Vinita Uttamsingh

Relative contributions of the five major human cytochromes P450, 1A2, 2C9, 2C19, 2D6, and 3A4, to the hepatic metabolism of the proteasome inhibitor bortezomib

VELCADE (bortezomib, PS-341), reversibly inhibits the 20S proteasome and exhibits cytotoxic and antitumor activities. Pretreatment of cancer cells with bortezomib increases the chemosensitivity of these cells, suggesting that bortezomib may be used in combination chemotherapy. The relative contributions of the five major human cytochromes P450 (P450s), 1A2, 2C9, 2C19, 2D6, and 3A4 (the focus of the present study), to the metabolism of bortezomib are an important aspect of potential drug interactions. Relative activity factor (RAF), chemical inhibition, and immunoinhibition using monoclonal antibodies were three approaches employed to determine the relative contributions of the major human P450s to the net hepatic metabolism of bortezomib. RAFs for the P450 isoform-selective substrates were determined; the ratio of the rate of metabolism of bortezomib with cDNA-expressed P450s versus rate of metabolism with human liver microsomes was normalized with respect to the RAF for each P450 isoform to determine the percentage contributions of the P450s to the net hepatic metabolism of bortezomib. CYP3A4 followed by CYP2C19 were determined to be the major contributors to the metabolism of bortezomib. Chemical inhibition and immunoinhibition confirmed that CYP3A4 and CYP2C19 were the major P450s responsible for the hepatic metabolism of bortezomib. The studies were conducted with 2 μM bortezomib, and the disappearance of bortezomib, rather than appearance of a specific metabolite, was quantified to determine the contributions of the P450s to the overall hepatic metabolism of bortezomib in humans.

Comparison of Intrinsic Clearance in Liver Microsomes and Hepatocytes from Rats and Humans: Evaluation of Free Fraction and Uptake in Hepatocytes

Apparent intrinsic clearance (CLint,app) of 7-ethoxycoumarin, phenacetin, propranolol, and midazolam was measured using rat and human liver microsomes and freshly isolated and cryopreserved hepatocytes to determine factors responsible for differences in rates of metabolism in these systems. The cryopreserved and freshly isolated hepatocytes generally provided similar results, although there was greater variability using the latter system. The CLint,app values in hepatocytes are observed to be lower than that in microsomes, and this difference becomes greater for compounds with high CLint,app. This could partly be attributed to the differences in the free fraction (fu). The fu in hepatocyte incubations (fu,hep-inc) was influenced not only by the free fraction of compounds in the incubation buffer (fu,buffer) but also by the rate constants of uptake (kup) and metabolism (kmet). This report provides a new derivation for fu,hep-inc, which can be expressed as fu,hep-inc = [kup/(kmet + kup)]/[1 + (Chep/Cbuffer) × (Vhep/Vbuffer)], where the Chep, Cbuffer, Vhep, and Vbuffer represent the concentrations of a compound in hepatocytes and buffer and volumes of hepatocytes and buffer, respectively. For midazolam, the fu,hep-inc was calculated, and the maximum metabolism rate in hepatocytes was shown to be limited by the uptake rate.

Optimization of the in Vitro Cardiac Safety of Hydroxamate-Based Histone Deacetylase Inhibitors

Histone deacetylase (HDAC) inhibitors have shown promise in treating various forms of cancer. However, many HDAC inhibitors from diverse structural classes have been associated with QT prolongation in humans. Inhibition of the human ether a-go-go related gene (hERG) channel has been associated with QT prolongation and fatal arrhythmias. To determine if the observed cardiac effects of HDAC inhibitors in humans is due to hERG blockade, a highly potent HDAC inhibitor devoid of hERG activity was required. Starting with dacinostat (LAQ824), a highly potent HDAC inhibitor, we explored the SAR to determine the pharmacophores required for HDAC and hERG inhibition. We disclose here the results of these efforts where a high degree of pharmacophore homology between these two targets was discovered. This similarity prevented traditional strategies for mitigating hERG binding/modulation from being successful and novel approaches for reducing hERG inhibition were required. Using a hERG homology model, two compounds, 11r and 25i, were discovered to be highly efficacious with weak affinity for the hERG and other ion channels.

Investigation of drug-drug interaction potential of bortezomib in vivo in female Sprague-Dawley rats and in vitro in human liver microsomes.

Bortezomib (Velcade, PS-341), a dipeptidyl boronic acid, is a first-in-class proteasome inhibitor approved in 2003 for the treatment of multiple myeloma. In a preclinical toxicology study, bortezomib-treated rats resulted in liver enlargement (35%). Ex vivo analyses of the liver samples showed an 18% decrease in cytochrome P450 (P450) content, a 60% increase in palmitoyl coenzyme A β-oxidation activity, and a 41 and 23% decrease in CYP3A protein expression and activity, respectively. Furthermore, liver samples of bortezomib-treated rats had little change in CYP2B and CYP4A protein levels and activities. To address the likelihood of clinical drug-drug interactions, the P450 inhibition potential of bortezomib and its major deboronated metabolites M1 and M2 and their dealkylated metabolites M3 and M4 was evaluated in human liver microsomes for the major P450 isoforms 1A2, 2C9, 2C19, 2D6, and 3A4/5. Bortezomib, M1, and M2 were found to be mild inhibitors of CYP2C19 (IC50 ∼ 18.0, 10.0, and 13.2 μM, respectively), and M1 was also a mild inhibitor of CYP2C9 (IC50 ∼ 11.5 μM). However, bortezomib, M1, M2, M3, and M4 did not inhibit other P450s (IC50 values > 30 μM). There also was no time-dependent inhibition of CYP3A4/5 by bortezomib or its major metabolites. Based on these results, no major P450-mediated clinical drug-drug interactions are anticipated for bortezomib or its major metabolites. To our knowledge, this is the first report on P450-mediated drug-drug interaction potential of proteasome inhibitors or boronic acid containing therapeutics.

Altering Metabolic Profiles of Drugs by Precision Deuteration 2: Discovery of a Deuterated Analog of Ivacaftor With Differentiated Pharmacokinetics for Clinical Development

Ivacaftor is currently used for the treatment of cystic fibrosis as both monotherapy (Kalydeco; Vertex Pharmaceuticals, Boston, MA) and combination therapy with lumacaftor (Orkambi; Vertex Pharmaceuticals). Each therapy targets specific patient populations: Kalydeco treats patients carrying one of nine gating mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein, whereas Orkambi treats patients homozygous for the F508del CFTR mutation. In this study, we explored the pharmacological and metabolic effects of precision deuteration chemistry on ivacaftor by synthesizing two novel deuterated ivacaftor analogs, CTP-656 (d9-ivacaftor) and d18-ivacaftor. Ivacaftor is administered twice daily and is extensively converted in humans to major metabolites M1 and M6; therefore, the corresponding deuterated metabolites were also prepared. Both CTP-656 and d18-ivacaftor showed in vitro pharmacologic potency similar to that in ivacaftor, and the deuterated M1 and M6 metabolites showed pharmacology equivalent to that in the corresponding metabolites of ivacaftor, which is consistent with the findings of previous studies of deuterated compounds. However, CTP-656 exhibited markedly enhanced stability when tested in vitro. The deuterium isotope effects for CTP-656 metabolism (DV = 3.8, DV/K = 2.2) were notably large for a cytochrome P450–mediated oxidation. The pharmacokinetic (PK) profile of CTP-656 and d18-ivacaftor were assessed in six healthy volunteers in a single-dose crossover study, which provided the basis for advancing CTP-656 in development. The overall PK profile, including the 15.9-hour half-life for CTP-656, suggests that CTP-656 may be dosed once daily, thereby enhancing patient adherence. Together, these data continue to validate deuterium substitution as a viable approach for creating novel therapeutic agents with properties potentially differentiated from existing drugs.

Cytochrome P450 3A4 and CYP3A5-Catalyzed Bioactivation of Lapatinib

Metabolic activation of the dual-tyrosine kinase inhibitor lapatinib by cytochromes CYP3A4 and CYP3A5 has been implicated in lapatinib-induced idiosyncratic hepatotoxicity; however, the relative enzyme contributions have not been established. The objective of this study was to examine the roles of CYP3A4 and CYP3A5 in lapatinib bioactivation leading to a reactive, potentially toxic quinoneimine. Reaction phenotyping experiments were performed using individual human recombinant P450 enzymes and P450-selective chemical inhibitors. Lapatinib metabolites and quinoneimine-glutathione (GSH) adducts were analyzed using liquid chromatography-tandem mass spectrometry. A screen of cDNA-expressed P450s confirmed that CYP3A4 and CYP3A5 are the primary enzymes responsible for quinoneimine-GSH adduct formation using lapatinib or O-dealkylated lapatinib as the substrate. The mean kinetic parameters (Km and kcat) of lapatinib O-dealkylation revealed that CYP3A4 was 5.2-fold more efficient than CYP3A5 at lapatinib O-dealkylation (CYP3A4 kcat/Km = 6.8 μM−1 min−1 versus CYP3A5 kcat/Km = 1.3 μM−1 min−1). Kinetic analysis of GSH adduct formation indicated that CYP3A4 was also 4-fold more efficient at quinoneimine-GSH adduct formation as measured by kcat (maximum relative GSH adduct levels)/Km (CYP3A4 = 0.0082 vs. CYP3A5 = 0.0021). In human liver microsomal (HLM) incubations, CYP3A4-selective inhibitors SR-9186 and CYP3cide reduced formation of GSH adducts by 78% and 72%, respectively, compared with >90% inhibition by the pan-CYP3A inhibitor ketoconazole. The 16%–22% difference between CYP3A- and CYP3A4-selective inhibition indicates the involvement of remaining CYP3A5 activity in generating reactive metabolites from lapatinib in pooled HLMs. Collectively, these findings support the conclusion that both CYP3A4 and CYP3A5 are quantitatively important contributors to lapatinib bioactivation.

In Vitro Metabolism of Oxymetazoline: Evidence for Bioactivation to a Reactive Metabolite

Oxymetazoline (6-tert-butyl-3-(2-imidazolin-2-ylmethyl)-2,4-dimethylphenol) has been widely used as a nonprescription nasal vasoconstrictor for >40 years; however, its metabolic pathway has not been investigated. This study describes the in vitro metabolism of oxymetazoline in human, rat, and rabbit liver postmitochondrial supernatant fraction from homogenized tissue (S9) fractions and their microsomes supplemented with NADPH. The metabolites of oxymetazoline identified by liquid chromatography (LC)/UV/tandem mass spectrometry (MS/MS), included M1 (monohydroxylation of the t-butyl group), M2 (oxidative dehydrogenation of the imidazoline to an imidazole moiety), M3 (monohydroxylation of M2), M4 (dihydroxylation of oxymetazoline), and M5 (dihydroxylation of M2). Screening with nine human expressed cytochromes P450 (P450s) identified CYP2C19 as the single P450 isoform catalyzing the formation of M1, M2, and M3. Glutathione conjugates of oxymetazoline (M6) and M2 (M7) were identified in the liver S9 fractions, indicating the capability of oxymetazoline to undergo bioactivation to reactive intermediate species. M6 and M7 were not detected in those liver S9 incubations without NADPH. Cysteine conjugates (M8 and M9) derived from glutathione conjugates and hydroxylated glutathione conjugates (M10 and M11) were also identified. The reactive intermediate of oxymetazoline was trapped with glutathione and N-acetyl cysteine and identified by LC/MS/MS. M6 was isolated and identified by one-dimensional or two-dimensional NMR as the glutathione conjugate of a p-quinone methide. We have shown the tendency of oxymetazoline to form p-quinone methide species via a bioactivation mechanism involving a CYP2C19-catalyzed two-electron oxidation. Nevertheless, we conclude that the formation of this reactive species might not be a safety concern for oxymetazoline nasal products because of the typical low-dose and brief dosage regimen limited to nasal delivery.

Altering Metabolic Profiles of Drugs by Precision Deuteration: Reducing Mechanism-Based Inhibition of CYP2D6 by Paroxetine

Selective deuterium substitution as a means of ameliorating clinically relevant pharmacokinetic drug interactions is demonstrated in this study. Carbon-deuterium bonds are more stable than corresponding carbon-hydrogen bonds. Using a precision deuteration platform, the two hydrogen atoms at the methylenedioxy carbon of paroxetine were substituted with deuterium. The new chemical entity, CTP-347 [(3S,4R)-3-((2,2-dideuterobenzo[d][1,3]dioxol-5-yloxy)methyl)-4-(4-fluorophenyl)piperidine], demonstrated similar selectivity for the serotonin receptor, as well as similar neurotransmitter uptake inhibition in an in vitro rat synaptosome model, as unmodified paroxetine. However, human liver microsomes cleared CTP-347 faster than paroxetine as a result of decreased inactivation of CYP2D6. In phase 1 studies, CTP-347 was metabolized more rapidly in humans and exhibited a lower pharmacokinetic accumulation index than paroxetine. These alterations in the metabolism profile resulted in significantly reduced drug-drug interactions between CTP-347 and two other CYP2D6-metabolized drugs: tamoxifen (in vitro) and dextromethorphan (in humans). Our results show that precision deuteration can improve the metabolism profiles of existing pharmacotherapies without affecting their intrinsic pharmacologies.

Abstract 3357: CTP-221, a deuterated S-enantiomer of lenalidomide, is greatly stabilized to epimerization and results in a more desirable pharmacokinetic profile than racemic lenalidomide.

IMiD-class compounds, including thalidomide, lenalidomide, and pomalidomide, have been developed as racemic mixtures of S- and R-enantiomers. The isolated enantiomers of thalidomide are known to have distinct biological activities. For example, the well-documented sedative effects of thalidomide are correlated with the R-enantiomer (Eriksson et al., 2000), whereas S-thalidomide exhibits enhanced potency for TNF-α inhibition compared to the R-enantiomer (Wnendt et al., 1996; Moreira et al., 1993). We have demonstrated that S-lenalidomide is more potent than racemic or R-lenalidomide in biological activities that are believed to be important for clinical efficacy of lenalidomide. Due to facile in vivo conversion, isolated S-enantiomers of IMiDs have not been developed clinically. Lenalidomide (Revlimid®) is a racemic mixture of S- and R-enantiomers that interconvert through epimerization. Revlimid is labeled for the treatment of 5q-myelodysplastic syndromes (MDS) and multiple myeloma. CTP-221 is a deuterium-modified analog of S-lenalidomide containing deuterium atoms at key positions including lenalidomide9s chiral center. Deuterium modification has the potential, albeit unpredictably, to alter the metabolic fate and hence the pharmacokinetic disposition of drugs, especially those that are biotransformed via the cleavage of carbon-hydrogen bonds (Fisher et al, 2006). The effect of deuterium modification on the epimerization rate and pharmacokinetic profile of CTP-221 was investigated in vitro and in vivo in mice, rats and monkeys. The rates of epimerization of CTP-221 and S-lenalidomide were compared in vitro in whole blood from mouse, rat, monkey, and human. It was found that CTP-221 was 2- to 3-fold more stable to epimerization than S-lenalidomide in the four species. To compare the in vivo pharmacokinetic profiles of CTP-221 and racemic lenalidomide, the compounds (10 mg/kg, PO) were administered to mice, rats and monkeys. In all three species, the exposure (AUC) of S- and R-lenalidomide following administration of racemic lenalidomide was about 50-57% and 43-50%, respectively, of the sum of the AUC of the individual enantiomers. However, when CTP-221 was administered, the AUC of CTP-221 and the deuterated R-enantiomers formed in vivo were about 96-99% and 1-4% respectively, of the sum of the AUC of the two enantiomers. Thus, CTP-221 epimerizes to a minimal extent in vivo and its administration provides exposure to very low levels of the R-enantiomer. In conclusion, the stabilization of CTP-221 via deuterium substitution resulted in maximal exposure to the more potent S-enantiomer and minimal exposure to the R-enantiomer. As a result, CTP-221 has the potential for improved potency and therapeutic index in comparison to racemic lenalidomide. Citation Format: Vinita Uttamsingh, Richard Gallegos, Changfu Cheng, Ara Aslanian, Julie Fields Liu, Roger Tung, Lijun Wu. CTP-221, a deuterated S-enantiomer of lenalidomide, is greatly stabilized to epimerization and results in a more desirable pharmacokinetic profile than racemic lenalidomide. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3357. doi:10.1158/1538-7445.AM2013-3357

Case History — Use of ADME Studies for Optimization of Drug Candidates

Efficacy and safety are the two key elements in the drug discovery and development processes. The primary goal for pharmaceutical research companies is to identify and manufacture therapeutic agents that are safe and efficacious for patients. In principle, benefits versus risks have to be considered for target patient populations. The risks are relatively high in life threatening diseases, e.g. cancer, compared to general areas, e.g. inflammation. Pharmacology, medicinal chemistry, pharmaceutical sciences, safety assessment, drug metabolism and pharmacokinetics (DMPK), clinical research, etc. are the essential multidisciplinary R&D functions assembled within the pharmaceutical R&D engine to accomplish the aforementioned mission. Pharmacokinetics (PK) is generally viewed as the universal biomarker which reflects the processes of how a drug molecule is absorbed (e.g. ka), distributed (e.g. Vd) in the body, and cleared from the body through metabolism and excretion. The area under the drug plasma concentration versus time curve (AUC) provides an indirect assessment of the exposure level and duration of action of the therapeutic agent at the site of action (e.g. synovial fluid, tumor, brain). An ideal drug candidate should possess a plasma drug level which is above the therapeutic concentration (i.e. efficacious) and below the toxic concentration (i.e. safe). In general, the therapeutic index is calculated by dividing the plasma exposure at the NO (toxic) Effect Level (NOEL), or NO Adverse Effect Level (NOAEL), by the minimum plasma concentration required for efficacy (e.g. EC50) and the safety margin is calculated by dividing NOEL (or NOAEL) plasma concentration by the maximum plasma drug concentration (Cmax) achieved at an efficacious dose.