Identification of novel glutathione conjugates of terbinafine in liver microsomes and hepatocytes across species.

1. Terbinafine (TBF), a common antifungal agent, has been associated with rare incidences of hepatotoxicity. It is hypothesized that bioactivation of TBF to reactive intermediates and subsequent binding to critical cellular proteins may contribute to this toxicity. In the present study, we have characterized the bioactivation pathways of TBF extensively in human, mouse, monkey, dog and rat liver microsomes and hepatocytes. 2. A total of twenty glutathione conjugates of TBF were identified in hepatocytes; thirteen of these conjugates were also detected in liver microsomes. To the best of our knowledge, only two of these conjugates have been reported previously. The conjugates were categorized into three groups based on their mechanism of formation: (a) alkene/alkyne oxidation followed by glutathione conjugation, with or without N-demethylation, (b) arene oxidation followed by glutathione conjugation, with or without N-demethylation, and (c) N-dealkylation followed by glutathione conjugation of the allylic aldehyde, alcohol and acid intermediates. 3. Differences were observed across species in the contributions of these pathways toward overall metabolic turnover. We conclude that, in addition to the glutathione conjugates known to form by Michael addition to the allylic aldehyde, there are other pathways involving the formation of arene oxides and alkene/alkyne epoxides that may be relevant to the discussion of TBF-mediated idiosyncratic drug reactions.

Metabolite Identification, Reaction Phenotyping, and Retrospective Drug-Drug Interaction Predictions of 17-Deacetylnorgestimate, the Active Component of the Oral Contraceptive Norgestimate.

Ortho Tri-Cyclen, a two-drug cocktail comprised of ethinylestradiol and norgestimate (13-ethyl-17-acetoxy-18, 19-dinor-17α-pregn-4-en-20yn-3 oxime), is commonly prescribed to avert unwanted pregnancies in women of reproductive age. In vivo, norgestimate undergoes extensive and rapid deacetylation to produce 17-deacetylnorgestimate (NGMN), an active circulating metabolite that likely contributes significantly to norgestimate efficacy. Despite being of primary significance, the metabolism and reaction phenotyping of NGMN have not been previously reported. Hence, detailed biotransformation and reaction phenotyping studies of NGMN with recombinant cytochrome P450 (P450), recombinant uridine 5'-diphospho-glucuronosyltransferases, and human liver microsomes in the presence and absence of selective P450 inhibitors were conducted. It was found that CYP3A4 plays a key role in NGMN metabolism with a fraction metabolized (fm) of 0.57. CYP2B6 and to an even lesser extent CYP2C9 were also observed to catalyze NGMN metabolism. Using this CYP3A4 fm value, the predicted plasma concentration versus time area under the curve (AUC) change in NGMN using a basic/mechanistic static model was found to be within 1.3-fold of the reported NGMN AUC changes for four modulators of CYP3A4. In addition to NGMN, we have also elucidated the biotransformation of norgestrel (NG), a downstream norgestimate and NGMN metabolite, and found that CYP3A4 and UGT1A1 have a major contribution to the elimination of NG with a combined fm value of 1. The data presented in this paper will lead to better understanding and management of NGMN-based drug-drug interactions when norgestimate is coadministered with CYP3A4 modulators.

Use of Hybrid Capillary Tube Apparatus on 400 MHz NMR for Quantitation of Crucial Low-Quantity Metabolites Using aSICCO Signal.

Metabolites of new chemical entities can influence safety and efficacy of a molecule and often times need to be quantified in preclinical studies. However, synthetic standards of metabolites are very rarely available in early discovery. Alternate approaches such as biosynthesis need to be explored to generate these metabolites. Assessing the quantity and purity of these small amounts of metabolites with a nondestructive analytical procedure becomes crucial. Quantitative NMR becomes the method of choice for these samples. Recent advances in high-field NMR (>500 MHz) with the use of cryoprobe technology have helped to improve sensitivity for analysis of small microgram quantity of such samples. However, this type of NMR instrumentation is not routinely available in all laboratories. To analyze microgram quantities of metabolites on a routine basis with lower-resolution 400 MHz NMR instrument fitted with a broad band fluorine observe room temperature probe, a novel hybrid capillary tube setup was developed. To quantitate the metabolite in the sample, an artificial signal insertion for calculation of concentration observed (aSICCO) method that introduces an internally calibrated mathematical signal was used after acquiring the NMR spectrum. The linearity of aSICCO signal was established using ibuprofen as a model analyte. The limit of quantification of this procedure was 0.8 mM with 10 K scans that could be improved further with the increase in the number of scans. This procedure was used to quantify three metabolites-phenytoin from fosphenytoin, dextrophan from dextromethorphan, and 4-OH-diclofenac from diclofenac-and is suitable for minibiosynthesis of metabolites from in vitro systems.

Comprehensive evaluation of liver microsomal cytochrome P450 3A (CYP3A) inhibition: comparison of cynomolgus monkey and human.

1. Members of the cytochrome P450 3A (CYP3A) subfamily metabolize numerous compounds and serve as the loci of drug-drug interactions (DDIs). Because of high amino acid sequence identity with human CYP3A, the cynomolgus monkey has been proposed as a model species to support DDI risk assessment. 2. Therefore, the objective of this study was to evaluate 35 known inhibitors of human CYP3A using human (HLM) and cynomolgus monkey (CLM) liver microsomes. Midazolam was employed as substrate to generate IC50 values (concentration of inhibitor rendering 50% inhibition) in the absence and presence of a preincubation (30 mins) with NADPH. 3. In the absence of preincubation, the IC50 values generated with CLM were similar to those obtained with HLM (86% within 2-fold; 100% within 3-fold difference). However, significant differences (up to 48-fold) in preincubation IC50 were observed with 17% of the compounds (raloxifene, bergamottin, nicardipine, mibefradil, ritonavir, and diltiazem). 4. Our results indicate that in most cases the cynomolgus monkey can be a viable DDI model. However, significant species differences in time-dependent CYP3A inhibition can be observed for some compounds. In the case of raloxifene, such a difference can be ascribed to a specific CYP3A4 amino acid residue.

Absorption and cleavage of enalapril, a carboxyl ester prodrug, in the rat intestine: in vitro, in situ intestinal perfusion and portal vein cannulation models.

In recent years prodrug strategy has been used extensively to improve the pharmacokinetic properties of compounds exhibiting poor bioavailability. Mechanistic understanding of the absorption and the role of intestine and liver in the activation of oral prodrugs is crucial. Enalapril, a carboxyl ester prodrug, is reported to be metabolized by human carboxylesterase-1 (CES1) but not by carboxylesterase-2 (CES2) to its active metabolite enalaprilat. Further, it has been reported that the small intestines of both rat and human contain mainly CES2. The objective of this work was to understand whether enalapril remains unchanged as it is absorbed through the intestine into the portal circulation. This was evaluated using different intestinal preparations, an in situ intestinal perfusion experiment and a portal vein cannulated rat model. No turnover of enalapril was seen with commercial rat intestinal S9 and microsomes, but reasonable turnover was observed with freshly prepared rat intestinal and mucosal homogenate and S9. In the intestinal perfusion study, both enalapril and enalaprilat were observed in the mesenteric plasma with the data suggesting 32% hydrolysis of enalapril in the intestine. In the portal vein cannulated rat, about 51% of enalapril absorbed into intestine was converted to enalaprilat. Overall, it was demonstrated that even though enalapril has been shown to be a specific substrate for CES1, it is converted to enalaprilat to a significant extent in the intestine. Such experimental techniques can be applied by other scientific groups who are working on prodrugs to determine the region and extent of activation. Copyright © 2015 John Wiley & Sons, Ltd.

Expression and characterization of cynomolgus monkey cytochrome CYP3A4 in a novel human embryonic kidney cell-based mammalian system.

Cynomolgus monkeys are a commonly used species in preclinical drug discovery, and have high genetic similarity to humans, especially for the drug-metabolizing cytochrome P450s. However, species differences are frequently observed in the metabolism of drugs between cynomolgus monkeys and humans, and delineating these differences requires expressed CYPs. Toward this end, cynomolgus monkey CYP3A4 (c3A4) was cloned and expressed in a novel human embryonic kidney 293-6E cell suspension system. Following the preparation of microsomes, the kinetic profiles of five known human CYP3A4 (h3A4) substrates (midazolam, testosterone, terfenadine, nifedipine, and triazolam) were determined. All five substrates were found to be good substrates of c3A4, although some differences were observed in the Km values. Overall, the data suggest a strong substrate similarity between c3A4 and h3A4. Additionally, c3A4 exhibited no activity against non-h3A4 probe substrates, except for a known human CYP2D6 substrate (bufuralol), which suggests potential metabolism of human cytochrome CYP2D6-substrates by c3A4. Ketoconazole and troleandomycin showed similar inhibitory potencies toward c3A4 and h3A4, whereas non-h3A4 inhibitors did not inhibit c3A4 activity. The availability of a c3A4 preparation, in conjunction with commercially available monkey liver microsomes, will support further characterization of the cynomolgus monkey as a model to assess CYP3A-dependent clearance and drug-drug interactions.

Role of hepatic blood flow and metabolism in the pharmacokinetics of ten drugs in lean, aged and obese rats.

1. The effect of age and obesity on the pharmacokinetics (PK), hepatic blood flow (HBF) and liver metabolism of 10 compounds was determined in rats. The animals fed a high-fat diet were defined as the diet-induced obese (DIO) group, while the animals that were aged similar to the DIO rats but not fed with high-fat diet were called the age-matched (AM) group. 2. The clearance (CL) values of high CL compounds (CL > 50 mL/min/kg, namely propranolol, diazepam, phenytoin, ethinylestradiol, lorcaserin and fenfluramine) decreased significantly (1.5- to 6-fold) in DIO and AM rats as compared to lean rats, while there was no clear trend for change in CL for the low-to-moderate CL compounds (CL < 50 mL/min/kg, namely atenolol, chlorzoxazone, vancomycin and sibutramine). Hepatocytes incubations revealed a change in half life (t1/2) only for phenytoin. The body weight normalized liver weights and HBF of AM and DIO rats were found to be 2- to 3-fold lower than in lean rats. 3. Our findings suggest that age, and diet to a lesser extent, can reduce HBF and body normalized liver weights and, hence, also reduce CL values for high CL compounds in rats.

Characterization of recombinantly expressed rat and monkey intestinal alkaline phosphatases: in vitro studies and in vivo correlations.

Intestinal alkaline phosphatases (IALPs) are widely expressed in the brush border of epithelial cells of the intestinal mucosa. Although their physiologic role is unclear, they are very significant when it comes to the release of bioactive parent from orally dosed phosphate prodrugs. Such prodrugs can be resistant to cleavage by IALP, or alternatively undergo rapid cleavage leading to the release and precipitation of the less soluble parent. Because purified IALPs from preclinical species are not commercially available, and species differences have not been investigated to date, an effort was made to recombinantly express, purify, and characterize rat and cynomolgus monkey IALP (rIALP). Specifically, recombinant IALP (rIALP)-catalyzed cleavage of five prodrugs (fosphenytoin, clindamycin phosphate, dexamethasone phosphate, ritonavir phosphate, and ritonavir oxymethyl phosphate) was tested in vitro and parent exposure was assessed in vivo (rat only) following an oral dose of each prodrug. It was determined that the rate of phosphate cleavage in vitro varied widely; direct phosphates were more resistant to bioconversion, whereas faster conversion was observed with oxymethyl-linked prodrugs. Overall, the rat rIALP-derived data were qualitatively consistent with in vivo data; prodrugs that were readily cleaved in vitro rendered higher parent drug exposure in vivo. Of the five prodrugs tested, one (ritonavir phosphate) showed no conversion in vitro and no in vivo parent exposure. Finally, the apparent K(m) values obtained for fosphenytoin and clindamycin phosphate in vitro suggest that IALP is not likely to be saturated at therapeutic doses.

Sliding principal component and dynamic reward reinforcement learning based IIoT attack detection.

The Internet of Things (IoT) involves the gathering of all those devices that connect to the Internet with the purpose of collecting and sharing data. The application of IoT in the different sectors, including health, industry has also picked up the threads to augment over the past few years. The IoT and, by integrity, the IIoT, are found to be highly susceptible to different types of threats and attacks owing to the networks nature that in turn leads to even poor outcomes (i.e., increasing error rate). Hence, it is critical to design attack detection systems that can provide the security of IIoT networks. To overcome this research work of IIoT attack detection in large amount of evolutions is failed to determine the certain attacks resulting in a minimum detection performance, reinforcement learning-based attack detection method called sliding principal component and dynamic reward reinforcement learning (SPC-DRRL) for detecting various IIoT network attacks is introduced. In the first stage of this research methodology, preprocessing of raw TON_IoT dataset is performed by employing min-max normalization scaling function to obtain normalized values with same scale. Next, with the processed sample data as output, to extract data from multi-sources (i.e., different service profiles from the dataset), a robust log likelihood sliding principal component-based feature extraction algorithm is applied with an arbitrary size sliding window to extract computationally-efficient features. Finally, dynamic reward reinforcement learning-based IIoT attack detection model is presented to control the error rate involved in the design. Here, with the design of dynamic reward function and introducing incident repository that not only generates the reward function in an arbitrary fashion but also stores the action results in the incident repository for the next training, therefore reducing the attack detection error rate. Moreover, an IIoT attack detection system based on SPC-DRRL is constructed. Finally, we verify the algorithm on the ToN_IoT dataset of University of New South Wales Australia. The experimental results show that the IIoT attack detection time and overhead along with the error rate are reduced considerably with higher accuracy than that of traditional reinforcement learning methods.

App-based oral health promotion interventions on modifiable risk factors associated with early childhood caries: A systematic review.

Background: Early childhood caries (ECC) is a preventable chronic disease. Parents' knowledge and attitudes toward oral healthcare have been associated with higher caries experience in their children. Mobile apps within the context of mHealth interventions are a potential tool for raising awareness and informing parents about their children's oral health. Objectives: The aim of this systematic review was to examine the effectiveness of mobile health apps, targeted at parents and caregivers, for the prevention of ECC. Data sources: A systematic search was carried out in five scientific databases; Embase, CINAHL, MEDLINE, PsycINFO and Web of Science. Study selection and data extraction: Original studies, delivering oral health interventions to parents of children <6 years via smartphones, were included. Both quantitative and qualitative findings from the included studies were extracted. Synthesis: A convergent segregated approach was used to integrate the quantitative and qualitative evidence, followed by side-by-side display and narrative synthesis. Results: Out of 5,953 retrieved articles, five met the inclusion criteria and were included in the review. Three articles reported quantitative findings, while two reported both quantitative and qualitative findings. Four studies reported that a mobile app can be an effective tool to improve the oral health knowledge of parents/caregivers, aiding them in incorporating good oral health habits into their children's daily routines. Conclusion: This review demonstrated that oral health promotion programs delivered through mobile apps to parents could be effective in improving child oral health awareness among parents. There is a need for more high-quality studies with a large number of participants to find out which features of mHealth interventions with parents could effectively be employed to reduce the prevalence of ECC. Further studies and apps should be developed based on evidence-based behaviour change techniques and incorporate features such as gamification to increase the effectiveness and engagement of the target population. Systematic Review Registration: [https://www.crd.york.ac.uk/prospero/display_record.php?], identifier [CRD42021268331].

Effect of P450 oxidoreductase variants on the metabolism of model substrates mediated by CYP2C9.1, CYP2C9.2, and CYP2C9.3.

OBJECTIVES: CYP2C9 is a microsomal cytochrome P450 that receives electrons from P450 oxidoreductase (POR) to metabolize about 15% of clinically used drugs. Similar to many P450 enzymes, CYP2C9 is polymorphic, with the hypomorphic *2 and *3 variants accounting for about 20% of White alleles. POR is also polymorphic, with the amino acid sequence variant A503V accounting for 19-37% of alleles in different populations. We aimed to understand how polymorphisms in these two interacting proteins might affect drug metabolism. METHODS: We assayed the activities of CYP2C9.1, CYP2C9.2, and CYP2C9.3 to metabolize diclofenac, flurbiprofen, and tolbutamide using a wild type or one of four POR variants (Q153R, A287P, R457H, and A503V). Human CYP2C9 and POR variants were expressed in bacteria, purified, and reconstituted in vitro and the Michaelis constant and maximum velocity were measured with each CYP2C9/POR combination and each substrate. RESULTS: With wild-type POR, the CYP2C9 activities were CYP2C9.1>CYP2C9.2>>CYP2C9.3 with all three substrates. Both the common A503V polymorphism and the rare Q153R variant showed modest increases in activity with all three CYP2C9 isoforms and all three substrates. This is in contrast to previous studies in which A503V showed a modest loss of function with CYP1A2, CYP2C19, CYP2D6, CYP3A4, and CYP17A1. The disease-causing POR variants A287P and R457H had a very low or unmeasurable activity with all CYP2C9 isoforms and all substrates, which is consistent with their low activities with other CYPs. CONCLUSION: POR variants affect CYP2C9 activities. The impact of a POR variant on catalysis varies with the isoform of CYP2C9 and the assay substrate.

The Relationship between Fluoride Exposure and Cognitive Outcomes from Gestation to Adulthood-A Systematic Review.

Chronic exposure to high levels of fluoride may cause health concerns, including in cognitive function. This study reviewed the evidence on the association between fluoride exposure and cognitive outcomes in children from gestation up to 18 years old. A literature search was conducted for studies on pregnant women and children below 18, exposed to any source of fluoride, and assessed with a validated cognitive tool. The data were analyzed using a systematic narrative synthesis approach and by subgroup: study design, age of participants, levels of fluoride exposure and methodological quality. Our search retrieved 15,072 articles, of which 46 met the inclusion criteria. Only 6 of the studies had a longitudinal design; the remainder were cross-sectional. The levels of fluoride exposure were ≥2 mg/L in 27 studies and <2 mg/L in 13 studies; 6 studies did not report levels of fluoride exposure. Only 1 of 5 studies graded as excellent quality showed a negative association between fluoride exposure and cognitive outcomes, whereas 30 of 34 poor and fair quality studies reported a negative association. The overall evidence from this review suggests that high fluoride exposure might be associated with negative cognitive outcomes in children. However, more longitudinal studies with high methodological quality are needed on this topic.

Improving Drug Delivery While Tailoring Prodrug Activation to Modulate Cmax and Cmin by Optimization of (Carbonyl)oxyalkyl Linker-Based Prodrugs of Atazanavir.

Structure-property relationships associated with a series of (carbonyl)oxyalkyl amino acid ester prodrugs of the marketed HIV-1 protease inhibitor atazanavir (1), designed to enhance the systemic drug delivery, were examined. Compared to previously reported prodrugs, optimized candidates delivered significantly enhanced plasma exposure and trough concentration (Cmin at 24 h) of 1 in rats while revealing differentiated PK paradigms based on the kinetics of prodrug activation and drug release. Prodrugs incorporating primary amine-containing amino acid promoieties offered the benefit of rapid bioactivation that translated into low circulating levels of the prodrug while delivering a high Cmax value of 1. Interestingly, the kinetic profile of prodrug cleavage could be tailored for slower activation by structural modification of the amino terminus to either a tertiary amine or a dipeptide motif, which conferred a circulating depot of the prodrug that orchestrated a sustained release of 1 along with substantially reduced Cmax and a further enhanced Cmin.

Identification of 2-Pyridinylindole-Based Dual Antagonists of Toll-like Receptors 7 and 8 (TLR7/8).

The toll-like receptors (TLRs) play key roles in activation of the innate immune system. Aberrant activation of TLR7 and TLR8 pathways can occur in the context of autoimmune disorders due to the elevated presence and recognition of self-RNA as activating ligands. Control of this unintended activation via inhibition of TLR7/8 signaling holds promise for the treatment of diseases such as psoriasis, arthritis, and lupus. Optimization of a 2-pyridinylindole series of compounds led to the identification of potent dual inhibitors of TLR7 and TLR8, which demonstrated good selectivity against TLR9 and other family members. The in vitro characterization and in vivo evaluation in rodent pharmacokinetic/pharmacodynamic and efficacy studies of BMS-905 is detailed, along with structural information obtained through X-ray cocrystallographic studies.

CYP2C9*1B promoter polymorphisms, in linkage with CYP2C19*2, affect phenytoin autoinduction of clearance and maintenance dose.

The commonly prescribed antiepileptic drug phenytoin has a narrow therapeutic range and wide interindividual variability in clearance explained in part by CYP2C9 and CYP2C19 coding variants. After finding a paradoxically low urinary phenytoin metabolite (S)/(R) ratio in subjects receiving phenytoin maintenance therapy with a CYP2C9*1/*1 and CYP2C19*1/*2 genotype, we hypothesized that CYP2C9 regulatory polymorphisms (rPMs), G-3089A and -2663delTG, in linkage disequilibrium with CYP2C19*2 were responsible. These rPMs explained as much as 10% of the variation in phenytoin maintenance dose in epileptic patients, but were not correlated with other patients' warfarin dose requirements or with phenytoin metabolite ratio in human liver microsomes. We hypothesized the rPMs affected CYP2C9 induction by phenytoin, a pregnane X receptor (PXR), and constitutive androstane receptor (CAR) activator. Transfection studies showed that CYP2C9 reporters with wild-type versus variant alleles had similar basal activity but significantly greater phenytoin induction by cotransfected PXR, CAR, and Nrf2 and less Yin Yang 1 transcription factor repression. Phenytoin induction of CYP2C9 was greater in human hepatocytes with the CYP2C9 wild type versus variant haplotype. Therefore, CYP2C9 rPMs affect phenytoin-dependent induction of CYP2C9 and phenytoin metabolism in humans, with an effect size comparable with that for CYP2C9*2 and 2C9*3. These findings may also be relevant to the clinical use of other PXR, CAR, and Nrf2 activators.

CYP2C9-CYP3A4 protein-protein interactions: role of the hydrophobic N terminus.

Cytochromes P450 (P450s) interact with redox transfer proteins, including P450 reductase (CPR) and cytochrome b(5) (b5), all being membrane-bound. In multiple in vitro systems, P450-P450 interactions also have been observed, resulting in alterations in enzymatic activity. The current work investigated the effects and mechanisms of interaction between CYP2C9 and CYP3A4 in a reconstituted system. CYP2C9-mediated metabolism of S-naproxen and S-flurbiprofen was inhibited up to 80% by coincubation with CYP3A4, although K(m) values were unchanged. Increasing CYP3A4 concentrations increased the degree of inhibition, whereas increasing CPR concentrations resulted in less inhibition. Addition of b5 only marginally affected the magnitude of inhibition. In contrast, CYP2C9 did not alter the CYP3A4-mediated metabolism of testosterone. The potential role of the hydrophobic N terminus on these interactions was assessed by incubating truncated CYP2C9 with full-length CYP3A4, and vice versa. In both cases, the inhibition was fully abolished, indicating an important role for hydrophobic forces in CYP2C9-CYP3A4 interactions. Finally, a CYP2C9/CYP3A4 heteromer complex was isolated by coimmunoprecipitation techniques, confirming the physical interaction of the proteins. These results show that the N-terminal membrane binding domains of CYP2C9 and CYP3A4 are involved in heteromer complex formation and that at least one consequence is a reduction in CYP2C9 activity.

(Carbonyl)oxyalkyl linker-based amino acid prodrugs of the HIV-1 protease inhibitor atazanavir that enhance oral bioavailability and plasma trough concentration.

We describe the design, synthesis and pharmacokinetic (PK) evaluation of a series of amino acid-based prodrugs of the HIV-1 protease inhibitor atazanavir (1) derivatized on the pharmacophoric secondary alcohol using a (carbonyl)oxyalkyl linker. Prodrugs of 1 incorporating simple (carbonyl)oxyalkyl-based linkers and a primary amine in the promoiety were found to exhibit low chemical stability. However, chemical stability was improved by modifying the primary amine moiety to a tertiary amine, resulting in a 2-fold enhancement of exposure in rats following oral dosing compared to dosing of the parent drug 1. Further refinement of the linker resulted in the discovery of 22 as a prodrug that delivered the parent 1 to rat plasma with a 5-fold higher AUC and 67-fold higher C24 when compared to oral administration of the parent drug. The PK profile of 22 indicated that plasma levels of this prodrug were higher than that of the parent, providing a more sustained release of 1 in vivo.

Discovery of Potent and Orally Bioavailable Small Molecule Antagonists of Toll-like Receptors 7/8/9 (TLR7/8/9).

The toll-like receptor (TLR) family is an evolutionarily conserved component of the innate immune system, responsible for the early detection of foreign or endogenous threat signals. In the context of autoimmunity, the unintended recognition of self-motifs as foreign promotes initiation or propagation of disease. Overactivation of TLR7 and TLR9 have been implicated as factors contributing to autoimmune disorders such as psoriasis, arthritis, and lupus. In our search for small molecule antagonists of TLR7/9, 7f was identified as possessing excellent on-target potency for human TLR7/9 as well as for TLR8, with selectivity against other representative TLR family members. Good pharmacokinetic properties and a relatively balanced potency against TLR7 and TLR9 in mouse systems (systems which lack functional TLR8) made this an excellent in vivo tool compound, and efficacy from oral dosing in preclinical models of autoimmune disease was demonstrated.

CYP2D6-CYP2C9 protein-protein interactions and isoform-selective effects on substrate binding and catalysis.

Cytochrome P450 (P450) protein-protein interactions have been observed with various in vitro systems. It is interesting to note that these interactions seem to be isoform-dependent, with some combinations producing no effect and others producing increased or decreased catalytic activity. With some exceptions, most of the work to date has involved P450s from rabbit, rat, and other animal species, with few studies including human P450s. In the studies presented herein, the interactions of two key drug-metabolizing enzymes, CYP2C9 and CYP2D6, were analyzed in a purified, reconstituted enzyme system for changes in both substrate-binding affinity and rates of catalysis. In addition, an extensive study was conducted as to the "order of mixing" for the reconstituted enzyme system and the impact on the observations. CYP2D6 coincubation inhibited CYP2C9-mediated (S)-flurbiprofen metabolism in a protein concentration-dependent manner. V(max) values were reduced by up to 50%, but no appreciable effect on K(m) was observed. Spectral binding studies revealed a 20-fold increase in the K(S) of CYP2C9 toward (S)-flurbiprofen in the presence of CYP2D6. CYP2C9 coincubation had no effect on CYP2D6-mediated dextromethorphan O-demethylation. The order of combination of the proteins (CYP2C9, CYP2D6, and cytochrome P450 reductase) influenced the magnitude of catalysis inhibition as well as the ability of increased cytochrome P450 reductase to attenuate the change in activity. A simple model, congruent with current results and those of others, is proposed to explain oligomer formation. In summary, CYP2C9-CYP2D6 interactions can alter catalytic activity and, thus, influence in vitro-in vivo correlation predictions.

Electrocatalytic drug metabolism by CYP2C9 bonded to a self-assembled monolayer-modified electrode.

Cytochrome P450 (P450) enzymes typically require the presence of at least cytochrome P450 reductase (CPR) and NADPH to carry out the metabolism of xenobiotics. To address whether the need for redox transfer proteins and the NADPH cofactor protein could be obviated, CYP2C9 was bonded to a gold electrode through an 11-mercaptoundecanoic acid and octanethiol self-assembled monolayer (SAM) through which a current could be applied. Cyclic voltammetry demonstrated direct electrochemistry of the CYP2C9 enzyme bonded to the electrode and fast electron transfer between the heme iron and the gold electrode. To determine whether this system could metabolize warfarin analogous to microsomal or expressed enzyme systems containing CYP2C9, warfarin was incubated with the CYP2C9-SAM-gold electrode and a controlled potential was applied. The expected 7-hydroxywarfarin metabolite was observed, analogous to expressed CYP2C9 systems, wherein this is the predominant metabolite. Current-concentration data generated with increasing concentrations of warfarin were used to determine the Michaelis-Menten constant (K(m)) for the hydroxylation of warfarin (3 microM), which is in good agreement with previous literature regarding K(m) values for this reaction. In summary, the CYP2C9-SAM-gold electrode system was able to carry out the metabolism of warfarin only after application of an electrical potential, but in the absence of either CPR or NADPH. Furthermore, this system may provide a unique platform for both studying P450 enzyme electrochemistry and as a bioreactor to produce metabolites without the need for expensive redox transfer proteins and cofactors.