HLM chip - A microfluidic approach to study the mechanistic basis of cytochrome P450 inhibition using immobilized human liver microsomes.

Cytochrome P450 (CYP) system is a critical elimination route to most pharmaceuticals in human, but also prone to drug-drug interactions arising from the fact that concomitantly administered pharmaceuticals inhibit one another's CYP metabolism. The most severe form of CYP interactions is irreversible inhibition, which results in permanent inactivation of the critical CYP pathway and is only restored by de novo synthesis of new functional enzymes. In this study, we conceptualize a microfluidic approach to mechanistic CYP inhibition studies using human liver microsomes (HLMs) immobilized onto the walls of a polymer micropillar array. We evaluated the feasibility of these HLM chips for CYP inhibition studies by establishing the stability and the enzyme kinetics for a CYP2C9 model reaction under microfluidic flow and determining the half-maximal inhibitory concentrations (IC50) of three human CYP2C9 inhibitors (sulfaphenazole, tienilic acid, miconazole), including evaluation of their inhibition mechanisms and nonspecific microsomal binding on chip. Overall, the enzyme kinetics of CYP2C9 metabolism on the HLM chip (KM = 127 ± 55 µM) was shown to be similar to that of static HLM incubations (KM = 114 ± 14 µM) and the IC50 values toward CYP2C9 derived from the microfluidic assays (sulfaphenazole 0.38 ± 0.09 µM, tienilic acid 3.4 ± 0.6 µM, miconazole 0.54 ± 0.09 µM) correlated well with those determined using current standard IC50 shift assays. Most importantly, the HLM chip could distinguish between reversible (sulfaphenazole) and irreversible (tienilic acid) enzyme inhibitors in a single, automated experiment, indicating the great potential of the HLM chip to simplify current workflows used in mechanistic CYP inhibition studies. Furthermore, the results suggest that the HLM chip can also identify irreversible enzyme inhibitors, which are not necessarily resulting in a time-dependent inhibition (like suicide inhibitors), but whose inhibition mechanism is based on other kind of covalent or irreversible interaction with the CYP system. With our HLM chip approach, we could identify miconazole as such a compound that nonselectively inhibits the human CYP system with a prolonged, possibly irreversible impact in vitro, even if it is not a time-dependent inhibitor according to the IC50 shift assay.

Pharmacokinetic study on the interaction between succinic acid and irbesartan in rats and its potential mechanism.

CONTEXT: Succinic acid and irbesartan are commonly used drugs in cardiovascular disease treatment. The interaction might occur during their co-administration, which was still unclear. OBJECTIVE: To reveal the effect of succinic acid on the metabolism of irbesartan and its potential mechanism. MATERIALS AND METHODS: The Sprague-Dawley rats (n = 6) were treated with a single dose of 30 mg/kg irbesartan (control) or the co-administration with the pre-treatment of 200 mg/kg succinic acid for 7 d. The effect of succinic acid on the metabolic stability and the activity of CYP2C9 was evaluated in rat liver microsomes. RESULTS: Succinic acid increased the AUC (5328.71 ± 959.31 µg/L × h vs. 3340.23 ± 737.75 µg/L × h) and prolonged the half-life of irbesartan (from 12.79 ± 0.73 h to 20.59 ± 6.35 h). The Tmax (2.83 ± 0.75 h vs. 3.83 ± 1.10 h) and clearance rate (3.46 ± 1.13 L/h/kg vs. 6.91 ± 1.65 L/h/kg) of irbesartan was reduced by succinic acid. Consistently, succinic acid improved the metabolic stability (half-life from 23.32 ± 3.46 to 27.35 ± 2.15 min, intrinsic clearance rate from 59.43 ± 6.12 to 50.68 ± 5.64 µL/min/mg protein). Succinic acid was also found to inhibit the activity of CYP2C9 with the IC50 value of 13.87 µM. DISCUSSION AND CONCLUSIONS: Succinic acid increased the system exposure of irbesartan via inhibiting CYP2C9. The experiment design of this study also provides a reference for the further validation of this interaction in humans.

Ruxolitinib exposure in patients with acute and chronic graft versus host disease in routine clinical practice-a prospective single-center trial.

PURPOSE: Knowledge on Ruxolitinib exposure in patients with graft versus host disease (GvHD) is scarce. The purpose of this prospective study was to analyze Ruxolitinib concentrations of GvHD patients and to investigate effects of CYP3A4 and CYP2C9 inhibitors and other covariates as well as concentration-dependent effects. METHODS: 262 blood samples of 29 patients with acute or chronic GvHD who were administered Ruxolitinib during clinical routine were analyzed. A population pharmacokinetic model obtained from myelofibrosis patients was adapted to our population and was used to identify relevant pharmacokinetic properties and covariates on drug exposure. Relationships between Ruxolitinib exposure and adverse events were assessed. RESULTS: Median of individual mean trough serum concentrations was 39.9 ng/mL at 10 mg twice daily (IQR 27.1 ng/mL, range 5.6-99.8 ng/mL). Applying a population pharmacokinetic model revealed that concentrations in our cohort were significantly higher compared to myelofibrosis patients receiving the same daily dose (p < 0.001). Increased Ruxolitinib exposure was caused by a significant reduction in Ruxolitinib clearance by approximately 50%. Additional comedication with at least one strong CYP3A4 or CYP2C9 inhibitor led to a further reduction by 15% (p < 0.05). No other covariate affected pharmacokinetics significantly. Mean trough concentrations of patients requiring dose reduction related to adverse events were significantly elevated (p < 0.05). CONCLUSION: Ruxolitinib exposure is increased in GvHD patients in comparison to myelofibrosis patients due to reduced clearance and comedication with CYP3A4 or CYP2C9 inhibitors. Elevated Ruxolitinib trough concentrations might be a surrogate for toxicity.

Risk prediction of drug-drug interaction potential of phenytoin and miconazole topical formulations.

The drug-drug interaction (DDI) risk of phenytoin with several topical formulations of miconazole is still unclear. The present investigation conducted in vitro-in vivo extrapolation to predict the potential risks. Our data indicated that miconazole potently inhibited phenytoin hydroxylation in both pooled human liver microsomes (HLMs) and recombinant cytochrome P450 2C9 (CYP2C9) with the Ki values of 125 ± 7 nM and 30 ± 2 nM, respectively. Quantitative prediction of DDI risk suggests that, beside intravenous administration or swallowed tablet, combination of phenytoin and miconazole high dose oral gel or buccal tablet may also result in a clinically significant increase of phenytoin AUC (>53%) by the inhibition of miconazole against phenytoin hydroxylation, consequently a higher frequency of adverse events, while the coadministration of miconazole vaginal formulation and phenytoin will be safe.

Mechanistic study of bergamottin-induced inactivation of CYP2C9.

Bergamottin (BGM) is a major furanocoumarin constituent of grapefruit and is reported to have inhibitory effects on cytochrome P450 enzymes. This study investigated the chemical interactions between BGM and the enzyme CYP2C9. BGM exhibited time-, concentration-, and NADPH-dependent inhibition of CYP2C9. Co-incubation with diclofenac, a reversible inhibitor of CYP2C9, attenuated the time-dependent enzyme inhibition. Exhaustive dialysis did not restore enzyme activity post-inhibition. Glutathione (GSH) and catalase/superoxide dismutase failed to reverse BGM-induced CYP2C9 inactivation. A GSH trapping study suggested that BGM was metabolized to an epoxide and/or γ-ketoenal that may have been responsible for the enzyme inactivation. In conclusion, BGM can be characterized as a mechanism-based inactivator of CYP2C9 acting via the formation of an epoxide and/or γ-ketoenal.

Pogostone inhibits the activity of CYP3A4, 2C9, and 2E1 in vitro.

CONTEXT: Pogostone possesses various pharmacological activities, which makes it widely used in the clinic. Its effect on the activity of cytochrome P450 enzymes (CYP450s) could guide its clinical combination. OBJECTIVE: To investigate the effect of pogostone on the activity of human CYP450s. MATERIALS AND METHODS: The effect of pogostone on the activity of CYP450s was evaluated in human liver microsomes (HLMs) compared with blank HLMs (negative control) and specific inhibitors (positive control). The corresponding parameters were obtained with 0-100 µM pogostone and various concentrations of substrates. RESULTS: Pogostone was found to inhibit the activity of CYP3A4, 2C9, and 2E1 with the IC50 values of 11.41, 12.11, and 14.90 µM, respectively. The inhibition of CYP3A4 by pogostone was revealed to be performed in a non-competitive and time-dependent manner with the Ki value of 5.69 µM and the KI/Kinact value of 5.86/0.056/(µM/min). For the inhibition of CYP2C9 and 2E1, pogostone acted as a competitive inhibitor with the Ki value of 6.46 and 7.67 µM and was not affected by the incubation time. DISCUSSION AND CONCLUSIONS: The inhibitory effect of pogostone on the activity of CYP3A4, 2C9, and 2E1 has been disclosed in this study, implying the potential risk during the co-administration of pogostone and drugs metabolized by these CYP450s. The study design provides a reference for further in vivo investigations to validate the potential interaction.

The inhibition of CYP1A2, CYP2C9, and CYP2D6 by pterostilbene in human liver microsomes.

This study used human liver microsomes to assess pterostilbene's effect on the metabolic activity of cytochrome P450 (CYP) 1A2, CYP2C9, and CYP2D6. The metabolism of their substrates (phenacetin, tolbutamide, and dextromethorphan) was assayed by quantifying their relevant metabolites by HPLC. The IC50 value was used to express the strength of inhibition, and the value of a volume per dose index (VDI) was used to indicate the metabolic ability of the enzyme. In this study, pterostilbene inhibited CYP1A2, CYP2C9, and CYP2D6's metabolic activities in vitro. CYP2C9's activity was most significantly inhibited by pterostilbene; its IC50 value was 0.12±0.04 µM. The IC50 value of CYP1A2 and CYP2D6 was 56.3±10.4 µM and 62.33±11.4 µM, respectively. The finding that suggests that pterostilbene has the potential to interact with CYP2C9 substrates in vivo. These results warrant clinical studies to assess the in vivo significance of these interactions.

The optimization and characterization of functionalized sulfonamides derived from sulfaphenazole against Mycobacterium tuberculosis with reduced CYP 2C9 inhibition.

In this study, a series of sulfonamide compounds was designed and synthesized through the systematic optimization of the antibacterial agent sulfaphenazole for the treatment of Mycobacterium tuberculosis (M. tuberculosis). Preliminary results indicate that the 4-aminobenzenesulfonamide moiety plays a key role in maintaining antimycobacterial activity. Compounds 10c, 10d, 10f and 10i through the optimization on phenyl ring at the R2 site on the pyrazole displayed promising antimycobacterial activity paired with low cytotoxicity. In particular, compound 10d displayed good activity (MIC = 5.69 µg/mL) with low inhibition of CYP 2C9 (IC50 > 10 µM), consequently low potential risk of drug-drug interaction. These promising results provide new insight into the combination regimen using sulfonamide as one component for the treatment of M. tuberculosis.

Amentoflavone Induces Cell-cycle Arrest, Apoptosis, and Invasion Inhibition in Non-small Cell Lung Cancer Cells.

BACKGROUND/AIM: Amentoflavone, an effective compound derived from medicinal plants, has been shown to boost therapeutic efficacy of chemotherapy in non-small cell lung cancer (NSCLC). However, anti-NSCLC effect of amentoflavone is ambiguous. The major purpose of the present study was to verify the inhibitory effects of amentoflavone in NSCLC cells. MATERIALS AND METHODS: The effects of amentoflavone on growth and invasion of NSCLC CL-1-5-F4 cells were evaluated by cell viability assay, flow cytometry, colony formation assay, nuclear factor-kappa B (NF-κB) reporter gene assay, immunofluorescence staining, transwell invasion, and western blot assay. RESULTS: Amentoflavone effectively induced cell growth inhibition, G1 cell-cycle arrest, apoptosis, and suppression of invasion. Furthermore, amentoflavone not only triggered expression of p27, cleaved caspase-3, -8 also reduced NF-κB signaling, protein levels of matrix metalloproteinase (MMP)-2, -9, Cyclin-D1, and vascular endothelial growth factor (VEGF). CONCLUSION: Cell-cycle arrest, apoptosis induction, NF-κB signaling inhibition are associated with amentoflavone-inhibited growth and invasion of NSCLC cells.

Modulation of CYP3A4 and CYP2C9 activity by Bulbine natalensis and its constituents: An assessment of HDI risk of B. natalensis containing supplements.

BACKGROUND: Bulbine natalensis is an African-folk medicinal plant used as a dietary supplement for enhancing sexual function and muscle strength in males by presumably boosting testosterone levels, but no scientific information is available about the possible herb-drug interaction (HDI) risk when bulbine-containing supplements are concomitantly taken with prescription drugs. PURPOSE: This study was aimed to investigate the HDI potential of B. natalensis in terms of the pregnane X receptor (PXR)-mediated induction of major drug-metabolizing cytochrome P450 enzyme isoforms (i.e., CYP3A4 and CYP2C9) as well as inhibition of their catalytic activity. RESULTS: We found that a methanolic extract of B. natalensis activated PXR (EC50 6.2 ± 0.6 µg/ml) in HepG2 cells resulting in increased mRNA expression of CYP3A4 (2.40 ± 0.01 fold) and CYP2C9 (3.37 ± 0.3 fold) at 30 µg/ml which was reflected in increased activites of the two enzymes. Among the constituents of B. natalensis, knipholone was the most potent PXR activator (EC50 0.3 ± 0.1 µM) followed by bulbine-knipholone (EC50 2.0 ± 0.5 µM), and 6'-methylknipholone (EC50 4.0 ± 0.5 µM). Knipholone was also the most effective in increasing the expression of CYP3A4 (8.47 ± 2.5 fold) and CYP2C9 (2.64 ± 0.3 fold) at 10 µM. Docking studies further confirmed the unique structural features associated with knipholones for their superior inductive potentials in the activation of PXR compared to other anthraquinones. In a CYP inhibition assay, the methanolic extract as well as the anthraquinones strongly inhibited the catalytic activity of CYP2C9 while, inhibition of CYP3A4 was weak. CONCLUSIONS: These results suggest that consumption of B. natalensis may pose a potential risk for HDI if taken with conventional medications that are substrates of CYP3A4 and CYP2C9 and may contribute to unanticipated adverse reactions or therapeutic failures. Further studies are warranted to validate these findings and establish their clinical relevancy.

Succinic acid inhibits the activity of cytochrome P450 (CYP450) enzymes.

CONTEXT: Succinic acid, extracted from amber, is widely used in cardiovascular therapy. OBJECTIVE: The effect of succinic acid on the activity of cytochrome P450 (CYP450) enzymes was investigated in this study. MATERIALS AND METHODS: The effect of succinic acid (100 µM) on the activity of eight isoforms of CYP450 (i.e., 1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19 and 2C8) was investigated compared to the specific inhibitor and blank controls in pooled human liver microsomes in vitro. The inhibition of CYPs was fitted with competitive or non-competitive inhibition models and corresponding parameters were also obtained. RESULTS: Succinic acid exerted inhibitory effect on the activity of CYP3A4, 2D6, and 2C9 with the IC50 values of 12.82, 14.53, and 19.60 µM, respectively. Succinic acid inhibited the activity of CYP3A4 in a non-competitive manner with the Ki value of 6.18 µM, and inhibited CYP2D6 and 2C9 competitively with Ki values of 7.40 and 9.48 µM, respectively. Furthermore, the inhibition of CYP3A4 was found to be time-dependent with the KI/Kinact value of 6.52/0.051 min-1·µM-1. DISCUSSION AND CONCLUSIONS: Succinic acid showed in vitro inhibitory effects on the activity of CYP3A4, 2D6, and 2C9, which indicated the potential drug-drug interactions. Succinic acid should be carefully co-administrated with the drugs metabolized by CYP3A4, 2D6, and 2C9.

Identification of a novel orally bioavailable NLRP3 inflammasome inhibitor.

NLRP3 inflammasome mediated release of interleukin-1ß (IL-1ß) has been implicated in various diseases, including COVID-19. In this study, rationally designed alkenyl sulfonylurea derivatives were identified as novel, potent and orally bioavailable NLRP3 inhibitors. Compound 7 was found to be potent (IL-1ß IC50 = 35 nM; IL-18 IC50 = 33 nM) and selective NLRP3 inflammasome inhibitor with excellent pharmacokinetic profile having oral bioavailability of 99% in mice.

Inhibition of CYP2C9 by natural products: insight into the potential risk of herb-drug interactions.

Due to the rapidly increasing global interest in the use of herbs, phytomedicines and other natural products as medical or complementary remedies, concerns about the clinical medication safety have drawn much attention worldwide. Particularly, many natural ingredients exhibit inhibitory effects on cytochrome P450 (CYP) enzymes, which are the most important Phase I metabolism enzymes in liver. CYP2C9 is one of the most abundant CYP enzymes and responsible for the metabolism of over 15% clinical drugs, including oral sulfonylurea hypoglycemics, nonsteroidal anti-inflammatory agents, selective cyclooxygenase-2 inhibitors, antiepileptics, angiotensin II receptor inhibitors and anticoagulants. Diclofenac (4'-hydroxylase) and tolbutamide (methylhydroxylation) are widely used as probe substrates for CYP2C9. To date, numerous natural products have been reported to have the capabilities of inhibiting the catalytic activity of CYP2C9 and further influencing the pharmacokinetic and pharmacodynamic behaviors of drugs that are mainly metabolized by CYP2C9, leading to potential herb-drug interactions. Moreover, some fatal adverse interactions may occur for drugs with a narrow therapeutic window when they are coadministered with a CYP2C9 inhibitor, especially irreversible inactivators. For the purpose of better understanding the interactions of natural products with CYP2C9, we comprehensively reviewed the characteristics of CYP2C9, the natural ingredients that inhibit CYP2C9, the related research approaches and strategies, the types of inhibition and the underlying mechanisms.

Amentoflavone induces cell cycle arrest, apoptosis, and autophagy in BV-2 cells.

Previous studies have shown that amentoflavone (AF) elicits anti-inflammatory and neuroprotective effects. To further investigate the effects of AF on the microglia cell line BV-2, proteomic analysis was performed to screen potential key regulators. The top 5 canonical pathways associated with AF treatment were EIF2 signaling, regulation of eIF4 and p70s6k signaling, mTOR signaling, protein ubiquitination pathway and phagosome maturation. The top up-regulated genes were DOCK2, SEC23A, ME1, UGGT1 and STOM, while the most down-regulated molecules were IGF2R, ATP5O, DDX47, WBP11 and IKBIP. AF significantly decreased BV-2 cell proliferation. It induced cell cycle arrest at G2/M, increased CDK2, p27Kip1 and p53/p-p53, and decreased CDK1/CDC2 and cyclin B1. Cell apoptosis was induced, with increased levels of BAX, c-caspase-3 and c-caspase-9, and decreased levels of BCL-XL. Increased level of autophagosome induced by AF was observed, and increased Beclin-1 and decreased phosphorylation of PI3K and Erk1 were found as well. In conclusion, AF induces cell cycle arrest at G2/M, promotes apoptosis and autophagy in BV-2 cells, which may account for the anti-inflammatory effect of AF in epilepsy.

Miconazole alleviates peripheral nerve crush injury by mediating a macrophage phenotype change through the NF-κB pathway.

BACKGROUND: Peripheral nerve injury (PNI) causes motor and sensory defects, has strong impact on life quality and still has no effective therapy. Miconazole is one of the most widely used antifungal drugs; the aims of the study were to investigate the effects of miconazole during sciatic nerve regeneration in a mouse model of sciatic nerve crush injury. METHODS: We established peripheral nerve crush model and investigated the effects of miconazole by multiple aspects. We further studied the potential mechanism of action of miconazole by Western blotting, fluorescence immunohistochemistry, and PCR analysis. RESULTS: Miconazole improves the symptoms of crushed nerve by improving inflammatory cell infiltration and demyelinating myelin of sciatic nerve. Affected by miconazole, the proportion of inflammatory M1 macrophages in the distal part of the sciatic nerve was reduced, and the proportion of anti-inflammatory M2 macrophages was increased. Finally, the neuroprotective properties of miconazole may be regulated by the nuclear factor (NF)-κB pathway. CONCLUSIONS: Our data suggest that miconazole can effectively alleviate PNI, and the mechanism involves mediating a phenotype change of M1/ M2 macrophages. Thus, miconazole may represent a potential therapeutic intervention for nerve crush injury.

Prediction of the Impact of Cytochrome P450 2C9 Genotypes on the Drug-Drug Interaction Potential of Siponimod With Physiologically-Based Pharmacokinetic Modeling: A Comprehensive Approach for Drug Label Recommendations.

We predicted the drug-drug interaction (DDI) potential of siponimod in presence of cytochrome P450 (CYP)2C9/CYP3A4 inhibitors/inducers in subjects with different CYP2C9 genotypes by physiologically-based pharmacokinetic (PK) modeling. The model was established using in vitro and clinical PK data and verified by adequately predicting siponimod PK when coadministered with rifampin. With strong and moderate CYP3A4 inhibitors, an increased DDI risk for siponimod was predicted for CYP2C9*3/*3 genotype vs. other genotypes area under the curve ratio (AUCR): 3.03-4.20 vs. ≤ 1.49 for strong; 2.42 vs. 1.14-1.30 for moderate. AUCRs increased with moderate (2.13-2.49) and weak (1.12-1.42) CYP3A4/CYP2C9 inhibitors to the same extent for all genotypes. With strong CYP3A4/moderate CYP2C9 inducers and moderate CYP3A4 inducers, predicted AUCRs were 0.21-0.32 and 0.35-0.71, respectively. This complementary analysis to the clinical PK-DDI studies confirmed the relevant influence of CYP2C9 polymorphism on the DDI behavior of siponimod and represented the basis for the DDI labeling recommendations.

Effect of 95% Ethanol Khat Extract and Cathinone on in vitro Human Recombinant Cytochrome P450 (CYP) 2C9, CYP2D6, and CYP3A4 Activity.

BACKGROUND AND OBJECTIVE: A significant number of people worldwide consume khat on daily basis. Long term of khat chewing has shown negative impact on several organ systems. It is likely that these people are co-administered khat preparations and conventional medication, which may lead to khat-drug interactions. This study aimed to reveal the inhibitory potencies of khat ethanol extract (KEE) and its major active ingredient (cathinone) on human cytochrome P450 (CYP) 2C9, CYP2D6, and CYP3A4 enzymes activities, which are collectively responsible for metabolizing 70-80% clinically used drugs. METHODS: In vitro fluorescence-based enzyme assays were developed and the CYP enzyme activities were quantified in the presence and absence of KEE and cathinone employing Vivid® CYP450 Screening Kits. RESULTS: KEE inhibited human CYP2C9, CYP2D6, and CYP3A4 enzyme activities with IC50 of 42, 62, and 18 µg/ml. On the other hand, cathinone showed negligible inhibitory effect on these CYPs. Further experiments with KEE revealed that KEE inhibited CYP2C9 via non-competitive or mixed mode with Ki of 14.7 µg/ml, CYP2D6 through competitive or mixed mode with Ki of 17.6 µg/ml, CYP3A4 by mixed inhibition mode with Ki of 12.1 µg/ml. CONCLUSION: Khat-drug interactions are possible due to administration of clinical drugs metabolized by CYP2C9/CYP2D6/CYP3A4 together with khat chewing. Further in vivo studies are required to confirm our findings and identify the causative constituents of these inhibitory effects.

Interaction between warfarin and cannabis.

Delta-9-tetrahydrocannabinol (THC), the main psychoactive cannabinoid in cannabis, may inhibit the cytochrome P450 enzyme CYP2C9. Consequently, cannabis use might infer a risk of drug-drug interaction with substrates for this enzyme, which includes drugs known to have a narrow therapeutic window. In this study, we describe a case report of a 27-year-old man treated with warfarin due to mechanical heart valve replacement who presented with elevated international normalized ratio (INR) value (INR = 4.6) following recreational cannabis use. We conducted a review of the available literature, using the PubMed and EMBASE databases while following PRISMA guidelines. Following screening of 85 articles, three eligible articles were identified, including one in vitro study and two case reports. The in vitro study indicated that THC inhibits the CYP2C9-mediated metabolism of warfarin. One case study reported of a man who on two occasions of increased marijuana use experienced INR values above 10 as well as bleeding. The other case study reported of a patient who initiated treatment with a liquid formulation of cannabidiol for the management of epilepsy, ultimately necessitating a 30% reduction in warfarin dose to maintain therapeutic INR values. The available, although sparse, data suggest that use of cannabinoids increases INR values in patients receiving warfarin. Until further data are available, we suggest patients receiving warfarin be warned against cannabis use.

Identification of Selective Dual ROCK1 and ROCK2 Inhibitors Using Structure-Based Drug Design.

A HTS campaign identified compound 1, an excellent hit-like molecule to initiate medicinal chemistry efforts to optimize a dual ROCK1 and ROCK2 inhibitor. Substitution (2-Cl, 2-NH2, 2-F, 3-F) of the pyridine hinge binding motif or replacement with pyrimidine afforded compounds with a clean CYP inhibition profile. Cocrystal structures of an early lead compound were obtained in PKA, ROCK1, and ROCK2. This provided critical structural information for medicinal chemistry to drive compound design. The structural data indicated the preferred configuration at the central benzylic carbon would be ( R), and application of this information to compound design resulted in compound 16. This compound was shown to be a potent and selective dual ROCK inhibitor in both enzyme and cell assays and efficacious in the retinal nerve fiber layer model after oral dosing. This tool compound has been made available through the AbbVie Compound Toolbox. Finally, the cocrystal structures also identified that aspartic acid residues 176 and 218 in ROCK2, which are glutamic acids in PKA, could be targeted as residues to drive both potency and kinome selectivity. Introduction of a piperidin-3-ylmethanamine group to the compound series resulted in compound 58, a potent and selective dual ROCK inhibitor with excellent predicted drug-like properties.

Interactions of casticin, ipriflavone, and resveratrol with serum albumin and their inhibitory effects on CYP2C9 and CYP3A4 enzymes.

Polyphenols are abundant molecules in the plant kingdom. They interact with several proteins in the body resulting in their complex biological effects. Previous studies demonstrated that polyphenols can interfere significantly with the pharmacokinetics of drugs by acting on their biotransformation, albumin-binding, and/or carrier-mediated transport. Casticin (CAS), ipriflavone (IPR), and resveratrol (RES) are well-known polyphenols often added to dietary supplements in high doses. In this study, we investigated the albumin-binding of these polyphenols by fluorescence spectroscopy, and their ability to displace the Sudlow's Site I ligand warfarin and the Site II ligand naproxen by ultrafiltration. Furthermore, the effects of CAS, IPR, and RES on CYP2C9 and CYP3A4 enzymes were examined, employing diclofenac and testosterone as substrates, respectively. Our main observations are the following: (1) Polyphenols formed stable complexes with albumin (K = 104-105 L/mol); (2) CAS and RES slightly displaced naproxen from human albumin, while albumin-binding of warfarin was not affected; (3) CAS and RES significantly inhibited CYP2C9, with CAS being as potent as the positive control warfarin; (4) each polyphenol significantly inhibited CYP3A4, with RES being stronger and CAS slightly weaker than the known inhibitor naringenin. Our results suggest that high intake of CAS and RES may interfere with the albumin-binding of Site II ligands as well as the metabolism of drugs by CYP2C9 and/or CYP3A4 enzymes, while large doses of IPR may affect the CYP3A4-catalyzed biotransformation of some drugs.