Human Liver Microsomal Activity Research Articles

Construction of humanized CYP1A2 rats using CRISPR/Cas9 to promote drug metabolism and pharmacokinetic research.

Cytochrome P450 family 1 subfamily A member 2 (CYP1A2), performs an indispensable role in metabolism of both exogenous and endogenous substances. What is more, CYP1A2 functions in human diseases by regulating homeostasis of cholesterol. Despite the emergence of gene-editing animal models, genetically humanized animals that overcome species differences for further exploring the role of CYP1A2 in drug metabolism and human diseases have not yet been constructed. In this study, we inserted human CYP1A2 cDNA into the rat Cyp1a2 gene by using CRISPR/Cas9 technology. Results showed that human CYP1A2 was successfully expressed in humanized rat liver and there were no statistically significant differences of physiological symptoms compared with wild-type (WT) rats. In vitro incubation results indicated the different inhibition of furafylline on CYP1A2 activity in human liver microsomes, humanized CYP1A2 (hCYP1A2) rat liver microsomes, and WT rat liver microsomes, with IC50 values of 7.1 μM, 36.5 μM, and 285.8 μM, respectively. Meanwhile, pharmacokinetic characteristics of clozapine were conducted, and the results suggested that in hCYP1A2 rats, clozapine tended to be metabolized into norclozapine. Both the in vitro and in vivo results demonstrated the different metabolic functions of CYP1A2 in humanized and WT rats. We successfully constructed a novel humanized CYP1A2 rat model using the CRISPR/Cas9 system, providing a powerful tool for better predicting CYP1A2-mediated drug metabolism and pharmacokinetics. Significance Statement Human CYP1A2 takes active part both in the biotransformation of exogenous substances and endogenous substances. Meanwhile, it plays a regulatory role in human diseases, including hypercholesterolemia, hypertension as well as various malignant tumors. This study successfully constructed humanized CYP1A2 rat model by CRISPR/Cas9 technology, providing a powerful model for promoting drug development and safety evaluation, as well as further exploring the role of CYP1A2 in human diseases.

METTL7A (TMT1A) and METTL7B (TMT1B) Are Responsible for Alkyl S-Thiol Methyl Transferase Activity in Liver.

S-methylation of drugs containing thiol-moieties often alters their activity and results in detoxification. Historically, scientists attributed methylation of exogenous aliphatic and phenolic thiols to a putative S-adenosyl-L-methionine (SAM)-dependent membrane-associated enzyme referred to as thiol methyltransferase (TMT). This putative TMT appeared to have a broad substrate specificity and methylated the thiol metabolite of spironolactone, mertansine, ziprasidone, captopril, and the active metabolites of the thienopyridine prodrugs, clopidogrel, and prasugrel. Despite TMT's role in the S-methylation of clinically relevant drugs, the enzyme(s) responsible for this activity remained unknown. We recently identified methyltransferase-like protein 7B (METTL7B) as an alkyl thiol methyltransferase. METTL7B is an endoplasmic reticulum-associated protein with similar biochemical properties and substrate specificity to the putative TMT. Yet, the historic TMT inhibitor 2,3-dichloro-α-methylbenzylamine (DCMB) did not inhibit METTL7B, indicating that multiple enzymes contribute to TMT activity. Here we report that methyltransferase-like protein 7A (METTL7A), an uncharacterized member of the METTL7 family, is also a SAM-dependent thiol methyltransferase. METTL7A exhibits similar biochemical properties to METTL7B and putative TMT, including inhibition by DCMB (IC50 = 1.17 μM). Applying quantitative proteomics to human liver microsomes and gene modulation experiments in HepG2 and HeLa cells, we determined that TMT activity correlates closely with METTL7A and METTL7B protein levels. Furthermore, purification of a novel His-GST-tagged recombinant protein and subsequent activity experiments prove that METTL7A can selectively methylate exogenous thiol-containing substrates, including 7α-thiospironolactone, dithiothreitol, 4-chlorothiophenol, and mertansine. We conclude that the METTL7 family encodes for two enzymes, METTL7A and METTL7B, which are now renamed thiol methyltransferase 1A (TMT1A) and thiol methyltransferase 1B (TMT1B), respectively, that are responsible for thiol methylation activity in human liver microsomes. SIGNIFICANCE STATEMENT: We identified methyltransferase-like protein 7A (thiol methyltransferase 1A) and methyltransferase-like protein 7B (thiol methyltransferase 1B) as the enzymes responsible for the microsomal alkyl thiol methyltransferase (TMT) activity. These are the first two enzymes directly associated with microsomal TMT activity. S-methylation of commonly prescribed thiol-containing drugs alters their pharmacological activity and/or toxicity, and identifying the enzymes responsible for this activity will improve our understanding of the drug metabolism and pharmacokinetic (DMPK) properties of alkyl- or phenolic thiol-containing therapeutics.

In Vitro-In Vivo Extrapolation and Scaling Factors for Clearance of Human and Preclinical Species with Liver Microsomes and Hepatocytes

In vitro-in vivo extrapolation ((IVIVE) and empirical scaling factors (SF) of human intrinsic clearance (CLint) were developed using one of the largest dataset of 455 compounds with data from human liver microsomes (HLM) and human hepatocytes (HHEP). For extended clearance classification system (ECCS) class 2/4 compounds, linear SFs (SFlin) are approximately 1, suggesting enzyme activities in HLM and HHEP are similar to those in vivo under physiological conditions. For ECCS class 1A/1B compounds, a unified set of SFs was developed for CLint. These SFs contain both SFlin and an exponential SF (SFβ) of fraction unbound in plasma (fu,p). The unified SFs for class 1A/1B eliminate the need to identify the transporters involved prior to clearance prediction. The underlying mechanisms of these SFs are not entirely clear at this point, but they serve practical purposes to reduce biases and increase prediction accuracy. Similar SFs have also been developed for preclinical species. For HLM-HHEP disconnect (HLM > HHEP) ECCS class 2/4 compounds that are mainly metabolized by cytochrome P450s/FMO, HLM significantly overpredicted in vivo CLint, while HHEP slightly underpredicted and geometric mean of HLM and HHEP slightly overpredicted in vivo CLint. This observation is different than in rats, where rat liver microsomal CLint correlates well with in vivo CLint for compounds demonstrating permeability-limited metabolism. The good CLint IVIVE developed using HLM and HHEP helps build confidence for prospective predictions of human clearance and supports the continued utilization of these assays to guide structure-activity relationships to improve metabolic stability.

The Disconnect in Intrinsic Clearance Determined in Human Hepatocytes and Liver Microsomes Results from Divergent Cytochrome P450 Activities.

Candidate drugs may exhibit higher unbound intrinsic clearances (CLint,u) in human liver microsomes (HLMs) relative to human hepatocytes (HHs), posing a challenge as to which value is more predictive of in vivo clearance (CL). This work was aimed at better understanding the mechanism(s) underlying this 'HLM:HH disconnect' via examination of previous explanations, including passive permeability limited CL or cofactor exhaustion in hepatocytes. A series of structurally related, passively permeable (Papps > 5 × 10-6 cm/s), 5-azaquinazolines were studied in different liver fractions, and metabolic rates and routes were determined. A subset of these compounds demonstrated a significant HLM:HH (CLint,u ratio 2-26) disconnect. Compounds were metabolized via combinations of liver cytosol aldehyde oxidase (AO), microsomal cytochrome P450 (CYP) and flavin monooxygenase (FMO). For this series, the lack of concordance between CLint,u determined in HLM and HH contrasted with an excellent correlation of AO dependent CLint,u determined in human liver cytosol[Formula: see text], r2 = 0.95, P < 0.0001). The HLM:HH disconnect for both 5-azaquinazolines and midazolam was as a result of significantly higher CYP activity in HLM and lysed HH fortified with exogenous NADPH relative to intact HH. Moreover, for the 5-azaquinazolines, the maintenance of cytosolic AO and NADPH-dependent FMO activity in HH, relative to CYP, supports the conclusion that neither substrate permeability nor intracellular NADPH for hepatocytes were limiting CLint,u Further studies are required to identify the underlying cause of the lower CYP activities in HH relative to HLM and lysed hepatocytes in the presence of exogenous NADPH. SIGNIFICANCE STATEMENT: Candidate drugs may exhibit higher intrinsic clearance in human liver microsomes relative to human hepatocytes, posing a challenge as to which value is predictive of in vivo clearance. This work demonstrates that the difference in activity determined in liver fractions results from divergent cytochrome P450 but not aldehyde oxidase or flavin monooxygenase activity. This is inconsistent with explanations including substrate permeability limitations or cofactor exhaustion and should inform the focus of further studies to understand this cytochrome P450 specific disconnect phenomenon.

Study on In Vitro Metabolism and In Vivo Pharmacokinetics of Beauvericin.

Beauvericin (BEA) is a well-known mycotoxin produced by many fungi, including Beaveria bassiana. The purpose of this study was to evaluate the in vitro distribution and metabolism characteristics as well as the in vivo pharmacokinetic (PK) profile of BEA. The in vitro metabolism studies of BEA were performed using rat, dog, mouse, monkey and human liver microsomes, cryopreserved hepatocytes and plasma under conditions of linear kinetics to estimate the respective elimination rates. Additionally, LC-UV-MSn (n = 1~2) was used to identify metabolites in human, rat, mouse, dog and monkey liver microsomes. Furthermore, cytochrome P450 (CYP) reaction phenotyping was carried out. Finally, the absolute bioavailability of BEA was evaluated by intravenous and oral administration in rats. BEA was metabolically stable in the liver microsomes and hepatocytes of humans and rats; however, it was a strong inhibitor of midazolam 1′-hydroxylase (CYP3A4) and mephenytoin 4′-hydroxylase (CYP2C19) activities in human liver microsomes. The protein binding fraction values of BEA were >90% and the half-life (T1/2) values of BEA were approximately 5 h in the plasma of the five species. The absolute bioavailability was calculated to be 29.5%. Altogether, these data indicate that BEA has great potential for further development as a drug candidate. Metabolic studies of different species can provide important reference values for further safety evaluation.

Toxicokinetics of β-Amanitin in Mice and In Vitro Drug-Drug Interaction Potential.

The toxicokinetics of β-amanitin, a toxic bicyclic octapeptide present abundantly in Amanitaceae mushrooms, was evaluated in mice after intravenous (iv) and oral administration. The area under plasma concentration curves (AUC) following iv injection increased in proportion to doses of 0.2, 0.4, and 0.8 mg/kg. β-amanitin disappeared rapidly from plasma with a half-life of 18.3–33.6 min, and 52.3% of the iv dose was recovered as a parent form. After oral administration, the AUC again increased in proportion with doses of 2, 5, and 10 mg/kg. Absolute bioavailability was 7.3–9.4%, which resulted in 72.4% of fecal recovery from orally administered β-amanitin. Tissue-to-plasma AUC ratios of orally administered β-amanitin were the highest in the intestine and stomach. It also readily distributed to kidney > spleen > lung > liver ≈ heart. Distribution to intestines, kidneys, and the liver is in agreement with previously reported target organs after acute amatoxin poisoning. In addition, β-amanitin weakly or negligibly inhibited major cytochrome P450 and 5′-diphospho-glucuronosyltransferase activities in human liver microsomes and suppressed drug transport functions in mammalian cells that overexpress transporters, suggesting the remote drug interaction potentials caused by β-amanitin exposure.

In Vitro and In Vivo Assessment of Atemoya Fruit (Annona atemoya) for Food-Drug Interactions.

Atemoya (Annona atemoya) is increasingly being consumed worldwide because of its pleasant taste. However, only limited information is available concerning possible atemoya-drug interactions. In the present study, the issue of whether atemoya shows food-drug interactions with substrate drugs of the major drug-metabolizing cytochrome P450s (i.e., CYP1A2, CYP2C9, and CYP3A) is addressed. The ability of atemoya juice to inhibit the activities of phenacetin O-deethylase (CYP1A2), diclofenac 4'-hydroxylase (CYP2C9), and midazolam 1'-hydroxylase (CYP3A) was examined in vitro using human and rat liver microsomes. The in vivo pharmacokinetics of phenacetin and metabolites derived from it in rats when atemoya juice or fluvoxamine (a CYP1A2 inhibitor) was preadministered were also investigated. Atemoya juice significantly inhibited CYP1A2 activity in human liver microsomes, but not the activities of CYP2C9 and CYP3A. In spite of this inhibition, preadministration of atemoya had no effect on the pharmacokinetics of phenacetin, a CYP1A2 substrate, in rats. Meanwhile, preadministration of fluvoxamine significantly extended the time needed for the elimination of phenacetin, possibly due to the inhibition of CYP1A2. This suggests that the intake of an excess amount of atemoya juice is necessary to cause a change in the pharmacokinetics of phenacetin when the IC50 values for CYP1A2 inhibition by atemoya and fluvoxamine are taken into account. The results indicate that a daily intake of atemoya would not change the pharmacokinetics of CYP1A2 substrates such as phenacetin as well as CYP2C9- and CYP3A-substrate drugs.

In vitro modulatory effects of ginsenoside compound K, 20(S)-protopanaxadiol and 20(S)-protopanaxatriol on uridine 5′-diphospho-glucuronosyltransferase activity and expression

We explored the inhibitory effect of ginsenoside compound K (CK), 20(S)-protopanaxadiol (PPD), and 20(S)-protopanaxatriol (PPT) on six uridine 5′-diphospho-glucuronosyltransferase (UGT) enzyme (UGT1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) activities in human liver microsomes (HLMs) and 10 UGT enzyme (UGT1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B10, 2B15, and 2B17) activities in recombinant UGT isoforms. PPD was a potent inhibitor of UGT1A3 activity with half-maximal inhibitory concentration values of 5.62 and 3.38 μM in HLMs and recombinant UGT1A3, respectively. UGT1A3 inhibition by CK and PPD was competitive with inhibitory constant (K i) values of 17.4 and 1.21 μM, respectively, and inhibition by PPT was non-competitive with a K i value of 8.07 μM in HLMs. PPD exhibited more than 3.4-fold selectivity for UGT1A3 inhibition compared with other UGT isoforms inhibition, while CK and PPT showed more than 2.16- and 2.21-fold selectivity, respectively. PPD did not significantly increase the mRNA expression of UGT1A1, 1A3, 1A4, 1A9, and 2B7 in hepatocytes. Given the low plasma concentrations of PPD in healthy human subjects and the absence of induction potential on UGT isoforms, we conclude that PPD cause no pharmacokinetic interactions with other co-administered drugs metabolised by UGT1A3.

In vitro Measurement and In vivo Prediction of Time-Dependent Inhibitory Effects of Three Tyrosine Kinase Inhibitors on CYP3A Activity.

Imatinib, sunitinib, and gefitinib are the three most common tyrosine kinase inhibitors (TKIs). However, their quantitative drug-drug interaction potentials In vivo and the relationship between their structure and inhibitory activity remain unknown. This study aimed to investigate the potential drug-drug interaction risk of three TKIs based on CYP3A. 6β-Hydroxylated testosterone formation was selected to probe the CYP3A activity in human liver microsomes. A molecular docking simulation was performed to explore the potential structural alerts. Imatinib exhibited the strongest inhibitory effect towards CYP3A, while the inhibitory potential of gefitinib and sunitinib were comparable to each other but weaker than imatinib. IC50 shift assays demonstrated that the inhibitory potential of all three TKIs was significantly increased after a 30-min preincubation with NADPH. The KI and Kinact values of imatinib, sunitinib, and gefitinib were 3.75 μM and 0.055 min-1, 1.96 μM and 0.037 min-1, and 9.94 μM and 0.031 min-1, respectively. IVIVE results showed that there was a 1.3- to 43.1-fold increase in the AUC of CYP3A-metabolizing drugs in the presence of the TKIs. All three TKIs exhibited a typical irreversible inhibitory effect towards CYP3A. The presence of more N-heterocycles and the resulting better binding confirmation of imatinib may have been responsible for its stronger inhibitory effect than sunitinib and gefitinib. Therefore, caution should be taken when CYP3A-metabolizing drugs are co-administrated with imatinib, sunitinib, or gefitinib.

Pharmacokinetics of α-amanitin in mice using liquid chromatography-high resolution mass spectrometry and in vitro drug–drug interaction potentials

ABSTRACT The aim of this study was to determine pharmacokinetics of α-amanitin, a toxic bicyclic octapeptide isolated from the poisonous mushrooms, following intravenous (iv) or oral (po) administration in mice using a newly developed and validated liquid chromatography-high resolution mass spectrometry. The iv injected α-amanitin disappeared rapidly from the plasma with high a clearance rate (26.9–30.4 ml/min/kg) at 0.1, 0.2, or 0.4 mg/kg doses, which was consistent with a rapid and a major excretion of α-amanitin via the renal route (32.6%). After the po administration of α-amanitin at doses of 2, 5, or 10 mg/kg to mice, the absolute bioavailability of α-amanitin was 3.5–4.8%. Due to this low bioavailability, 72.5% of the po administered α-amanitin was recovered from the feces. When α-amanitin is administered po, the tissue to plasma area under the curve ratio was higher in stomach > large intestine > small intestine > lung ~ kidneys > liver but not detected in brain, heart, and spleen. The high distribution of α-amanitin to intestine, kidneys, and liver is in agreement with the previously reported major intoxicated organs following acute α-amanitin exposure. In addition, α-amanitin weakly or negligibly inhibited cytochrome P450 and 5′-diphospho-glucuronosyltransferase enzymes activity in human liver microsomes as well as major drug transport functions in mammalian cells overexpressing transporters. Data suggested remote drug interaction potential may be associated with α-amanitin exposure.

Epicatechin gallate and epigallocatechin gallate are potent inhibitors of human arylacetamide deacetylase

Recently, in addition to carboxylesterases (CESs), we found that arylacetamide deacetylase (AADAC) plays an important role in the metabolism of some clinical drugs. In this study, we screened for food-related natural compounds that could specifically inhibit human AADAC, CES1, or CES2. AADAC, CES1, and CES2 activities in human liver microsomes were measured using phenacetin, fenofibrate, and procaine as specific substrates, respectively. In total, 43 natural compounds were screened for their inhibitory effects on each of these enzymes. Curcumin and quercetin showed strong inhibitory effects against all three enzymes, whereas epicatechin, epicatechin gallate (ECg), and epigallocatechin gallate (EGCg) specifically inhibited AADAC. In particular, ECg and EGCg showed strong inhibitory effects on AADAC (IC50 values: 3.0 ± 0.5 and 2.2 ± 0.2 μM, respectively). ECg and EGCg also strongly inhibited AADAC-mediated rifampicin hydrolase activity in human liver microsomes with IC50 values of 2.2 ± 1.4 and 1.7 ± 0.4 μM, respectively, whereas it weakly inhibited p-nitrophenyl acetate hydrolase activity, which is catalyzed by AADAC, CES1, and CES2. Our results indicate that ECg and EGCg are potent inhibitors of AADAC.

Tetrahydrofurofuranoid Lignans, Eudesmin, Fargesin, Epimagnolin A, Magnolin, and Yangambin Inhibit UDP-Glucuronosyltransferase 1A1 and 1A3 Activities in Human Liver Microsomes.

Eudesmin, fargesin, epimagnolin A, magnolin, and yangambin are tetrahydrofurofuranoid lignans with various pharmacological activities found in Magnoliae Flos. The inhibition potencies of eudesmin, fargesin, epimagnolin A, magnolin, and yangambin on six major human uridine 5′-diphospho-glucuronosyltransferase (UGT) activities in human liver microsomes were evaluated using liquid chromatography–tandem mass spectrometry and cocktail substrates. Eudesmin, fargesin, epimagnolin A, magnolin, and yangambin inhibited UGT1A1 and UGT1A3 activities, but showed negligible inhibition of UGT1A4, UGT16, UGT1A9, and UGT2B7 activities at 200 μM in pooled human liver microsomes. Moreover, eudesmin, fargesin, epimagnolin A, magnolin, and yangambin noncompetitively inhibited UGT1A1-catalyzed SN38 glucuronidation with Ki values of 25.7, 25.3, 3.6, 26.0, and 17.1 μM, respectively, based on kinetic analysis of UGT1A1 inhibition in pooled human liver microsomes. Conversely, the aforementioned tetrahydrofurofuranoid lignans competitively inhibited UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation with 39.8, 24.3, 15.1, 37.6, and 66.8 μM, respectively in pooled human liver microsomes. These in vitro results suggest the necessity of evaluating whether the five tetrahydrofurofuranoid lignans can cause drug–drug interactions with UGT1A1 and UGT1A3 substrates in vivo.

Characterization of CYP3A pharmacogenetic variation in American Indian and Alaska Native communities, targeting CYP3A4*1G allele function.

The frequencies of genetic variants in the CYP3A4 and CYP3A5 genes differ greatly across global populations, leading to profound differences in the metabolic activity of these enzymes and resulting drug metabolism rates, with important consequences for therapeutic safety and efficacy. Yet, the impact of genetic variants on enzyme activity are incompletely described, particularly in American Indian and Alaska Native (AIAN) populations. To characterize genetic variation in CYP3A4 and CYP3A5 and its effect on enzyme activity, we partnered with AIAN people living in two regions of Alaska: Yup’ik Alaska Native people living in the Yukon‐Kuskokwim Delta region of rural southwest Alaska and AIAN people receiving care at the Southcentral Foundation in Anchorage, Alaska. We identified low frequencies of novel and known variation in CYP3A4 and CYP3A5, including low frequencies of the CYP3A4*1G and CYP3A5*1 variants, and linkage disequilibrium patterns that differed from those we previously identified in an American Indian population in western Montana. We also identified increased activity of the CYP3A4*1G allele in vitro and in vivo. We demonstrated that the CYP3A4*1G allele confers increased protein content in human lymphoblastoid cells and both increased protein content and increased activity in human liver microsomes. We confirmed enhanced CYP3A4‐mediated 4β‐vitamin D hydroxylation activity in Yup’ik people with the CYP3A4*1G allele. AIAN people in Alaska and Montana who carry the CYP3A4*1G allele—coupled with low frequency of the functional CYP3A5*1 variant—may metabolize CYP3A substrates more rapidly than people with the reference CYP3A4 allele.

Selective inhibitory effects of HYIpro-3-1 on CYP1A2 in human liver microsomes.

CYP1A2 is one of the main Cytochrome P450 enzymes in the human liver associated with the metabolism of several xenobiotics. CYP1A2 is especially involved in the metabolic activation of different procarcinogens. Therefore, the development of cancer may be inhibited by inhibiting CYP1A2 activity. Here, the inhibitory effect of HYIpro-3-1 and its derivatives on CYP1A2 activity in human liver microsomes (HLM) was studied through LC-MS/MS using a cocktail assay. Among the four compounds, HYIpro-3-1 showed the most selective and strongest inhibitory effect on CYP1A2 at IC50 values of 0.1 µM in HLMs and inhibition was confirmed using purified human CYP1A2. It was determined that inhibition is reversible because the inhibitory effect of HYIpro-3-1 is not dependent on preincubation time. HYIpro-3-1 showed a typical pattern of competitive inhibition for CYP1A2-catalyzed phenacetin O-deethylation, based on the Lineweaver-Burk plot, with a Ki value of 0.05 μM in HLMs; the secondary plot also showed a linear pattern. In our study, HYIpro-3-1 was proposed as a novel inhibitor with the capacity to selectively inhibit CYP1A activity in HLMs.

Effects of acid and lactone forms of statins on S-warfarin 7-hydroxylation catalyzed by human liver microsomes and recombinant CYP2C9 variants (CYP2C9.1 and CYP2C9.3)

The inhibition of CYP2C9-mediated warfarin metabolism by acid or lactone forms of statin converted in the body and effects of CYP2C9 genetic variants on their inhibition are not fully understood. Here, the effects of acid and lactone forms of statins on S-warfarin 7-hydroxylation were investigated in vitro. S-Warfarin 7-hydroxylase activities of human liver microsomes (HLMs), recombinant CYP2C9.1 (rCYP2C9.1), and rCYP2C9.3 (Ile359Leu variant) in the presence of statins were determined by high-performance liquid chromatography. Lactone forms of atorvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin inhibited the activity of HLMs more potently than the corresponding acid forms, whereas fluvastatin acid showed stronger inhibition than fluvastatin lactone. When the effects of statins on rCYP2C9 variants were examined, inhibition profiles of acid versus lactone forms of statins except for fluvastatin were similar between rCYP2C9.1 and rCYP2C9.3. However, the degrees of inhibition by atorvastatin lactone, fluvastatin acid, fluvastatin lactone, lovastatin lactone, and pitavastatin lactone (Ki values) were significantly different between these variants. These results indicated that lactone forms of statins other than fluvastatin showed more potent inhibition of CYP2C9-catalyzed S-warfarin 7-hydroxylation than the corresponding acid forms. Furthermore, our results indicated that Ile359Leu substitution in CYP2C9 affected the inhibitory potencies of statins.

Refined Prediction of Pharmacokinetic Kratom-Drug Interactions: Time-Dependent Inhibition Considerations.

Preparations from the leaves of the kratom plant (Mitragyna speciosa) are consumed for their opioid-like effects. Several deaths have been associated with kratom used concomitantly with some drugs. Pharmacokinetic interactions are potential underlying mechanisms of these fatalities. Accumulating in vitro evidence has demonstrated select kratom alkaloids, including the abundant indole alkaloid mitragynine, as reversible inhibitors of several cytochromes P450 (CYPs). The objective of this work was to refine the mechanistic understanding of potential kratom-drug interactions by considering both reversible and time-dependent inhibition (TDI) of CYPs in the liver and intestine. Mitragynine was tested against CYP2C9 (diclofenac 4'-hydroxylation), CYP2D6 (dextromethorphan O-demethylation), and CYP3A (midazolam 1'-hydroxylation) activities in human liver microsomes (HLMs) and CYP3A activity in human intestinal microsomes (HIMs). Comparing the absence to presence of NADPH during preincubation of mitragynine with HLMs or HIMs, an ∼7-fold leftward shift in IC50 (∼20 to 3 μM) toward CYP3A resulted, prompting determination of TDI parameters (HLMs: K I , 4.1 ± 0.9 μM; k inact , 0.068 ± 0.01 min-1; HIMs: K I , 4.2 ± 2.5 μM; k inact , 0.079 ± 0.02 min-1). Mitragynine caused no leftward shift in IC50 toward CYP2C9 (∼40 μM) and CYP2D6 (∼1 μM) but was a strong competitive inhibitor of CYP2D6 (K i , 1.17 ± 0.07 μM). Using a recommended mechanistic static model, mitragynine (2-g kratom dose) was predicted to increase dextromethorphan and midazolam area under the plasma concentration-time curve by 1.06- and 5.69-fold, respectively. The predicted midazolam area under the plasma concentration-time curve ratio exceeded the recommended cutoff (1.25), which would have been missed if TDI was not considered. SIGNIFICANCE STATEMENT: Kratom, a botanical natural product increasingly consumed for its opioid-like effects, may precipitate potentially serious pharmacokinetic interactions with drugs. The abundant kratom indole alkaloid mitragynine was shown to be a time-dependent inhibitor of hepatic and intestinal cytochrome P450 3A activity. A mechanistic static model predicted mitragynine to increase systemic exposure to the probe drug substrate midazolam by 5.7-fold, necessitating further evaluation via dynamic models and clinical assessment to advance the understanding of consumer safety associated with kratom use.

In situ target enzyme-activated near-infrared fluorescent probe: A case study of CYP2J2 using three-fragmentary molecular assembly engineering

Cytochrome P450 2J2 (CYP2J2) is overexpressed in human carcinoma cells lines and malignant tumors, and has been closely related to the pathogenesis of cancers and further deterioration of the tumor. However, the molecular tools used for In Situ monitoring CYP2J2 are still rarely reported due to the multiplex of CYP2J2 enzyme structure and biological sample. Herein, a near-infrared (NIR) fluorescent probe (DPBM) with large Stokes shift (118 nm) for detection CYP2J2 was developed based on three-fragmentary molecular assembly engineering. The DPBM exhibited excellent specificity, high sensitivity and rapid ‘turn-on’ NIR emission for CYP2J2 relative to other CYP isoforms in the simulated physiological environment. The limit of detection of DPBM was calculated to be 0.09 nM for CYP2J2, insinuating that DPBM would be sensitive enough to perceive endogenous CYP2J2 activity. Indeed, the DPBM was successfully applied for real-time and long-term monitoring and bio-imaging of CYP2J2 activity in human liver microsomes (HLMs), living cells, isolated organs and In Situ tumor-bearing BALB/c nude mice. These findings insinuate that DPBM can accurately sense CYP2J2 enzyme activity in vitro/vivo, which maybe facilitate further explorations of physiological and pathological processes of CYP2J2-associated diseases.

Modulation of CYP2C9 activity and hydrogen peroxide production by cytochrome b5

Cytochromes P450 (CYP) play a major role in drug detoxification, and cytochrome b5 (cyt b5) stimulates the catalytic cycle of mono-oxygenation and detoxification reactions. Collateral reactions of this catalytic cycle can lead to a significant production of toxic reactive oxygen species (ROS). One of the most abundant CYP isoforms in the human liver is CYP2C9, which catalyzes the metabolic degradation of several drugs including nonsteroidal anti-inflammatory drugs. We studied modulation by microsomal membrane-bound and soluble cyt b5 of the hydroxylation of salicylic acid to gentisic acid and ROS release by CYP2C9 activity in human liver microsomes (HLMs) and by CYP2C9 baculosomes. CYP2C9 accounts for nearly 75% of salicylic acid hydroxylation in HLMs at concentrations reached after usual aspirin doses. The anti-cyt b5 antibody SC9513 largely inhibits the rate of salicylic acid hydroxylation by CYP2C9 in HLMs and CYP2C9 baculosomes, increasing the KM approximately threefold. Besides, soluble human recombinant cyt b5 stimulates the Vmax nearly twofold while it decreases nearly threefold the Km value in CYP2C9 baculosomes. Regarding NADPH-dependent ROS production, soluble recombinant cyt b5 is a potent inhibitor both in HLMs and in CYP2C9 baculosomes, with inhibition constants of 1.04 ± 0.25 and 0.53 ± 0.06 µM cyt b5, respectively. This study indicates that variability in cyt b5 might be a major factor underlying interindividual variability in the metabolism of CYP2C9 substrates.

Modulation of Major Human Liver Microsomal Cytochromes P450 by Component Alkaloids of Goldenseal: Time-Dependent Inhibition and Allosteric Effects.

Botanical and other natural products (NPs) are often coconsumed with prescription medications, presenting a risk for cytochrome P450 (P450)-mediated NP-drug interactions. The NP goldenseal (Hydrastis canadensis) has exhibited antimicrobial activities in vitro attributed to isoquinoline alkaloids contained in the plant, primarily berberine, (-)-β-hydrastine, and to a lesser extent, hydrastinine. These alkaloids contain methylenedioxyphenyl rings, structural alerts with potential to inactivate P450s through formation of metabolic intermediate complexes. Time-dependent inhibition experiments were conducted to evaluate their ability to inhibit major P450 activities in human liver microsomes by using a cocktail of isozyme-specific substrate probes. Berberine inhibited CYP2D6 (dextromethorphan O-demethylation; K I = 2.7 μM, kinact = 0.065 minute-1) and CYP3A4/5 (midazolam 1'-hydroxylation; K I = 14.8 μM, kinact = 0.019 minute-1); (-)-β-hydrastine inhibited CYP2C9 (diclofenac 4'-hydroxylation; K I = 49 μM, kinact = 0.036 minute-1), CYP2D6 (K I > 250 μM, kinact > 0.06 minute-1), and CYP3A4/5 (K I = 28 μM, kinact = 0.056 minute-1); and hydrastinine inhibited CYP2D6 (K I = 37 μM, kinact = 0.049 minute-1) activity. Berberine additionally exhibited allosteric effects on midazolam hydroxylation, showing both positive and negative heterotropic cooperativity. Experiments with recombinant isozymes showed that berberine activated midazolam 1'-hydroxylation by CYP3A5, lowering K m(app), but showed mixed inhibition and negative cooperativity toward this reaction when catalyzed by CYP3A4. Berberine inactivated CYP3A4 at a much faster rate than CYP3A5 and was a noncompetitive inhibitor of midazolam 4-hydroxylation by CYP3A4 but a strong mixed inhibitor of the CYP3A5 catalyzed reaction. These complex kinetics should be considered when extrapolating the risk for NP-drug interactions involving goldenseal. SIGNIFICANCE STATEMENT: Robust kinetic parameters were determined for the reversible and time-dependent inhibition of CYP2C9, CYP2D6, and CYP3A4/5 activities in human liver microsomes by major component isoquinoline alkaloids contained in the botanical natural product goldenseal. The alkaloid berberine also exhibited opposing, isozyme-specific allosteric effects on midazolam hydroxylation mediated by recombinant CYP3A4 (inhibition) and CYP3A5 (activation). These data will inform the development of a physiologically based pharmacokinetic model that can be used to predict potential clinically relevant goldenseal-drug interactions.

Inhibitory effects of sesamin on CYP2C9-dependent 7-hydroxylation of S-warfarin

A recent report demonstrated that sesamin strongly and non-competitively inhibits S-warfarin 7-hydroxylation activity in human liver microsomes with a Ki value of 0.2 μM. This finding suggests that sesamin predominantly binds to CYP2C9 at another site for which it has a higher affinity than its affinity for the active site, thereby inhibiting the activity of CYP2C9 non-competitively. In this study, we found that sesamin competitively inhibited the 7-hydroxylation activity of S-warfarin in human liver microsomes with a Ki value of 15.7 μM. In addition, the recombinant CYP2C9-dependent 7-hydroxylation activity of S-warfarin was competitively inhibited by sesamin with a Ki value of 13.1 μM. These results are consistent with the fact that sesamin is a good substrate of CYP2C9, and its activity follows Michaelis-Menten kinetics. As the plasma concentration of sesamin after its administration is usually lower than 0.01 μM, the inhibition of S-warfarin metabolism by sesamin does not appear to be severe.