Cynomolgus monkey as a surrogate for human aldehyde oxidase metabolism of the EGFR inhibitor BIBX1382.

BIBX1382 was an epidermal growth factor receptor inhibitor under clinical investigation for treatment of cancer. This candidate possessed an attractive preclinical absorption, distribution, metabolism, and excretion profile, yet failed in clinical studies due in part to poor oral exposure, resulting from extensive metabolism by aldehyde oxidase (AO). In vitro metabolism studies were performed in liver cytosol and cryopreserved hepatocytes from multiple species. In addition, a pharmacokinetic study was performed in cynomolgus monkey for comparison with the reported human pharmacokinetics of BIBX1382. Estimated hepatic clearance of BIBX1382 in rhesus (42 ml/min per kg) and cynomolgus monkey (43 ml/min per kg) liver cytosol was comparable to human (≥93% of liver blood flow). Metabolite identification after incubation of BIBX1382 in liver cytosol fortified with the AO inhibitor raloxifene confirmed that AO is involved in the formation of the predominant metabolite (BIBU1476, M1) in cynomolgus monkey. After intravenous and oral administration of BIBX1382 to cynomolgus monkeys, high plasma clearance (118 ml/min per kg) and low oral exposure (C(max) = 12.7 nM and 6% oral bioavailability) was observed, with the exposure of M1 exceeding BIBX1382 after oral dosing. This pharmacokinetic profile compared favorably with the human clinical data of BIBX1382 (plasma clearance 25-55 ml/min per kg and 5% oral bioavailability). Thus, it appears that cynomolgus monkey represents a suitable surrogate for the observed human AO metabolism of BIBX1382. To circumvent clinical failures due to uncharacterized metabolism by AO, in vitro studies in the appropriate subcellular fraction, followed by pharmacokinetic and toxicokinetic studies in the appropriately characterized surrogate species should be conducted for substrates of AO.

Aldehyde oxidase activity in donor-matched fresh and cryopreserved human hepatocytes and assessment of variability in 75 donors.

Studies were conducted to evaluate the impact of time and cryopreservation on aldehyde oxidase (AO) activity in human hepatocytes isolated from 10 donor livers, using O(6)-benzylguanine as a probe substrate. In addition, variability in activity was assessed using cryopreserved hepatocytes from 75 donors. Substantial donor-dependent loss in AO activity within 24 hours after isolation of hepatocytes was observed (average loss of 42%, range 15%-81%). Meanwhile, AO activity in cryopreserved hepatocytes more closely represented the activity observed in fresh hepatocytes that were incubated immediately after isolation for the same donors (within 81% of fresh, range 48%-100%). Activity of AO in cryopreserved hepatocytes from 75 donors varied by at least 17-fold (≤ 5.4 to 90 ml/minute per kilogram of body weight), with 63% of the donors having higher activity than a pooled 19-donor lot (34.2 ml/minute per kilogram). Comparison of demographics such as gender, body mass index, age, and ethnicity showed no statistically significant correlations with activity. Evaluation of medical histories revealed that three of the five donors with no measurable activity had immediate histories of extensive alcohol abuse. Meanwhile, two single nucleotide polymorphisms (SNPs) for AOX1 (rs3731772 and rs55754655) were detected in our donor pool and showed allelic frequencies similar to those reported from other cohort studies. However, these SNPs did not correlate with a statistically significant difference in intrinsic clearance compared with wild-type donors. With a general lack of clarity about what causes highly variable AO activity, prescreening donors for AO activity and creating a custom high-activity pooled lot of cryopreserved hepatocytes are advised to minimize underpredictions of clearance.

Characterization of aldehyde oxidase enzyme activity in cryopreserved human hepatocytes.

Substrates of aldehyde oxidase (AO), for which human clinical pharmacokinetics are reported, were selected and evaluated in pooled mixed-gender cryopreserved human hepatocytes in an effort to quantitatively characterize AO activity. Estimated hepatic clearance (Cl(h)) for BIBX1382, carbazeran, O6-benzylguanine, zaleplon, and XK-469 using cryopreserved hepatocytes was 18, 17, 12, <4.3, and <4.3 ml · min⁻¹ · kg⁻¹, respectively. The observed metabolic clearance in cryopreserved hepatocytes was confirmed to be a result of AO-mediated metabolism via two approaches. Metabolite identification after incubations in the presence of H2¹8O confirmed that the predominant oxidative metabolite was generated by AO, as expected isotope patterns in mass spectra were observed after analysis by high-resolution mass spectrometry. Second, clearance values were efficiently attenuated upon coincubation with hydralazine, an inhibitor of AO. The low exposure after oral doses of BIBX1382 and carbazeran (∼5% F) would have been fairly well predicted using simple hepatic extraction (f(h)) values derived from cryopreserved hepatocytes. In addition, the estimated hepatic clearance value for O6-benzylguanine was within ∼80% of the observed total clearance in humans after intravenous administration (15 ml · min⁻¹ · kg⁻¹), indicating a reasonable level of quantitative activity from this in vitro system. However, a 3.5-fold underprediction of total clearance was observed for zaleplon, despite the 5-oxo metabolite being clearly observed. These data taken together suggest that the use of cryopreserved hepatocytes may be a practical approach for assessing AO-mediated metabolism in discovery and potentially useful for predicting hepatic clearance of AO substrates.

Differential modulation of cytochrome P450 activity and the effect of 1-aminobenzotriazole on hepatic transport in sandwich-cultured human hepatocytes.

Sandwich-cultured human hepatocytes (SCHH) have been widely used for in vitro assessments of biliary clearance. However, the modulation of metabolism enzymes has not been fully evaluated in this system. The present study was therefore undertaken to determine the activity of cytochrome P450 (P450) 1A2, 2C8, 2C9, 2C19, 2D6, and 3A and to evaluate the impact of 1-aminobenzotriazole (ABT) on hepatic uptake and biliary excretion in SCHH. The SCHH maintained integrity and viability as determined by lactate dehydrogenase release and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assays conducted over the culture period. Although all assessed P450 activity decreased in day 2 SCHH, the extent of the decrease and the subsequent rebound in activity varied across the different isoforms. Day 5 CYP1A2 activity was approximately 2.5-fold higher than day 1 activity, whereas the CYP3A and CYP2C9 activities were 90 and 60% of the day 1 levels, respectively. In contrast, the initial CYP2C8, CYP2C19, and CYP2D6 activity losses did not rebound over the 5-day culture period. Furthermore, ABT was not found to have an effect, whether directly or indirectly as a P450 inactivator, with respect to the hepatic transport of rosuvastatin, atrovastatin, and midazolam in SCHH. Taken together, these results suggest that the SCHH model is a reliable tool to characterize hepatic uptake and biliary excretion. Due to the differential modulation of P450 activity, SCHH may not be considered a suitable tool for metabolic stability assessments with compounds predominantly cleared by certain P450 enzymes.

Discovery of novel spirocyclic inhibitors of fatty acid amide hydrolase (FAAH). Part 2. Discovery of 7-azaspiro[3.5]nonane urea PF-04862853, an orally efficacious inhibitor of fatty acid amide hydrolase (FAAH) for pain.

Fatty acid amide hydrolase (FAAH) is an integral membrane serine hydrolase responsible for the degradation of fatty acid amide signaling molecules such as endocannabinoid anandamide (AEA), which has been shown to possess cannabinoid-like analgesic properties. Herein we report the optimization of spirocyclic 7-azaspiro[3.5]nonane and 1-oxa-8-azaspiro[4.5]decane urea covalent inhibitors of FAAH. Using an iterative design and optimization strategy, lead compounds were identified with a remarkable reduction in molecular weight and favorable CNS drug like properties. 3,4-Dimethylisoxazole and 1-methyltetrazole were identified as superior urea moieties for this inhibitor class. A dual purpose in vivo efficacy and pharmacokinetic screen was designed to be the key decision enabling experiment affording the ability to move quickly from compound synthesis to selection of preclinical candidates. On the basis of the remarkable potency, selectivity, pharmacokinetic properties and in vivo efficacy, PF-04862853 (15p) was advanced as a clinical candidate.

A humanized UGT1 mouse model expressing the UGT1A1*28 allele for assessing drug clearance by UGT1A1-dependent glucuronidation.

Humanized mice that express the human UDP-glucuronosyltransferase (UGT) 1 locus have been developed in a Ugt1-null background as a model to improve predictions of human UGT1A-dependent drug clearance. Enzyme kinetic parameters (K(m) and V(max)) and pharmacokinetic properties of three probe drugs were compared using wild-type and humanized UGT1 mice that express the Gilbert's UGT1A1*28 allele [Tg(UGT1(A1*28)) Ugt1(-/-) mice]. The well characterized substrate for UGT1A1, 7-ethyl-10-hydroxy-camptothecin (SN-38), showed the greatest difference in parent drug exposure ( approximately 3-fold increase) and clearance ( approximately 3-fold decrease) in Tg(UGT1(A1*28)) Ugt1(-/-) mice after intravenous administration compared with wild-type and phenobarbital-treated animals. In contrast, the clearance of the UGT2B7 substrate (-)-17-allyl-4, 5alpha-epoxy-3, 14-dihydroxymorphinan-6-one (naloxone) was not altered in Tg(UGT1(A1*28)) Ugt1(-/-) mice. In addition, pharmacokinetic parameters with 1-(4-fluorophenyl)3(R)-[3-(4-fluorophenyl)-3(S)-hydroxypropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone (ezetimibe, Zetia; Merck & Co., Whitehouse Station, NJ), considered to be a major substrate for UGT1A1, showed small to no dependence on UGT1A1-directed glucuronidation. Enzyme kinetic parameters assessed for SN-38, ezetimibe, and naloxone using liver microsomes prepared from wild-type and Tg(UGT1(A1*28)) Ugt1(-/-) mice showed patterns consistent with the in vivo pharmacokinetic data. For SN-38 glucuronidation, V(max) decreased 5-fold in Tg(UGT1(A1*28)) Ugt1(-/-) mouse liver microsomes compared with microsomes prepared from wild-type mice, and decreased 10-fold compared with phenobarbital-treated Tg(UGT1(A1*28)) Ugt1(-/-) mice. These differences are consistent with SN-38 glucuronidation activities using HLMs isolated from individuals genotyped as UGT1A1*1 or UGT1A1*28. For ezetimibe and naloxone the differences in V(max) were minimal. Thus, Tg(UGT1(A1*28)) Ugt1(-/-) mice can serve as a pharmacokinetic model to further investigate the effects of UGT1A1 expression on drug metabolism.

Effect of compound plate composition on measurement of hERG current IC(50) using PatchXpress.

Inhibition of the human ether-a-go-go-related gene (hERG) potassium channel by pharmaceutical agents can lead to acquired long QT syndrome and the generation of potentially lethal arrhythmias. Higher throughput automated patch clamp systems, such as PatchXpress, can greatly increase the speed and capacity of evaluation of pharmaceutical compounds for hERG blocking activity. A factor that may affect the IC(50) value of a compound measured in this system is the composition of the multi-well compound plate. Hydrophobic compounds may adsorb to the surfaces of multi-well plates resulting in a reduction in the effective concentration of the compound delivered to the cell and altered IC(50) values. In the present study, we investigated the effects of four different compound plates--glass vials, non-binding polystyrene, hydrophilic polystyrene, and polystyrene--on determination of IC(50)s for four compounds--sotalol, dofetilide, cisapride, and bepridil--which ranged in hydrophobicity. In addition, we investigated the effects of incubation time in the compound plate on determination of IC(50)s. hERG currents were measured using the PatchXpress 7000A Automated Parallel Patch Clamp System (Molecular Devices Corporation; Sunnyvale, CA) and hERG channels stably expressed in HEK293 cells. The results suggest that more hydrophobic compounds may adsorb to non-binding polystyrene, hydrophilic, and polystyrene compound plates versus glass plates, especially with increasing time on the plates, resulting in altered IC(50) values.

Structure-activity relationship (SAR): effort towards blocking N-glucuronidation of indazoles (PF-03376056) by human UGT1A enzymes.

GyrATPase is a cellular enzyme that has been used as an antibacterial target for treatment of nosocomial and community acquired bacterial infections. The leading chemical series targeted at inhibiting this enzyme, indazoles, were rapidly cleared in rats (CL > 70 mL/min/kg). The predominant metabolite identified in both urine and bile samples from a bile duct-cannulated study corresponded to direct glucuronidation of the parent compound and was excreted rapidly. Subsequently, a carefully designed analog was used to pinpoint the site of glucuronidation (N-glucuronidation) by incubation with rat hepatocytes and followed by mass spectrometry analysis. Reaction mapping with an array of recombinant UGT isozymes revealed that N-glucuronidation was predominantly catalyzed by the UGT1A family of enzymes. Based on the results, the following approaches were considered to reduce or eliminate glucuronidation: 1) adding sterically hindered substitutions on the phenyl ring of the indazole core; 2) changing the electron distribution by substituting with electron-donating or -withdrawing groups; 3) replacing the site of glucuronidation. The resulted compounds were evaluated in vitro in rat hepatocytes to assess their metabolic stabilities followed by in vivo efficacy studies in the murine peritonitis sepsis model (at 50 mg/kg) for selected compounds.

In vitro metabolism of phenoxypropoxybiguanide analogues in human liver microsomes to potent antimalarial dihydrotriazines.

Phenoxypropoxybiguanides, such as 1 (PS-15), are prodrugs analogous to the relationship of proguanil and its active metabolite cycloguanil. Unlike cycloguanil, however, 1a (WR99210), the active metabolite of 1, has retained in vitro potency against newly emerging antifolate-resistant malaria parasites. Unfortunately, manufacturing processes and gastrointestinal intolerance have prevented the clinical development of 1. In vitro antimalarial activity and in vitro metabolism studies have been performed on newly synthesized phenoxypropoxybiguanide analogues. All of the active dihydrotriazine metabolites exhibited potent antimalarial activity with in vitro IC(50) values less than 0.04 ng/mL. In vitro metabolism studies in human liver microsomes identified the production of not only the active dihydrotriazine metabolite, but also a desalkylation on the carbonyl chain, and multiple hydroxylated metabolites. The V(max) for production of the active metabolites ranged from 10.8 to 27.7 pmol/min/mg protein with the K(m) ranging from 44.8 to 221 microM. The results of these studies will be used to guide the selection of a lead candidate.