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The availability of assays that predict the contribution of cytochrome P450 (CYP) metabolism allows for the design of new chemical entities (NCEs) with minimal oxidative metabolism. These NCEs are often substrates of non-CYP drug-metabolizing enzymes (DMEs), such as UDP-glucuronosyltransferases (UGTs), sulfotransferases (SULTs), carboxylesterases (CESs), and aldehyde oxidase (AO). Nearly 30% of clinically approved drugs are metabolized by non-CYP enzymes. However, knowledge about the differential hepatic versus extrahepatic abundance of non-CYP DMEs is limited. In this study, we detected and quantified the protein abundance of eighteen non-CYP DMEs (AO, CES1 and 2, ten UGTs, and five SULTs) across five different human tissues. AO was most abundantly expressed in the liver and to a lesser extent in the kidney; however, it was not detected in the intestine, heart, or lung. CESs were ubiquitously expressed with CES1 being predominant in the liver, while CES2 was enriched in the small intestine. Consistent with the literature, UGT1A4, UGT2B4, and UGT2B15 demonstrated liver-specific expression, whereas UGT1A10 expression was specific to the intestine. UGT1A1 and UGT1A3 were expressed in both the liver and intestine; UGT1A9 was expressed in the liver and kidney; and UGT2B17 levels were significantly higher in the intestine than in the liver. All five SULTs were detected in the liver and intestine, and SULT1A1 and 1A3 were detected in the lung. Kidney abundance was the most variable among the studied tissues, and overall, high interindividual variability (15-fold) was observed for UGT2B17, CES2 (intestine), SULT1A1 (liver), UGT1A9, UGT2B7, and CES1 (kidney). These differential tissue abundance data can be integrated into physiologically based pharmacokinetic (PBPK) models for the prediction of non-CYP drug metabolism and toxicity in hepatic and extrahepatic tissues.

Current challenges in accurately predicting intestinal metabolism arise from the complex nature of the intestine, leading to limited applicability of available in vitro tools as well as knowledge deficits in intestinal physiology, including enzyme abundance. In particular, information on regional enzyme abundance along the small intestine is lacking, especially for non-cytochrome P450 enzymes such as carboxylesterases (CESs), UDP-glucuronosyltransferases (UGTs), and sulfotransferases (SULTs). We used cryopreserved human intestinal mucosa samples from nine donors as an in vitro surrogate model for the small intestine and performed liquid chromatography tandem mass spectrometry-based quantitative proteomics for 17 non-cytochrome P450 enzymes using stable isotope-labeled peptides. Relative protein quantification was done by normalization with enterocyte marker proteins, i.e., villin-1, sucrase isomaltase, and fatty acid binding protein 2, and absolute protein quantification is reported as picomoles per milligram of protein. Activity assays in glucuronidations and sequential metabolisms were conducted to validate the proteomics findings. Relative or absolute quantifications are reported for CES1, CES2, five UGTs, and four SULTs along the small intestine: duodenum, jejunum, and ileum for six donors and in 10 segments along the entire small intestine (A-J) for three donors. Relative quantification using marker proteins may be beneficial in further controlling for technical variabilities. Absolute quantification data will allow for scaling factor generation and in vivo extrapolation of intestinal clearance using physiologically based pharmacokinetic modeling. SIGNIFICANCE STATEMENT: Current knowledge gaps exist in intestinal protein abundance of non-cytochrome P450 enzymes. Here, we employ quantitative proteomics to measure non-cytochrome P450 enzymes along the human small intestine in nine donors using cryopreserved human intestinal mucosa samples. Absolute and relative abundances reported here will allow better scaling of intestinal clearance.

Remdesivir (RDV; GS-5734, Veklury), the first FDA-approved antiviral to treat COVID-19, is a single-diastereomer monophosphoramidate prodrug of an adenosine analogue. RDV is taken up in the target cells and metabolized in multiple steps to form the active nucleoside triphosphate (TP) (GS-443902), which, in turn, acts as a potent and selective inhibitor of multiple viral RNA polymerases. In this report, we profiled the key enzymes involved in the RDV metabolic pathway with multiple parallel approaches: (i) bioinformatic analysis of nucleoside/nucleotide metabolic enzyme mRNA expression using public human tissue and lung single-cell bulk mRNA sequence (RNA-seq) data sets, (ii) protein and mRNA quantification of enzymes in human lung tissue and primary lung cells, (iii) biochemical studies on the catalytic rate of key enzymes, (iv) effects of specific enzyme inhibitors on the GS-443902 formation, and (v) the effects of these inhibitors on RDV antiviral activity against SARS-CoV-2 in cell culture. Our data collectively demonstrated that carboxylesterase 1 (CES1) and cathepsin A (CatA) are enzymes involved in hydrolyzing RDV to its alanine intermediate MetX, which is further hydrolyzed to the monophosphate form by histidine triad nucleotide-binding protein 1 (HINT1). The monophosphate is then consecutively phosphorylated to diphosphate and triphosphate by cellular phosphotransferases. Our data support the hypothesis that the unique properties of RDV prodrug not only allow lung-specific accumulation critical for the treatment of respiratory viral infection such as COVID-19 but also enable efficient intracellular metabolism of RDV and its MetX to monophosphate and successive phosphorylation to form the active TP in disease-relevant cells.

Introduction Spleen tyrosine kinase (SYK) mediates signal transduction in multiple hematopoietic cells, including platelets. SYK signals downstream of immunoreceptors and SYK inhibition may ameliorate disease pathology in multiple autoimmune disorders; however, the impact of SYK inhibition in platelets and its potential relevance to bleeding is not fully understood. These studies evaluated the effect of an oral SYK inhibitor, GS-9876, on platelets in vitro and in vivo, and the impact of GS-9876 plus non-steroidal anti-inflammatory drugs (NSAIDs) on platelet aggregation. Material and methods The effect of GS-9876 on platelet activation, aggregation, and binding was characterized by western blotting, aggregometry, fluorescence-activated cell sorting, and microscopy techniques. The effect of GS-9876 on in vivo bleeding time (BT) was determined in cynomolgus monkeys and humans. Results GS-9876 inhibited glycoprotein VI (GPVI)-induced phosphorylation of linker for activation of T cells and phospholipase Cγ2, platelet activation and aggregation in human whole blood, and platelet binding to collagen under arterial flow. Ex vivo, GPVI-stimulated platelet aggregation was inhibited in GS-9876-treated monkeys without a concomitant increase in BT. Similarly, orally administered GS-9876 did not increase BT in humans. No in vitro additive effects on inhibition of platelet aggregation were observed with GS-9876 plus NSAIDs in human blood. Conclusions GS-9876 inhibited SYK activity in platelets via the GPVI receptor without prolonging BT in monkeys or humans. Furthermore, GS-9876 did not increase inhibition of platelet aggregation by NSAIDs in vitro, suggesting that these agents can potentially be combined without increasing bleeding risk in humans.

Cyclophilins are a family of peptidyl-prolyl isomerases that are implicated in a wide range of diseases including hepatitis C. Our aim was to discover through total synthesis an orally bioavailable, non-immunosuppressive cyclophilin (Cyp) inhibitor with potent anti-hepatitis C virus (HCV) activity that could serve as part of an all oral antiviral combination therapy. An initial lead 2 derived from the sanglifehrin A macrocycle was optimized using structure based design to produce a potent and orally bioavailable inhibitor 3. The macrocycle ring size was reduced by one atom, and an internal hydrogen bond drove improved permeability and drug-like properties. 3 demonstrates potent Cyp inhibition (Kd = 5 nM), potent anti-HCV 2a activity (EC50 = 98 nM), and high oral bioavailability in rat (100%) and dog (55%). The synthetic accessibility and properties of 3 support its potential as an anti-HCV agent and for interrogating the role of Cyp inhibition in a variety of diseases.

The pharmacokinetics and safety of velpatasvir, a potent pangenotypic hepatitis C virus NS5A inhibitor, were evaluated in two hepatic impairment studies: a phase I study in hepatitis C virus-uninfected subjects and a phase III study (ASTRAL-4) in hepatitis C virus-infected patients. In the phase I study, subjects with moderate or severe hepatic impairment (Child-Pugh-Turcotte Class B or C), and demographically matched subjects with normal hepatic function received a single dose of velpatasvir 100 mg. Pharmacokinetics and safety assessments were performed, and pharmacokinetic parameters were calculated using non-compartmental methods and summarized using descriptive statistics and compared statistically by geometric least-squares mean ratios and 90% confidence intervals. In ASTRAL-4, subjects with decompensated cirrhosis (Child-Pugh-Turcotte Class B) were randomized to receive treatment with either sofosbuvir/velpatasvir ± ribavirin for 12 weeks or sofosbuvir/velpatasvir for 24 weeks. Pharmacokinetic and safety assessments were performed and pharmacokinetic parameters were calculated using a non-compartmental analysis and summarized using descriptive statistics and were compared to pharmacokinetics from ASTRAL-1 [subjects without cirrhosis or with compensated (Child-Pugh-Turcotte Class A) cirrhosis]. In the phase I study, plasma exposures (area under the concentration–time curve) were similar in subjects with Child-Pugh-Turcotte Class B (n = 10) or Child-Pugh-Turcotte Class C hepatic impairment (n = 10) compared with normal hepatic function (n = 13). Percent free velpatasvir was similar in subjects without or with any degree of hepatic impairment. In the phase III study, velpatasvir overall exposure (area under the concentration–time curve over the 24-h dosing interval; AUCtau) was similar and sofosbuvir exposures were higher (~ 100%) for patients with Child-Pugh-Turcotte Class B hepatic impairment compared with the ASTRAL-1 population, which was not considered clinically relevant. No sofosbuvir/velpatasvir dose modification is warranted for patients with any degree of hepatic impairment.

Momelotinib (MMB), a small-molecule inhibitor of Janus kinase (JAK)1/2 and of activin A receptor type 1 (ACVR1), is in clinical development for the treatment of myeloproliferative neoplasms. The pharmacokinetics and disposition of [14C]MMB were characterized in a single-dose, human mass-balance study. Metabolism and the pharmacologic activity of key metabolites were elucidated in multiple in vitro and in vivo experiments. MMB was rapidly absorbed following oral dosing with approximately 97% of the radioactivity recovered, primarily in feces with urine as a secondary route. Mean blood-to-plasma [14C] area under the plasma concentration-time curve ratio was 0.72, suggesting low association of MMB and metabolites with blood cells. [14C]MMB-derived radioactivity was detectable in blood for ≤48 hours, suggesting no irreversible binding of MMB or its metabolites. The major circulating human metabolite, M21 (a morpholino lactam), is a potent inhibitor of JAK1/2 and ACVR1 in vitro. Estimation of pharmacological activity index suggests M21 contributes significantly to the pharmacological activity of MMB for the inhibition of both JAK1/2 and ACVR1. M21 was observed in disproportionately higher amounts in human plasma than in rat or dog, the rodent and nonrodent species used for the general nonclinical safety assessment of this molecule. This discrepancy was resolved with additional nonclinical studies wherein the circulating metabolites and drug-drug interactions were further characterized. The human metabolism of MMB was mediated primarily by multiple cytochrome P450 enzymes, whereas M21 formation involved initial P450 oxidation of the morpholine ring followed by metabolism via aldehyde oxidase.

[Image: see text] Spleen tyrosine kinase (SYK) is a critical regulator of signaling in a variety of immune cell types such as B-cells, monocytes, and macrophages. Accordingly, there have been numerous efforts to identify compounds that selectively inhibit SYK as a means to treat autoimmune and inflammatory diseases. We previously disclosed GS-9973 (entospletinib) as a selective SYK inhibitor that is under clinical evaluation in hematological malignancies. However, a BID dosing regimen and drug interaction with proton pump inhibitors (PPI) prevented development of entospletinib in inflammatory diseases. Herein, we report the discovery of a second-generation SYK inhibitor, GS-9876 (lanraplenib), which has human pharmacokinetic properties suitable for once-daily administration and is devoid of any interactions with PPI. Lanraplenib is currently under clinical evaluation in multiple autoimmune indications.

Background:Janus kinase (JAK) inhibitors (JAKinibs) show similar efficacy in rheumatoid arthritis (RA). However, in vitro studies have shown differences in JAK selectivity profiles for baricitinib (BARI), tofacitinib (TOFA), upadacitinib (UPA) and filgotinib (FIL).1,2 These lead to distinct pharmacologic profiles in cellular signaling assays that may impact clinical efficacy or safety1. NK cells are innate lymphocytes important in anti-pathogen responses and immune surveillance, which function via production of cytokines and cell killing3. NK cell proliferation and IFNγ production are JAK-dependent pathways and may be modulated by JAKinibs. Clinical findings show transient decreases in NK cell numbers in patients treated with JAKinibs, but the link to safety is unclear4Objectives:To extend upon findings in proximal cell signaling assays, we compared the selectivity and potency of clinical JAKinibs on NK cell function by assessing proliferation mediated by IL-15 (JAK1/3) and IFN-γ production driven by IL-12 (JAK2/TYK2)+IL-18.Methods:NK cells were isolated from healthy donor PBMC, incubated in vitro with 8 concentrations of each evaluated JAKinib (TOFA, BARI, FIL, FIL metabolite, UPA) and stimulated with IL-15 for proliferation or IL-12/18 for IFNγ production. Proliferation was assessed by Cell Trace dye dilution after 6 days and IFNγ production by intracellular flow cytometry 4hrs post-stimulation. Half maximal inhibitory concentration (IC50) values were calculated for CD56bright, CD56dim, and total NK cells. Steady-state pharmacologic profile over a clinical dosing interval was modeled using concentration-time profiles from JAKinib population pharmacokinetic data in RA subjects under the therapeutic dose5-7. For each JAKinib, the time above IC50 and average daily inhibition of IFNγ or proliferation were calculated for each NK cell population in each donor.Results:Cellular assays in purified NK cells showed dose-dependent inhibition of IL-15-induced proliferation by all JAKinibs with TOFA showing the highest average inhibition and time above IC50 (35-60% inhibition for 8-15 hrs; TOFA>UPA>BARI≈FIL). The differences between JAKinibs are in line with differences in pSTAT inhibition downstream of IL-151. Interestingly, IL-12/18-induced production of IFNγ, which is mediated via JAK2/TYK2 (IL-12) and non-JAK dependent pathways (IL-18), showed weaker inhibition for all compounds. Moreover, all JAKinibs showed 50 for IFNγ production or pSTAT4 inhibition at clinical doses. CD56dim and CD56bright sub-populations of NK cells are proposed to have distinct functions and unique expression of surface receptors. Analysis of the IC50 for pSTAT4 and IFNγ production showed ~2-10-fold weaker inhibition by JAKinibs in CD56bright NK cells, suggesting less dependence on JAK-dependent signals in CD56bright NK cells than CD56dim NK cells.Conclusion:NK cell proliferation depends on JAK1 and JAK3-mediated signaling and is differentially inhibited at clinical doses of distinct JAKinibs. In contrast, functional responses downstream of JAK2/TYK2-dependent IL-12/18 were not substantially inhibited by any of the JAKinibs studied. Inhibition of functional and proliferative responses in purified NK cells aligned well with proximal pSTAT inhibition. JAKinib modulation of NK cell proliferation, but not response to IL-12, reflects unique pharmacologic profiles of the drugs studied and could be one component underlying clinical safety observations, including increased risk of viral infections or malignancy4.References:[1]Traves PG et al. Ann Rheum Dis 2021 (in press)[2]McInnes IB, et al. Arthritis Res Ther 2019;21:183.[3]Cooper MA, Fehniger TA, Caligiuri MA. Trends Immunol 2001 Nov;22(11):633-40.[4]Winthrop KL. Nat Rev Rheumatol 2017; 13(4):234-243[5]Zhang X, et al. CPT Pharmacometrics Syst Pharmacol 2017;6(12):804-13.[6]CDER. Application Number: 203214Orig1s000. NDA 203214: Tofacitinib.[7]Klunder B et al. Clin Pharmacokinet 2019;58(8):1045-58.Disclosure of Interests:Paqui Gonzalez-Traves Shareholder of: Gilead Sciences, Employee of: Gilead Sciences, Laura Simpson Shareholder of: Gilead Sciences, Employee of: Gilead Sciences, Bernard Murray Shareholder of: Gilead Sciences, Employee of: Gilead Sciences, Amy Meng Shareholder of: Gilead Sciences, Employee of: Gilead Sciences, Julie A. Di Paolo Shareholder of: Gilead Sciences, Employee of: Gilead Sciences, Ethan Grant Shareholder of: Gilead Sciences, Employee of: Gilead Sciences, Gundula Min-Oo Shareholder of: Gilead Sciences, Employee of: Gilead Sciences

Late sodium current (late INa) is enhanced during ischemia by reactive oxygen species (ROS) modifying the Nav 1.5 channel, resulting in incomplete inactivation. Compound 4 (GS-6615, eleclazine) a novel, potent, and selective inhibitor of late INa, is currently in clinical development for treatment of long QT-3 syndrome (LQT-3), hypertrophic cardiomyopathy (HCM), and ventricular tachycardia-ventricular fibrillation (VT-VF). We will describe structure-activity relationship (SAR) leading to the discovery of 4 that is vastly improved from the first generation late INa inhibitor 1 (ranolazine). Compound 4 was 42 times more potent than 1 in reducing ischemic burden in vivo (S-T segment elevation, 15 min left anteriorior descending, LAD, occlusion in rabbits) with EC50 values of 190 and 8000 nM, respectively. Compound 4 represents a new class of potent late INa inhibitors that will be useful in delineating the role of inhibitors of this current in the treatment of patients.

Background:Janus kinase 1 (JAK1) inhibitors are efficacious in rheumatoid arthritis (RA). Despite having similar efficacy, in vitro studies have shown differences in JAK selectivity profiles for the small-molecule JAK inhibitors (JAKi) baricitinib (BARI), tofacitinib (TOFA), and upadacitinib (UPA).1For example, BARI and UPA are JAK1/JAK2 selective, while TOFA is JAK1/JAK3 selective, but each JAKi has some activity against other JAKs. As JAKs form signaling pairs, differences in selectivity could lead to distinct pharmacologic profiles that may impact clinical efficacy and safety.Objectives:As a first step to understand the basis of potential differences at therapeutic doses, we compared the selectivity and potency of filgotinib (FIL) and its major metabolite (MET) to those of BARI, TOFA, and UPA in cytokine-stimulated peripheral blood mononuclear cells (PBMCs) and whole blood (WB).Methods:PBMCs and WB from healthy donors were incubated in vitro with 8 doses of each JAKi, and levels of signal transducer and activator of transcription phosphorylation (pSTAT) were measured following cytokine stimulation. Half maximal inhibitory concentration (IC50) values were calculated in phenotypically sorted leukocyte populations by flow cytometry. Therapeutic dose relevance of the in vitro analyses was assessed using calculated mean concentration-time profiles from JAKi population pharmacokinetic data in RA subjects. For each JAKi, the time above IC50and average daily pSTAT inhibition were calculated for each cytokine/STAT pair in B cells, CD4+ T cells, CD8+ T cells, monocytes, and/or NK cells.Results:Cellular assays in PBMCs and WB showed dose-dependent inhibition of cytokine-induced pSTATs with all JAKi (correlation between the protein-adjusted IC50values from PBMCs and IC50values from WB, r2=0.98). Among the most potently inhibited pathways were JAK1/TYK2-dependent cytokine, interferon alpha (IFNα), and the JAK1/2-dependent cytokine, interleukin (IL)-6. FIL and MET had weaker potencies against JAK2/TYK2 (G-CSF/pSTAT3), JAK1/2 (IFNƴ/pSTAT1), and JAK2/2 (granulocyte-macrophage colony-stimulating factor [GM-CSF])-dependent pathways compared to JAK1/TYK2 (IFNα/pSTAT5). FIL and MET showed the greatest selectivity vs the JAK2/2 pathway (GM-CSF/pSTAT3) in monocytes.The mean concentration-time profiles and time above IC50over 24 hr for each cytokine/STAT pathway showed that JAK1/2 (IL-6/pSTAT1) and JAK1/TYK2 (IFNα/pSTAT1) pathways were strongly modulated with all tested JAKi. FIL (200 mg) showed similar activity in average target coverage and time above IC50to the approved low doses of TOFA (5 mg) and UPA (15 mg); conversely, FIL had reduced mean average inhibition and time above IC50levels against JAK1/2 (IFNƴ/pSTAT1), JAK1/3-dependent cytokines (IL-2, -4, and -15), JAK2/TYK2 (G-CSF/pSTAT3), and JAK2/2 (GM-CSF/pSTAT5)-dependent pathways compared to TOFA and UPA, and in certain cases to BARI (2 mg).Conclusion:Different JAKi modulate distinct cytokine pathways to varying degrees, and no agent potently and continuously inhibited an individual cytokine signaling pathway throughout the dosing interval. FIL (200 mg) showed a similar inhibition profile to TOFA, BARI, and UPA against the JAK1/TYK2- (IFNα/pSTAT1) or JAK1/2-dependent (IL-6/pSTAT1) responses, consistent with the role of these pathways in clinical efficacy.2However, FIL displayed a differentiated pharmacologic profile from the other JAKi, showing biologically reduced activity on the JAK1/2 (IFNγ)-, JAK1/3 (IL-2, -4 and -15)-, JAK2/TYK2 (G-CSF)-, and JAK2/2 (GM-CSF)-dependent pathways, which play important roles in hematopoiesis and immune function. These data suggest that FIL (200 mg) may have less impact on a subset of homeostatic immune functions signaling via JAK2 and JAK3 than those observed at the clinically approved doses of TOFA (5 mg and 10 mg), UPA (15 mg), and BARI (4 mg).References:[1]McInnes IB, et al. Arthritis Res Ther. 2019;21:183.[2]Banerjee S, et al. Drugs. 2017;77:521-546.Disclosure of Interests:Paqui Gonzalez-Traves Employee of: Gilead, Bernard Murray Employee of: Gilead, Federico Campigotto Employee of: Gilead, Amy Meng Shareholder of: Gilead Sciences, Employee of: Gilead, Julie A. Di Paolo Employee of: Gilead

Idelalisib, a potent phosphatidylinositol-3-kinase delta (PI3Kδ) inhibitor, is metabolized primarily by aldehyde oxidase to form GS-563117 and to a lesser extent by cytochrome P450 (CYP) 3A and uridine 5'-diphospho-glucuronosyltransferase 1A4. In vitro, idelalisib inhibits P-glycoprotein (P-gp) and organic anion transporting polypeptides 1B1 and 1B3, and GS-563117 is a time-dependent CYP3A inhibitor. This study enrolled 24 healthy subjects and evaluated (1) the effect of idelalisib on the pharmacokinetics (PK) of digoxin, a P-gp probe substrate, rosuvastatin, a breast cancer resistance protein, and OATP1B1/OATP1B3 substrate, and midazolam, a CYP3A substrate; and (2) the effect of a strong inducer, rifampin, on idelalisib PK. On treatment, the most common clinical adverse events (AEs) were headache and pyrexia. Grade 3 transaminase increases were observed in 5 of 24 subjects and were reversible. Two subjects had serious AEs after treatment completion (grade 3 pyrexia and/or drug-induced liver injury). Idelalisib coadministration did not affect digoxin and rosuvastatin PK. Coadministration with idelalisib increased plasma exposures of midazolam (138% and 437% for maximum observed plasma concentration [Cmax ] and area under the plasma concentration-time curve from time 0 extrapolated to infinity [AUCinf ], respectively), consistent with the in vitro finding of CYP3A inhibition by GS-563117. Rifampin caused a substantial decrease in idelalisib (58% and 75%, Cmax and AUCinf , respectively) and GS-563117 exposures, indicating an enhanced contribution of CYP3A to idelalisib metabolism under a strongly induced state.

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in young children. Presatovir (previously GS-5806) is a novel, orally administered RSV fusion inhibitor with a favorable safety profile and proven antiviral efficacy in preclinical and clinical studies. In vitro, presatovir is a substrate of the efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) and hepatic uptake transporters organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 and is slowly metabolized by cytochrome P450 (CYP) 3A4 and CYP3A5. This study enrolled 64 healthy subjects to evaluate the effect of cyclosporine, a P-gp, BCRP, and OATP1B1/1B3 inhibitor; rifampin, a strong CYP3A4 and P-gp inducer; efavirenz, a moderate CYP3A4 inducer; and cobicistat, a potent CYP3A inhibitor, on presatovir pharmacokinetics. Presatovir plasma exposures (maximum observed plasma concentration [Cmax ] and area under the plasma concentration-time curve from time 0 extrapolated to infinity [AUCinf ]) were not affected by coadministration of cyclosporine, suggesting presatovir is not a sensitive substrate of P-gp, BCRP, or OATP1B1/1B3. As expected, based on the role of CYP3A in presatovir metabolism, presatovir exposure was increased by cobicistat (122% in AUCinf ), and decreased by rifampin (40.3% in Cmax and 82.5% in AUCinf ) and efavirenz (55.7% in AUCinf ). These data support coadministration of presatovir with inhibitors of P-gp, BCRP, OATP1B1/1B3, or CYP3A, but not with moderate or strong CYP3A4 inducers. Presatovir was well-tolerated with the most common drug-related adverse events of dizziness (n = 12) and somnolence (n = 4) reported during efavirenz treatment.

We hereby disclose the discovery of inhibitors of CaMKII (7h and 7i) that are highly potent in rat ventricular myocytes, selective against hERG and other off-target kinases, while possessing good CaMKII tissue isoform selectivity (cardiac γ/δ vs. neuronal α/β). In vitro and in vivo ADME/PK studies demonstrated the suitability of these CaMKII inhibitors for PO (7h rat F = 73%) and IV pharmacological studies.

The experimental pharmacoenhancer cobicistat (COBI), a potent mechanism-based inhibitor of cytochrome P450 3A enzymes, was found to inhibit the intestinal efflux transporters P-glycoprotein and breast cancer resistance protein. Consistent with its transporter inhibition, COBI significantly increased the absorptive flux of potential candidates for clinical coadministration, including the HIV protease inhibitors atazanavir and darunavir and the lymphoid cell- and tissue-targeted prodrug of the nucleotide analog tenofovir, GS-7340, through monolayers of Caco-2 cells in vitro .

GS-8374 is a novel bis-tetrahydrofuran HIV-1 protease (PR) inhibitor (PI) with a unique diethylphosphonate moiety. It was selected from a series of analogs containing various di(alkyl)phosphonate substitutions connected via a linker to the para position of a P-1 phenyl ring. GS-8374 inhibits HIV-1 PR with high potency ( K i = 8.1 pM) and with no known effect on host proteases. Kinetic and thermodynamic analysis of GS-8374 binding to PR demonstrated an extremely slow off rate for the inhibitor and favorable contributions of both the enthalpic and entropic components to the total free binding energy. GS-8374 showed potent antiretroviral activity in T-cell lines, primary CD4 + T cells (50% effective concentration [EC 50 ] = 3.4 to 11.5 nM), and macrophages (EC 50 = 25.5 nM) and exhibited low cytotoxicity in multiple human cell types. The antiviral potency of GS-8374 was only moderately affected by human serum protein binding, and its combination with multiple approved antiretrovirals showed synergistic effects. When it was tested in a PhenoSense assay against a panel of 24 patient-derived viruses with high-level PI resistance, GS-8374 showed lower mean EC 50 s and lower fold resistance than any of the clinically approved PIs. Similar to other PIs, in vitro hepatic microsomal metabolism of GS-8374 was efficiently blocked by ritonavir, suggesting a potential for effective pharmacokinetic boosting in vivo . In summary, results from this broad in vitro pharmacological profiling indicate that GS-8374 is a promising candidate to be further assessed as a new antiretroviral agent with potential for clinical efficacy in both treatment-naïve and -experienced patients.

Katz and colleagues summarize knowledge on associations between drug pharmacokinetics and variations in genes coding for proteins involved in drug disposition. They propose a novel strategy in which pharmacogenetic data from early clinical studies is used both to feed back to furtherin vitrostudies of drug pharmacokinetics and to feed forward to optimize later-stage clinical trials, and discuss how this could improve drug development. In preclinical and early clinical drug development, information about the factors influencing drug disposition is used to predict drug interaction potential, estimate and understand population pharmacokinetic variability, and select doses for clinical trials. However, both in vitro drug metabolism studies and pharmacogenetic association studies on human pharmacokinetic parameters have focused on a limited subset of the proteins involved in drug disposition. Furthermore, there has been a one-way information flow, solely using results of in vitro studies to select candidate genes for pharmacogenetic studies. Here, we propose a two-way pharmacogenetic–pharmacokinetic strategy that exploits the dramatic recent expansion in knowledge of functional genetic variation in proteins that influence drug disposition, and discuss how it could improve drug development.

Nav1.8 (also known as PN3) is a tetrodotoxin-resistant (TTx-r) voltage-gated sodium channel (VGSC) that is highly expressed on small diameter sensory neurons and has been implicated in the pathophysiology of inflammatory and neuropathic pain. Recent studies using an Nav1.8 antisense oligonucleotide in an animal model of chronic pain indicated that selective blockade of Nav1.8 was analgesic and could provide effective analgesia with a reduction in the adverse events associated with nonselective VGSC blocking therapeutic agents. Herein, we describe the preparation and characterization of a series of 5-substituted 2-furfuramides, which are potent, voltage-dependent blockers (IC5010 nM) of the human Nav1.8 channel. Selected derivatives, such as 7 and 27, also blocked TTx-r sodium currents in rat dorsal root ganglia (DRG) neurons with comparable potency and displayed100-fold selectivity versus human sodium (Nav1.2, Nav1.5, Nav1.7) and human ether-a-go-go (hERG) channels. Following systemic administration, compounds 7 and 27 dose-dependently reduced neuropathic and inflammatory pain in experimental rodent models.

Cytochrome P450, CYP3A4, is the dominant human liver endoplasmic reticulum (ER) hemoprotein enzyme, responsible for the metabolism of over 60% of clinically relevant drugs. We have previously shown that mechanism-based suicide inactivation of CYP3A4 and its rat liver ER orthologs, CYPs 3A, via heme-modification of their protein moieties, results in their ubiquitin (Ub)-dependent 26S proteasomal degradation (Korsmeyer et al. (1999) Arch. Biochem. Biophys. 365, 31; Wang et al. (1999) Arch. Biochem. Biophys. 365, 45). This is not surprising given that the heme-modified CYP3A proteins are structurally damaged. To determine whether the turnover of the native enzyme similarly recruited this pathway, we heterologously expressed this protein in wild-type Saccharomyces cerevisiae and mutant strains (hrd1Delta, hrd2-1, and hrd3Delta) previously shown to be deficient in the Ub-dependent 26S proteasomal degradation of the polytopic ER protein 3-hydroxy-3-methylglutaryl-CoA reductase (isoform Hmg2p), the rate-limiting enzyme in sterol biosynthesis, as well as in strains deficient in ER-associated Ub-conjugating enzymes, Ubc6p and/or Ubc7p (Hampton et al. (1996) Mol. Biol. Cell 7, 2029; Hampton and Bhakta (1997) Proc. Natl. Acad. Sci. USA 94, 12,944). Our findings reveal that in common with the degradation of Hmg2p, that of native CYP3A4 also requires Hrd2p (a subunit of the 19S cap complex of the 26S proteasome) and Ubc7p, and to a much lesser extent Hrd3p, a component of the ER-associated Ub-ligase complex. In contrast to Hmg2p-degradation, that of native CYP3A4 does not appear to absolutely require Hrd1p, another component of the ER-associated Ub-ligase complex. Furthermore, studies in a S. cerevisiae pep4Delta strain proven to be deficient in the vacuolar degradation of carboxypeptidase Y indicated that CYP3A4 degradation is also largely independent of vacuolar (lysosomal) proteolytic function. The degradation of two other native ER proteins, Sec61p and Sec63p, normal components of the ER translocon, were also examined in parallel and found to be stabilized to some extent in HRD2- and UBC7-deficient strains. Together these findings attest to the remarkable mechanistic diversity in the normal degradation of ER proteins.

The HIV protease inhibitor (PI) ritonavir (RTV) has been widely used as a pharmacoenhancer for other PIs, which are substrates of cytochrome P450 3A (CYP3A). However the potent anti-HIV activity of ritonavir may limit its use as a pharmacoenhancer with other classes of anti-HIV agents. Ritonavir is also associated with limitations such as poor physicochemical properties. To address these issues a series of compounds with replacements at the P2 and/or P3 region was designed and evaluated as novel CYP3A inhibitors. Through these efforts, a potent and selective inhibitor of CYP3A, GS-9350 (cobicistat) with improved physiochemical properties was discovered.

Ritonavir (RTV), an HIV-1 protease inhibitor (PI), is also a potent mechanism-based inhibitor of human cytochrome P450 3A (CYP3A) and has been widely prescribed as a pharmacoenhancer. As a boosting agent for marketed PIs, it reduces pill burden, and improves compliance. Removal of the hydroxyl group from RTV reduces, but does not eliminate HIV PI activity and does not affect CYP3A inhibition. Herein we report the discovery of a novel series of CYP3A inhibitors that are devoid of antiviral activity. The synthesis and evaluation of analogs with extensive modifications of the 1,4-diamine core along with the structure activity relationships with respect to anti-HIV activity, CYP3A inhibitory activity, selectivity against other CYP enzymes and the human pregnane X receptor (PXR) will be discussed.

Investigation of thiophene-2-carboxylic acid HCV NS5B site II inhibitors, guided by measurement of cell culture medium binding, revealed the structure–activity relationships for intrinsic cellular potency. The pharmacokinetic profile was enhanced through incorporation of heterocyclic ethers on the N-alkyl substituent. Hydroxyl groups were incorporated to modulate protein binding. Intrinsic potency was further improved through enantiospecific introduction of an olefin in the N-acyl motif, resulting in the discovery of the phase 2 clinical candidate GS-9669. The unexpected activity of this compound against the clinically relevant NS5B M423T mutant, relative to the wild type, was shown to arise from both the N-alkyl substituent and the N-acyl group.

GS-9669 is a highly optimized thumb site II nonnucleoside inhibitor of the hepatitis C virus (HCV) RNA polymerase, with a binding affinity of 1.35 nM for the genotype (GT) 1b protein. It is a selective inhibitor of HCV RNA replication, with a mean 50% effective concentration (EC 50 ) of ≤11 nM in genotype 1 and 5 replicon assays, but lacks useful activity against genotypes 2 to 4. The M423T mutation is readily generated clinically upon monotherapy with the thumb site II inhibitors filibuvir and lomibuvir, and it is notable that GS-9669 exhibited only a 3-fold loss in potency against this variant in the genotype 1b replicon. Rather than M423T, resistance predominantly tracks to residues R422K and L419M and residue I482L in GT 1b and 1a replicons, respectively. GS-9669 exhibited at least additive activity in combination with agents encompassing four other direct modes of action (NS3 protease, NS5A, NS5B via an alternative allosteric binding site, and NS5B nucleotide) as well as with alpha interferon or ribavirin in replicon assays. It exhibited high metabolic stability in in vitro human liver microsomal assays, which, in combination with its pharmacokinetic profiles in rat, dog, and two monkey species, is predictive of good human pharmacokinetics. GS-9669 is well suited for combination with other orally active, direct-acting antiviral agents in the treatment of genotype 1 chronic HCV infection. (This study has been registered at ClinicalTrials.gov under registration number NCT01431898.)

1Heterocyclic amines are formed in parts per billion levels when meat is cooked. 2The heterocyclic amines MeIQx and PhIP are efficiently absorbed into the systemic circulation after ingestion of cooked food. 3We have shown that MeIQx and PhIP, both in vitro and in vivo, are substrates for human hepatic CYP1A2, which exclusively and efficiently catalyses their conversion to genotoxic hydroxylamines. 4MeIQx and PhIP are promutagens. MeIQx is a very powerful bacterial mutagen whereas PhIP is a more potent mammalian cell mutagen. Using a mammalian cell target gene, hprt, we have shown that PhIP induces a characteristic mutational ‘fingerprint’. 5MeIQx and PhIP are carcinogenic in bioassays. The PhIP mutational ‘fingerprint’ has been detected in the Apc gene of 5/8 colonic tumours induced by PhIP in rats.

Background Spleen Tyrosine Kinase (SYK) mediates signaling in a range of hematopoietic cells involved in the initiation and progression of RA including B cells, monocytes, macrophages, dendritic cells, and osteoclasts. There is strong preclinical validation for SYK as a therapeutic target for RA based on cellular data and animal models of disease. We have identified GS-9876 as a potent and selective SYK inhibitor with pharmaceutical properties compatible with once daily oral dosing in human. Objectives To characterize the cellular activity of GS-9876 on pathologically relevant pathways in RA, and establish target inhibition requireements for efficacy in rat models of arthritis. Methods The potency and selectivity of GS-9876 were characterized in biochemical and cellular assays. Effects in B cells were measured by inhibition of BCR-mediated protein phosphorylation, CD69 and CD86 expression, and in macrophages by inhibition of immune-complex (IC)-stimulated cytokine release. Cellular selectivity was demonstrated by comparing the inhibition of B cell proliferation versus T cell proliferation. GS-9876 potency in human blood was evaluated by inhibition of phosphorylated SYK (pSYK), CD63 expression on basophils, and CD69 expression on B cells. The in vivo efficacy of GS-9876 was tested in rat models of collagen-induced arthritis (CIA). Results GS-9876 is a potent SYK inhibitor (IC50=9.5±4.3 nM) and is highly selective against a panel of 456 other kinases. GS-9876 inhibited anti-IgM stimulated phosphorylation of AKT, BLNK, BTK, ERK, MEK, and PKCδ in human B cells with EC50 values of 24–51 nM. Functionally, GS-9876 inhibited anti-IgM mediated CD69 and CD86 expression on B-cells (EC50=112±10 nM and 164±15 nM, respectively) and anti-IgM /anti-CD40 co-stimulated B cell proliferation (EC50=108±55 nM). In human macrophages, GS-9876 inhibited IC-stimulated TNFα and IL-1β release (EC50=121±77 nM and 9±17 nM, respectively). Anti-CD3/anti-CD28 stimulated T cell proliferation was weakly inhibited (EC50=1291±398 nM), with selectivity >10-fold versus the inhibition of B cell proliferation. In human blood, GS-9876 blocked SYK phosphorylation, CD69 expression on B cells, and CD63 expression in basophils. GS-9876 demonstrated a dose-dependent improvement in clinical score and histopathology parameters with once-daily dosing in short and long term rat CIA models. Significant efficacy could be achieved with GS-9876 doses that produced trough pSYK inhibition of Conclusions GS-9876 is a novel SYK inhibitor that potently inhibits multiple cellular events implicated in RA pathogenesis and displays excellent in vivo efficacy in rat CIA models after once-daily dosing. GS-9876 has markedly improved selectivity over competitor SYK programs. Our data support the development of GS-9876 in inflammatory diseases, with potential for an improved safety profile. Disclosure of Interest J. Di Paolo Shareholder of: Gilead Sciences, P. Blomgren Shareholder of: Gilead Sciences, M. Dolton Shareholder of: Gilead Sciences, R. Jones Shareholder of: Gilead Sciences, J. Kropf Shareholder of: Gilead Sciences, T. Lee Shareholder of: Gilead Sciences, S. Mitchell Shareholder of: Gilead Sciences, B. Murray Shareholder of: Gilead Sciences, K. Suekawa-Pirrone Shareholder of: Gilead Sciences, S. Wise Shareholder of: Gilead Sciences, J. Xu Shareholder of: Gilead Sciences, Z. Zhao Shareholder of: Gilead Sciences, K. Currie Shareholder of: Gilead Sciences

An antibody was raised against a synthetic peptide (Ser-Glu-Asn-Tyr-Lys-Asp-Asn) corresponding to residues 290–296 of the cytochrome P450 enzyme, CYP1A2, of both rat and mouse. A cysteine residue attached to the N-terminus of the peptide during synthesis allowed coupling in a specific orientation via the thiol group to the carrier protein, keyhole limpet haemocyanin. Antiserum raised in rabbits bound specifically to CYP1A2 in the rat and mouse. To determine those amino acid residues involved in binding of the antibody, related peptides of various lengths were synthesised and the binding of the antibody was determined by an enzyme-linked immunosorbent assay. These studies show that the minimum epitope is the C-terminal tripeptide sequence, Lys-Asp-Asn. Other than in rat and mouse CYP1A2, this tripeptide is found as an internal sequence in a large number of proteins including bovine: fibronectin, chicken gizzard myosin heavy chain, and the P450 enzymes, rabbit CYP3A6 and human CYP3A4, but the antibody did not bind to any of these proteins. However, the antibody did bind to yeast glucose-6-phosphate dehydrogenase in which the tripeptide sequence is the C-terminus. Antibodies raised against a truncated peptide (Tyr-Lys-Asp-Asn), representing the C-terminal half of the peptide, also bound to glucose-6-phosphate dehydrogenase, but failed to bind to CYP1A2; thus although the C-terminal region of the peptide 290–296 is strongly immunogenic, it appears that it is not this population of antibodies that binds to CYP1A2. As antibodies were found to bind strongly to the C-terminus of glucose-6-phosphate dehydrogenase, the C-termini of proteins as targets for anti-peptide antibodies were investigated further by immunising rabbits with four 5-residue peptides which represent the C-termini of the P450 enzymes, CYP1A1, CYP1A2, CYP2E1 and CYP2A6. The peptides were coupled to keyhole limpet haemocyanin through their N-termini via cysteine residues added to the sequences. All four antisera bound specifically to their respective target proteins, as demonstrated by immunoblotting using hepatic microsomal fractions from rat, rabbit and human. It is suggested that this method of antibody production could be of general use for the reliable production of antisera against proteins where their sequence at the C-terminus is known, and such antibodies can be highly specific as they do not bind to internal sequences.

The activation of heterocyclic amines to mutagenic products by hepatic microsomal fractions from cynomolgus monkey, marmoset monkey and man was compared with the respective levels of cytochrome P450 enzymes CYP1A1 and CYP1A2. The rate of activation of 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to mutagens by hepatic microsomal fraction from cynomolgus monkey was very low. This was associated with a lack of constitutive expression of CYP1A1 and CYP1A2. In contrast, human hepatic microsomal fraction readily activates these heterocyclic amines and this is associated with constitutive expression of CYP1A2. Treatment of cynomolgus monkey with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes a very modest induction of CYP1A2, and a small increase in the activation of MeIQx and IQ. However, there was marked induction of CYP1A1 which was accompanied by > 10-fold increases in PhIP activation and 7-ethoxyresorufin O-deethylase (EROD), 7-methoxyresorufin O-demethylase (MROD) and aryl hydrocarbon hydroxylase activities. Following treatment of cynomolgus monkey with 3-methylcholanthrene, induction of CYP1A1, but not CYP1A2, was evident. In untreated marmoset monkey the activations of MeIQx and PhIP, as well as phenacetin O-deethylase, EROD, MROD and aryl hydrocarbon hydroxylase activities, are similar to those in man, although the activations of IQ and coumarin 7-hydroxylase activity are lower than in man. The presence of constitutive CYP1A2, and the absence of CYP1A1, in the liver of this species correspond to the situation in man. Treatment of marmoset monkey with TCDD results in increased CYP1A2 levels (4-fold), accompanied by proportional increases in the activation of MeIQx and IQ and phenacetin O-deethylase, EROD and MROD activities. The activation of PhIP is increased disproportionately, by 8-fold, most likely due to the activity of CYP1A1 which is also induced by TCDD in this species. Overall, the hepatic metabolism of heterocyclic amines by CYP1A enzymes in the untreated marmoset monkey resembles that in human more closely than that in the cynomolgus monkey. Therefore, marmoset monkey may be a more suitable model than the cynomolgus monkey for carcinogenicity studies involving MeIQx and PhIP, but not IQ.

A novel series of HCV replication inhibitors based on a pyrido[3,2-d]pyrimidine core were optimized for pharmacokinetics (PK) in rats. Several associations between physicochemical properties and PK were identified and exploited to guide the design of compounds. In addition, a simple new metric that may aid in the prediction of bioavailability for compounds with higher polar surface area is described (3*HBD-cLogP).

A monoclonal antibody, 12/2/3/2, which was raised against purified rat CYP1A1 recognises specifically rat and mouse CYP1A1 and CYP1A2, but not any cytochrome P-450 present in hepatic microsomal fractions from rabbit, guinea pig, hamster or human. By comparing the primary sequences of cytochromes P-450 to which 12/2/3/2 does and does hot bind, 10 possible locations for its epitope were found. Of these, one was extremely hydrophilic and, hence, predicted to be the most antigenic in the native protein. An antibody was produced against the synthetic peptide corresponding to this region (Gly-Arg-Asp-Arg-GlnPro-Arg-Leu: residues 356–363 and 350–357 of rat CYP1A1 and CYP1A2, respectively). The antibody bound to rat, mouse and hamster CYP1A1 and to rat and mouse CYP1A2, but did hot bind to any protein present in hepatic microsomal fractions from the rabbit, guinea pig or human. The binding of the anti-peptide antibody to CYP1A1 or CYP1A2 was partially antagonised by the monoclonal antibody. However, whereas the monoclonal antibody inhibited both CYP1A1- (aryl hydrocarbon hydroxylase) and CYP1A2-(high-affinity phenacetin O- deethylase ) dependent monooxygenase activity, the anti-peptide antibody was without effect on these activities. Antigen denaturation by 8 M urea or 0.05% (w/v) SDS had no effect on binding of the anti-peptide antibody to cytochrome P-450 , whilst binding of the monocional antibody was reduced by more than 1000-fold. The anti-peptide antibody partially antagonised the binding of 12/2/3/2 to urea-denatured but not native cytochrome P-450 . These data suggest that whilst the complete binding site for the monoclonal antibody is discontinuous, sufficient of the epitope is linear, so that when the antigen is denatured the monoclonal antibody is still able to bind and this binding is antagonised by the anti-peptide antibody. However, inhibition of catalytic activity by the monoclonal antibody must require binding to discontinuous residues.

Cobicistat (3, GS-9350) is a newly discovered, potent, and selective inhibitor of human cytochrome P450 3A (CYP3A) enzymes. In contrast to ritonavir, 3 is devoid of anti-HIV activity and is thus more suitable for use in boosting anti-HIV drugs without risking selection of potential drug-resistant HIV variants. Compound 3 shows reduced liability for drug interactions and may have potential improvements in tolerability over ritonavir. In addition, 3 has high aqueous solubility and can be readily coformulated with other agents.

Publisher Summary Compounds that inactivate cytochromes P450 in a selective manner require metabolism by the enzyme and are most commonly known as “mechanism-based inhibitors (MBIs)/inactivators,” but they may also be termed as “suicide inhibitors” or “time-dependent inhibitors (TDIs).” The metabolism of a substrate can lead to the inhibition of an enzyme in various ways. While it is theoretically possible for a cytochrome P450 substrate to be metabolized to a form that interacts more potently with the enzyme, there are no clear examples of such an agent. This is likely because the products of metabolism by a cytochrome P450 are generally less hydrophobic than the substrate, and therefore binds less strongly to the enzyme. In addition, recent advances in the understanding of MBIs of cytochromes P450 have provided a framework for the prediction of in vivo effects from in vitro data and have given insight into the biology of the turnover of the proteins. The potent, long-lasting effects of MBIs of enzymes such as CYP3A4 continue to be a liability for some drugs and a positive advantage for others, and therefore a thorough understanding of the mechanism and kinetics of inhibition is important.

A region of rat cytochrome P450IA1 at residues 294-301 (Gln-Asp-Arg-Arg-Leu-Asp-Glu-Asn), equivalent to a proinhibitory region of cytochrome P450IA2, was identified by sequence alignment. Anti-peptide antibodies were successfully raised when the peptide was coupled through either its N- or its C-terminus to carrier protein, but no antibodies were produced against the so-called multiple peptide antigen, which consisted of eight copies of the peptide attached through its C-terminus to a synthetic base. Both of the anti-peptide antibodies bound specifically to cytochrome P450IA1 in the rat, as shown by e.l.i.s.a. and immunoblotting. They inhibited microsomal aryl hydrocarbon hydroxylase activity and the mutagenic activation of 2-acetylaminofluorene (these reactions are catalysed by cytochrome P450IA1), but not high-affinity phenacetin O-de-ethylation activity, which is catalysed by cytochrome P450IA2. However, there was differences in the properties of the two antisera in their binding to cytochromes P450IA1 in species other than the rat, their relative binding to the multiple peptide antigen, the yield of antibody following affinity purification using peptide coupled through its N-terminus to CNBr-activated Sepharose, and the binding of the purified preparations to N- and C-terminal-coupled peptide conjugates. These observations indicated that the antibodies were directed to the region of the peptide opposite to the end which was coupled to the carrier protein. Nevertheless, both of the antibody preparations bound equally well to the target cytochrome P450, thus indicating that, in the native protein, the whole of the peptide region is exposed on the surface of cytochrome P450IA1 and is available for binding by the antibodies. The role of this region appears to be the same in both cytochromes P450IA1 and P450IA2, despite the difference in its primary structure in the two cytochromes P450.

Pharmacogenomic (PGx) research on the absorption, distribution, metabolism, and excretion (ADME) properties of drugs has begun to have impact for both drug development and utilization. To provide a cross-industry perspective on the utility of ADME PGx, the Pharmaceutical Research and Manufacturers of America (PhRMA) conducted a survey of major pharmaceutical companies on their PGx practices and applications during 2003-2005. This white paper summarizes and interprets the results of the survey, highlights the contributions and applications of PGx by industrial scientists as reflected by original research publications, and discusses changes in drug labels that improve drug utilization by inclusion of PGx information. In addition, the paper includes a brief review on the clinically relevant genetic variants of drug-metabolizing enzymes and transporters most relevant to the pharmaceutical industry.

An anti-peptide antibody has been produced which binds to and specifically inhibits the activity of cytochrome P-450IA2 in rat hepatic microsomes. This was achieved by raising an antibody against a synthetic peptide (Ser-Glu-Asn-Tyr-Lys-Asp-Asn), the sequence of which occurs in cytochrome P-450IA2 at positions 290-296. The selection of this region of cytochrome P-450IA2 was based on several criteria, including prediction of surface and loop areas, identification of variable regions between cytochromes P-450IA2 and P-450IA1, and consideration of a site on cytochrome P-450IA1 where chemical modification has been shown to cause substantial enzyme inactivation. The specificity of antibody binding was determined by enzyme-linked immunosorbent assay and by immunoblotting using hepatic microsomal preparations and purified cytochrome P-450 isoenzymes. This showed that the antibody binds specifically to rat and mouse cytochrome P-450IA2 and to no other cytochrome P-450, as was predicted from the amino acid sequences of the peptide and the cytochromes P-450. The effect of the antibody upon enzyme activity was studied in hepatic microsomes from rats treated with 3-methylcholanthrene. The antibody was shown to inhibit specifically the activity of reactions catalysed by cytochrome P-450IA2 (phenacetin O-de-ethylase and 2-acetylaminofluorene activation), but had no effect on aryl hydrocarbon hydroxylase activity, which is catalysed by cytochrome P-450IA1, or on aflatoxin B1 activation.

Mammalian hepatic cytochromes P450 (P450s) are endoplasmic reticulum (ER)-anchored hemoproteins with highly variable half-lives. CYP3A4, the dominant human liver drug-metabolizing enzyme, and its rat liver orthologs undergo ubiquitin (Ub)-dependent 26S proteasomal degradation after suicide inactivation or after heterologous expression in Saccharomyces cerevisiae. In contrast, rat liver CYP2C11 is degraded by the vacuolar "lysosomal" pathway when similarly expressed in yeast. The structural determinants that commit P450s to proteasomal or lysosomal degradation are unknown. To further validate S. cerevisiae as a model for exploring mammalian P450 turnover, the degradation of phenobarbital-inducible liver CYP2B1, an enzyme reportedly degraded via the rat hepatic autophagic-lysosomal pathway, was examined in a yeast strain (pep4delta) deficient in vacuolar degradation and its isogenic wild-type control (PEP4). Although CYP2B1 was equivalently expressed in both strains during early logarithmic growth, its degradation was retarded in pep4delta strain, remaining at a level 5-fold higher than that in PEP4 yeast when monitored at the stationary phase. No comparable CYP2B1 stabilization was detected in yeast genetically deficient in the ER Ub-conjugating enzyme Ubc6p or Ubc7p or defective in 19S proteasomal subunit Hrd2p. Thus, as in the rat liver, CYP2B1 is a target of vacuolar/lysosomal rather than proteasomal degradation in yeast, thereby further validating this model for mammalian P450 turnover. It is intriguing that a chimeric protein, CYP2B1-3A4CT, with the CYP3A4 C-terminal heptapeptide grafted onto the CYP2B1 C terminus, was proteasomally degraded after similar expression. Such diversion of CYP2B1 from its predominantly vacuolar degradation suggests that the CYP3A4 heptapeptide could either actively signal its proteasomal degradation or block its vacuolar proteolysis.

Cytochromes P450 (P450s) are hemoprotein enzymes committed to the metabolism of chemically diverse endo- and xenobiotics. They are anchored to the endoplasmic reticulum (ER) membrane with the bulk of their catalytic domain exposed to the cytosol, and thus they constitute excellent examples of integral monotopic ER proteins. Physiologically they are known to turn over asynchronously, but the determinants that trigger their proteolytic disposal and the pathways for such cellular disposal are not well defined. We recently showed that CYP3A4, the dominant human liver drug-metabolizing enzyme, and its rat liver orthologs undergo ubiquitin-dependent 26S proteasomal degradation not only after suicide inactivation, but also when CYP3A4 is expressed in Saccharomyces cerevisiae, presumably in its "native" form. The latter findings, obtained by the use of strains either with compromised proteasomal degradation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) or deficient in ubiquitin-conjugating enzymes (Ubc; UBC), revealed that this native monotopic P450 enzyme, in common with the polytopic HMGR, required the function of certain HRD (HMGR degradation) and UBC genes. In this study, we examined the degradation of CYP2C11, a male rat liver-specific P450, by heterologous expression in S. cerevisiae under comparable conditions. We report that unlike CYP3A4 and HMGR, the degradation of CYP2C11 in S. cerevisiae is independent of either HRD or UBC gene function, but it is largely dependent on vacuolar (lysosomal) proteolysis. These findings with two monotopic ER hemoproteins, CYP2C11 and CYP3A4, and the polytopic ER protein HMGR attest to the remarkable mechanistic diversity of cellular proteolytic disposal of ER proteins.

The liver cytosolic enzyme tryptophan 2,3-dioxygenase (TDO) catalyzes the oxidation of L-tryptophan to formylkynurenine and controls the physiological flux of tryptophan into both the serotonergic and kynureninic pathways. This hemoprotein enzyme is composed of four noncovalently bound subunits of equivalent mass and contains two heme moieties per molecule. Electron paramagnetic resonance analyses have indicated that a histidyl nitrogen is involved in heme ligation [Henry et al., (1976) J. Biol. Chem. 251, 1578], but the identity of the His residue(s) is unknown. In an attempt to characterize the active site of the enzyme we have substituted each of the 12 His residues in the rat TDO subunit with Ala, to determine their relative importance in heme binding. Sequence alignment of the rat liver protein with that of known or putative TDO sequences from other organisms reveals that four of the His residues are conserved in eukaryotes, two of which are also conserved in prokaryotes. Our findings indicate that replacement of the evolutionarily conserved His 76 and 328 residues resulted in a dramatic reduction of TDO activity, whereas that of the eukaryotically conserved His70 resulted in a significant reduction relative to that of the wild-type enzyme. On the other hand, replacement of the other eukaryotically conserved His273 residue, while affecting the relative expression of the enzyme, had little effect on its specific activity. Size-exclusion analyses revealed that the His76Ala and His328Ala mutants retained little or no heme, suggesting that these may be key residues in ligating the prosthetic heme moieties. Whether these His residues are both provided by the same TDO subunit or a different TDO subunit remains to be determined.

An anti-peptide antibody was raised against the sequence Thr-Gly-Ala-Leu-Phe-Lys-His-Ser-Glu-Asn-Tyr-Lys which occurs at positions 283–294 in the rat cytochrome P450 enzyme CYP1A2. Compared with its binding to the peptide used for immunization, the antibody bound with only slightly reduced affinity to the truncated peptides Thr-Gly-Ala-Leu-Phe-Lys-His-Ser and Leu-Phe-Lys-His-Ser. However, binding to the peptide Ser-Glu-Asn-Tyr-Lys-Asp-Asn, which overlaps with the C-terminal region of the immunizing peptide, was very low. Thus, a major epitope for the anti-peptide antibody is Leu-Phe-Lys-His-Ser, which corresponds to a region of CYP1A2 that is conserved in many species. The antibody was tested by immunoblotting for its ability to bind to hepatic microsomal fractions from a number of species. Where possible animals were treated with compounds which induce CYP1A2 and the results compared with those with untreated animals. It was found that the antibody bound to rat, mouse, rabbit, hamster, guinea pig, pig, marmoset monkey and human CYP1A2. No evidence was found for binding to dog CYP1A2. The region corresponding to the major epitope at residues 286–290 of rat CYP1A2 was identical in mouse, hamster, rabbit and human CYP1A2. The sequence of marmoset and guinea pig CYP1A2 are not known but are predicted to be very similar to the sequence in the rat. The lack of binding of the antibody to dog CYP1A2 may be explained by two differences in this region compared with rat CYP1A2. Maximum inhibition of CYP1A2 activity by this antibody, as measured by high-affinity phenacetin O-deethylase activity, was 20%. This is in contrast to a previously described anti-peptide antibody directed to an adjacent region which caused 65% inhibition of this activity. Thus, the edge of an inhibitory region on the surface of cytochrome P450 has been identified. The ability of the antibody to bind to CYP1A2 from a number of animals should make this antibody of use for studying the levels of CYP1A2 apoprotein in many species.

Mono-specific antibodies targeted to human CYP1A1 and CYP1A2 have been produced by immunizing rabbits with protein conjugates of short synthetic peptides corresponding to residues 290-297 and 284-296 respectively, of these enzymes. The antibody targeted to CYP1A1 bound in immunoblotting to the recombinant protein expressed in yeast but did not bind to any human hepatic microsomal protein, whereas the antibody targeted to CYP1A2 bound only to this enzyme in immunoblotting of human hepatic microsomal fractions and did not recognize recombinant human CYP1A1. The intensity of hepatic microsomal CYP1A2 immunoreactivity (n = 5) correlated significantly with a number of activities characteristic of this enzyme: phenacetin O-deethylase (POD), ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase activities and the ability to activate the dietary carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), to a mutagen. The anti-CYP1A2 anti-peptide antibody consistently inhibited both POD and EROD activities, but inhibition was incomplete (28%). In view of the known (> 90%) contribution of CYP1A2 to these activities and the correlation with antibody binding, this is consonant with an epitope for the anti-CYP1A2 anti-peptide antibody that forms the edge of a functionally important proinhibitory surface region previously identified in rat cytochromes CYP1A. CYP1A2 immunoreactivity determined by immunoblotting correlated significantly with the ability of human hepatic microsomal fractions to activate 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP), another dietary carcinogen, to a mutagen. It is concluded that CYP1A1 is absent from human liver and that CYP1A2 is likely to be a major catalyst in the hepatic activation of PhIP.