Paul G. Pearson

Enzyme kinetics of cytochrome P450-mediated reactions.

The most common drug-drug interactions may be understood in terms of alterations of metabolism, associated primarily with changes in the activity of cytochrome P450 (CYP) enzymes. Kinetic parameters such as Km, Vmax, Ki and Ka, which describe metabolism-based drug interactions, are usually determined by appropriate kinetic models and may be used to predict the pharmacokinetic consequences of exposure to one or multiple drugs. According to classic Michaelis-Menten (M-M) kinetics, one binding site models can be employed to simply interpret inhibition (pure competitive, non-competitive and uncompetitive) or activation of the enzyme. However, some cytochromes P450, in particular CYP3A4, exhibit unusual kinetic characteristics. In this instance, the changes in apparent kinetic constants in the presence of inhibitor or activator or second substrate do not obey the rules of M-M kinetics, and the resulting kinetics are not straightforward and hamper mechanistic interpretation of the interaction in question. These unusual kinetics include substrate activation (autoactivation), substrate inhibition, partial inhibition, activation, differential kinetics and others. To address this problem, several kinetic models can be proposed, based upon the assumption that multiple substrate binding sites exist at the active site of a particular P450, and the resulting kinetic constants are, therefore, solved to adequately describe the observed interaction between multiple drugs. The following is an overview of some cytochrome P450-mediated classic and atypical enzyme kinetics, and the associated kinetic models. Applications of these kinetic models can provide some new insights into the mechanism of P450-mediated drug-drug interactions.

Disposition of Caspofungin: Role of Distribution in Determining Pharmacokinetics in Plasma

ABSTRACT The disposition of caspofungin, a parenteral antifungal drug, was investigated. Following a single, 1-h, intravenous infusion of 70 mg (200 μCi) of [3H]caspofungin to healthy men, plasma, urine, and feces were collected over 27 days in study A (n = 6) and plasma was collected over 26 weeks in study B (n = 7). Supportive data were obtained from a single-dose [3H]caspofungin tissue distribution study in rats (n = 3 animals/time point). Over 27 days in humans, 75.4% of radioactivity was recovered in urine (40.7%) and feces (34.4%). A long terminal phase (t1/2 = 14.6 days) characterized much of the plasma drug profile of radioactivity, which remained quantifiable to 22.3 weeks. Mass balance calculations indicated that radioactivity in tissues peaked at 1.5 to 2 days at ∼92% of the dose, and the rate of radioactivity excretion peaked at 6 to 7 days. Metabolism and excretion of caspofungin were very slow processes, and very little excretion or biotransformation occurred in the first 24 to 30 h postdose. Most of the area under the concentration-time curve of caspofungin was accounted for during this period, consistent with distribution-controlled clearance. The apparent distribution volume during this period indicated that this distribution process is uptake into tissue cells. Radioactivity was widely distributed in rats, with the highest concentrations in liver, kidney, lung, and spleen. Liver exhibited an extended uptake phase, peaking at 24 h with 35% of total dose in liver. The plasma profile of caspofungin is determined primarily by the rate of distribution of caspofungin from plasma into tissues.

Disposition of Caspofungin, a Novel Antifungal Agent, in Mice, Rats, Rabbits, and Monkeys

ABSTRACT The metabolism, excretion, and pharmacokinetics of caspofungin (Cancidas; Merck & Co., Inc.) were investigated after administration of a single intravenous dose to mice, rats, rabbits, and monkeys. Caspofungin had a low plasma clearance (0.29 to 1.05 ml/min/kg) and a long terminal elimination half-life (11.7 h to 59.7 h) in all preclinical species. The elimination kinetics of caspofungin were multiphasic and displayed an initial distribution phase followed by a dominant β-elimination phase. The presence of low levels of prolonged radioactivity in plasma was observed and was partially attributable to the chemical degradation product M0. Excretion studies with [3H]caspofungin indicated that the hepatic and renal routes play an important role in the elimination of caspofungin, as a large percentage of the radiolabeled dose was recovered in urine and feces. Excretion of radioactivity in all species studied was slow, and low levels of radioactivity were detected in daily urine and fecal samples throughout a prolonged collection period. Although urinary profiles indicated the presence of several metabolites (M0, M1, M2, M3, M4, M5, and M6), the majority of the total radioactivity was associated with the polar metabolites M1 [4(S)-hydroxy-4-(4-hydroxyphenyl)-l-threonine] and M2 [N-acetyl-4(S)-hydroxy-4-(4-hydroxyphenyl)-l-threonine]. Caspofungin was thus primarily eliminated by metabolic transformation; however, the rate of metabolism was slow. These results suggest that distribution plays a prominent role in determining the plasma pharmacokinetics and disposition of caspofungin, as very little excretion or biotransformation occurred during the early days after dose administration, a period during which concentrations in plasma fell substantially. The disposition of caspofungin in preclinical species was similar to that reported previously in humans.

An Inhibitory Metabolite Leads to Dose- and Time-Dependent Pharmacokinetics of (R)-N-{1-(3-(4-Ethoxy-phenyl)-4-oxo-3,4- dihydro-pyrido(2,3-d)pyrimidin-2-yl)-ethyl}-N-pyridin-3-yl-methyl-2- (4-trifluoromethoxy-phenyl)-acetamide (AMG 487) in Human Subjects After Multiple Dosing

(R)-N-{1-[3-(4-Ethoxy-phenyl)-4-oxo-3,4-dihydro-pyrido[2,3-d]-pyrimidin-2-yl]-ethyl}-N-pyridin-3-yl-methyl-2-(4-trifluoromethoxyphenyl)-acetamide (AMG 487) is a potent and selective orally bioavailable chemokine (C-X-C motif) receptor 3 (CXCR3) antagonist that displays dose- and time-dependent pharmacokinetics in human subjects after multiple oral dosing. Although AMG 487 exhibited linear pharmacokinetics on both days 1 and 7 at the 25-mg dose, dose- and time-dependent kinetics were evident at the two higher doses. Nonlinear kinetics were more pronounced after multiple dosing. Area under the plasma concentration-time curve from 0 to 24 h [AUC(0–24 h)] increased 96-fold with a 10-fold increase in dose on day 7 compared with a 28-fold increase in AUC(0–24 h) on day 1. These changes were correlated with time- and dose-dependent decreases in the metabolite to parent plasma concentrations, suggesting that these changes result from a decrease in the oral clearance (CL) of AMG 487 (e.g., intestinal/hepatic first-pass metabolism and systemic CL). The biotransformation of AMG 487 is dependent on CYP3A and results in the formation of two primary metabolites, a pyridyl N-oxide AMG 487 (M1) and an O-deethylated AMG 487 (M2). One of these metabolites, M2, undergoes further metabolism by CYP3A. M2 has also been demonstrated to inhibit CYP3A in a competitive (Ki = 0.75 μM) manner as well as via mechanism-based inhibition (unbound KI = 1.4 μM, kinact = 0.041 min–1). Data from this study implicate M2-mediated CYP3A mechanism-based inhibition as the proximal cause for the time-dependent pharmacokinetics of AMG 487. However, the sequential metabolism of M2, nonlinear AMG 487 pharmacokinetics, and the inability to accurately determine the role of intestinal AMG 487 metabolism complicates the correlation between M2 plasma concentrations and the time-dependent AMG 487 pharmacokinetic changes.

An Inhibitory Metabolite Leads To Dose- And Time-Dependent Pharmacokinetics Of AMG 487 In Human Subjects Following Multiple Dosing

(R)-N-{1-[3-(4-Ethoxy-phenyl)-4-oxo-3,4-dihydro-pyrido[2,3-d]-pyrimidin-2-yl]-ethyl}-N-pyridin-3-yl-methyl-2-(4-trifluoromethoxyphenyl)-acetamide (AMG 487) is a potent and selective orally bioavailable chemokine (C-X-C motif) receptor 3 (CXCR3) antagonist that displays dose- and time-dependent pharmacokinetics in human subjects after multiple oral dosing. Although AMG 487 exhibited linear pharmacokinetics on both days 1 and 7 at the 25-mg dose, dose- and time-dependent kinetics were evident at the two higher doses. Nonlinear kinetics were more pronounced after multiple dosing. Area under the plasma concentration-time curve from 0 to 24 h [AUC(0–24 h)] increased 96-fold with a 10-fold increase in dose on day 7 compared with a 28-fold increase in AUC(0–24 h) on day 1. These changes were correlated with time- and dose-dependent decreases in the metabolite to parent plasma concentrations, suggesting that these changes result from a decrease in the oral clearance (CL) of AMG 487 (e.g., intestinal/hepatic first-pass metabolism and systemic CL). The biotransformation of AMG 487 is dependent on CYP3A and results in the formation of two primary metabolites, a pyridyl N-oxide AMG 487 (M1) and an O-deethylated AMG 487 (M2). One of these metabolites, M2, undergoes further metabolism by CYP3A. M2 has also been demonstrated to inhibit CYP3A in a competitive (Ki = 0.75 μM) manner as well as via mechanism-based inhibition (unbound KI = 1.4 μM, kinact = 0.041 min–1). Data from this study implicate M2-mediated CYP3A mechanism-based inhibition as the proximal cause for the time-dependent pharmacokinetics of AMG 487. However, the sequential metabolism of M2, nonlinear AMG 487 pharmacokinetics, and the inability to accurately determine the role of intestinal AMG 487 metabolism complicates the correlation between M2 plasma concentrations and the time-dependent AMG 487 pharmacokinetic changes.

Hormonal Effects on Tirilazad Clearance in Women: Assessment of the Role of CYP3A

This study assessed whether the previously reported difference in tirilazad clearance between pre‐ and postmenopausal women is reversed by hormone replacement and whether this observation can be explained by differences in CYP3A4 activity. Ten healthy women from each group were enrolled: premenopausal (ages 18–35), postmenopausal (ages 50–70), postmenopausal receiving estrogen, and postmenopausal women receiving estrogen and progestin. Volunteers received 0.0145 mg/kg midazolam and 3.0 mg/kg tirilazad mesylate intravenously on separate days. Plasma tirilazad and midazolam were measured by HPLC/dual mass spectrophotometry (MS/MS) assays. Tirilazad clearance was significantly higher in premenopausal women (0.51 ± 0.09 L/hr/kg) than in postmenopausal groups (0.34 ± 0.07, 0.32 ± 0.06, and 0.36 ± 0.08 L/hr/kg, respectively) (p = 0.0001). Midazolam clearance (0.64 ± 0.12 L/hr/kg) was significantly higher in premenopausal women compared to postmenopausal groups (0.47 ± 0.11, 0.49 ± 0.11, and 0.53 ± 0.19 L/hr/kg, respectively) (p = 0.037). Tirilazad clearance was weakly correlated with midazolam clearance (r2 = 0.129, p = 0.02). Tirilazad clearance is faster in premenopausal women than in postmenopausal women, but the effect of menopause on clearance is not reversed by hormone replacement. Tirilazad clearance in these women is weakly related to midazolam clearance, a marker of CYP3A activity.

Acalabrutinib (ACP-196): A Covalent Bruton Tyrosine Kinase Inhibitor with a Differentiated Selectivity and In Vivo Potency Profile

Several small-molecule Bruton tyrosine kinase (BTK) inhibitors are in development for B cell malignancies and autoimmune disorders, each characterized by distinct potency and selectivity patterns. Herein we describe the pharmacologic characterization of BTK inhibitor acalabrutinib [compound 1, ACP-196 (4-[8-amino-3-[(2S)-1-but-2-ynoylpyrrolidin-2-yl]imidazo[1,5-a]pyrazin-1-yl]-N-(2-pyridyl)benzamide)]. Acalabrutinib possesses a reactive butynamide group that binds covalently to Cys481 in BTK. Relative to the other BTK inhibitors described here, the reduced intrinsic reactivity of acalabrutinib helps to limit inhibition of off-target kinases having cysteine-mediated covalent binding potential. Acalabrutinib demonstrated higher biochemical and cellular selectivity than ibrutinib and spebrutinib (compounds 2 and 3, respectively). Importantly, off-target kinases, such as epidermal growth factor receptor (EGFR) and interleukin 2-inducible T cell kinase (ITK), were not inhibited. Determination of the inhibitory potential of anti-immunoglobulin M–induced CD69 expression in human peripheral blood mononuclear cells and whole blood demonstrated that acalabrutinib is a potent functional BTK inhibitor. In vivo evaluation in mice revealed that acalabrutinib is more potent than ibrutinib and spebrutinib. Preclinical and clinical studies showed that the level and duration of BTK occupancy correlates with in vivo efficacy. Evaluation of the pharmacokinetic properties of acalabrutinib in healthy adult volunteers demonstrated rapid absorption and fast elimination. In these healthy individuals, a single oral dose of 100 mg showed approximately 99% median target coverage at 3 and 12 hours and around 90% at 24 hours in peripheral B cells. In conclusion, acalabrutinib is a BTK inhibitor with key pharmacologic differentiators versus ibrutinib and spebrutinib and is currently being evaluated in clinical trials.

The Impact of Drug Metabolism in Contemporary Drug Discovery: New Opportunities and Challenges for Mass Spectrometry

Current developments in contemporary drug discovery techniques offer the potential to discover and develop new chemical entities to treat a spectrum of diseases or medical conditions for which either no current therapies exist, or for which existing therapies are inadequate. In particular, advances in synthetic medicinal chemistry (i. e., combinatorial chemistry and rapid analog synthesis), the availability of new molecular targets derived from genomics-based discovery, and high-throughput screening technology have the potential to identify new chemical entities (NCEs) with potent and selective pharmacological properties. To realize the full potential of these techniques, it is absolutely essential that a contemporary pharmacokinetics and drug metabolism program be implemented as a fully-integrated component of the drug-discovery process.