Cross-platform comparison of immune-related gene expression to assess intratumor immune responses following cancer immunotherapy.

Neoadjuvant immunotherapy can induce immune responses within the tumor microenvironment. Gene expression can be used to assess responses with limited amounts of conventionally-fixed patient-derived samples. We aim to assess the cross-platform concordance of immune-related gene expression data. We performed comparisons across three panels in two platforms: Nanostring nCounter® PanCancer Immune Profiling Panel (nS), HTG EdgeSeq Oncology Biomarker Panel (HTG OBP) and Precision Immuno-Oncology Panel (HTG PIP). All tissue samples of 14 neoadjuvant GM-CSF treated, 14 neoadjuvant Provenge treated, and 12 untreated prostate cancer patients were radical prostatectomy (RP) tissues, while 6 prostatitis patients and 6 non-prostatitis subjects were biopsies. For all 52 patients, more than 90% of the common genes were significantly correlated (p < 0.05) and more than 76% of the common genes were highly correlated (r > 0.5) between any two panels. Co-inertia analysis also demonstrated high overall dataset structure similarity (correlation>0.84). Although both dimensionality reduction visualization analysis and unsupervised hierarchical cluster analysis for highly correlated common genes (r > 0.9) suggested a high-level of consistency across the panels, there were subsets of genes that were differentially expressed across the panels. In addition, while the effect size of the differential testing for neoadjuvant treated vs. untreated localized prostate cancer patients across the panels were significantly correlated, some genes were only differentially expressed in the HTG panels. Finally, the HTG PIP panel had the best classification performance among the 3 panels. These differences detected may be a result of the different panels or platforms due to their technical setting and focus. Thus, researchers should be aware of those potential differences when deciding which platform and panel to use.

Pharmacokinetics of trastuzumab deruxtecan (T-DXd), a novel anti-HER2 antibody-drug conjugate, in HER2-positive tumour-bearing mice.

Trastuzumab deruxtecan (T-DXd, DS-8201a) is an antibody-drug conjugate (ADC), comprising an anti-HER2 antibody (Ab) at a drug-to-Ab ratio of 7-8 with the topoisomerase I inhibitor DXd. In this study, we investigated the pharmacokinetics (PK), biodistribution, catabolism, and excretion profiles of T-DXd in HER2-positive tumour-bearing mice.Following intravenous (iv) administration of T-DXd, the PK profiles of T-DXd and total Ab (the sum of conjugated and unconjugated Ab) were almost similar, indicating that the linker is stable during circulation. Biodistribution studies using radiolabelled T-DXd demonstrated tumour-specific distribution and long-term retention. DXd was the main catabolite released from T-DXd in tumours, with exposure levels at least five times higher than those in normal tissues and seven times higher than those achieved by non-targeted control ADC. Following iv administration of DXd, it was rapidly cleared from the circulation (T1/2; 1.35 h) and excreted mainly through faeces as its intact form.The PK profiles reveal that T-DXd effectively delivers the expected payload, DXd, to tumours, while minimising payload exposure to the systemic circulation and normal tissues. The released DXd is rapidly cleared from systemic circulation, presumably via the bile with negligible metabolism, and excreted through the faeces.

Clonal Deletion of Tumor-Specific T Cells by Interferon-γ Confers Therapeutic Resistance to Combination Immune Checkpoint Blockade.

Resistance to checkpoint-blockade treatments is a challenge in the clinic. We found that although treatment with combined anti-CTLA-4 and anti-PD-1 improved control of established tumors, this combination compromised anti-tumor immunity in the low tumor burden (LTB) state in pre-clinical models as well as in melanoma patients. Activated tumor-specific T cells expressed higher amounts of interferon-γ (IFN-γ) receptor and were more susceptible to apoptosis than naive T cells. Combination treatment induced deletion of tumor-specific T cells and altered the T cell repertoire landscape, skewing the distribution of T cells toward lower-frequency clonotypes. Additionally, combination therapy induced higher IFN-γ production in the LTB state than in the high tumor burden (HTB) state on a per-cell basis, reflecting a less exhausted immune status in the LTB state. Thus, elevated IFN-γ secretion in the LTB state contributes to the development of an immune-intrinsic mechanism of resistance to combination checkpoint blockade, highlighting the importance of achieving the optimal magnitude of immune stimulation for successful combination immunotherapy strategies.

Neoadjuvant sipuleucel-T induces both Th1 activation and immune regulation in localized prostate cancer.

Sipuleucel-T is the only FDA-approved immunotherapy for metastatic castration-resistant prostate cancer. The mechanism by which this treatment improves survival is not fully understood. We have previously shown that this treatment can induce the recruitment of CD4 and CD8 T cells to the tumor microenvironment. In this study, we examined the functional state of these T cells through gene expression profiling. We found that the magnitude of T cell signatures correlated with the frequency of T cells as measured by immunohistochemistry. Sipuleucel-T treatment was associated with increased expression of Th1-associated genes, but not Th2-, Th17 - or Treg-associated genes. Post-treatment tumor tissues with high CD8+T cell infiltration was associated with high levels of CXCL10 expression. On in situ hybridization, CXCL10+ cells colocalized with CD8+T cells in post-treatment prostatectomy tumor tissue. Neoadjuvant sipuleucel-T was also associated with upregulation of immune inhibitory checkpoints, including CTLA4 and TIGIT, and downregulation of the immune activation marker, dipeptidylpeptidase, DPP4. Treatment-associated declines in serum PSA were correlated with induction of Th1 response. In contrast, rises in serum PSA while on treatment were associated with the induction of multiple immune checkpoints, including CTLA4, CEACAM6 and TIGIT. This could represent adaptive immune resistance mechanisms induced by treatment. Taken together, neoadjuvant sipuleucel-T can induce both a Th1 response and negative immune regulation in the prostate cancer microenvironment.

Interaction of Nevirapine with the Peptide Binding Groove of HLA-DRB1*01:01 and Its Effect on the Conformation of HLA-Peptide Complex.

Human leukocyte antigen (HLA)-DRB1*01:01 has been shown to be involved in nevirapine-induced hepatic hypersensitivity reactions. In the present study, in silico docking simulations and molecular dynamics simulations were performed to predict the interaction mode of nevirapine with the peptide binding groove of HLA-DRB1*01:01 and its possible effect on the position and orientation of the ligand peptide derived from hemagglutinin (HA). In silico analyses suggested that nevirapine interacts with HLA-DRB1*01:01 around the P4 pocket within the peptide binding groove and the HA peptide stably binds on top of nevirapine at the groove. The analyses also showed that binding of nevirapine at the groove will significantly change the inter-helical distances of the groove. An in vitro competitive assay showed that nevirapine (1000 µM) increases the binding of the HA peptide to HLA-DRB1*01:01 in an allele-specific manner. These results indicate that nevirapine might interact directly with the P4 pocket and modifies its structure, which could change the orientation of loaded peptides and the conformation of HLA-DRB1*01:01; these changes could be distinctively recognized by T-cell receptors. Through this molecular mechanism, nevirapine might stimulate the immune system, resulting in hepatic hypersensitivity reactions.

In Silico and In Vitro Analysis of Interaction between Ximelagatran and Human Leukocyte Antigen (HLA)-DRB1*07:01.

Idiosyncratic ximelagatran-induced hepatotoxicity has been reported to be associated with human leukocyte antigen (HLA)-DRB1*07:01 and ximelagatran has been reported to inhibit the binding of the ligand peptide to HLA-DRB1*07:01 in vitro. In order to predict the possible interaction modes of ximelagatran with HLA-DR molecules, in silico docking simulations were performed. Molecular dynamics (MD) simulations were also performed to predict the effect of ximelagatran on the binding mode of the ligand peptide to HLA-DRB1*07:01. A series of in silico simulations supported the inhibitory effect of ximelagatran on the binding of the ligand peptide to HLA-DRB1*07:01 in vitro. Furthermore, direct interactions of ximelagatran with HLA-DR molecules were evaluated in vitro, which supported the simulated interaction mode of ximelagatran with HLA-DRB1*07:01. These results indicated that ximelagatran directly interacts with the peptide binding groove of HLA-DRB1*07:01 and competes with the ligand peptide for the binding site, which could alter the immune response and lead to the idiosyncratic ximelagatran-induced hepatotoxicity.

Bystander killing effect of DS-8201a, a novel anti-human epidermal growth factor receptor 2 antibody-drug conjugate, in tumors with human epidermal growth factor receptor 2 heterogeneity.

Antibody-drug conjugates deliver anticancer agents selectively and efficiently to tumor tissue and have significant antitumor efficacy with a wide therapeutic window. DS-8201a is a human epidermal growth factor receptor 2 (HER2)-targeting antibody-drug conjugate prepared using a novel linker-payload system with a potent topoisomerase I inhibitor, exatecan derivative (DX-8951 derivative, DXd). It was effective against trastuzumab emtansine (T-DM1)-insensitive patient-derived xenograft models with both high and low HER2 expression. In this study, the bystander killing effect of DS-8201a was evaluated and compared with that of T-DM1. We confirmed that the payload of DS-8201a, DXd (1), was highly membrane-permeable whereas that of T-DM1, Lys-SMCC-DM1, had a low level of permeability. Under a coculture condition of HER2-positive KPL-4 cells and negative MDA-MB-468 cells in vitro, DS-8201a killed both cells, whereas T-DM1 and an antibody-drug conjugate with a low permeable payload, anti-HER2-DXd (2), did not. In vivo evaluation was carried out using mice inoculated with a mixture of HER2-positive NCI-N87 cells and HER2-negative MDA-MB-468-Luc cells by using an in vivo imaging system. In vivo, DS-8201a reduced the luciferase signal of the mice, indicating suppression of the MDA-MB-468-Luc population; however, T-DM1 and anti-HER2-DXd (2) did not. Furthermore, it was confirmed that DS-8201a was not effective against MDA-MB-468-Luc tumors inoculated at the opposite side of the NCI-N87 tumor, suggesting that the bystander killing effect of DS-8201a is observed only in cells neighboring HER2-positive cells, indicating low concern in terms of systemic toxicity. These results indicated that DS-8201a has a potent bystander effect due to a highly membrane-permeable payload and is beneficial in treating tumors with HER2 heterogeneity that are unresponsive to T-DM1.

Component of Caramel Food Coloring, THI, Causes Lymphopenia Indirectly via a Key Metabolic Intermediate.

Caramel color is widely used in the food industry, and its many variations are generally considered to be safe. It has been known for a long time that THI (2-acetyl-4-(tetrahydroxybutyl)imidazole), a component of caramel color III, causes lymphopenia in animals through sphingosine 1-phosphate (S1P) lyase (S1PL) inhibition. However, this mechanism of action has not been fully validated because THI does not inhibit S1PL in vitro. To reconcile this situation, we examined molecular details of THI mechanism of action using "smaller" THI derivatives. We identified a bioactive derivative, A6770, which has the same lymphopenic effect as THI via S1PL inhibition. In the case of A6770 we observe this effect both in vitro and in vivo, and demonstrate that A6770 is phosphorylated and inhibits S1PL in the same way as 4-deoxypyridoxine. In addition, A6770 was detected in rat plasma following oral administration of THI, suggesting that A6770 is a key metabolic intermediate of THI.

DS-8201a, A Novel HER2-Targeting ADC with a Novel DNA Topoisomerase I Inhibitor, Demonstrates a Promising Antitumor Efficacy with Differentiation from T-DM1.

PURPOSE: An anti-HER2 antibody-drug conjugate with a novel topoisomerase I inhibitor, DS-8201a, was generated as a new antitumor drug candidate, and its preclinical pharmacologic profile was assessed. EXPERIMENTAL DESIGN: In vitro and in vivo pharmacologic activities of DS-8201a were evaluated and compared with T-DM1 in several HER2-positive cell lines and patient-derived xenograft (PDX) models. The mechanism of action for the efficacy was also evaluated. Pharmacokinetics in cynomolgus monkeys and the safety profiles in rats and cynomolgus monkeys were assessed. RESULTS: DS-8201a exhibited a HER2 expression-dependent cell growth-inhibitory activity and induced tumor regression with a single dosing at more than 1 mg/kg in a HER2-positive gastric cancer NCI-N87 model. Binding activity to HER2 and ADCC activity of DS-8201a were comparable with unconjugated anti-HER2 antibody. DS-8201a also showed an inhibitory activity to Akt phosphorylation. DS-8201a induced phosphorylation of Chk1 and Histone H2A.X, the markers of DNA damage. Pharmacokinetics and safety profiles of DS-8201a were favorable and the highest non-severely toxic dose was 30 mg/kg in cynomolgus monkeys, supporting DS-8201a as being well tolerated in humans. DS-8201a was effective in a T-DM1-insensitive PDX model with high HER2 expression. DS-8201a, but not T-DM1, demonstrated antitumor efficacy against several breast cancer PDX models with low HER2 expression. CONCLUSIONS: DS-8201a exhibited a potent antitumor activity in a broad selection of HER2-positive models and favorable pharmacokinetics and safety profiles. The results demonstrate that DS-8201a will be a valuable therapy with a great potential to respond to T-DM1-insensitive HER2-positive cancers and low HER2-expressing cancers. Clin Cancer Res; 22(20); 5097-108. ©2016 AACR.

Human Intestinal Raf Kinase Inhibitor Protein (RKIP) Catalyzes Prasugrel as a Bioactivation Hydrolase.

Prasugrel is a thienopyridine antiplatelet prodrug that undergoes rapid hydrolysis in vivo to a thiolactone metabolite by human carboxylesterase-2 (hCE2) during gastrointestinal absorption. The thiolactone metabolite is further converted to a pharmacologically active metabolite by cytochrome P450 isoforms. The aim of the current study was to elucidate hydrolases other than hCE2 involved in the bioactivation step of prasugrel in human intestine. Using size-exclusion column chromatography of a human small intestinal S9 fraction, another peak besides the hCE2 peak was observed to have prasugrel hydrolyzing activity, and this protein was found to have a molecular weight of about 20 kDa. This prasugrel hydrolyzing protein was successfully purified from a monkey small intestinal cytosolic fraction by successive four-step column chromatography and identified as Raf-1 kinase inhibitor protein (RKIP) by liquid chromatography-tandem mass spectrometry. Second, we evaluated the enzymatic kinetic parameters for prasugrel hydrolysis using recombinant human RKIP and hCE2 and estimated the contributions of these two hydrolyzing enzymes to the prasugrel hydrolysis reaction in human intestine, which were approximately 40% for hRKIP and 60% for hCE2. Moreover, prasugrel hydrolysis was inhibited by anti-hRKIP antibody and carboxylesterase-specific chemical inhibitor (bis p-nitrophenyl phosphate) by 30% and 60%, respectively. In conclusion, another protein capable of hydrolyzing prasugrel to its thiolactone metabolite was identified as RKIP, and this protein may play a significant role with hCE2 in prasugrel bioactivation in human intestine. RKIP is known to have diverse functions in many intracellular signaling cascades, but this is the first report describing RKIP as a hydrolase involved in drug metabolism.

The Possible Mechanism of Idiosyncratic Lapatinib-Induced Liver Injury in Patients Carrying Human Leukocyte Antigen-DRB1*07:01.

Idiosyncratic lapatinib-induced liver injury has been reported to be associated with human leukocyte antigen (HLA)-DRB1*07:01. In order to investigate its mechanism, interaction of lapatinib with HLA-DRB1*07:01 and its ligand peptide derived from tetanus toxoid, has been evaluated in vitro. Here we show that lapatinib enhances binding of the ligand peptide to HLA-DRB1*07:01. Furthermore in silico molecular dynamics analysis revealed that lapatinib could change the ß chain helix in the HLA-DRB1*07:01 specifically to form a tightly closed binding groove structure and modify a large part of the binding groove. These results indicate that lapatinib affects the ligand binding to HLA-DRB1*07:01 and idiosyncratic lapatinib-induced liver injury might be triggered by this mechanism. This is the first report showing that the clinically available drug can enhance the binding of ligand peptide to HLA class II molecules in vitro and in silico.

Hepatic microsomal thiol methyltransferase is involved in stereoselective methylation of pharmacologically active metabolite of prasugrel.

Prasugrel, a thienopyridine antiplatelet drug, is converted in animals and humans to the pharmacologically active metabolite R-138727 [(2Z)-{1-[(1RS)-2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-sulfanylpiperidin-3-ylidene}ethanoic acid], which has two chiral centers, occurring as a mixture of four isomers. The RS and RR isomers are more active than the SS and SR isomers (RS > RR > > SR = SS). The pharmacologically active metabolite is further metabolized to an S-methylated metabolite that is the major identified inactive metabolite in humans. In rat, dog, and human liver microsomes supplemented with S-adenosyl methione, the SS and SR isomers of the active metabolite were extensively S-methylated while the RS and RR isomers were not. Addition of 2,3-dichloromethyl benzylamine (50 µM) completely inhibited the S-methylation reaction, indicating that the microsomal and cytosolic thiol methyltransferase but not the cytosolic thiopurine S-methyltransferase is involved in the methylation. The hepatic intrinsic clearance values for methylation of the RS, RR, SS, and SR isomers (ml/min/kg) were 0, 0, 40.4, and 37.6, respectively, in rat liver microsomes, 0, 0, 11.6, and 2.5, respectively, in dog liver microsomes, and 0, 0, 17.3, and 17.7, respectively, in human liver microsomes, indicating that the RS and RR isomers are not methylated in vitro and that the methylation of SS and SR isomers is high with rat > human > dog. This finding in vitro agreed well with the in vivo observation in rats and dogs, where the S-methylated SS and SR isomers were the major metabolites in the plasma whereas negligible amounts of S-methylated RS and RR isomers were detected after intravenous administration of the pharmacologically active metabolites.

Glutaredoxin is involved in the formation of the pharmacologically active metabolite of clopidogrel from its GSH conjugate.

Clopidogrel is a thienopyridine antiplatelet agent that is converted to the active metabolite, R-361015, in vivo. Clopidogrel is first oxidized to a thiolactone intermediate R-115991. R-115991 is thought to be metabolized to a GSH conjugate of R-361015 (R-361015-SG) and then is reduced to R-361015 in the presence of GSH. In this study, we investigated the enzyme-mediated formation of R-361015 from R-361015-SG in human liver microsomes and cytosols. After incubation of R-115991 in human liver microsomes, the formation of R-361015-SG, and subsequently of R-361015, was observed. The apparent formation rate of R-361015-SG was markedly decreased when human liver cytosols were added. Fitting the data to the kinetic model showed that the rate constant of R-361015-SG reduction to R-361015 in human liver microsomes was approximately 20-fold higher in the presence of human liver cytosols (6.56 min⁻¹) than in the absence of cytosols (0.326 min⁻¹). In addition, the formation rate of R-361015 from R-361015-SG was higher in human liver cytosols (2843 ± 1176 pmol · min⁻¹ · mg⁻¹) compared with in human liver microsomes (508 ± 396 pmol · min⁻¹ · mg⁻¹). The formation of R-361015 from R-361015-SG in human liver microsomes or cytosols was inhibited by anti-human glutaredoxin antibody in a concentration-dependent manner. Recombinant human glutaredoxin mediated the formation of R-361015 from R-361015-SG with the K(m) and V(max) values of 30.0 ± 1.3 µM and 381.6 ± 209.8 pmol · min⁻¹ · µg⁻¹, respectively. The intrinsic clearance value (V(max)/K(m)) was 12.9 ± 7.5 µl · min⁻¹ · µg⁻¹. In conclusion, we found that human glutaredoxin is a main contributor to the formation of the pharmacologically active metabolite of clopidogrel from its GSH conjugate in human liver.

The intestine as an important contributor to prasugrel active metabolite formation in vivo.

Prasugrel [2-acetoxy-5-(α-cyclopropylcarbonyl-2-fluorobenzyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine], a thienopyridine antiplatelet agent, undergoes rapid hydrolysis in vivo to a thiolactone intermediate, 2-[2-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl]-1-cyclopropyl-2-(2-fluorophenyl)ethanone (R-95913), which is further converted to a pharmacologically active metabolite, 2-[1-2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-mercapto-3-piperidinylidene acetic acid (R-138727), by oxidation via cytochromes P450. In this study, we investigated how much the intestine and liver contribute to the formation of R-95913 and R-138727 after intraduodenal administration of prasugrel (1 mg/kg) to portal vein- and hepatic vein-cannulated dogs. The areas under the plasma concentration-time curve up to 2 h of R-95913 in the portal, hepatic, and systemic veins were 525, 32, and 17 ng · h/ml, respectively, and those of R-138727 were 564, 529, and 495 ng · h/ml, respectively. The dose of prasugrel was absorbed and then converted to R-95913 and R-138727 by 93 and 13%, respectively, in the intestine. In the liver, 23% of the R-95913, which passed through the intestine, was converted to R-138727. In conclusion, this is the first report to directly demonstrate that the conversion of prasugrel to R-138727 in the intestine is comparable to that converted in the liver of dogs.

Glutaredoxin and thioredoxin can be involved in producing the pharmacologically active metabolite of a thienopyridine antiplatelet agent, prasugrel.

A thienopyridine antiplatelet agent, prasugrel, is rapidly hydrolyzed to a thiolactone metabolite (R-95913, 2-[2-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl]-1-cyclopropyl-2-(2-fluorophenyl)ethanone). R-95913 is oxidized by hepatic cytochromes P450 to the pharmacologically active metabolite R-138727 (2-[1-2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-mercapto-3-piperidinylidene]acetic acid). One possible intermediate in the in vitro bioactivation pathway is a glutathione conjugate, R-133490, which could be reduced to generate R-138727 in the presence of a reducing agent such as glutathione. In this study, enzymes in human liver cytosols were found to accelerate reduction of R-133490 leading to the formation of R-138727. To explore the possible reductive enzymes, we separated the various proteins in human liver cytosol based on size using gel filtration chromatography. Two active peaks were detected and found to contain thioredoxin and glutaredoxin, respectively. In addition, recombinant human glutaredoxin and thioredoxin promoted the formation of R-138727 from R-133490 with much higher activity for glutaredoxin than for thioredoxin. This study is the first in vitro observation indicating that glutaredoxin and thioredoxin in human liver are active in reducing the mixed disulfide formed between xenobiotics and glutathione.

Biotransformation of prasugrel, a novel thienopyridine antiplatelet agent, to the pharmacologically active metabolite.

Prasugrel, a novel thienopyridine antiplatelet agent, undergoes rapid hydrolysis in vivo to a thiolactone, R-95913, which is further converted to its thiol-containing, pharmacologically active metabolite, R-138727, by oxidation via cytochromes P450 (P450). We trapped a sulfenic acid metabolite as a mixed disulfide with 2-nitro-5-thiobenzoic acid in an incubation mixture containing the thiolactone R-95913, expressed CYP3A4, and NADPH. Further experiments investigated one possible mechanism for the conversion of the sulfenic acid to the active thiol metabolite in vitro. A mixed disulfide form of R-138727 with glutathione was found to be a possible precursor of R-138727 in vitro when glutathione was present. The rate constant for the reduction of the glutathione conjugate of R-138727 to R-138727 was increased by addition of human liver cytosol to the human liver microsomes. Thus, one possible mechanism for the ultimate formation of R-138727 in vitro can be through formation of a sulfenic acid mediated by P450s followed possibly by a glutathione conjugation to a mixed disulfide and reduction of the disulfide to the active metabolite R-138727.

Identification of the human cytochrome P450 enzymes involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically active metabolite.

The aim of the current study is to identify the human cytochrome P450 (P450) isoforms involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically active metabolite. In the in vitro experiments using cDNA-expressed human P450 isoforms, clopidogrel was metabolized to 2-oxo-clopidogrel, the immediate precursor of its pharmacologically active metabolite. CYP1A2, CYP2B6, and CYP2C19 catalyzed this reaction. In the same system using 2-oxo-clopidogrel as the substrate, detection of the active metabolite of clopidogrel required the addition of glutathione to the system. CYP2B6, CYP2C9, CYP2C19, and CYP3A4 contributed to the production of the active metabolite. Secondly, the contribution of each P450 involved in both oxidative steps was estimated by using enzyme kinetic parameters. The contribution of CYP1A2, CYP2B6, and CYP2C19 to the formation of 2-oxo-clopidogrel was 35.8, 19.4, and 44.9%, respectively. The contribution of CYP2B6, CYP2C9, CYP2C19, and CYP3A4 to the formation of the active metabolite was 32.9, 6.76, 20.6, and 39.8%, respectively. In the inhibition studies with antibodies and selective chemical inhibitors to P450s, the outcomes obtained by inhibition studies were consistent with the results of P450 contributions in each oxidative step. These studies showed that CYP2C19 contributed substantially to both oxidative steps required in the formation of clopidogrel active metabolite and that CYP3A4 contributed substantially to the second oxidative step. These results help explain the role of genetic polymorphism of CYP2C19 and also the effect of potent CYP3A inhibitors on the pharmacokinetics and pharmacodynamics of clopidogrel in humans and on clinical outcomes.

A possible mechanism for the differences in efficiency and variability of active metabolite formation from thienopyridine antiplatelet agents, prasugrel and clopidogrel.

The efficiency and interindividual variability in bioactivation of prasugrel and clopidogrel were quantitatively compared and the mechanisms involved were elucidated using 20 individual human liver microsomes. Prasugrel and clopidogrel are converted to their thiol-containing active metabolites through corresponding thiolactone metabolites. The formation rate of clopidogrel active metabolite was much lower and more variable [0.164 + or - 0.196 microl/min/mg protein, coefficient of variation (CV) = 120%] compared with the formation of prasugrel active metabolite (8.68 + or - 6.64 microl/min/mg protein, CV = 76%). This result was most likely attributable to the less efficient and less consistent formation of clopidogrel thiolactone metabolite (2.24 + or - 1.00 microl/min/mg protein, CV = 45%) compared with the formation of prasugrel thiolactone metabolite (55.2 + or - 15.4 microl/min/mg protein, CV = 28%). These differences may be attributed to the following factors. Clopidogrel was largely hydrolyzed to an inactive acid metabolite (approximately 90% of total metabolites analyzed), and the clopidogrel concentrations consumed were correlated to human carboxylesterase 1 activity in each source of liver microsomes. In addition, 48% of the clopidogrel thiolactone metabolite formed was converted to an inactive thiolactone acid metabolite. The oxidation of clopidogrel to its thiolactone metabolite correlated with variable activities of CYP1A2, CYP2B6, and CYP2C19. In conclusion, the active metabolite of clopidogrel was formed with less efficiency and higher variability than that of prasugrel. This difference in thiolactone formation was attributed to hydrolysis of clopidogrel and its thiolactone metabolite to inactive acid metabolites and to variability in cytochrome P450-mediated oxidation of clopidogrel to its thiolactone metabolite, which may contribute to the poorer and more variable active metabolite formation for clopidogrel than prasugrel.

Mechanism-based inhibition of human cytochrome P450 2B6 by ticlopidine, clopidogrel, and the thiolactone metabolite of prasugrel.

Mechanism-based inhibition of CYP2B6 in human liver microsomes by thienopyridine antiplatelet agents ticlopidine and clopidogrel and the thiolactone metabolites of those two agents plus that of prasugrel were investigated by determining the time- and concentration-dependent inhibition of the activity of bupropion hydroxylase as the typical CYP2B6 activity. By comparing the ratios of k(inact) (maximal inactivation rate constant)/K(I) (the inactivator concentration producing a half-maximal rate of inactivation), it was found that the thiolactone metabolite of prasugrel is 10- and 22-fold less potent, respectively, in the mechanism-based inhibition of CYP2B6 than ticlopidine and clopidogrel. The k(inact)/K(I) ratio of the thiolactone metabolite of ticlopidine was comparable with that of the parent compound, whereas this ratio for the thiolactone metabolite of clopidogrel was significantly smaller than that of clopidogrel. In conclusion, ticlopidine, its thiolactone metabolite, and clopidogrel were more potent mechanism-based inhibitors of CYP2B6 than the thiolactone metabolite of prasugrel.

Comparison of human cytochrome P450 inhibition by the thienopyridines prasugrel, clopidogrel, and ticlopidine.

Differences in the inhibition of cytochrome P450 activities among thienopyridine antiplatelet agents, ticlopidine, clopidogrel, prasugrel, and the metabolites, 2-oxo-clopidogrel, clopidogrel acid metabolite, deacetylated metabolite of prasugrel (R-95913) and the pharmacologically active metabolites of clopidogrel and prasugrel, were examined using recombinant cytochromes P450 and fluorescent probe substrates. Ticlopidine and clopidogrel inhibited CYP2B6 with IC(50) values of 0.0517+/-0.0323 microM and 0.0182+/-0.0069 microM, respectively, and inhibited CYP2C19 with IC(50) values of 0.203+/-0.124 microM and 0.524+/-0.160 microM, respectively. Ticlopidine also inhibited CYP2D6 (IC(50) of 0.354+/-0.158 microM). In contrast, 2-oxo-clopidogrel, prasugrel and R-95913 were much weaker inhibitors of CYP2B6, CYP2C19 and CYP2D6. The inhibitory effects of all the compounds tested were much weaker on the isoforms other than those indicated above. The active metabolites of clopidogrel and prasugrel and clopidogrel acid metabolite also did not affect the activities of the P450s examined.