Lysosomal P-gp-MDR1 Confers Drug Resistance of Brentuximab Vedotin and Its Cytotoxic Payload Monomethyl Auristatin E in Tumor Cells.

Antibody-drug conjugates (ADCs) are composed of an antibody linked to cytotoxic anticancer payloads. ADCs recognize tumor-specific cell surface antigens and are internalized into lysosomes where proteolytic enzymes release the cytotoxic payloads. Efflux transporters on plasma membrane that play a significant role on multi-drug resistance in chemotherapy can be internalized on lysosomal membrane and sequester the cytotoxic payloads. In the present study, ATP binding cassette (ABC) efflux transporters including breast cancer resistance protein (BCRP), P-glycoprotein (P-gp-MDR1), multidrug resistance protein (MRP) 2, MRP3 and MRP4 in lysosomal, and plasma membrane of tumor cells were quantified by targeted quantitative proteomics. The cytotoxicity of brentuximab vedotin and its cytotoxic payload monomethyl auristatin E (MMAE) to the tumor cell lines in the presence and absence of elacridar (P-gp-MDR1 inhibitor) or chloroquine (lysosomotropic agent) were evaluated. MMAE is a substrate for P-gp-MDR1, as the apparent efflux ratio in MDR1 transfected MDCK cell monolayers was 44.5, and elacridar abolished the MMAE efflux. Cell lines that highly express P-gp-MDR1 show higher EC50s toward the cell killing effects of MMAE. Co-incubation with chloroquine or elacridar resulted in left shift of MMAE EC50 by 2.9-16-fold and 4.2-22-fold, respectively. Similarly co-incubation with chloroquine or elacridar or in combination of chloroquine and elacridar increased cytotoxic effects of brentuximab vedotin by 2.8- to 21.4-fold on KM-H2 cells that express a specific tumor antigen CD30 and P-gp-MDR1. These findings demonstrate important roles of P-gp-MDR1 on cytotoxic effects of brentuximab vedotin and its payload MMAE. Collectively, ABC transporter-mediated drug extrusion and/or sequestration needs to be early assessed for selection of optimal payloads and linkers when developing ADCs.

Diclofenac and Its Acyl Glucuronide: Determination of In Vivo Exposure in Human Subjects and Characterization as Human Drug Transporter Substrates In Vitro.

Although the metabolism and disposition of diclofenac (DF) has been studied extensively, information regarding the plasma levels of its acyl-ß-d-glucuronide (DF-AG), a major metabolite, in human subjects is limited. Therefore, DF-AG concentrations were determined in plasma (acidified blood derived) of six healthy volunteers following a single oral DF dose (50 mg). Levels of DF-AG in plasma were high, as reflected by a DF-AG/DF ratio of 0.62 ± 0.21 (Cmax mean ± S.D.) and 0.84 ± 0.21 (area under the concentration-time curve mean ± S.D.). Both DF and DF-AG were also studied as substrates of different human drug transporters in vitro. DF was identified as a substrate of organic anion transporter (OAT) 2 only (Km = 46.8 µM). In contrast, DF-AG was identified as a substrate of numerous OATs (Km = 8.6, 60.2, 103.9, and 112 µM for OAT2, OAT1, OAT4, and OAT3, respectively), two organic anion-transporting polypeptides (OATP1B1, Km = 34 µM; OATP2B1, Km = 105 µM), breast cancer resistance protein (Km = 152 µM), and two multidrug resistance proteins (MRP2, Km = 145 µM; MRP3, Km = 196 µM). It is concluded that the disposition of DF-AG, once formed, can be mediated by various candidate transporters known to be expressed in the kidney (basolateral, OAT1, OAT2, and OAT3; apical, MRP2, BCRP, and OAT4) and liver (canalicular, MRP2 and BCRP; basolateral, OATP1B1, OATP2B1, OAT2, and MRP3). DF-AG is unstable in plasma and undergoes conversion to parent DF. Therefore, caution is warranted when assessing renal and hepatic transporter-mediated drug-drug interactions with DF and DF-AG.

Metabolic chiral inversion of brivanib and its relevance to safety and pharmacology.

Brivanib alaninate is an orally administered alanine prodrug of brivanib, a dual inhibitor of the vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) signaling pathways. It is currently in clinical trials for the treatment of hepatocellular carcinoma and colorectal cancer. Brivanib has a single asymmetric center derived from a secondary alcohol. The potential for chiral inversion was investigated in incubations with liver subcellular fractions and in animals and humans after oral doses of brivanib alaninate. Incubations of [¹4C]brivanib alaninate with liver microsomes and cytosols from rats, monkeys, and humans followed by chiral chromatography resulted in two radioactive peaks, corresponding to brivanib and its enantiomer. The percentage of the enantiomeric metabolite relative to brivanib in microsomal and cytosolic incubations of different species in the presence of NADPH ranged from 11.6 to 15.8 and 0.8 to 3.1%, respectively. The proposed mechanism of inversion involves the oxidation of brivanib to a ketone metabolite, which is subsequently reduced to brivanib and its enantiomer. After oral doses of brivanib alaninate to rats and monkeys, the enantiomeric metabolite was a prominent drug-related component in plasma, with the percentages of area under the curve (AUC) at 94.7 and 39.7%, respectively, relative to brivanib. In humans, the enantiomeric metabolite was a minor circulating component, with the AUC <3% of brivanib. Pharmacological studies indicated that brivanib and its enantiomer had similar potency toward the inhibition of VEGF receptor-2 and FGF receptor-1 kinases. Because of low plasma concentration in humans, the enantiomeric metabolite was not expected to contribute significantly to target-related pharmacology of brivanib. Moreover, adequate exposure in the toxicology species suggested no specific safety concerns with respect to exposure to the enantiomeric metabolite.

Identification of the oxidative and conjugative enzymes involved in the biotransformation of brivanib.

Brivanib alaninate, the L-alanine ester prodrug of brivanib, is currently being developed as an anticancer agent. In humans, brivanib alaninate is rapidly hydrolyzed to brivanib. Prominent biotransformation pathways of brivanib included oxidation and direct sulfate conjugation. A series of in vitro studies were conducted to identify the human esterases involved in the prodrug hydrolysis and to identify the primary human cytochrome P450 and sulfotransferase (SULT) enzymes involved in the metabolism of brivanib. Brivanib alaninate was efficiently converted to brivanib in the presence of either human carboxylesterase 1 or carboxylesterase 2. Because esterases are ubiquitous, it is likely that multiple esterases are involved in the hydrolysis. Oxidation of brivanib in human liver microsomes (HLM) primarily formed a hydroxylated metabolite (M7). Incubation of brivanib with human cDNA-expressed P450 enzymes and with HLM in the presence of selective chemical inhibitors and monoclonal P450 antibodies demonstrated that CYP1A2 and CYP3A4 were the major contributors for the formation of M7. Direct sulfation of brivanib was catalyzed by multiple SULT enzymes, including SULT1A1, SULT1B1, SULT2A1, SULT1A3, and SULT1E1. Because the primary in vitro oxidative metabolite (M7) was not detected in humans after oral doses of brivanib alaninate, further metabolism studies of M7 in HLM and human liver cytosol were performed. The data demonstrated that M7 was metabolized to the prominent metabolites observed in humans. Overall, multiple enzymes are involved in the metabolism of brivanib, suggesting a low potential for drug-drug interactions either through polymorphism or through inhibition of a particular drug-metabolizing enzyme.

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, 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.

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.

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.

Acivicin-induced alterations in renal and hepatic glutathione concentrations and in gamma-glutamyltransferase activities.

gamma-Glutamyltransferase (gamma-GT) catalyzes the hydrolysis of glutathione, glutathione S-conjugates, and gamma-substituted l-glutamate derivatives. Acivicin is an irreversible inhibitor of gamma-GT that has been used to study the role of gamma-GT in glutathione homeostasis and glutathione-dependent bioactivation reactions. The present studies were undertaken because of reported conflicting effects of acivicin on the nephrotoxicity of some haloalkenes that undergo glutathione-dependent bioactivation. The objective of this study was to test the hypothesis that acivicin may alter renal glutathione concentrations; acivicin-induced changes in renal glutathione concentrations may alter the susceptibility of the kidney to the nephrotoxic effects of haloalkenes. Hence, diurnal and acivicin-induced changes in renal and hepatic glutathione concentrations along with renal and hepatic gamma-GT activities were investigated. The previously observed diurnal variations in hepatic glutathione concentrations in fed rats were confirmed, but no diurnal variations were observed in renal glutathione concentrations or in renal or hepatic gamma-GT activities. Renal and hepatic glutathione concentrations and gamma-GT activities were measured in tissue homogenates from rats given 0, 0.1, or 0.2 mmol acivicin/kg (i.p.) and killed 0, 2, 4, 8, 12, or 24 hr later. Renal glutathione concentrations were increased above control values in acivicin-treated rats, whereas acivicin had no effect on hepatic glutathione concentrations. Renal gamma-GT activities decreased within 2 hr after giving acivicin and remained decreased for 24 hr. Acivicin had no effect on hepatic gamma-GT activities, except at 24 hr after treatment when values in acivicin-treated rats were elevated compared with controls. Although the present studies do not afford an explanation of the mechanism whereby acivicin increases the nephrotoxicity of some haloalkenes, they do indicate that acivicin is not a reliable probe to investigate the role of gamma-GT in haloalkene-induced nephrotoxicity.

Comparative biotransformation of radiolabeled [(14)C]omapatrilat and stable-labeled [(13)C(2)]omapatrilat after oral administration to rats, dogs, and humans.

Omapatrilat, a novel vasopeptidase inhibitor, is under development for the treatment of hypertension and congestive heart failure. This study describes the comparative biotransformation of radiolabeled [(14)C]- and stable-labeled [(13)C(2)]omapatrilat after administration of single oral doses to rats, dogs, and humans. The metabolites were identified by a combination of methods including reduction, hydrolysis, and comparison of high performance liquid chromatography retention times with those of the synthetic standards. Urinary metabolites were further characterized by liquid chromatography tandem mass spectrometry analysis. Prominent metabolites identified in human plasma, which were also present in rat and dog plasma, were S-methyl omapatrilat and S-2-thiomethyl-3-phenylpropionic acid. Omapatrilat accounted for only a small portion of the extractable radioactivity in plasma in all three species. A portion of the plasma radioactivity was unextractable in all three species (27-53%). The majority of unextractable radioactivity in plasma was characterized after dithiothreitol reduction to be omapatrilat and (S)-2-thio-3-phenylpropionic acid, both apparently bound to plasma proteins by reversible disulfide bonds. The major human urinary metabolites were the amine hydrolysis product, diasteromeric sulfoxide of (S)-2-thiomethyl-3-phenylpropionic acid, acyl glucuronide of S-methyl omapatrilat, and S-methyl omapatrilat. The minor metabolites were acyl glucuronide of (S)-2-thiomethyl-3-phenylpropionic acid, L-cysteine mixed disulfide of omapatrilat, diastereomers of S-methyl sulfoxide of omapatrilat, and S-methyl omapatrilat ring sulfoxide. The metabolic profiles of dog and human urine were qualitatively similar whereas rat urine showed only metabolites arising from hydrolysis of omapatrilat. Unchanged omapatrilat was not found in rat, dog, or human urine samples indicating extensive metabolism in vivo.