Siamak Cyrus Khojasteh

Chemical inhibitors of cytochrome P450 isoforms in human liver microsomes: a re-evaluation of P450 isoform selectivity

The majority of marketed small-molecule drugs undergo metabolism by hepatic Cytochrome P450 (CYP) enzymes (Rendic 2002). Since these enzymes metabolize a structurally diverse number of drugs, metabolism-based drug–drug interactions (DDIs) can potentially occur when multiple drugs are coadministered to patients. Thus, a careful in vitro assessment of the contribution of various CYP isoforms to the total metabolism is important for predicting whether such DDIs might take place. One method of CYP phenotyping involves the use of potent and selective chemical inhibitors in human liver microsomal incubations in the presence of a test compound. The selectivity of such inhibitors plays a critical role in deciphering the involvement of specific CYP isoforms. Here, we review published data on the potency and selectivity of chemical inhibitors of the major human hepatic CYP isoforms. The most selective inhibitors available are furafylline (in co-incubation and pre-incubation conditions) for CYP1A2, 2-phenyl-2-(1-piperidinyl)propane (PPP) for CYP2B6, montelukast for CYP2C8, sulfaphenazole for CYP2C9, (–)-N-3-benzyl-phenobarbital for CYP2C19 and quinidine for CYP2D6. As for CYP2A6, tranylcypromine is the most widely used inhibitor, but on the basis of initial studies, either 3-(pyridin-3-yl)-1H-pyrazol-5-yl)methanamine (PPM) and 3-(2-methyl-1H-imidazol-1-yl)pyridine (MIP) can replace tranylcypromine as the most selective CYP2A6 inhibitor. For CYP3A4, ketoconazole is widely used in phenotyping studies, although azamulin is a far more selective CYP3A inhibitor. Most of the phenotyping studies do not include CYP2E1, mostly because of the limited number of new drug candidates that are metabolized by this enzyme. Among the inhibitors for this enzyme, 4-methylpyrazole appears to be selective.

Absorption, Metabolism, Excretion, and the Contribution of Intestinal Metabolism to the Oral Disposition of [14C]Cobimetinib, a MEK Inhibitor, in Humans

The pharmacokinetics, metabolism, and excretion of cobimetinib, a MEK inhibitor, were characterized in healthy male subjects (n = 6) following a single 20 mg (200 μCi) oral dose. Unchanged cobimetinib and M16 (glycine conjugate of hydrolyzed cobimetinib) were the major circulating species, accounting for 20.5% and 18.3% of the drug-related material in plasma up to 48 hours postdose, respectively. Other circulating metabolites were minor, accounting for less than 10% of drug-related material in plasma. The total recovery of the administered radioactivity was 94.3% (±1.6%, S.D.) with 76.5% (±2.3%) in feces and 17.8% (±2.5%) in urine. Metabolite profiling indicated that cobimetinib had been extensively metabolized with only 1.6% and 6.6% of the dose remaining as unchanged drug in urine and feces, respectively. In vitro phenotyping experiments indicated that CYP3A4 was predominantly responsible for metabolizing cobimetinib. From this study, we concluded that cobimetinib had been well absorbed (fraction absorbed, Fa = 0.88). Given this good absorption and the previously determined low hepatic clearance, the systemic exposures were lower than expected (bioavailability, F = 0.28). We hypothesized that intestinal metabolism had strongly attenuated the oral bioavailability of cobimetinib. Supporting this hypothesis, the fraction escaping gut wall elimination (Fg) was estimated to be 0.37 based on F and Fa from this study and the fraction escaping hepatic elimination (Fh) from the absolute bioavailability study (F = Fa × Fh × Fg). Physiologically based pharmacokinetics modeling also showed that intestinal clearance had to be included to adequately describe the oral profile. These collective data suggested that cobimetinib was well absorbed following oral administration and extensively metabolized with intestinal first-pass metabolism contributing to its disposition.

Going Beyond Common Drug Metabolizing Enzymes: Case Studies of Biotransformation Involving Aldehyde Oxidase, γ-Glutamyl Transpeptidase, Cathepsin B, Flavin-Containing Monooxygenase, and ADP-Ribosyltransferase

The significant roles that cytochrome P450 (P450) and UDP-glucuronosyl transferase (UGT) enzymes play in drug discovery cannot be ignored, and these enzyme systems are commonly examined during drug optimization using liver microsomes or hepatocytes. At the same time, other drug-metabolizing enzymes have a role in the metabolism of drugs and can lead to challenges in drug optimization that could be mitigated if the contributions of these enzymes were better understood. We present examples (mostly from Genentech) of five different non-P450 and non-UGT enzymes that contribute to the metabolic clearance or bioactivation of drugs and drug candidates. Aldehyde oxidase mediates a unique amide hydrolysis of GDC-0834 (N-[3-[6-[4-[(2R)-1,4-dimethyl-3-oxopiperazin-2-yl]anilino]-4-methyl-5-oxopyrazin-2-yl]-2-methylphenyl]-4,5,6,7-tetrahydro-1-benzothiophene-2-carboxamide), leading to high clearance of the drug. Likewise, the rodent-specific ribose conjugation by ADP-ribosyltransferase leads to high clearance of an interleukin-2-inducible T-cell kinase inhibitor. Metabolic reactions by flavin-containing monooxygenases (FMO) are easily mistaken for P450-mediated metabolism such as oxidative defluorination of 4-fluoro-N-methylaniline by FMO. Gamma-glutamyl transpeptidase is involved in the initial hydrolysis of glutathione metabolites, leading to formation of proximate toxins and nephrotoxicity, as is observed with cisplatin in the clinic, or renal toxicity, as is observed with efavirenz in rodents. Finally, cathepsin B is a lysosomal enzyme that is highly expressed in human tumors and has been targeted to release potent cytotoxins, as in the case of brentuximab vedotin. These examples of non-P450- and non-UGT-mediated metabolism show that a more complete understanding of drug metabolizing enzymes allows for better insight into the fate of drugs and improved design strategies of molecules in drug discovery.

Evaluation of Metabolism and Disposition of GDC-0152 in Rats Using 14C Labeling Strategy at Two Different Positions: A Novel Formation of Hippuric Acid from 4-Phenyl-5-Amino-1,2,3-Thiadiazole

The compound (S)-1-[(S)-2-cyclohexyl-2-([S]-2-[methylamino]propanamido)acetyl]-N-(4-phenyl-1,2,3-thiadiazol-5-yl)pyrrolidine-2-carboxamide (GDC-0152) is a peptidomimetic small molecule antagonist of inhibitor of apoptosis (IAP) proteins with antitumor activity. The mass balance, pharmacokinetics, tissue distribution and metabolism of GDC-0152 was investigated in rats following intravenous administration of 15 mg/kg of [14C]GDC-0152, labeled either at the terminal phenyl ring (A) or at the carbonyl of the 2-amino-2-cyclohexylacetyl moiety (B). In rats, 92.2%–95.1% of the radiolabeled GDC-0152 dose was recovered. Approximately 62.3% and 25.1% of A was excreted in urine and feces, respectively. By contrast, B was excreted almost equally in urine (27.2%), feces (32.2%), and expired air (27.5%). GDC-0152 underwent extensive metabolism, with less than 9% of the dose recovered as parent in excreta. Similarly, in plasma, GDC-0152 represented 16.7% and 7.5% of the area under the curve of the total radioactivity for A and B, respectively. The terminal half-life (t1/2) for total radioactivity was longer for B (21.2 hours) than for A (4.59 hours). GDC-0152 was highly metabolized via oxidation and amide hydrolysis, followed by subsequent sulfation and glucuronidation. The most abundant circulating metabolites were the amide hydrolyzed products, M26, M28, M30, M31, and M34, which ranged from 3.5% to 9.0% of total radioactivity. In quantitative whole-body autoradiography studies, the residence of radioactivity in tissues was longer for B than for A, which is consistent with the t1/2 of the total radioactivity in circulation. A novel 4-phenyl-5-amino-1,2,3-thiadiazole (M28) oxidative cleavage resulted in the formation of hippuric acid (M24). This biotransformation was also observed in rat hepatocyte incubations with para-substituted M28 analogs. In addition, the formation of M24 was inhibited by 1-aminobenzotriazole, which points to the involvement of P450 enzymes.

Antibody Drug Conjugates Differentiate Uptake and DNA Alkylation of Pyrrolobenzodiazepines in Tumors from Organs of Xenograft Mice

Pyrrolobenzodiazepine (PBD)-dimer is a DNA minor groove alkylator, and its CD22 THIOMAB antibody drug conjugate (ADC) demonstrated, through a disulfide linker, an efficacy in tumor reduction for more than 7 weeks with minimal body weight loss in xenograft mice after a single 0.5–1 mg/kg i.v. dose. The DNA alkylation was investigated here in tumors and healthy organs of mice to understand the sustained efficacy and tolerability. The experimental procedures included the collection of tumors and organ tissues of xenograft mice treated with the ADC followed by DNA isolation/hydrolysis/quantitation and payload recovery from reversible DNA alkylation. PBD-dimer formed a considerable amount of adducts with tissue DNA, representing approximately 98% (at 24 hours), and 99% (at 96 hours) of the total PBD-dimer in tumors, and 78–89% in liver and lung tissues, suggesting highly efficient covalent binding of the released PBD-dimer to tissue DNA. The amount of PBD-DNA adducts in tumor tissues was approximately 24-fold (at 24 hours) and 70-fold (at 96 hours) greater than the corresponding amount of adducts in liver and lung tissues. In addition, the DNA alkylation levels increased 3-fold to 4-fold from 24 to 96 hours in tumors [41/106 base pairs (bp) at 96 hours] but remained at the same level (1/106 bp) in livers and lungs. These results support the typical target-mediated cumulative uptake of ADC into tumors and payload release that offers an explanation for its sustained antitumor efficacy. In addition, the low level of DNA alkylation in normal tissues is consistent with the tolerability observed in mice.

1-Aminobenzotriazole Coincubated with (S)-Warfarin Results in Potent Inactivation of CYP2C9

1-Aminobenzotriazole (ABT) is a nonselective, mechanism-based inactivator of cytochrome P450 (P450) and a useful tool compound to discern P450- from non-P450-mediated metabolism. ABT effectively inactivates major human P450 isoforms, with the notable exception of CYP2C9. Here we propose that ABT preferentially binds to the warfarin-binding pocket in the CYP2C9 active-site cavity; thus, ABT bioactivation and subsequent inactivation is not favored. Therefore, coincubation with (S)-warfarin would result in displacement of ABT from the warfarin-binding pocket and subsequent binding to the active site, converting ABT into a potent inactivator of CYP2C9. To test this hypothesis, in vitro studies were conducted using various coincubation combinations of ABT and (S)-warfarin or diclofenac to modulate the effectiveness of CYP2C9 inactivation by ABT. Coincubation of ABT with (S)-warfarin (diclofenac probe substrate) resulted in potent inactivation, whereas weak inactivation was observed following coincubation of ABT with diclofenac [(S)-warfarin probe substrate]. The kinetic parameters of time-dependent inhibition of ABT for CYP2C9 in the absence and presence of (S)-warfarin (20 μM) were 0.0826 and 0.273 min−1 for kinact and 3.49 and 0.157 mM for KI, respectively. In addition, a 73.4-fold shift was observed in the in vitro potency (kinact/KI ratio), with an increase from 23.7 ml/min/mmol (ABT alone) to 1740 ml/min/mmol [ABT with (S)-warfarin (20 μM)]. These findings were supported by in silico structural modeling, which showed ABT preferentially binding to the warfarin-binding pocket and the displacement of ABT to the active site in the presence of (S)-warfarin.

Absorption, metabolism and excretion of cobimetinib, an oral MEK inhibitor, in rats and dogs

Abstract 1. The absorption, metabolism and excretion of cobimetinib, an allosteric inhibitor of MEK1/2, was characterized in mass balance studies following single oral administration of radiolabeled (14C) cobimetinib to Sprague–Dawley rats (30 mg/kg) and Beagle dogs (5 mg/kg). 2. The oral dose of cobimetinib was well absorbed (81% and 71% in rats and dogs, respectively). The maximal plasma concentrations for cobimetinib and total radioactivity were reached at 2–3 h post-dose. Drug-derived radioactivity was fully recovered (∼90% of the administered dose) with the majority eliminated in feces via biliary excretion (78% of the dose for rats and 65% for dogs). The recoveries were nearly complete after the first 48 h following dosing. 3. The metabolic profiles indicated extensive metabolism of cobimetinib prior to its elimination. For rats, the predominant metabolic pathway was hydroxylation at the aromatic core. Lower exposures for cobimetinib and total radioactivity were observed in male rats compared with female rats, which was consistent to in vitro higher clearance of cobimetinib for male rats. For dogs, sequential oxidative reactions occurred at the aliphatic portion of the molecule. Though rat metabolism was well-predicted in vitro with liver microsomes, dog metabolism was not. 4. Rats and dogs were exposed to the two major human circulating Phase II metabolites, which provided relevant metabolite safety assessment. In general, the extensive sequential oxidative metabolism in dogs, and not the aromatic hydroxylation in rats, was more indicative of the metabolism of cobimetinib in humans.

Elucidating the Mechanism of Cytochrome P450–Mediated Pyrimidine Ring Conversion to Pyrazole Metabolites with the BACE1 Inhibitor GNE-892 in Rats

We investigated an uncommon biotransformation of pyrimidine during the metabolism of GNE-892 ((R)-2-amino-1,3′,3′-trimethyl-7′-(pyrimidin-5-yl)-3′,4′-dihydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-one), a β-secretase 1 inhibitor. Three novel metabolites, formed by conversion of pyrimidine to pyrazole, were observed in the 14C-radiolabeled mass balance study in rats. Their structures were characterized by high-resolution mass spectrometry and nuclear magnetic resonance. Although these metabolites accounted for <5% of the administered dose, their unique nature prompted us to conduct further investigations. The pyrazole-containing metabolites were formed in vitro with rat hepatocytes and liver microsomes, which supported that they were formed during hepatic metabolism. Further, their generation was inhibited by 1-aminobenzotriazole, indicating involvement of cytochrome P450s. Studies with rat recombinant enzymes identified that CYP2D2 generated the N-hydroxypyrazole metabolite from GNE-892. This biotransformation proceeded through multiple steps from the likely precursor, pyrimidine N-oxide. On the basis of these data, we propose a mechanism in which the pyrimidine is activated via N-oxidation, followed by a second oxidative process that opens the pyrimidine ring to form a formamide intermediate. After hydrolysis of the formamide, a carbon is lost as formic acid, together with ring closure to form the pyrazole ring. This article highlights a mechanistic approach for determining the biotransformation of the pyrimidine to a pyrazole for GNE-892.

Investigations into the Mechanisms of Pyridine Ring Cleavage in Vismodegib

Vismodegib (Erivedge, GDC-0449) is a first-in-class, orally administered small-molecule Hedgehog pathway inhibitor that is approved for the treatment of advanced basal cell carcinoma. Previously, we reported results from preclinical and clinical radiolabeled mass balance studies in which we determined that metabolism is the main route of vismodegib elimination. The metabolites of vismodegib are primarily the result of oxidation followed by glucuronidation. The focus of the current work is to probe the mechanisms of formation of three pyridine ring-cleaved metabolites of vismodegib, mainly M9, M13, and M18, using in vitro, ex vivo liver perfusion and in vivo rat studies. The use of stable-labeled (13C2,15N)vismodegib on the pyridine ring exhibited that the loss of carbon observed in both M9 and M13 was from the C-6 position of pyridine. Interestingly, the source of the nitrogen atom in the amide of M9 was from the pyridine. Evidence for the formation of aldehyde intermediates was observed using trapping agents as well as 18O-water. Finally, we conclude that cytochrome P450 is involved in the formation of M9, M13, and M18 and that M3 (the major mono-oxidative metabolite) is not the precursor for the formation of these cleaved products; rather, M18 is the primary cleaved metabolite.

ADME Properties and Their Dependence on Physicochemical Properties

Absorption, distribution, metabolism, and excretion (ADME) properties such as absorption, clearance, and volume of distribution are strongly influenced by physicochemical parameters. A lot of retrospective analyses have been performed to identify those attributes that give rise to favorable ADME parameters. These attributes should be incorporated in compound design to increase the chance of identifying a compound with superior ADME properties.