Novel Mechanism of Decyanation of GDC-0425 by Cytochrome P450.

GDC-0425 [5-((1-ethylpiperidin-4-yl)oxy)-9H-pyrrolo[2,3-b:5,4-c']dipyridine-6-carbonitrile] is an orally bioavailable small-molecule inhibitor of checkpoint kinase 1 that was investigated as a novel cotherapy to potentiate chemotherapeutic drugs, such as gemcitabine. In a radiolabeled absorption, distribution, metabolism, and excretion study in Sprague-Dawley rats, trace-level but long-lived C-labeled thiocyanate was observed in circulation. This thiocyanate originated from metabolic decyanation of GDC-0425 and rapid conversion of cyanide to thiocyanate.

Linker Immolation Determines Cell Killing Activity of Disulfide-Linked Pyrrolobenzodiazepine Antibody-Drug Conjugates.

Disulfide bonds could be valuable linkers for a variety of therapeutic applications requiring tunable cleavage between two parts of a molecule (e.g., antibody-drug conjugates).

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.

Transporter Expression in Liver Tissue from Subjects with Alcoholic or Hepatitis C Cirrhosis Quantified by Targeted Quantitative Proteomics.

Although data are available on the change of expression/activity of drug-metabolizing enzymes in liver cirrhosis patients, corresponding data on transporter protein expression are not available. Therefore, using quantitative targeted proteomics, we compared our previous data on noncirrhotic control livers (n = 36) with the protein expression of major hepatobiliary transporters, breast cancer resistance protein (BCRP), bile salt export pump (BSEP), multidrug and toxin extrusion protein 1 (MATE1), multidrug resistance-associated protein (MRP)2, MRP3, MRP4, sodium taurocholate-cotransporting polypeptide (NTCP), organic anion-transporting polypeptides (OATP)1B1, 1B3, 2B1, organic cation transporter 1 (OCT1), and P-glycoprotein (P-gp) in alcoholic (n = 27) and hepatitis C cirrhosis (n = 30) livers. Compared with control livers, the yield of membrane protein from alcoholic and hepatitis C cirrhosis livers was significantly reduced by 56 and 67%, respectively.

Chemical Structure and Concentration of Intratumor Catabolites Determine Efficacy of Antibody Drug Conjugates.

Despite recent technological advances in quantifying antibody drug conjugate (ADC) species, such as total antibody, conjugated antibody, conjugated drug, and payload drug in circulation, the correlation of their exposures with the efficacy of ADC outcomes in vivo remains challenging. Here, the chemical structures and concentrations of intratumor catabolites were investigated to better understand the drivers of ADC in vivo efficacy. Anti-CD22 disulfide-linked pyrrolobenzodiazepine (PBD-dimer) conjugates containing methyl- and cyclobutyl-substituted disulfide linkers exhibited strong efficacy in a WSU-DLCL2 xenograft mouse model, whereas an ADC derived from a cyclopropyl linker was inactive.

An Automated High-Throughput Metabolic Stability Assay Using an Integrated High-Resolution Accurate Mass Method and Automated Data Analysis Software.

Advancement of in silico tools would be enabled by the availability of data for metabolic reaction rates and intrinsic clearance (CLint) of a diverse compound structure data set by specific metabolic enzymes. Our goal is to measure CLint for a large set of compounds with each major human cytochrome P450 (P450) isozyme. To achieve our goal, it is of utmost importance to develop an automated, robust, sensitive, high-throughput metabolic stability assay that can efficiently handle a large volume of compound sets.

A clinical drug-drug interaction study to evaluate the effect of a proton-pump inhibitor, a combined P-glycoprotein/cytochrome 450 enzyme (CYP)3A4 inhibitor, and a CYP2C9 inhibitor on the pharmacokinetics of vismodegib.

Purpose: The Hedgehog pathway inhibitor vismodegib exhibits pH-dependent solubility, and in vitro studies have shown that vismodegib is a substrate of P-glycoprotein (P-gp) and is metabolized by cytochrome P450 (CYP) 2C9 and 3A4. The objective of this four-arm parallel study in healthy subjects was to evaluate the effect of the proton-pump inhibitor rabeprazole, the P-gp/CYP3A4 inhibitor itraconazole, and the CYP2C9 and 3A4 inhibitor fluconazole on vismodegib steady-state pharmacokinetics.

Methods: Cohorts included a control arm (n = 22), in which vismodegib 150 mg was administered once daily (QD) for 7 days, and 3 arms in which vismodegib was co-administered QD for 7 days with rabeprazole 20 mg (including a 4-day lead-in; n = 24); itraconazole 200 mg (n = 22); or fluconazole 400 mg (n = 22).

Elucidating the Mechanism of Tofacitinib Oxidative Decyanation.

Background: Tofacitinib is known to generate two metabolites M2 (alcohol) and M4 (acid), which are formed as the result of oxidation and loss of the nitrile [1].

Method: Systematic in vitro investigation into generation of M2 and M4 from tofacitinib.

Results: In vitro using human liver microsomes, we found a new geminal diol metabolite of tofacitinib (MX) that lost the nitrile.

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

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 (C) cobimetinib to Sprague-Dawley rats (30 mg/kg) and Beagle dogs (5 mg/kg). 2.

Simultaneous determination of itraconazole, hydroxy itraconazole, keto itraconazole and N-desalkyl itraconazole concentration in human plasma using liquid chromatography with tandem mass spectrometry.

A high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) assay was developed and validated for simultaneous determination of itraconazole (ITZ), hydroxy-itraconazole (OH-ITZ), keto-itraconazole (keto-ITZ) and N-desalkyl itraconazole (ND-ITZ) concentration in human plasma. One hundred and fifty microliters of human plasma were extracted using a solid-supported liquid extraction (SLE) method and the final extracts were analyzed using reverse-phase chromatography and positive electrospray ionization mass spectrometry. The standard curve range is 5-2500 ng/mL for ITZ and OH-ITZ and 0.

Evaluation of Time Dependent Inhibition Assays for Marketed Oncology Drugs: Comparison of Human Hepatocytes and Liver Microsomes in the Presence and Absence of Human Plasma.

Purpose: To evaluate an alternative in vitro system which can provide more quantitatively accurate drug drug interaction (DDI) prediction for 10 protein kinase inhibitors for which DDI risk was over-predicted by inhibition data generated in human liver microsomes (HLM).

Methods: Three cryopreserved human hepatocyte (hHEP) systems: 1) plated hHEPs; 2) hHEPs suspended in Dulbecco's Modified Eagle Medium (DMEM) and 3) hHEPs suspended in human plasma (plasma hHEPs) were developed to detect CYP3A time dependent inhibition, and the static mechanistic model was used to predict clinical outcomes.

Results: A general trend was observed in the CYP3A inactivation potency (k inact /K I, app ) as HLM > plated > DMEM ≥ plasma hHEPs.

Surrogate analyte approach for quantitation of endogenous NAD(+) in human acidified blood samples using liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

A high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for the quantitative determination of NAD(+) in human whole blood using a surrogate analyte approach was developed and validated. Human whole blood was acidified using 0.5N perchloric acid at a ratio of 1:3 (v:v, blood:perchloric acid) during sample collection.

Development of a Physiologically Based Pharmacokinetic Model for Itraconazole Pharmacokinetics and Drug-Drug Interaction Prediction.

Background And Objectives: Physiologically based pharmacokinetic (PBPK) modeling for itraconazole has been challenging due to highly variable in vitro d ata used for 'bottom-up' model building. Under-prediction of pharmacokinetics and drug-drug interactions (DDIs) following multiple doses of itraconazole has limited the use of PBPK model simulation to aid an itraconazole clinical DDI study design. The aim of this work is to develop an itraconazole PBPK model predominantly using a 'top-down' approach to enable a more accurate pharmacokinetic and DDI prediction.

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.

A novel reaction mediated by human aldehyde oxidase: amide hydrolysis of GDC-0834.

GDC-0834, a Bruton's tyrosine kinase inhibitor investigated as a potential treatment of rheumatoid arthritis, was previously reported to be extensively metabolized by amide hydrolysis such that no measurable levels of this compound were detected in human circulation after oral administration. In vitro studies in human liver cytosol determined that GDC-0834 (R)-N-(3-(6-(4-(1,4-dimethyl-3-oxopiperazin-2-yl)phenylamino)-4-methyl-5-oxo- 4,5-dihydropyrazin-2-yl)-2-methylphenyl)-4,5,6,7-tetrahydrobenzo[b] thiophene-2-carboxamide) was rapidly hydrolyzed with a CLint of 0.511 ml/min per milligram of protein.

Prediction of drug distribution in subcutaneous xenografts of human tumor cell lines and healthy tissues in mouse: application of the tissue composition-based model to antineoplastic drugs.

Advanced tissue composition-based models can predict the tissue-plasma partition coefficient (Kp ) values of drugs under in vivo conditions on the basis of in vitro and physiological input data. These models, however, focus on healthy tissues and do not incorporate data from tumors. The objective of this study was to apply a tissue composition-based model to six marketed antineoplastic drugs (docetaxel, DOC; doxorubicin, DOX; gemcitabine, GEM; methotrexate, MTX; topotecan, TOP; and fluorouracil, 5-FU) to predict their Kp values in three human tumor xenografts (HCT-116, H2122, and PC3) as well as in healthy tissues (brain, muscle, lung, and liver) under steady-state in vivo conditions in female NCR nude mice.

Interspecies variability in expression of hepatobiliary transporters across human, dog, monkey, and rat as determined by quantitative proteomics.

We quantified, by liquid chromatography tandem mass spectrometry, transporter protein expression of BSEP, MATE1, MRP3, MRP4, NTCP, and OCT1 in our human liver bank (n = 55) and determined the relationship between protein expression and sex, age and genotype. These data complement our previous work in the same liver bank where we quantified the protein expression of OATPs, BCRP, MDR1, and MRP2. In addition, we quantified and compared the interspecies differences in expression of the hepatobiliary transporters, corresponding to the above human transporters, in liver tissue and hepatocytes of male beagle dogs, cynomolgus monkeys, Sprague-Dawley rats, and Wistar rats.

Quantitative transporter proteomics by liquid chromatography with tandem mass spectrometry: addressing methodologic issues of plasma membrane isolation and expression-activity relationship.

To predict transporter-mediated drug disposition using physiologically based pharmacokinetic models, one approach is to measure transport activity and relate it to protein expression levels in cell lines (overexpressing the transporter) and then scale these to via in vitro to in vivo extrapolation (IVIVE). This approach makes two major assumptions. First, that the expression of the transporter is predominantly in the plasma membrane.

Physiologically based pharmacokinetic modeling to predict drug-drug interactions involving inhibitory metabolite: a case study of amiodarone.

Evaluation of drug-drug interaction (DDI) involving circulating inhibitory metabolites of perpetrator drugs has recently drawn more attention from regulatory agencies and pharmaceutical companies. Here, using amiodarone (AMIO) as an example, we demonstrate the use of physiologically based pharmacokinetic (PBPK) modeling to assess how a potential inhibitory metabolite can contribute to clinically significant DDIs. Amiodarone was reported to increase the exposure of simvastatin, dextromethorphan, and warfarin by 1.

Rational use of plasma protein and tissue binding data in drug design.

It is a commonly accepted assumption that only unbound drug molecules are available to interact with their targets. Therefore, one of the objectives in drug design is to optimize the compound structure to increase in vivo unbound drug concentration. In this review, theoretical analyses and experimental observations are presented to illustrate that low plasma protein binding does not necessarily lead to high in vivo unbound plasma concentration.

Measuring NAD(+) levels in mouse blood and tissue samples via a surrogate matrix approach using LC-MS/MS.

Background: NAD(+) is an endogenous analyte and is unstable during blood sample collection, both of which present obstacles for quantitation. Moreover, current procedures for NAD(+) sample collection require onsite treatment with strong acid to stabilize the NAD(+) in mouse blood cells.

Results: NAD(+) can be stabilized by addition of acid before the frozen mouse blood sample was thawed.

Dose-dependent exposure and metabolism of GNE-892, a β-secretase inhibitor, in monkeys: contributions by P450, AO, and P-gp.

(R)-2-Amino-1,3',3'-trimethyl-7'-(pyrimidin-5-yl)-3',4'-dihydro-2'H-spiro[imidazole-4,1'-naphthalen]-5(1H)-one (GNE-892) is an orally administered inhibitor of β-secretase 1 (β-site amyloid precursor protein cleaving enzyme 1, BACE1) that was developed as an intervention therapy against Alzheimer's disease. A clinical microdosing strategy was being considered for de-risking the potential pharmacokinetic liabilities of GNE-892. We tested whether dose-proportionality was observed in cynomolgus monkey as proof-of-concept for a human microdosing study.

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 (14)C-radiolabeled mass balance study in rats. Their structures were characterized by high-resolution mass spectrometry and nuclear magnetic resonance.

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.

Assessment of the hepatic CYP reductase null mouse model and its potential application in drug discovery.

CYP Oxidoreductase (Por) is the essential electron donor for all CYP enzymes and is responsible for the activation of CYP. The Taconic Hepatic CYP Reductase Null (HRN) mouse model possesses a targeted mutation that results in liver-specific deletion of the Por gene thereby resulting in a disruption of CYP metabolism in the liver. The objectives of these studies were to further characterize the HRN mouse using probe drugs metabolized by CYP.