Natural Products Inhibition of Cytochrome P450 2B6 Activity and Methadone Metabolism.

Methadone is cleared predominately by hepatic cytochrome P450 (CYP) 2B6-catalyzed metabolism to inactive metabolites. CYP2B6 also catalyzes the metabolism of several other drugs. Methadone and CYP2B6 are susceptible to pharmacokinetic drug-drug interactions. Use of natural products such as herbals and other botanicals is substantial and growing, and concomitant use of prescription medicines and non-prescription herbals is common and may result in interactions, often precipitated by CYP inhibition. Little is known about herbal product effects on CYP2B6 activity, and CYP2B6-catalyzed methadone metabolism. We screened a family of natural product compounds used in traditional medicines, herbal teas, and synthetic analogs of compounds found in plants, including kavalactones, flavokavains, chalcones and gambogic acid, for inhibition of expressed CYP2B6 activity and specifically inhibition of CYP2B6-mediated methadone metabolism. An initial screen evaluated inhibition of CYP2B6-catalyzed 7-ethoxy-4-(trifluoromethyl) coumarin O-deethylation. Hits were further evaluated for inhibition of racemic methadone metabolism, including mechanism of inhibition and kinetic constants. In order of decreasing potency, the most effective inhibitors of methadone metabolism were dihydromethysticin (competitive, K i 0.074 µM), gambogic acid (noncompetitive, K i 6 µM), and 2,2'-dihydroxychalcone (noncompetitive, K i 16 µM). Molecular modeling of CYP2B6-methadone and inhibitor binding showed substrate and inhibitor binding position and orientation and their interactions with CYP2B6 residues. These results show that CYP2B6 and CYP2B6-catalyzed methadone metabolism are inhibited by certain natural products, at concentrations which may be clinically relevant. SIGNIFICANCE STATEMENT: This investigation identified several natural product constituents which inhibit in vitro human recombinant CYP2B6 and CYP2B6-catalyzed N-demethylation of the opioid methadone. The most potent inhibitors (K i) were dihydromethysticin (0.074 µM), gambogic acid (6 µM) and 2,2'-dihydroxychalcone (16 µM). Molecular modeling of ligand interactions with CYP2B6 found that dihydromethysticin and 2,2'-dihydroxychalcone bound at the active site, while gambogic acid interacted with an allosteric site on the CYP2B6 surface. Natural product constituents may inhibit CYP2B6 and methadone metabolism at clinically relevant concentrations.

Methadone pharmacogenetics in vitro and in vivo: Metabolism by CYP2B6 polymorphic variants and genetic variability in paediatric disposition.

AIMS: Methadone metabolism and clearance are determined principally by polymorphic cytochrome P4502B6 (CYP2B6). Some CYP2B6 allelic variants affect methadone metabolism in vitro and disposition in vivo. We assessed methadone metabolism by CYP2B6 minor variants in vitro. We also assessed the influence of CYP2B6 variants, and P450 oxidoreductase (POR) and CYP2C19 variants, on methadone clearance in surgical patients in vivo. METHODS: CYP2B6 and P450 oxidoreductase variants were coexpressed with cytochrome b5 . The metabolism of methadone racemate and enantiomers was measured at therapeutic concentrations and intrinsic clearances were determined. Adolescents receiving methadone for surgery were genotyped for CYP2B6, CYP2C19 and POR, and methadone clearance and metabolite formation clearance were determined. RESULTS: In vitro, CYP2B6.4 was more active than wild-type CYP2B6.1. CYPs 2B6.5, 2B6.6, 2B6.7, 2B6.9, 2B6.17, 2B6.19 and 2B6.26 were less active. CYPs 2B6.16 and 2B6.18 were inactive. CYP2B6.1 expressed with POR variants POR.28, POR.5 and P228L had lower rates of methadone metabolism than wild-type reductase. In vivo, methadone clinical clearance decreased linearly with the number of CYP2B6 slow metabolizer alleles, but was not different in CYP2C19 slow or rapid metabolizer phenotypes, or in carriers of the POR*28 allele. CONCLUSIONS: Several CYP2B6 and POR variants were slow metabolizers of methadone in vitro. Polymorphisms in CYP2B6, but not CYP2C19 or P450 reductase, affected methadone clearance in vivo. CYP2B6 polymorphisms 516G>T and 983T>C code for canonical loss of function variants and should be assessed when considering genetic influences on clinical methadone disposition. These complementary translational in vitro and in vivo results inform on pharmacogenetic variability affecting methadone disposition in patients.

Stereoselective Bupropion Hydroxylation by Cytochrome P450 CYP2B6 and Cytochrome P450 Oxidoreductase Genetic Variants.

Bioactivation of the antidepressant and smoking cessation drug bupropion is catalyzed predominantly by CYP2B6. The metabolite hydroxybupropion derived from t-butylhydroxylation is considered to contribute to the antidepressant and smoking-cessation effects of the parent drug. Bupropion hydroxylation is the canonical in vitro and in vivo probe for CYP2B6 activity. P450 also requires obligate partnership with P450 oxidoreductase (POR). Human CYP2B6 and POR genes are highly polymorphic. Some CYP2B6 variants affect bupropion disposition. This investigation evaluated the influence of several human CYP2B6 and POR genetic variants on stereoselective bupropion metabolism, using an insect cell coexpression system containing CYP2B6, POR, and cytochrome b 5 Based on intrinsic clearances (Clints), relative activities for S,S-hydroxybupropion formation were in the order CYP2B6.4 > CYP2B6.1 > CYP2B6.17 > CYP2B6.5 > CYP2B6.6 ≈ CYP2B6.26 ≈ CYP2B6.19 > CYP2B6.7 > CYP2B6.9 > > CYP2B6.16 and CYP2B6.18; relative activities for R,R-hydroxybupropion formation were in the order CYP2B6.17 > CYP2B6.4 > CYP2B6.1 > CYP2B6.5 ≈ CYP2B6.19 ≈ CYP2B6.26 > CYP2B6.6 > CYP2B6.7 ≈ CYP2B6.9 > > CYP2B6.16 and CYP2B6.18. Bupropion hydroxylation was not influenced by POR variants. CYP2B6-catalyzed bupropion hydroxylation is stereoselective. Though Vmax and Km varied widely among CYP2B6 variants, stereoselectivity was preserved, reflected by similar Clint(S,S-hydroxybupropion)/Clint(R,R-hydroxybupropion) ratios (1.8-2.9), except CYP2B6.17, which was less enantioselective. Established concordance between human bupropion hydroxylation in vitro and in vivo, together with these new results, suggests additional CYP2B6 variants may influence human bupropion disposition. SIGNIFICANCE STATEMENT: Bupropion pharmacokinetics, metabolism, and clinical effects are affected by the CYP2B6*6 polymorphism. Other expressed CYP2B6 polymorphisms had diminished (*5, *6, *7, *9, *19, *26) or defective (*16, *18) in vitro bupropion hydroxylation. P450 oxidoreductase genetic variants had no effect on metabolism, suggesting no clinical consequence of this polymorphism. These CYP2B6 polymorphisms may portend diminished in vivo bupropion hydroxylation and predict additional clinically important variant alleles.

Efavirenz Metabolism: Influence of Polymorphic CYP2B6 Variants and Stereochemistry.

Efavirenz (more specifically the S-enantiomer) is a cornerstone antiretroviral therapy for treatment of HIV infection. The major primary metabolite is S-8-hydroxyefavirenz, which does not have antiretroviral activity but is neurotoxic. Cytochrome P450 2B6 (CYP2B6) is the major enzyme catalyzing S-8-hydroxyefavirenz formation. CYP2B6 genetics and drug interactions are major determinants of clinical efavirenz disposition and dose adjustment. In addition, as a prototypic CYP2B6 substrate, S-efavirenz and analogs can inform on the structure, activity, catalytic mechanisms, and stereoselectivity of CYP2B6. Metabolism of R-efavirenz by CYP2B6 remains unexplored. This investigation assessed S-efavirenz metabolism by clinically relevant CYP2B6 genetic variants. This investigation also evaluated R-efavirenz hydroxylation by wild-type CYP2B6.1 and CYP2B6 variants. S-Efavirenz 8-hydroxylation by wild-type CYP2B6.1 and variants exhibited positive cooperativity and apparent cooperative substrate inhibition. On the basis of Clmax values, relative activities for S-efavirenz 8-hydroxylation were in the order CYP2B6.4 > CYP2B6.1 ≈ CYP2B6.5 ≈ CYP2B6.17 > CYP2B6.6 ≈ CYP2B6.7 ≈ CYP2B6.9 ≈ CYP2B6.19 ≈ CYP2B6.26; CYP2B6.16 and CYP2B6.18 showed minimal activity. Rates of R-efavirenz metabolism were approximately 1/10 those of S-efavirenz for wild-type CYP2B6.1 and variants. On the basis of Clmax values, there was 14-fold enantioselectivity (S > R-efavirenz) for wild-type CYP2B6.1, and 5- to 22-fold differences for other CYP2B6 variants. These results show that both CYP2B6 516G > T (CYP2B6*6 and CYP2B6*9) and 983T > C (CYP2B6*16 and CYP2B6*18) polymorphisms cause canonical diminishment or loss-of-function variants for S-efavirenz 8-hydroxylation, provide a mechanistic basis for known clinical pharmacogenetic differences in efavirenz disposition, and may predict additional clinically important variant alleles. Efavirenz is the most stereoselective CYP2B6 drug substrate yet identified and may be a useful probe for the CYP2B6 active site and catalytic mechanisms. SIGNIFICANCE STATEMENT: Clinical disposition of the antiretroviral S-efavirenz is affected by CYP2B6 polymorphisms. Expressed CYP2B6 with 516G>T (CYP2B6*6 and CYP2B6*9), and 983T>C (CYP2B6*16 and CYP2B6*18) polymorphisms had a diminishment or loss of function for efavirenz 8-hydroxylation. This provides a mechanistic basis for efavirenz clinical pharmacogenetics and may predict additional clinically important variant alleles. Efavirenz metabolism showed both cooperativity and cooperative substrate inhibition. With greater than 10-fold enantioselectivity (S- vs. R- metabolism), efavirenz is the most stereoselective CYP2B6 drug substrate yet identified. These findings may provide mechanistic insights.

Halogen Substitution Influences Ketamine Metabolism by Cytochrome P450 2B6: In Vitro and Computational Approaches.

Ketamine is analgesic at anesthetic and subanesthetic doses, and it has been used recently to treat depression. Biotransformation mediates ketamine effects, influencing both systemic elimination and bioactivation. CYP2B6 is the major catalyst of hepatic ketamine N-demethylation and metabolism at clinically relevant concentrations. Numerous CYP2B6 substrates contain halogens. CYP2B6 readily forms halogen-protein (particularly Cl-π) bonds, which influence substrate selectivity and active site orientation. Ketamine is chlorinated, but little is known about the metabolism of halogenated analogs. This investigation evaluated halogen substitution effects on CYP2B6-catalyzed ketamine analogs N-demethylation in vitro and modeled interactions with CYP2B6 using various computational approaches. Ortho phenyl ring halogen substituent changes caused substantial (18-fold) differences in Km, on the order of Br (bromoketamine, 10 µM) < Cl < F < H (deschloroketamine, 184 µM). In contrast, Vmax varied minimally (83-103 pmol/min/pmol CYP). Thus, apparent substrate binding affinity was the major consequence of halogen substitution and the major determinant of N-demethylation. Docking poses of ketamine and analogs were similar, sharing a π-stack with F297. Libdock scores were deschloroketamine < bromoketamine < ketamine < fluoroketamine. A Bayesian log Km model generated with Assay Central had a ROC of 0.86. The probability of activity at 15 µM for ketamine and analogs was predicted with this model. Deschloroketamine scores corresponded to the experimental Km, but the model was unable to predict activity with fluoroketamine. The binding pocket of CYP2B6 also suggested a hydrophobic component to substrate docking, on the basis of a strong linear correlation ( R2 = 0.92) between lipophilicity ( Alog P) and metabolism (log Km) of ketamine and analogs. This property may be the simplest design criteria to use when considering similar compounds and CYP2B6 affinity.

Stereoselective Ketamine Metabolism by Genetic Variants of Cytochrome P450 CYP2B6 and Cytochrome P450 Oxidoreductase.

WHAT WE ALREADY KNOW ABOUT THIS TOPIC: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Human ketamine N-demethylation to norketamine in vitro at therapeutic concentrations is catalyzed predominantly by the cytochrome P4502B6 isoform (CYP2B6). The CYP2B6 gene is highly polymorphic. CYP2B6.6, the protein encoded by the common variant allele CYP2B6*6, exhibits diminished ketamine metabolism in vitro compared with wild-type CYP2B6.1. The gene for cytochrome P450 oxidoreductase (POR), an obligatory P450 coenzyme, is also polymorphic. This investigation evaluated ketamine metabolism by genetic variants of human CYP2B6 and POR. METHODS: CYP2B6 (and variants), POR (and variants), and cytochrome b5 (wild-type) were coexpressed in a cell system. All CYP2B6 variants were expressed with wild-type POR and b5. All POR variants were expressed with wild-type CYP2B6.1 and b5. Metabolism of R- and S-ketamine enantiomers, and racemic RS-ketamine to norketamine enantiomers, was determined using stereoselective high-pressure liquid chromatography-mass spectrometry. Michaelis-Menten kinetic parameters were determined. RESULTS: For ketamine enantiomers and racemate, metabolism (intrinsic clearance) was generally wild-type CYP2B6.1 > CYP2B6.4 > CYP2B6.26, CYP2B6.19, CYP2B6.17, CYP2B6.6 > CYP2B6.5, CYP2B6.7 > CYP2B6.9. CYP2B6.16 and CYP2B6.18 were essentially inactive. Activity of several CYP2B6 variants was less than half that of CYP2B6.1. CYP2B6.9 was 15 to 35% that of CYP2B6.1. The order of metabolism was wild-type POR.1 > POR.28, P228L > POR.5. CYP2B6 variants had more influence than POR variants on ketamine metabolism. Neither CYP2B6 nor POR variants affected the stereoselectivity of ketamine metabolism (S > R). CONCLUSIONS: Genetic variants of CYP2B6 and P450 oxidoreductase have diminished ketamine N-demethylation activity, without affecting the stereoselectivity of metabolism. These results suggest candidate genetic polymorphisms of CYP2B6 and P450 oxidoreductase for clinical evaluation to assess consequences for ketamine pharmacokinetics, elimination, bioactivation, and therapeutic effects.

Neurotoxins subvert the allosteric activation mechanism of SARM1 to induce neuronal loss.

SARM1 is an inducible TIR-domain NAD+ hydrolase that mediates pathological axon degeneration. SARM1 is activated by an increased ratio of NMN to NAD+, which competes for binding to an allosteric activating site. When NMN binds, the TIR domain is released from autoinhibition, activating its NAD+ hydrolase activity. The discovery of this allosteric activating site led us to hypothesize that other NAD+-related metabolites might activate SARM1. Here, we show the nicotinamide analog 3-acetylpyridine (3-AP), first identified as a neurotoxin in the 1940s, is converted to 3-APMN, which activates SARM1 and induces SARM1-dependent NAD+ depletion, axon degeneration, and neuronal death. In mice, systemic treatment with 3-AP causes rapid SARM1-dependent death, while local application to the peripheral nerve induces SARM1-dependent axon degeneration. We identify 2-aminopyridine as another SARM1-dependent neurotoxin. These findings identify SARM1 as a candidate mediator of environmental neurotoxicity and suggest that SARM1 agonists could be developed into selective agents for neurolytic therapy.

Stereoselective Steady-State Disposition and Bioequivalence of Brand and Generic Bupropion in Adults.

The antidepressant bupropion is stereoselectively metabolized and metabolite enantiomers have differential pharmacologic effects, but steady-state enantiomeric disposition is unknown. Controversy persists about bupropion XL 300 mg generic equivalence to brand product, and whether generics might have different stereoselective disposition leading to enantiomeric non-bioequivalence and, thus, clinical nonequivalence. This preplanned follow-on analysis of a prospective, randomized, double-blinded, crossover study of brand and 3 generic bupropion XL 300 mg products measured steady-state enantiomeric plasma and urine parent bupropion and primary and secondary metabolite concentrations and evaluated bioequivalence and pharmacokinetics. Steady-state plasma and urine bupropion disposition was markedly stereoselective, with up to 40-fold differences in plasma concentrations of the active metabolite S,S-hydroxybupropion vs. R,R,-hydroxybupropion. Urine metabolite glucuronides were prominent, but glucuronidation was metabolite-specific and enantioselective. There were no differences between any generic and brand, or between generics, in plasma enantiomer concentrations of bupropion or the major metabolites. All generic products satisfied formal bioequivalence criteria (peak plasma concentration (Cmax ) and area under the plasma concentration-time curve over 24 hours (AUC0-24 )) using enantiomers for bupropion as well as for metabolites, and generics were comparable to each other, and were considered bioequivalent, based on enantiomeric analysis. Enantiomeric bioequivalence explains the previously observed therapeutic equivalence of bupropion generics and brand in treating major depression. These results have important implications for understanding the clinical therapeutic effects of bupropion based on complex and stereoselective metabolism.

Bioequivalence and Therapeutic Equivalence of Generic and Brand Bupropion in Adults With Major Depression: A Randomized Clinical Trial.

Controversy persists about bupropion XL 300 mg generic equivalence to brand product. A prospective, randomized, double-blinded crossover in 70 adults with major depression in stable remission taking any bupropion XL 300 mg tested bioequivalence and therapeutic equivalence of available XL 300 mg products. After a 4-week lead-in on patients' existing bupropion, four 6-week phases evaluated brand and three generics. Patients were uninformed of switching. Drug overencapsulation ensured blinding. There were no differences between any generic and brand, or between generics, in peak plasma concentration (Cmax ) and area under the plasma concentration-time curve over the 24-hour dosing interval (AUC0-24 ) for racemic bupropion or major metabolites. All generics met formal bioequivalence criteria for bupropion and metabolites. There were no differences between generics and brand, or between generics, in depression symptoms or side effects, assessed by every 3-week in-person interview and daily smartphone-based self-report. There were no differences in patients' perceptions of bupropion products. Results show three bupropion XL 300 mg generic products are both bioequivalent and not therapeutically different from brand drug and each other.

How central is central poststroke pain? The role of afferent input in poststroke neuropathic pain: a prospective, open-label pilot study.

Central poststroke pain (CPSP) is a neuropathic pain disorder, the underlying mechanisms of which are not well understood. It has been suggested that stroke-associated loss of inhibitory neurons in the spinothalamic tract causes disinhibition of thalamic neurons, which autonomously generate ectopic nociceptive action potentials responsible for the pain experience. We hypothesized that CPSP is a result of misinterpretation of afferent sensory input by the sensitized neurons within the brain, rather than generated spontaneously by the damaged central nervous system (CNS) neurons. To test this hypothesis, we prospectively recruited 8 patients with definite CPSP affecting at least 1 extremity. In an open-label intervention, an ultrasound-guided peripheral nerve block with lidocaine was performed to block afferent sensory input from a painful extremity. Spontaneous and evoked pain, neuropathic pain descriptors, and lidocaine plasma concentrations were measured. The blockade of peripheral sensory input resulted in complete abolition of pain in 7 of the 8 subjects within 30 minutes (the primary outcome measure of the study), and >50% pain relief in the remaining participant. Median (interquartile range) spontaneous pain intensity changed from 6.5 (4.3-7.0) at baseline to 0 (0-0) after the block (P = 0.008). All mechanical/thermal hypersensitivity was abolished by the nerve block. The results suggest that it is unlikely that CPSP is autonomously generated within the CNS. Rather, this pain is dependent on afferent input from the painful region in the periphery, and may be mediated by misinterpretation of peripheral sensory input by sensitized neurons in the CNS.

Metal-Organic Framework Encapsulation Preserves the Bioactivity of Protein Therapeutics.

Protein therapeutics are prone to lose their structure and bioactivity under various environmental stressors. This study reports a facile approach using a nanoporous material, zeolitic imidazolate framework-8 (ZIF-8), as an encapsulant for preserving the prototypic protein therapeutic, insulin, against different harsh conditions that may be encountered during storage, formulation, and transport, including elevated temperatures, mechanical agitation, and organic solvent. Both immunoassay and spectroscopy analyses demonstrate the preserved chemical stability and structural integrity of insulin offered by the ZIF-8 encapsulation. Biological activity of ZIF-8-preserved insulin after storage under accelerated degradation conditions (i.e., 40 °C) is evaluated in vivo using a diabetic mouse model, and shows comparable bioactivity to refrigeration-stored insulin (-20 °C). It is also demonstrated that ZIF-8-preserved insulin has low cytotoxicity in vitro and does not cause side effects in vivo. Furthermore, ZIF-8 residue can be completely removed by a simple purification step before insulin administration. This biopreservation approach is potentially applicable to diverse protein therapeutics, thus extending the benefits of advanced biologics to resource-limited settings and underserved populations/regions.