J. Matthew Hutzler

Is 1-Aminobenzotriazole an Appropriate in Vitro Tool as a Nonspecific Cytochrome P450 Inactivator?

1-Aminobenzotriazole (1-ABT) is generally considered to be a nonselective mechanism-based inactivator of both human and non-human cytochrome P450 (P450) enzymes. Thus, 1-ABT is routinely used when conducting in vitro reaction phenotyping studies with new chemical entities in drug discovery to decipher P450 from non-P450-mediated metabolism. Experiments with pooled human liver microsomes (HLMs) demonstrated that carbon monoxide binding, although substantially reduced after a 30-min preincubation with 1-ABT, was still measurable. Thus, remaining activity of nine major human P450s (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4) in HLMs was determined using established selective probe substrates after 30-min preincubation with either 1-ABT (1 mM), a positive control time-dependent inhibitor, or a competitive inhibitor. Whereas P450 2A6 and 3A4 activity was essentially eliminated upon 30-min pretreatment with 1-ABT, the other human P450s were less affected, with at least 20% activity remaining after pretreatment. In contrast, most of the known P450 selective time-dependent inhibitors were more effective inactivators than 1-ABT at lower concentrations. A particularly interesting finding was that 1-ABT was quite ineffective at inactivating P450 2C9, with roughly 60% activity remaining after pretreatment, which suggests that 1-ABT is much less selective for certain human P450s. This collection of data clearly demonstrates that assuming 1-ABT is a nonselective P450 inhibitor in vitro is risky, and false conclusions regarding remaining metabolic activity being non-P450 mediated after 1-ABT pretreatment may be made.

Visible spectra of type II cytochrome P450-drug complexes: evidence that "incomplete" heme coordination is common.

The visible spectrum of a ligand-bound cytochrome P450 is often used to determine the nature of the interaction between the ligand and the P450. One particularly characteristic form of spectra arises from the coordination of nitrogen-containing ligands to the P450 heme iron. These type II ligands tend to be inhibitors because they stabilize the low reduction potential P450 and prevent oxygen binding to the heme. Yet, several type II ligands containing aniline, imidazole, and triazole moieties are also known to be substrates of P450, although P450 binding spectra are not often scrutinized to make this distinction. Therefore, the three nitrogenous ligands aniline, imidazole, and triazole were used as binding spectra standards with purified human CYP3A4 and CYP2C9, because their small size should not present any steric limitations in their accessing the heme prosthetic group. Next, the spectra of P450 with drugs containing the three nitrogenous groups were collected for comparison. The absolute spectra demonstrated that the red-shift of the low-spin Soret band is mostly dependent on the electronic properties of the nitrogen ligand since they tended to match their respective standards, aniline, imidazole, and triazole. On the other hand, difference spectra seemed to be more sensitive to the steric properties of the ligand because they facilitated comparison of the spectral amplitudes achieved with the drugs versus those with the standard nitrogen ligands. Therefore, difference spectra may help reveal “weak” coordination to the heme that results from suboptimal orientation or ligand binding to more remote locations within the P450 active sites.

Mechanism-Based Inactivation of Cytochrome P450 2C9 by Tienilic Acid and (±)-Suprofen: A Comparison of Kinetics and Probe Substrate Selection

In vitro experiments were conducted to compare kinact, KI and inactivation efficiency (kinact/KI) of cytochrome P450 (P450) 2C9 by tienilic acid and (±)-suprofen using (S)-flurbiprofen, diclofenac, and (S)-warfarin as reporter substrates. Although the inactivation of P450 2C9 by tienilic acid when (S)-flurbiprofen and diclofenac were used as substrates was similar (efficiency of ∼9 ml/min/μmol), the inactivation kinetics were characterized by a sigmoidal profile. (±)-Suprofen inactivation of (S)-flurbiprofen and diclofenac hydroxylation was also described by a sigmoidal profile, although inactivation was markedly less efficient (∼1 ml/min/μmol). In contrast, inactivation of P450 2C9-mediated (S)-warfarin 7-hydroxylation by tienilic acid and (±)-suprofen was best fit to a hyperbolic equation, where inactivation efficiency was moderately higher (10 ml/min/μmol) and ∼3-fold higher (3 ml/min/μmol), respectively, relative to that of the other probe substrates, which argues for careful consideration of reporter substrate when mechanism-based inactivation of P450 2C9 is assessed in vitro. Further investigations into the increased inactivation seen with tienilic acid relative to that with (±)-suprofen revealed that tienilic acid is a higher affinity substrate with a spectral binding affinity constant (Ks) of 2 μM and an in vitro half-life of 5 min compared with a Ks of 21 μM and a 50 min in vitro half-life for (±)-suprofen. Lastly, a close analog of tienilic acid with the carboxylate functionality replaced by an oxirane ring was devoid of inactivation properties, which suggests that an ionic binding interaction with a positively charged residue in the P450 2C9 active site is critical for recognition and mechanism-based inactivation by these close structural analogs.

Discovery of ((1S,3R)-1-isopropyl-3-((3S,4S)-3-methoxy-tetrahydro-2H-pyran-4-ylamino)cyclopentyl)(4-(5-(trifluoromethyl)pyridazin-3-yl)piperazin-1-yl)methanone, PF-4254196, a CCR2 antagonist with an improved cardiovascular profile.

We describe the systematic optimization, focused on the improvement of CV-TI, of a series of CCR2 antagonists. This work resulted in the identification of 10 (((1S,3R)-1-isopropyl-3-((3S,4S)-3-methoxy-tetrahydro-2H-pyran-4-ylamino)cyclopentyl)(4-(5-(trifluoromethyl)pyridazin-3-yl)piperazin-1-yl)methanone) which possessed a low projected human dose 35-45mg BID and a CV-TI=3800-fold.

Drug–Drug Interactions: Designing Development Programs and Appropriate Product Labeling

Drug–drug interactions can represent a major public health issue. Drug metabolism science has evolved to the point where interactions with cytochrome P-450 isozymes can be predicted and potentially avoided or managed, but much work remains to allow accurate prediction of non-P-450 mediated interactions. Based on preclinical data, rational clinical plans can be developed to study potential drug–drug interactions in humans and develop labeling that allows optimal usage of new drugs.

Assessment of the Metabolism and Intrinsic Reactivity of a Novel Catechol Metabolite

PH-302 ( 1) demonstrates potent inhibitory activity against the inducible form of nitric oxide synthase (iNOS). The primary metabolite of PH-302 is a catechol ( 2) resulting from oxidative demethylenation of the methylenedioxyphenyl moiety by cytochrome P450 3A4. Concerns regarding subsequent two-electron oxidation of 2 to an electrophilic quinone species and the potential for resulting toxicity prompted additional studies to examine the reactivity and metabolic fate of this metabolite. Contrary to literature reports of catechol reactivity, 2 appeared to be resistant to quinone formation in human liver microsomal incubations, as determined by the lack of detectable glutathione (GSH) adducts and no covalent binding to microsomal proteins. In addition, 2 showed no evidence of depletion of intracellular glutathione or cytotoxicity at concentrations up to 1 mM in primary human and rat hepatocytes. In the presence of tyrosinase, spectral evidence indicated that 2 was oxidized to the ortho-quinone, and upon incubation in the presence of GSH, two conjugates were detected and characterized by LC/MS/MS. Lastly, the metabolic pathways of 2 were investigated in rat and human hepatocytes and found to be similar, proceeding via glucuronidation, sulfation, and methylation of the catechol. Collectively, these studies demonstrate that 2 appears to be resistant to further oxidation to quinone in liver microsomes, as well as spontaneous redox cycling, while the formation of phase II metabolites in hepatocytes suggests that multiple detoxication pathways may provide added protection against toxicity in the liver.

Assessment of the Biotransformation of Low-Turnover Drugs in the HµREL Human Hepatocyte Coculture Model

Metabolic profiles of four drugs possessing diverse metabolic pathways (timolol, meloxicam, linezolid, and XK469) were compared following incubations in both suspended cryopreserved human hepatocytes and the HμREL hepatocyte coculture model. In general, minimal metabolism was observed following 4-hour incubations in both suspended hepatocytes and the HμREL model, whereas incubations conducted up to 7 days in the HμREL coculture model resulted in more robust metabolic turnover. In the case of timolol, in vivo human data suggest that 22% of the dose is transformed via multistep oxidative opening of the morpholine moiety. Only the first-step oxidation was detected in suspended hepatocytes, whereas the relevant downstream metabolites were produced in the HµREL model. For meloxicam, both the hydroxymethyl and subsequent carboxylic acid metabolites were abundant following incubation in the HμREL model, while only a trace amount of the hydroxymethyl metabolite was observed in suspension. Similar to timolol, linezolid generated substantially higher levels of morpholine ring-opened carboxylic acid metabolites in the HμREL model. Finally, while the major aldehyde oxidase–mediated mono-oxidative metabolite of XK469 was minimally produced in hepatocyte suspension, the HμREL model robustly produced this metabolite, consistent with a pathway reported to account for 54% of the total urinary excretion in human. In addition, low-level taurine and glycine conjugates were identified in the HµREL model. In summary, continuous metabolite production was observed for up to 7 days of incubation in the HµREL model, covering cytochrome P450, aldehyde oxidase, and numerous conjugative pathways, while predominant metabolites correlated with relevant metabolites reported in human in vivo studies.

CHAPTER 5:Non-Cytochrome P450 Enzymes and Glucuronidation

While the metabolism of small molecule drugs has been dominated by the cytochrome P450 family of enzymes, many other enzyme families exist that help facilitate the conversion of lipophilic drug molecules to metabolites that may be readily excreted from the body. A shift in the chemical space that medicinal chemists are interrogating has led to generally more polar drug molecules, which has in turn has caused an increase in the prevalence of non-cytochrome P450 metabolic pathways. It is thus critical that drug metabolism scientists are aware of in vitro methods for identifying the role of these enzymes. For example, the role of the thermally labile metabolic enzyme flavin monooxygenase (FMO) is likely under-diagnosed due to the way in which in vitro incubations in human liver microsomes are conducted, with pre-incubations at 37 °C often devoid of NADPH. In addition, interest in the oxidative enzyme aldehyde oxidase (AO) has surged in recent years in response to its direct negative impact on clinical programs. Lastly, the UDP-glucuronosyltransferase (UGT) family of enzymes are highly problematic, with the extrapolation from in vitro systems to predict clearance to in vivo being a challenge. While many non-cytochrome P450 enzymes exist, the focus of this chapter will be on these three important enzyme systems.