dogs. After administration of a single oral dose of [(14)C]BMS-690514 to rats and dogs, the majority of the radioactive dose (61-71%) was recovered in the feces, whereas 18 to 20% was eliminated in urine. In bile duct-cannulated rats, 83 and 17% of the administered radioactivity was recovered in the bile and urine, respectively, suggesting that biliary secretion was a major route for the elimination of BMS-690514-derived radioactivity in rats. The parent compound underwent extensive metabolism in both species, with <12% of the administered radioactivity recovered as BMS-690514 in the excreta samples. Metabolite profiles in plasma were qualitatively similar in rats, rabbits, and dogs. Unchanged BMS-690514 was a prominent drug-related component in the plasma profiles from all the species. However, multiple metabolites contributed significantly to the circulating radioactivity, particularly for rabbit and dog, in which metabolites comprised 73 to 93% of the area under the time curve (0-8 h). Circulating metabolites included M6, a direct O-glucuronide conjugate; M1, a hydroxylated metabolite; and glucuronide conjugates of hydroxylated and O-demethylated metabolites. Overall, the results from these studies suggested that BMS-690514 was well absorbed and highly metabolized through multiple pathways in these preclinical species.

Metabolism and disposition of [14C]BMS-690514, an ErbB/vascular endothelial growth factor receptor inhibitor, after oral administration to humans.

(3R,4R)-4-Amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)-3-piperidinol (BMS-690514), an oral selective inhibitor of human epidermal growth factor receptors 1 (or epidermal growth factor receptor), 2, and 4, and vascular endothelial growth factor receptors 1, 2, and 3, is being developed as a treatment for patients with non-small-cell lung cancer and metastatic breast cancer. The disposition of [(14)C]BMS-690514 was investigated in nine healthy male subjects (group 1, n = 6; group 2, n = 3) after oral administration of a 200-mg dose. Urine, feces, and plasma were collected from all subjects for up to 12 days postdose. In group 2 subjects, bile was collected from 3 to 8 h postdose. Across groups, approximately 50 and 34% of administered radioactivity was recovered in the feces and urine, respectively. An additional 16% was recovered in the bile of group 2 subjects. Less than 28% of the dose was recovered as parent drug in the combined excreta, suggesting that BMS-690514 was highly metabolized. BMS-690514 was rapidly absorbed (median time of maximum observed concentration 0.5 h) with the absorbed fraction estimated to be approximately 50 to 68%. BMS-690514 represented ≤7.9% of the area under the concentration-time curve from time 0 extrapolated to infinite time of plasma radioactivity, indicating that the majority of the circulating radioactivity was from metabolites. BMS-690514 was metabolized via multiple oxidation reactions and direct glucuronidation. Circulating metabolites included a hydroxylated rearrangement product (M1), a direct ether glucuronide (M6), and multiple secondary glucuronide conjugates. None of these metabolites is expected to contribute to the pharmacology of BMS-690514. In summary, BMS-690514 was well absorbed and extensively metabolized via multiple metabolic pathways in humans, with excretion of drug-related radioactivity in both bile and urine.

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 (< or =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.

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.

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, [(14)C]-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 K(m) 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 V(max) values were calculated with the predetermined K(m) values and the metabolite formation rates from a limited number of incubations with [(14)C]dasatinib. The intrinsic formation clearance values (V(max)/K(m)) of 52, 14, 274, and 20 microl/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.