Editor’s Perspective 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.

Protein therapeutics are prone to lose their structure and bioactivity under various environmental stressors. Herein, we report 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 analysis 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) was evaluated in vivo using a diabetic mouse model, and showed comparable bioactivity to refrigeration-stored insulin (−20°C). We also demonstrate that ZIF-8 preserved insulin had low cytotoxicity in vitro and did 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.

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 K m, on the order of Br (bromoketamine, 10 μM) < Cl < F < H (deschloroketamine, 184 μM). In contrast, V max 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 K m 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 K m, 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 (R 2 = 0.92) between lipophilicity (AlogP) and metabolism (log K m) of ketamine and analogs. This property may be the simplest design criteria to use when considering similar compounds and CYP2B6 affinity.

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.

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 Sefavirenz metabolism by clinically relevant CYP2B6 genetic variants. This investigation also evaluated R-efavirenz hydroxylation by wildtype 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 Cl max 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 wildtype CYP2B6.1 and variants. On the basis of Cl max 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. Rmetabolism), efavirenz is the most stereoselective CYP2B6 drug substrate yet identified. These findings may provide mechanistic insights.

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.

Bioactivation of the antidepressant and smoking cessation drug bupropion is catalyzed predominantly by cytochrome P4502B6 (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, 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 V max and K m varied widely among CYP2B6 variants, stereoselectivity was preserved, reflected by similar Cl int (S,Shydroxybupropion)/Cl int (R,R-hydroxybupropion) ratios (1.8 to 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.

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 (C max) and area under the plasma concentration-time curve over 24 hours (AUC 0-24)) 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.