Cytochrome P450 Inhibition Assay Research Articles

Discovery of HZ-L105, a Next Generation of Bcl-2 Inhibitor, Overcomes Bcl-2 Mutation and Exhibits Superior Antitumor Activity

Introduction: Bcl-2, a key regulator of apoptosis pathway, is over-expressed in many hematologic malignancies, which results in blocking of apoptosis and cell survival. Targeting Bcl-2 for the treatment of malignant hematological tumors has been well validated and demonstrated by FDA approval of the first generation Bcl-2 inhibitor Venetoclax for chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL) and acute myeloid leukemia (AML). Although Venetoclax provides significant clinical benefits, there are reports show that after long-term monotherapy patients develop disease progression , often driven by acquisition of Bcl-2 Gly101Val (G101V), Asp103Tyr (D103Y) resistance mutations. Here, we described the discovery of a next generation Bcl-2 inhibitor HZ-L105 as with high potency against Bcl-2, Bcl-2(G101V) and Bcl-2(D103Y) for treatment of Bcl-2 dependent and Venetoclax-resistant hematological cancers. Methods:Biochemical inhibition assays were conducted towards Bcl-2/Bcl-2 G101V/Bcl-2 D103Y/Bcl-xL by time-resolved fluorescence resonance energy transfer (TR-FRET);Cellular proliferation tests of RS4;11 and RS4;11-G101V overexpressed stable line were carried out using MTS assay;Human platelet viability was measured by Cell-titer Glo assay;Cytochrome P450 inhibition assays were performed using fluorometric assay;In vivo pharmacodynamic (PD) studies were performed in RS4;11-derived subcutaneous xenograft SCID mouse model;Preclinical pharmacokinetics (PK) studies were assayed in mice and dogs and parameters were determined by LC-MS. Results: In biochemical assays, HZ-L105 potently inhibited Bcl-2 WT, Bcl-2 G101V and Bcl-2 D103Y with IC50 of 0.54 nM, 6.16 nM and 5.57 nM，while Venetoclax's IC50 of 1.62 nM, 440.6 nM and 279.8 nM, respectively. Therefore, it is expected to overcome the acquisition of Bcl-2 resistance mutations. In cellular assays, HZ-L105 suppressed cell growth not only in RS4;11 but also in RS4;11-G101V stable line (IC50=0.30 nM in RS4;11 and 243.36 nM in RS4;11-G101V), which was much more superior to Venetoclax (IC50= 2.91 nM in RS4;11 and 3360.79 nM in RS4;11-G101V). HZ-L105 was also highly selective against Bcl-2, showing >1000 folds selectivity to Bcl-xL. Furthermore, platelet viability assay in vitro showed HZ-L105 induced hypotoxicity as similarly to Venetoclax, suggesting its good potential to avoid thrombocytopenia due to Bcl-xL inhibition. In the preclinical Pharmacodynamics studies, oral administration of HZ-L105 induced rapid and robust antitumor effect in RS4;11 ALL xenografts. HZ-L105 at a dose of 5 mg/kg demonstrated significantly excellent efficacy than Venetoclax at a dose 15mg/kg in RS4;11-derived xenograft mouse model. In the preclinical pharmacokinetic studies, HZ-L105 showed excellent pharmacokinetic properties in ICR mice and beagle dogs, with long T1/2 and high dose-normalized AUC. HZ-L105 did not reduced hERG tail current (% current blocked 2.87% at 30μM), indicating the compound has a low potential to induce QT prolongation. HZ-L105 has less drug-drug interaction potential, as evidenced by no inhibitory effect on CYPs, especially in CYP2C9 (IC50 >20 μM) compared with Venetoclax (IC50=1.12 μM). Besides, in 14-days oral gavage toxicity study in mice, no compound related deaths occurred at 15, 50 and 300 mg/kg/day, and only leukopenia was observed similarly to Venetoclax. Conclusion: In summary, HZ-L105 as a next generation Bcl-2 inhibitor, showed great potency on wild type and acquisition resistance mutations of Bcl-2 in vitro, excellent oral bioavailability, superior anti-tumor activities on preclinical xenograft model, and safety profiles,. It may provide new thoughts on treatment for a wide range of Bcl-2-dependent and Venetoclax-resistant hematological cancers. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Potential Inhibitory and Inducing Effects of Triphala Formulation on Cytochrome P450 Enzymes

Triphala formulation is one of the most common traditional medicines used for several health conditions. The study aimed to investigate the inhibitory and inducing potentials of Triphala formulation, including its active compounds gallic acid and ellagic acid, on cytochrome P450 (CYP450) enzymes. The inhibitory effects of Triphala formulation, gallic acid and ellagic acid on the 5 major human CYP450 enzymes were evaluated using a bioluminescent CYP450 inhibition assay. Evaluation of inducing effect of Triphala formulation on CYP1A2 and CYP3A1 mRNA expression following daily oral doses of 1,000 and 5,000 mg/kg body weight for 28 days was evaluated using RT-PCR. Gallic acid potently inhibited CYP1A2, CYP2C9 and CYP2C19 with a non-competitive nature, while the inhibitory potencies on CYP2D6 and CYP3A4 were weak. Chronic dosing of Triphala formulation at 1,000 and 3,000 mg/kg body weight significantly induced mRNA expression of CYP1A2 but not CYP3A1. Results suggest the propensity of metabolic drug interactions when Triphala formulation was concurrently administered with other conventional drugs or herbal medicines. 
 HIGHLIGHTS
 
 Gallic acid potently inhibited CYP1A2, CYP2C9 and CYP2C19 with a non-competitive nature
 Triphala formulation at the dose of 1,000 and 3,000 mg/kg body weight significantly induced mRNA expression of CYP1A2
 Results suggest the propensity of metabolic drug interactions when Triphala formulation is concurrently administered with conventional drugs or herbal products by human CYP450 enzymes, particularly CYP1A2, CYP2C9 and CYP2C19
 
 GRAPHICAL ABSTRACT

Diagnosis of Parkinson's disease by investigating the inhibitory effect of serum components on P450 inhibition assay

Parkinson’s disease (PD) is the second most common neurodegenerative disease, and diagnostic methods and biomarkers for patients without subjective motor symptoms have not yet been established. Previously, we developed a cytochrome P450 inhibition assay that detects alterations in metabolite levels associated with P450s caused by inflammation and exposure to endogenous or exogenous substances. However, it is unknown whether the P450 inhibition assay can be applied in PD diagnosis. Here, we determined whether the P450 inhibition assay can discriminate sera between patients with PD and healthy individuals. The results of the assay revealed that the P450 inhibition assay can discriminate PD with an area under the receiver operating characteristic curve (AUC) value of 0.814–0.914 in rats and an AUC value of 0.910 in humans. These findings demonstrate that the P450 inhibition assay can aid in the future development of liquid biopsy-based diagnostic methods for PD.

Optimization of the CYP inhibition assay using LC-MS/MS

The data presented in this article are related to the research article entitled “Cytochrome P450 inhibition activities of non-standardized botanical products” [1], in which the possible CYP inhibitory properties of botanical products were investigated. This article describes the optimization and bioanalytical method validation of the CYP (Cytochrome P450 inhibition assay) inhibition assays, namely, phenacetin O-deethylase assay, testosterone 6β-hydroxylase assay, felodipine dehydrogenase assay and midazolam 1’-hydroxylase assay using LC-MS/MS.

Development of Robust Quantitative Structure-Activity Relationship Models for CYP2C9, CYP2D6, and CYP3A4 Catalysis and Inhibition.

Cytochrome P450 enzymes are responsible for the metabolism of >75% of marketed drugs, making it essential to identify the contributions of individual cytochromes P450 to the total clearance of a new candidate drug. Overreliance on one cytochrome P450 for clearance levies a high risk of drug-drug interactions; and considering that several human cytochrome P450 enzymes are polymorphic, it can also lead to highly variable pharmacokinetics in the clinic. Thus, it would be advantageous to understand the likelihood of new chemical entities to interact with the major cytochrome P450 enzymes at an early stage in the drug discovery process. Typical screening assays using human liver microsomes do not provide sufficient information to distinguish the specific cytochromes P450 responsible for clearance. In this regard, we experimentally assessed the metabolic stability of ∼5000 compounds for the three most prominent xenobiotic metabolizing human cytochromes P450, i.e., CYP2C9, CYP2D6, and CYP3A4, and used the data sets to develop quantitative structure-activity relationship models for the prediction of high-clearance substrates for these enzymes. Screening library included the NCATS Pharmaceutical Collection, comprising clinically approved low-molecular-weight compounds, and an annotated library consisting of drug-like compounds. To identify inhibitors, the library was screened against a luminescence-based cytochrome P450 inhibition assay; and through crossreferencing hits from the two assays, we were able to distinguish substrates and inhibitors of these enzymes. The best substrate and inhibitor models (balanced accuracies ∼0.7), as well as the data used to develop these models, have been made publicly available (https://opendata.ncats.nih.gov/adme) to advance drug discovery across all research groups. SIGNIFICANCE STATEMENT: In drug discovery and development, drug candidates with indiscriminate cytochrome P450 metabolic profiles are considered advantageous, since they provide less risk of potential issues with cytochrome P450 polymorphisms and drug-drug interactions. This study developed robust substrate and inhibitor quantitative structure-activity relationship models for the three major xenobiotic metabolizing cytochromes P450, i.e., CYP2C9, CYP2D6, and CYP3A4. The use of these models early in drug discovery will enable project teams to strategize or pivot when necessary, thereby accelerating drug discovery research.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method development for screening of potential tamoxifen-drug/herb interaction via in vitro cytochrome P450 inhibition assay

Screening for potential drug-drug interaction (DDI) or herb-drug interaction (HDI) using in vitro cytochrome P450 inhibition (IVCI) assays requires robust analytical methods with high sensitivity and reproducibility. Utilization of liquid chromatography-mass spectrometry (LC-MS) for analyte quantification is often hampered by the presence of non-volatile IVCI sample buffer constituents that often results in ion suppression. In this study, to enable screening of drug interactions involving tamoxifen (TAM) metabolism using IVCI-LC-MS/MS, a liquid–liquid extraction (LLE) method was developed and optimized for sample clean-up. Utilization of chloroform as extraction solvent and adjustment of sample pH to 11 was found to result in satisfactory recovery (>70%) and low ion suppression (<19%). A LC-MS/MS method was subsequently developed and validated for simultaneous quantification of major TAM metabolites, such as N-desmethyltamoxifen (NDT), endoxifen (EDF) and 4-hydroxytamoxifen (HTF) to enable IVCI sample analysis. Satisfactory separation of E-/Z-isomers of endoxifen with peak resolution (Rs) of 1.9 was achieved. Accuracy and precision of the method was verified within the linear range of 0–50 ng/mL for NDT, 0–25 ng/mL for HTF and 0–25 ng/mL for EDF (E/Z isomers). Inhibitory potency (IC50, Ki and mode of inhibition) of known CYP inhibitors and Strobilanthes crispus extract was then evaluated using the validated method. In summary, the results demonstrated applicability of the developed LLE and validated LC-MS/MS method for in vitro screening of DDI and HDI involving TAM metabolism.

Development and Validation of a Higher-Throughput Cytochrome P450 Inhibition Assay with the Novel Cofactor-Supplemented Permeabilized Cryopreserved Human Hepatocytes (MetMax Human Hepatocytes).

Here, we report the application of a novel hepatocyte system, the cofactor-supplemented permeabilized cryopreserved human hepatocytes [MetMax human hepatocytes (MMHHs)] in a higher-throughput 384-well plate assay for the evaluation of cytochrome P450 (P450) inhibition. The assay was created to develop physiologically relevant P450 inhibition information, taking advantage of the complete organelle composition and their associated drug-metabolizing enzymes of the MMHH but with the ease of use of human liver microsomes, including storage at -80°C instead of in liquid nitrogen, and thaw and use without centrifugation and microscopic evaluation as required for intact hepatocytes. Nine key P450 isoforms for drug metabolism (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) were evaluated using multiple isoform-selective inhibitors. Results with MMHH were found to be comparable to those obtained with intact cryopreserved human hepatocytes (CHHs). Isoform-selective drug-metabolizing enzyme pathways evaluated were phenacetin O-deethylation (CYP1A2), coumarin 7-hydroxylation (CYP2A6), bupropion hydroxylation (CYP2B6), amodiaquine N-deethylation (CYP2C8), diclofenac 4-hydroxylation (CYP2C9), s-mephenytoin 4'-hydroxylation (CYP2C19), dextromethorphan O-demethylation (CYP2D6), chlorzoxazone 6-hydroxylation (CYP2E1), and midazolam 1'-hydroxylation and testosterone 6β-hydroxylation (CYP3A4). The Km values obtained with MMHHs were comparable with those reported in the literature for CHHs. Using substrate concentrations at or near Km values, the IC50 values for the standard inhibitors against the P450 activities were found to be comparable between MMHHs and CHHs, with 73% and 84% of values falling within 2-fold and 3-fold, respectively, from the line of unity. The results indicate that MMHHs can be an efficient experimental system for the evaluation of P450 inhibition in hepatocytes. SIGNIFICANCE STATEMENT: MetMax human hepatocytes (MMHHs) are cofactor-supplemented cryopreserved human hepatocytes with the complete drug-metabolizing enzyme pathways of the conventional hepatocytes but with the convenience of human liver microsomes, including storage at -80°C instead of in liquid nitrogen, and direct thaw and use without a need for centrifugation and microscopic examination. Here, we report the application of MMHH in a high-throughput assay in a 384-well plate format for the evaluation of cytochrome P450 (P450) inhibition. Our results show that data obtained with MMHH are similar to those with conventional hepatocytes, suggesting that the MMHH 384-well P450 inhibition assay can be used routinely for the evaluation of drug-drug interaction potential of new chemical entities in drug development.

Evaluation of Herb-Drug Interaction of Synacinn™ and Individual Biomarker through Cytochrome 450 Inhibition Assay.

SynacinnTM contains five standardized herbal extracts of Orthosiphon Stamineus (OS), Syzygium polyanthum (SZ), Curcuma xantorrizza (CX), Cinnamomum zeylanicum (CZ) and Andrographis paniculata (AP) and is standardized against phytochemical markers of rosmarinic acid, gallic acid, curcumin, catechin and andrographolide respectively. This herbal medicine has been used as health supplement for diabetes. SynacinnTM is recommended to be consumed as supplement to the diabetic drugs. However, herb-drug interaction of SynacinnTM polyherbal with present drugs is unknown. This study was designed to investigate the effect of SynacinnTM and its individual biomarkers on drug metabolizing enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4 (Midazolam), CYP3A4 (Testosteron)), to assess its herb-drug interaction potential through cytochrome P450 inhibition assay. This study was conducted using liquid chromatography- tandem mass spectroscopy (LC-MS/MS) using probe substrates using human liver microsomes against CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4 (Midazolam) and CYP3A4 (Testosteron). Result showed that SynacinnTM at maximum concentration (5000 µg/ml) 100% inhibit CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4 (Midazolam) and CYP3A4 (Testosteron). IC50 values determined were 0.23, 0.60, 0.47, 0.78, 1.23, 0.99, 1.01, and 0.91 mg/ml for CYP 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4 (midazolam) and 3A4 (testosterone), respectively. Meanwhile, all individual biomarkers showed no, less or moderate inhibitory effect towards all the tested CYP450 except for curcumin that showed inhibition of CYP2C8 (91%), CYP2C9 (81%) and CYP2C19 (72%) at 10µM. Curcumin was found to be an active constituent that might contribute to the inhibition of SynacinnTM against CYP2C8, CYP2C9 and CYP2C19. It can be suggested that SynacinnTM can be consumed separately from a drug known to be metabolized by all tested CYP450 enzymes.

Automated cytochrome P450 inhibition assay using IC50 shift analysis and cocktail LC–MS/MS analysis

Automated cytochrome P450 inhibition assay using IC50 shift analysis and cocktail LC–MS/MS analysis

Pharmacokinetic studies of a three-component complex that repurposes the front line antibiotic isoniazid against Mycobacterium tuberculosis

The frontline tuberculosis (Tb) antibiotic isoniazid has been repurposed using a three component complex aimed at increasing the delivery efficiency and adding new avenues to its mechanism of action. This study focuses on pharmacokinetic studies of the isoniazid-sucrose–copper (II)-PEG-3350 complex. The assays include the Plasma Protein Binding Assay (85.8%), Caco-2 Permeability Assay (B→APapp, 0.13 × 10−6 cm/s), Cytochrome P450 Inhibition Assay (i.e. CYP2B6, IC50 = 7.26 μM), In vitro microsomal Stability Assay (t1/2 NADPH-Dependent > 240 min), and HepG2 Cytotoxicity (no toxicity). The National Cancer Institute's 60 cell line panel is used to measure activity against cancer cells. The percent growth values averaged over all 60 cell lines indicates the complex has no anti-cancer activity, which also suggests a lack of general toxicity. It also provides data for the complexes specificity against Mycobacterium tuberculosis.

In vitro evaluation of the inhibition and induction potential of olaparib, a potent poly(ADP-ribose) polymerase inhibitor, on cytochrome P450

1. In vitro studies were conducted to evaluate potential inhibitory and inductive effects of the poly(ADP-ribose) polymerase (PARP) inhibitor, olaparib, on cytochrome P450 (CYP) enzymes. Inhibitory effects were determined in human liver microsomes (HLM); inductive effects were evaluated in cultured human hepatocytes.2. Olaparib did not inhibit CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2D6 or CYP2E1 and caused slight inhibition of CYP2C9, CYP2C19 and CYP3A4/5 in HLM up to a concentration of 100 μM. However, olaparib (17–500 μM) inhibited CYP3A4/5 with an IC50 of 119 μM. In time-dependent CYP inhibition assays, olaparib (10 μM) had no effect against CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1 and a minor effect against CYP3A4/5. In a further study, olaparib (2–200 μM) functioned as a time-dependent inhibitor of CYP3A4/5 (KI, 72.2 μM and Kinact, 0.0675 min−1). Assessment of the CYP induction potential of olaparib (0.061–44 μM) showed minor concentration-related increases in CYP1A2 and more marked increases in CYP2B6 and CYP3A4 mRNA, compared with positive control activity; however, no significant change in CYP3A4/5 enzyme activity was observed.3. Clinically significant drug–drug interactions due to olaparib inhibition or induction of hepatic or intestinal CYP3A4/5 cannot be excluded. It is recommended that olaparib is given with caution with narrow therapeutic range or sensitive CYP3A substrates, and that prescribers are aware that olaparib may reduce exposure to substrates of CYP2B6.

Herb–drug interaction potential of Berberis aristata through cytochrome P450 inhibition assay

Abstract Herbal medicines have been used from ancient times for the management of several disease and ailments along with prescribed medicine without consulting the physicians. Berberis aristata DC. has been widely used in management of diabetes as alternative therapy in India and other Asian countries. An approach has been made to evaluate the possible cytochrome P450 (CYP450) enzyme inhibition potential with the bark of the anti-diabetic herb Berberis aristata DC. along with combination of oral hypoglycaemic agents like glimepiride and gliclazide. Bioactive compound was quantified through RP-HPLC, in order to standardize the plant extract and interaction potential of standardized extract and bioactive compound with oral hypoglycaemic drugs were evaluated. Herb–drug interaction potential of the test samples were evaluated by CYP450-carbonmonoxide complex (CYP450-CO) assay with pooled rat liver microsome. Influence on individual recombinant human CYP450 isoforms such as CYP3A4, CYP2D6, CYP2C9 and CYP1A2 isozymes were analyzed through fluorescence microplate screening assay. The combination index (CI)—isobologram method was applied for the prediction of combination effects. Plant extract showed significantly higher IC50 value than respective positive control against CYP3A4, 2D6, 2C9 and 1A2. Combination of the test extract and the phytoconstituent present therein along with oral hypoglycemic drugs also showed significantly less (P

An improved substrate cocktail for assessing direct inhibition and time-dependent inhibition of multiple cytochrome P450s.

The substrate cocktail is frequently used to evaluate cytochrome P450 (CYP) enzyme-mediated drug interactions and potential interactions among the probe substrates. Here, we re-optimized the substrate cocktail method to increase the reliability and accuracy of screening for candidate compounds and expanded the method from a direct CYP inhibition assay to a time-dependent inhibition (TDI) assay. In the reaction mixtures containing human liver microsome (0.1 mg/mL), both the concentrations of a substrate cocktail (phenacetin for 1A2, coumarin for 2A6, bupropion for 2B6, diclofenac for 2C9, dextromethorphan for 2D6, and testosterone for 3A4) and the incubation time were optimized. Metabolites of the substrate probes were simultaneously analyzed by multiple-reaction monitoring (MRM) using a routine LC/MS/MS. Direct CYP inhibition was validated using 7 inhibitors (α-naphthoflavone, tranylcypromine, ticlopidine, fluconazole, quinidine, ketoconazole and 1-ABT). The time-dependent inhibition was partially validated with 5 inhibitors (ketoconazole, verapamil, quinidine, paroxetine and 1-ABT). The inhibition curve profiles and IC50 values of 7 CYP inhibitors were approximate when a single substrate and the substrate cocktail were tested, and were consistent with the previously reported values. Similar results were obtained in the IC50 shifts of 5 inhibitors when a single substrate and the substrate cocktail were tested in the TDI assay. The 6-in-1 substrate cocktail (for 1A2, 2A6, 2B6, 2C9, 2D6 and 3A) is reliable for assessing CYP inhibition and time-dependent inhibition of drug candidates.

Effect of Dioscorea bulbifera and its Major Bioactive Compound, Diosgenin on CYP450 Mediated Drug Metabolism

Cytochrome P450 (CYP450) inhibition by the bioactive molecules of herbs leading to greater potential for toxicity of co-administered drugs. Diosgenin is an important bioactive steroidal sapogenin from Dioscorea bulbifera belonging to the triterpene group and is of great interest to the pharmaceutical industry. The purpose of this study was, to find whether D. bulbifera and Diosgenin influences the effect on rat CYP450 enzymes and its important isoforms (CYP3A4, CYP2D6) by using high through put screening. CYP450-CO Complex assay was performed in rat pooled liver microsomes to calculate percentage inhibition of test samples on CYP450. IC values were determined by fluorometric assays of CYP3A4 and CYP2D6. Mode of inhibition of Diosgenin on these CYPs were further assessed by molecular docking studies with the Glide (Schrodinger Inc. U.S.A.). In CYP450 inhibition assay, the inhibitory potential of herb extract (40.44±1.28 %) was found to be less than positive control (72.08±1.96 %). In fluorometric assay, herb extract exhibited higher IC values (96.21 ± 1.32 to 180.42±0.12 µg/ml) when compared to positive inhibitors and lower than Diosgenin (172.54±0.52 to 201.86±1.49 µg/ml) on CYP3A4 and CYP2D6. Based on the inhibitory potential, test substances exhibited very less interaction capacity, thereby leading to less significant herb-drug interaction with co-administered drugs.Further clinical studies are required to fully assess the safety of Diosgenin in terms of CYP450.

Comparison of liquid chromatography and supercritical fluid chromatography coupled to compact single quadrupole mass spectrometer for targeted in vitro metabolism assay

The goal of this study was to evaluate the combination of powerful chromatographic methods and compact single quadrupole MS device for simple in vitro cytochrome P450 (CYP) inhibition assay, instead of more expensive triple quadrupole MS/MS detectors. For this purpose, two modern chromatographic approaches (ultra-high pressure liquid chromatography (UHPLC) and ultra-high performance supercritical fluid chromatography (UHPSFC)) were tested in combination with simple MS detector. To show the applicability for an in vitro CYP-mediated metabolism assay using the cocktail approach, a method was first developed in UHPLC–MS to separate a mixture of 8 probe substrates and 8 CYP-specific metabolites. A screening procedure was initially applied to determine the best combination of a column, an organic modifier and a mobile-phase pH, followed by fine tuning of the conditions (i.e., gradient profile, temperature and pH) using HPLC modelling software. A similar sequential method development procedure was also evaluated for UHPSFC–MS. For method development, where peak tracking is necessary, the use of single quadrupole MS was found to be extremely valuable for following the investigated analytes. Ultimately, a baseline separation of the 16 compounds was achieved in both UHPLC–MS and UHPSFC–MS with an analysis time of less than 7min. In a second series of experiments, sensitivity was evaluated, and LOQ values were between 2 and 100ng/mL in UHPLC–MS, while they ranged from 2 to 200ng/mL in UHPSFC–MS. Based on the concentrations employed for the current in vitro phase I metabolism assay, these LOQ values were appropriate for this type of application. Finally, the two analytical methods were applied to in vitro CYP-dependent metabolism testing. Two well-known phytochemical inhibitors, yohimbine and resveratrol, were investigated, and reliable conclusions were drawn from these experiments with both UHPLC–MS and UHPSFC–MS. At the end, the proposed strategy of optimized chromatography combined with simple MS device has been shown to potentially compete with the widely used combination of generic chromatography and highly selective MS/MS device for simple in vitro CYP inhibition assays. In addition, our analytical method may be easier to use in a routine environment; the instrument cost is significantly reduced and the two developed methods fit for purpose.

Development and validation of a liquid-chromatography high-resolution tandem mass spectrometry approach for quantification of nine cytochrome P450 (CYP) model substrate metabolites in an in vitro CYP inhibition cocktail.

Knowledge about the cytochrome P450 (CYP) inhibition potential of new drug candidates is important for drug development because of its risk of interactions. For novel psychoactive substances (NPS), corresponding data are not available. For developing a general drug inhibition cocktail assay, a liquid-chromatography high-resolution tandem mass spectrometry multi-analyte approach was developed and validated for quantifying low concentrations of O-diethyl phenacetin for CYP 1A2, 7-hydroxy coumarin for CYP 2A6, 4-hydroxy bupropion for CYP 2B6, N-diethyl amodiaquine for CYP 2C8, 4-hydroxy diclofenac for CYP 2C9, 5-hydroxy omeprazole for CYP 2C19, O-dimethyl dextromethorphan for CYP 2D6, 6-hydroxy chlorzoxazone for CYP 2E1, and 6-beta-hydroxy testosterone for CYP 3A in the incubation mixture in the presence of substrates and inhibitors. The tested matrix effects ranged from 63 to 141 % and the recoveries from 95 to 110 %. Time-saving one-point calibration allowed sufficient quantification, although some of the validation results for 7-hydroxy coumarin, 4-hydroxy bupropion, 4-hydroxy diclofenac, and 6-beta-hydroxy testosterone were outside the acceptance criteria (AC) but without influence of the IC50 calculation. Validation showed also that the approach was sensitive and selective using mass spectral multiplexing. In conclusion, the presented assay was suitable for the quantification of the model substrate metabolites and could be used for the development of a CYP inhibition assay for testing most CYPs and a wide range of drugs of abuse.

Cytochrome P450 inhibitory potential and RP-HPLC standardization of trikatu—A Rasayana from Indian Ayurveda

Ethnopharmacological relevanceTrikatu is a very well known ‘Rasayana’ in Ayurveda and widely used as a polyherbal ayurvedic formulation in India. It consists of three well known plants, viz., Piper longum (PL), Piper nigrum (PN) and Zingiber officinale (ZO) in equal ratio. Trikatu has been prescribed for cough, cold, fever, asthma, respiratory problems and improvement of digestive disorders. The aim of the present study was to investigate the effect of individual ingredients of trikatu namely PL, PN, and ZO and formulations [Marketed formulation (MF) and laboratory formulation (LF)] on drug metabolizing enzymes (CYP3A4 and CYP2D6), to assess its herb–drug interaction potential through cytochrome P450 inhibition assays. Further this work was aimed to develop an RP-HPLC method for the identification and quantification of piperine and 6-gingerol in the crude drug trikatu. Materials and methodsEnzyme inhibition effect of LF, MF, PL, PN and ZO was explored through CYP450–CO complex assay using rat liver microsomes (RLM) and a fluorescence screening method using individual isoenzymes (CYP3A4 and CYP2D6). The RP-HPLC method was developed for the identification and quantification of piperine and 6-gingerol in LF, MF and individual plant materials at the concentration of 1mg/mL. ResultsRP-HPLC analysis confirmed the presence of piperine and 6-gingerol in LF and MF [Piperine: 7.89±2.12% (w/w) (MF), 6.70±2.13% (w/w) (LF)]; [6-gingerol: 5.3±1.21% (w/w) (MF), 4.95±2.34% (w/w) (LF)]. Inhibitory potential of MF and LF in CYP450–CO complex assay was found to be 37.54±3.12% (MF) and 35.12±2.31% (LF) and against CYP2D6 and CYP3A4 was estimated to be IC50 251.30±3.98 and 245.23±1.92μg/mL and IC50 225.50±1.02 and 223.254±0.92μg/mL respectively. ConclusionsDifferent concentrations of the trikatu formulation and its individual components showed significantly (p<0.001) less inhibitory activity on individual isoenzymes as compared to the positive control. The crude drug exhibited inhibitory potential against the CYP450 enzymes in a concentration dependent manner. Outcome of the present study demonstrated that trikatu has less interaction potential with drug metabolizing enzymes.

In vitro inhibitory effect of piperlonguminine isolated from Piper longum on human cytochrome P450 1A2

Piperlonguminine (PL), a major alkaloid isolated from Piper longum fruits, shows several biological activities including anti-tumor, anti-hyperlipidemic and anti-inflammatory effects. Although there have been studies of the biological effects of PL, the potential drug-interaction effect of PL following evaluation of inhibitory effects of cytochrome P450 (CYP) activities was not investigated. Here, to investigate the inhibitory effects of PL on the activities of CYP isoforms, CYP inhibition assays were conducted using a cocktail of probe substrates in pooled human liver microsome (HLMs) and human recombinant cDNA-expressed CYP. PL strongly inhibited CYP1A2-mediated phenacetin O-deethylation with an IC50 value of 8.8 μM, as NADPH-independent inhibition, while other CYPs were not significantly inhibited. A Lineweaver-Burk plot resulted in the inhibition mechanism of PL being divided into two different modes, reversible competitive inhibition in a low concentration range of 0-16 μM with a Ki value of 1.39 μM and uncompetitive inhibitory behavior at a high concentration range of 16-40 μM. In addition, PL only decreased CYP 1A2-catalyzed phenacetin O-deethylase activity with IC50 values of 10.0 μM in human recombinant cDNA-expressed 1A2, not 1A1. Overall, this is the first investigation of potential herb-drug interactions associated with PL conducted by identifying the competitive inhibitory effects of PL on CYP1A2 in HLMs.

Utility of a column-switching LC/MS/MS method in cytochrome P450 inhibition assays using human liver microsomes

Liquid chromatography-tandem mass spectrometry (LC/MS/MS)-based in vitro cytochrome P450 (CYP) inhibition assays in pooled human liver microsomes using therapeutically relevant probe drugs are recommended by the US Food and Drug Administration to assess the potential for drug-drug interactions. As these assays are used routinely in pharmaceutical drug discovery screening of new chemical entities for drug interaction liabilities, there is a need to have higher analytical throughput. Column-switching methods may offer increased chromatographic throughput while maintaining the quality of data generated. In this study, the CYP3A4 inhibition assay was used as a potential application to demonstrate the performance of a dual-column parallel chromatographic system in a column-switching mode. Testosterone 6β-hydroxylation was monitored and IC50 values of known CYP3A4 inhibitors were determined using conventional as well as column-switching LC/MS/MS methods. Mean IC50 values of ketoconazole, itraconazole and verapamil were 0.056, 0.061 and 23 μM (conventional method) compared to 0.05, 0.057 and 26 μM (column-switching method), respectively. The two different chromatographic methods resulted in IC50 values that were not statistically different and were within a twofold range, demonstrating reproducibility of results. Further, the column-switching method saved nearly 50% of analytical time in comparison to the conventional chromatographic method, indicating increased throughput leading to better utilization of mass spectrometer time without compromising the quality of data. Similar column-switching methods may be used for other isoforms as well and offer a convenient increased analytical throughput in CYP inhibition assays.

Evaluation of the effect of compound aqueous solubility in cytochrome P450 inhibition assays

It is a common practice in drug discovery organizations to screen new chemical entities in order to predict future drug-drug interactions. For this purpose, there are two main assay strategies, one based on recombinant cytochrome P450 (rCYP) enzymes and fluorescent detection, and other on human liver microsomes (HLM) and liquid chromatography coupled to mass spectrometry. Many authors have reported a poor correlation between both technologies, giving rise to concerns about the usefulness of fluorometric methods for predicting drug-drug interactions. In this study, we investigated the role that compound aqueous kinetic solubility may play in this lack of correlation. We found that drug discovery compounds with unacceptable kinetic solubility, measured by a turbidimetric solutibility assay, tended to yield higher IC50 values in in vitro models based on human liver microsomes, whereas compounds with kinetic solubility values higher than 50 μM showed very similar IC50 values in both in vitro models. Our results show that the turbidimetric solubility assay is a useful tool to identify those discovery compounds that may require further investigation in order to avoid overlooking future drug-drug interactions.