Effects of Fostamatinib on the Pharmacokinetics of Oral Contraceptive, Warfarin, and the Statins Rosuvastatin and Simvastatin: Results From Phase I Clinical Studies.

BACKGROUND AND OBJECTIVES: Fostamatinib is a spleen tyrosine kinase inhibitor that has been investigated as therapy for rheumatoid arthritis and immune thrombocytopenic purpura. The present studies assessed the potential for pharmacokinetic interaction between fostamatinib and the commonly prescribed medications oral contraceptive (OC), warfarin, and statins (rosuvastatin, simvastatin) in healthy subjects. METHODS: The OC study was a crossover study over two 28-day treatment periods (Microgynon(®) 30 plus placebo or fostamatinib). Concentrations of OC constituents (ethinyl estradiol/levonorgestrel) were measured. Effects on warfarin pharmacokinetics and pharmacodynamics were assessed (21-day study). Warfarin was administered on days 1 and 14, fostamatinib on days 8-20. The statin study was a two-period, fixed-sequence study of the effects of fostamatinib on exposure to rosuvastatin or simvastatin (single doses). Safety was assessed throughout. RESULTS: Fostamatinib co-administration with OC increased exposure to ethinyl estradiol [area under the plasma concentration-time curve at steady state (AUCss) 28% [confidence interval (CI 90%) 21-36]; maximum plasma concentration (Cmax) at steady state (Cmax,ss) 34% (CI 26-43)], but not levonorgestrel (AUCss 5%; Cmax,ss -3%), while exposure to luteinizing hormone and follicle-stimulating hormone decreased (≈ 20%). Fostamatinib did not affect the pharmacokinetics/pharmacodynamics of warfarin to a clinically relevant extent, but caused an upward trend in AUC for both R- and S-warfarin [18% (CI 13-23) and 13% (CI 7-19)]. Fostamatinib increased rosuvastatin AUC by 96% (CI 78-115) and Cmax by 88% (CI 69-110), and increased simvastatin acid AUC by 74% (CI 50-102) and Cmax by 83% (CI 57-113). CONCLUSION: Fostamatinib exhibits drug-drug interactions when co-administered with OC, simvastatin, or rosuvastatin, with the AUC of statins almost doubling. Fostamatinib did not exhibit a clinically relevant DDI on warfarin.

Validation of cell-based OATP1B1 assays to assess drug transport and the potential for drug-drug interaction to support regulatory submissions.

Transporters are carrier proteins that may influence pharmacokinetic, pharmacodynamic, and toxicological characteristics of drugs. The development of validated in vitro transporter models is imperative to support regulatory submissions of drug candidates. This study is focused on utilizing human embryonic kidney (HEK) 293 cell cultures genetically transfected with the human organic anion transporting polypeptides (OATP) 1B1 transporter to identify substrates and inhibitors in drug development. The kinetics of OATP1B1-mediated uptake of [(3)H]-oestradiol 17beta-glucuronide and inhibition of uptake by rifamycin SV were used to determine K(m), V(max), and IC(50) values over a range of passage numbers to investigate accuracy and precision. The mean K(m) and V(max) values were found to be 6.3 +/- 1.2 microM and 460 +/- 96 pmol min(-1) mg(-1), respectively. The mean IC(50) value for rifamycin SV was 0.23 +/- 0.07 microM on uptake of 1 microM [(3)H]-oestradiol 17beta-glucuronide. These data were similar to previously reported values (accuracy greater than 82%), reproducible (precision less than 29%) and exhibited low standard deviations (SDs) obviating the need to study test compounds on more than one occasion. [(3)H]-oestrone 3-sulfate and [(3)H]-pravastatin exhibited concentration-dependent OATP1B1 uptake, and statistically significant differences were observed at each concentration between uptake rates of HEK293-OATP1B1 and HEK293-MOCK cells (uptake ratios greater than or equal to 3). Propranolol showed no positive uptake ratio. Bezafibrate and gemfibrozil exhibited concentration-dependent inhibition of OATP1B1-mediated uptake of [(3)H]-oestradiol 17beta-glucuronide with mean IC(50) values of 16 and 27 microM, respectively. Based on the validation results, acceptance criteria to identify a test compound as a substrate and/or inhibitor using these specific cell lines were determined. These validated OATP1B1 assays were robust, reproducible, and suitable for routine in vitro evaluation of candidate drugs.

Validation and application of Caco-2 assays for the in vitro evaluation of development candidate drugs as substrates or inhibitors of P-glycoprotein to support regulatory submissions.

1. An understanding of the role that transporters, in particular P-glycoprotein (P-gp), can play in the absorption, distribution, metabolism and excretion (ADME) of candidate drugs, and an assessment of how these processes might impact on toxicity and the potential for drug-drug interactions in the clinic, is required to support drug development and registration. It is therefore necessary to validate preclinical assays for the in vitro evaluation of candidate drugs as substrates or inhibitors of human P-gp. 2. The present study has characterized a Caco-2 cell monolayer model by determining the bi-directional apparent permeabilities and efflux ratios of the known P-gp substrates ([(3)H]-digoxin, [(3)H]-ketoconazole, [(3)H]-verapamil, [(3)H]-quinidine, dipyridamole and loratidine; 1-100 microM) a non-substrate ([(3)H]-propranolol; 10 microM), or by determining the inhibitory potencies (IC(50)) of inhibitors (verapamil, ketoconazole, quinidine, dipyridamole and probenecid; 0.1-100 microM) on the basolateral-to-apical transport of [(3)H]-digoxin (5 microM), in order to validate methodologies for the identification of substrates or inhibitors of P-gp, respectively. 3. The reproducibility of the [(3)H]-digoxin or verapamil data determined from replicate monolayers across different cell passages indicates that the functional expression of P-gp is consistent across the range of passages (25-40) utilized for transport experiments and that the determination of bi-directional apparent permeability, or IC(50) for inhibition of P-gp, respectively, need only be performed on one occasion for a test compound. [(3)H]-digoxin and [(3)H]-propranolol or verapamil and probenecid were considered to be appropriate positive and negative controls of P-gp-mediated transport, or inhibition of P-gp, respectively, to ensure performance of the assays when assessing candidate drugs. Additionally, the low IC(50) values determined for ketoconazole and quinidine indicated that these inhibitors were suitable to use to confirm the role of P-gp in the efflux of a test compound. 4. These validated Caco-2 assays are robust, reproducible and suitable for routine in vitro evaluation of candidate drugs. They have been successfully applied to development projects resulting in the identification of two candidate drugs as substrates and inhibitors of P-gp, whereas a third was neither a substrate nor an inhibitor of this transporter.

Metabolism of 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin by human hepatic CYP isoforms: evidence for selectivity towards CYP3A4.

1. The metabolism of 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin (BFBFC) to 7-hydroxy-4-trifluoromethylcoumarin (HFC) was studied in human liver microsomes and in cDNA-expressed human liver CYP isoforms. For purposes of comparison, some limited studies were also performed with 7-benzyloxyquinoline (7BQ). 2. Initial interactive docking studies with a homology model of human CYP3A4 indicated that BFBFC was likely to be a selective substrate for CYP3A4 with a relatively high binding affinity, due to the presence of several key hydrogen bonds with active site amino acid residues. 3. Kinetic analysis of NADPH-dependent BFBFC metabolism to HFC in three preparations of pooled human liver microsomes revealed mean (+/- TSEM) Km and Vmax = 4.6 +/- 0.3 microM and 20.0 +/- 3.8 pmol/min/mg protein, respectively. 4. The metabolism of BFBFC to HFC was determined in a characterized bank of 24 individual human liver microsomal preparations employing a BFBFC substrate concentration of lO microM (i.e. around twice Km). Good correlations (r2 = 0.736-0.904) were observed between BFBFC metabolism and markers of CYP3A isoforms. 5. While 10O microM BFBFC was metabolized to HFC by cDNA-expressed CYP3A4, little or no metabolism was observed with cDNA-expressed CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. 6. The metabolism of 10 microM BFBFC in human liver microsomes was markedly inhibited by 5-50 microM troleandomycin and 0.2-5 microM ketoconazole, but stimulated by 0.2-10 microM alpha-naphthoflavone. The metabolism of 10 microM BFBFC in human liver microsomes was also markedly inhibited by an antibody to CYP3A4. 7. Kinetic analysis of NADPH-dependent 7BQ metabolism to 7-hydroxyquinoline (7HQ) in human liver microsomes revealed Km and Vmax = 70 microM and 3.39 nmol/min/mg protein, respectively. 8. While 80 microM 7BQ was metabolized to 7HQ by cDNA-expressed CYP3A4, only low rates of metabolism were observed with cDNA-expressed CYPIA2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. 9. In summary, by correlation analysis, the use of cDNA-expressed CYP isoforms, chemical inhibition and inhibitory antibodies, BFBFC metabolism in human liver microsomes appears to be primarily catalysed by CYP3A4. BFBFC may be a useful fluorescent probe substrate for human hepatic CYP3A4, but compared with 7BQ has only a low rate of metabolism in human liver microsomes.

Evaluation of 7-benzyloxy-4-trifluoromethylcoumarin, some other 7-hydroxy-4-trifluoromethylcoumarin derivatives and 7-benzyloxyquinoline as fluorescent substrates for rat hepatic cytochrome P450 enzymes.

1. The aim of this study was to evaluate a number of derivatives of 7-hydroxy-4-trifluoromethylcoumarin (HFC) and 7-benzyloxyquinoline (7BQ) as novel fluorescent substrates for monitoring rat hepatic cytochrome P450 (CYP) enzyme specificity in a 96- well plate format. The HFC derivatives examined comprised 7-benzyloxy-4-trifluoromethylcoumarin (BFC), 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin (BFBFC), 3,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin (BTBFC), 2-(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin (2TFBFC), 3-(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin (3TFBFC) and 3-(trifluoromethoxy)-7-benzyloxy-4-trifluoromethylcoumarin (3TFMeOBFC). 2. The CYP specificity of the fluorescent probe substrates was examined using characterized liver microsomes from male Sprague-Dawley rats treated with beta naphthoflavone (BNF), sodium phenobarbitone (NaPB), isoniazid, pregnenolone-16alpha-carbonitrile (PCN), dexamethasone (DEX) and methylclofenapate to induce CYP1A, CYP2B, CYP2E, CYP3A, CYP3A and CYP4A forms, respectively. Studies were also performed with microsomes from baculovirus-infected insect cells containing rat cDNA-expressed CYP1A1, CYP1A2, CYP2B1, CYP3A1 and CYP3A2. 3. BFC metabolism was most markedly induced by BNF and NaPB, whereas BFBFC metabolism was most markedly induced by PCN and DEX and BTBFC was not metabolized by rat liver microsomes. BFC was a high-affinity substrate for cDNA-expressed CYP1A1 and CYP2B1, whereas BFBFC exhibited a high affinity for CYP3A1 and CYP3A2. 4. The metabolism of 2TFBFC and 3TFBFC was induced by NaPB, PCN and DEX. 3TFBFC was a relatively specific substrate for cDNA-expressed CYP2B1, whereas 2TFBFC could be metabolized by CYP2B1, CYP3A1 and CYP3A2. 5. 3TFMeOBFC metabolism was markedly induced by BNF treatment and 3TFMeOBFC was extensively metabolized by cDNA-expressed CYP1A1. 6. The metabolism of 7BQ to 7-hydroxyquinoline was induced by treatment with PCN and DEX. 7BQ was a substrate for cDNA-expressed CYP3A2 and to a lesser extent for CYP3A1. 7. In summary, some of the HFC derivatives studied and 7BQ are useful fluorescent probe substrates for rat CYP enzymes. BFC appears to be a probe for CYP1A and CYP2B, 2TFBFC for CYP2B and CYP3A and 3TFBFC for CYP2B. While 3TFMeOBFC appears to be a relatively specific probe for CYP1A1, both BFBFC and 7BQ are good probes for the induction of CYP3A.

CYP isoform induction screening in 96-well plates: use of 7-benzyloxy-4-trifluoromethylcoumarin as a substrate for studies with rat hepatocytes.

1. In this study, 7-benzyloxy-4-trifluoromethylcoumarin (BFC) was evaluated as a substrate to assess the induction of cytochrome P450 (CYP) isoform enzyme activities in rat hepatocytes using a 96-well plate format. 2. BFC was metabolized by both untreated and sodium phenobarbitone (NaPB)-treated rat hepatocytes in a time- and concentration-dependent manner to the highly fluorescent product 7-hydroxy-4-trifluoromethylcoumarin (HFC). 3. HFC was extensively conjugated with D-glucuronic acid and/or sulphate in both untreated and NaPB-treated rat hepatocytes, thus necessitating the inclusion of an enzymatic deconjugation step in the assay procedure. 4. The time-course of induction of 7-ethoxyresorufin metabolism by the CYP1A inducer beta-naphthoflavone (BNF), 7-benzyloxyresorufin metabolism by the CYP2B inducer NaPB and BFC metabolism b both BNF and NaPB was studied in rat hepatocytes treated for 24-96 h. The optimal time for induction of metabolism of all three substrates was 72 h, with no medium changes being necessary during this period. 5. The effect of treatment with 0.5-20 microM BNF, 50-2000 microM NaPB, 2-20 microM dexamethasone (DEX), 20-100 microM methylclofenapate (MCP), and 50 and 200 microM isoniazid (ISN) for 72 h on BFC metabolism in cultured rat hepatocytes was studied. BFC metabolism was induced by treatment with BNF, NaPB and MCP, but not with either DEX or ISN. 6. The metabolism of BFC in liver microsomes from the control rat and rat treated with CYP isoform inducers was also studied. BFC metabolism was induced by treatment with NaPB, BNF and DEX. 7. The metabolism of BFC was also studied using microsomes from baculovirus-infected insect cells containing rat cDNA-expressed CYP1A, CYP2B, CYP2C and CYP3A isoforms. Whereas BFC was metabolized to some extent by all the rat cDNA-expressed CYP isoforms examined, at a substrate concentration of 2.5 microM the greatest rates of BFC metabolism were observed with the CYP1A1, CYP1A2 and CYP2B1 preparations. 8. In summary, the results demonstrate that BFC is a good substrate for assessing the induction of CYP1A and CYP2B isoforms in rat hepatocytes in a 96-well plate format.

Rapid determination of rat hepatocyte mRNA induction potential using oligonucleotide probes for CYP1A1, 1A2, 3A and 4A1.

1. A new assay to quantify mRNA levels in small numbers of rat hepatocytes has been developed for cytochrome P450 (CYP) isoforms 1A1, 1A2, 3A and 4A1. The assay uses sets of oligonucleotide probes end-labelled with [35S]-dATP to hybridize to mRNA in control- or drug-treated rat hepatocytes cultured on Cytostar-T 96-well scintillating microplates. 2. The rat hepatocyte induction potential (RHIP) assays for CYP3A, 1A1, 1A2 and 4A1 are sensitive and selective and have an excellent qualitative relationship with CYP induction data ex vivo. The robustness of the CYP3A assay was determined following a run of > 40 plates. The variation of the dexamethasone (DEX) response on each plate, calculated as %coefficient of variation, showed that there was no significant difference between the variability of the response to DEX. 3. Assay specificity for each CYP isoform was achieved by designing probes (four per isoform) antisense to coding regions of each CYP gene sequence. In the CYP3A RHIP assay, pregnenalone 16alpha-carbonitrile (PCN), DEX, clotrimazole (CLOT) and miconazole (MIC) were all good inducers of CYP3A mRNA; beta-napthoflavone (BNF) and methylclofenapate (MCP), however, did not induce CYP3A mRNA, further defining the specificity of this methodology. Specificity was similarly confirmed for the other CYP isoforms. 4. Ind50, the concentration of inducer required to elicit a 50% induction of CYP-specific mRNA, was derived for prototypical CYP inducers: BNF 0.54 and 0.17 microM (CYP1A1 and 1A2 respectively), 3-methylcholanthrene (3MC) 0.11 and 0.04 microM (CYP1A1 and 1A2 respectively), PCN 0.03 microM, DEX 0.17 microM, CLOT 0.48 microM, MIC 3 microM, TAO 3 microM (CYP3A), MCP 1.8 microM, clofibrate (CLOF) 65 microM and ciprofibrate (CIP) 1.9 microM (CYP4A1). Ind50 for BNF and 3MC at CYP1A2 was 3-fold lower than that at CYP1A1 indicating a subfamily difference in inducer potency. 5. Reducing the numbers of animals and the amount of compound required to study CYP induction is an important advantage of the RHIP assays over conventional evaluations in vivo. Typically four rats are dosed for 4 days using oral doses in the range 50-500 mg kg(-1) day(-1). In comparison, the amount of hepatocytes required to carry out all the studies reported herein may be obtained from a single animal (< 2 x 10(8) viable cells) and CYP induction investigated using microg rather than g quantities of drug substance. 6. With appropriately designed oligonucleotide probes, the RHIP technology can assess CYP induction in human hepatocytes, which together with preclinical data can contribute to improving the quality of compounds progressing into the expensive process of drug development.

Metabolism of 7-benzyloxy-4-trifluoromethyl-coumarin by human hepatic cytochrome P450 isoforms.

1. The metabolism of 7-benzyloxy-4-trifluoromethylcoumarin (BFC) to 7-hydroxy-4-trifluoromethylcoumarin (HFC) was studied in human liver microsomal preparations and in cDNA-expressed human cytochrome P450 (CYP) isoforms. 2. Kinetic analysis of the NADPH-dependent metabolism of BFC to HFC in four preparations of pooled human liver microsomes revealed mean (+/- SEM) Km and Vmax of 8.3 +/- 1.3 microM and 454 +/- 98 pmol/min/mg protein respectively. 3. The metabolism of BFC to HFC was determined in a characterized bank of 24 individual human liver microsomal preparations employing BFC substrate concentrations of 20 and 50 microM (i.e. about two and six times Km respectively). With 20 microM BFC the highest correlations were observed between BFC metabolism and markers of CYP1A2 (r2 = 0.784-0.797) and then with CYP3A (r2 = 0.434-0.547) isoforms, whereas with 50 microM BFC the highest correlations were observed between BFC metabolism and markers of CYP3A (r2 = 0.679-0.837) and then with CYP1A2 (r2 = 0.421-0.427) isoforms. At both BFC substrate concentrations, lower correlations were observed between BFC metabolism and enzymatic markers for CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP4A9/11. 4. Using human beta-lymphoblastoid cell microsomes containing cDNA-expressed CYP isoforms, 20 microM BFC was metabolized by CYP1A2 and CYP3A4, with lower rates of metabolism being observed with CYP2C9 and CYP2C19. Kinetic studies with the CYP1A2 and CYP3A4 preparations demonstrated a lower Km with the CYP1A2 preparation, but a higher Vmax with the CYP3A4 preparation. 5. The metabolism of 20 microM BFC in human liver microsomes was inhibited to 37-48% of control by 5-100 microM of the mechanism-based CYP1A2 inhibitor furafylline and to 64-69% of control by 5-100 microM of the mechanism-based CYP3A4 inhibitor troleandomycin. While some inhibition of BFC metabolism was observed in the presence of 100 and 200 microM diethyldithiocarbamate, the addition of 2-50 microM sulphaphenazole, 50-500 microm S-mephenytoin and 2-50 microM quinidine had little effect. 6. The metabolism of 20 microM BFC to HFC in human liver microsomes was also inhibited by an antibody to CYP3A4, whereas antibodies to CYP2C8/9 and CYP2D6 had no effect. 7. In summary, by correlation analysis, use of cDNA-expressed CYP isoforms, chemical inhibition and inhibitory antibodies, BFC appears metabolized by a number of CYP isoforms in human liver. BFC metabolism appears to be primarily catalysed by CYP1A2 and CYP3A4, with possibly some contribution by CYP2C9, CYP2C19 and perhaps other CYP isoforms. 8. The results also demonstrate the importance of the selection of an appropriate substrate concentration when conducting reaction phenotyping studies with human hepatic CYP isoforms.

High throughput ribonuclease protection assay for the determination of CYP3A mRNA induction in cultured rat hepatocytes.

1. A rapid 96-well plate based method for the determination of CYP3A mRNA induction in primary rat hepatocytes has been developed which has substantial advantages over current technologies including the ability to test the effect of relatively large numbers of new chemical entities on the expression of CYP3A mRNA in hepatocytes. 2. The ribonuclease protection assay detects changes in mRNA levels in small numbers of hepatocytes by the utilization of a radiolabelled antisense riboprobe that will hybridize CYP3A1 and CYP3A23. Using in situ hybridization techniques in conjunction with Amersham 96-well Cytostar-T scintillating microplates, there is no need for isolation of mRNA. A simple ribonuclease digestion step allows quantitative data to be generated easily within 1 week of hepatocyte isolation. 3. Rat hepatocytes were cultured for 48 h post-isolation on the Cytostar plates coated with a basal matrix of Matrigel. Prototypical CYP3A inducers (dexamethasone and pregnenolone 16alpha-carbonitrile) have been studied using various treatment periods from 0.5 to 24 h. Methylclofenapate and beta-naphthoflavone, prototypical inducers of CYP4A and CYP1A respectively, have been used as controls to show specificity of the [33P]-labelled riboprobe for the CYP3A family. 4. Time-dependent increases in CYP3A mRNA were demonstrated following exposure of hepatocytes to prototypical CYP3A inducers, but not for methylclofenapate or beta-naphthoflavone, so demonstrating specificity for CYP3A mRNA over CYP1A and CYP4A. Analysis of the 24-h induction data demonstrates that significant differences from controls can be determined and that induction potential can be assessed. The system has the potential to screen for overall CYP3A mRNA induction in response to compounds at an early stage in drug research.

Comparison of the effects of some CYP3A and other enzyme inducers on replicative DNA synthesis and cytochrome P450 isoforms in rat liver.

The aim of this study was to investigate the mitogenic effects of some inducers of cytochrome P450 (CYP) isoforms in rat liver. Female Sprague-Dawley CD rats were treated with 100 mg/kg per day of either sodium phenobarbitone (NaPB), barbituric acid (BA), isoniazid (ISN), beta-naphthoflavone (BNF), pregnenolone-16alpha-carbonitrile (PCN), miconazole (MIC) or clotrimazole (CLOT), 75 mg/kg per day methylclofenapate (MCP), 50 mg/kg per day dexamethasone (DEX) and 500 mg/kg per day troleandomycin (TAO) by daily oral gavage for four days. Treatment with all compounds except BA, ISN and MIC, significantly increased relative liver weight. Administration of NaPB, PCN, DEX, MIC, CLOT and TAO all induced total CYP content, and by Western immunoblotting, levels of CYP3A isoforms in hepatic microsomal fractions. Apart from CLOT, all these compounds induced microsomal testosterone 6beta-hydroxylase activity. By measurement of marker enzyme activities and Western immunoblotting with antibodies to CYP1A2, CYP2B1/2 and CYP2E1, BNF, NaPB, ISN and MCP were shown to induce CYP1A2, CYP2B1/2, CYP2E and CYP4A isoforms, respectively. Replicative DNA synthesis was studied by implanting osmotic pumps containing 5-bromo-2'-deoxyuridine 1 day before the commencement of treatment with the enzyme inducers. Hepatocyte labelling index values were significantly increased by treatment with NaPB, PCN, MCP, CLOT and TAO, but not by BA, ISN, BNF, DEX and MIC. These studies demonstrate that while CYP2B and CYP4A enzyme inducers may stimulate replicative DNA synthesis, only some CYP3A enzyme inducers are mitogenic agents in rat liver.