Quantitative prediction of CYP3A-mediated drug-drug interactions by correctly estimating fraction metabolized using human liver chimeric mice.

BACKGROUND AND PURPOSE: Fraction metabolized (fm ) and fraction transported (ft ) are important for understanding drug-drug interactions (DDIs) in drug discovery and development. However, current in vitro systems cannot accurately estimate in vivo fm due to inability to reflect the ft by efflux transporters (ft,efflux ). This study demonstrates how CYP3A-mediated DDI for CYP3A/P-gp substrates can be predicted using Hu-PXB mice as human liver chimeric mice. EXPERIMENTAL APPROACH: For estimating human in vitro fm by CYP3A enzyme (fm,CYP3A,in vitro ), six drugs, including CYP3A/P-gp substrates (alprazolam, cyclosporine, docetaxel, midazolam, prednisolone, and theophylline) and human hepatocytes were incubated with or without ketoconazole as a CYP3A inhibitor. We calculated fm,CYP3A,in vitro based on hepatic intrinsic clearance. To estimate human in vivo fm,CYP3A (fm,CYP3A,in vivo ), we collected information on clinical DDI caused by ketoconazole for these six drugs. We calculated fm,CYP3A,in vivo using the change of total clearance (CLtotal ). For evaluating the human DDI predictability, the six drugs were administered intravenously to Hu-PXB and SCID mice with or without ketoconazole. We calculated the change of CLtotal caused by ketoconazole. We compared the CLtotal change in humans with that in Hu-PXB and SCID mice. KEY RESULTS: The fm,CYP3A,in vitro was overestimated compared to the fm,CYP3A,in vivo . Hu-PXB mice showed much better correlation in the change of CLtotal with humans (R2 = 0.95) compared to SCID mice (R2 = 0.0058). CONCLUSIONS AND IMPLICATIONS: CYP3A-mediated DDI can be predicted by correctly estimating human fm,CYP3A,in vivo using Hu-PXB mice. These mice could be useful predicting hepatic fm and ft,efflux .

Enhancing drug-drug Interaction Prediction by Integrating Physiologically-Based Pharmacokinetic Model with Fraction Metabolized by CYP3A4.

BACKGROUND: Enhancing the precision of drug-drug interaction (DDI) prediction is essential for improving drug safety and efficacy. The aim is to identify the most effective fraction metabolized by CY3A4 (fm) for improving DDI prediction using physiologically based pharmacokinetic (PBPK) models. RESEARCH DESIGN AND METHODS: The fm values were determined for 33 approved drugs using a human liver microsome for in vitro measurements and the ADMET Predictor software for in silico predictions. Subsequently, these fm values were integrated into PBPK models using the GastroPlus platform. The PBPK models, combined with a ketoconazole model, were utilized to predict AUCR (AUCcombo with ketoconazole/AUCdosing alone), and the accuracy of these predictions was evaluated by comparison with observed AUCR. RESULTS: The integration of in vitro fm method demonstrates superior performance compared to the in silico fm method and fm of 100% method. Under the Guest-limits criteria, the integration of in vitro fm achieves an accuracy of 76%, while the in silico fm and fm of 100% methods achieve accuracies of 67% and 58%, respectively. CONCLUSIONS: Our study highlights the importance of in vitro fm data to improve the accuracy of predicting DDIs and demonstrates the promising potential of in silico fm in predicting DDIs.

In Vitro and In Vivo Metabolic Activation of Tolterodine Mediated by CYP3A.

Tolterodine (TOL) is an antimuscarinic drug used for the treatment of patients with overactive bladder presenting urinary frequency, urgency, and urge incontinence. During the clinical use of TOL, adverse events such as liver injury took place. The present study aimed at the investigation of the metabolic activation of TOL possibly associated with its hepatotoxicity. One GSH conjugate, two NAC conjugates, and two cysteine conjugates were found in both mouse and human liver microsomal incubations supplemented with TOL, GSH/NAC/cysteine, and NADPH. The detected conjugates suggest the production of a quinone methide intermediate. The same GSH conjugate was also observed in mouse primary hepatocytes and in the bile of rats receiving TOL. One of the urinary NAC conjugates was observed in rats administered TOL. One of the cysteine conjugates was found in a digestion mixture containing hepatic proteins from animals administered TOL. The observed protein modification was dose-dependent. CYP3A primarily catalyzes the metabolic activation of TOL. Ketoconazole (KTC) pretreatment reduced the generation of the GSH conjugate in mouse liver and cultured primary hepatocytes after TOL treatment. In addition, KTC reduced the susceptibility of primary hepatocytes to TOL cytotoxicity. The quinone methide metabolite may be involved in TOL-induced hepatotoxicity and cytotoxicity.

Relationships between Isomeric Metabolism and Regioselective Toxicity of Hydroxychrysenes in Embryos of Japanese Medaka (Oryzias latipes).

Oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) are ubiquitous contaminants that can be formed through oxidation of parent PAHs. Our previous studies found 2-hydroxychrysene (2-OHCHR) to be significantly more toxic to Japanese medaka embryos than 6-hydroxychrysene (6-OHCHR), an example of regioselective toxicity. We have also previously identified a sensitive developmental window to 2-OHCHR toxicity that closely coincided with liver development, leading us to hypothesize that differences in metabolism may play a role in the regioselective toxicity. To test this hypothesis, Japanese medaka embryos were treated with each isomer for 24 h during liver development (52-76 hpf). Although 6-OHCHR was absorbed 97.2 ± 0.18% faster than 2-OHCHR, it was eliminated 57.7 ± 0.36% faster as a glucuronide conjugate. Pretreatment with cytochrome P450 inhibitor, ketoconazole, reduced anemia by 96.8 ± 3.19% and mortality by 95.2 ± 4.76% in 2-OHCHR treatments. Formation of chrysene-1,2-diol (1,2-CAT) was also reduced by 64.4 ± 2.14% by ketoconazole pretreatment. While pretreatment with UDP-glucuronosyltransferase inhibitor, nilotinib, reduced glucuronidation of 2-OHCHR by 52.4 ± 2.55% and of 6-OHCHR by 63.7 ± 3.19%, it did not alter toxicity for either compound. These results indicate that CYP-mediated activation, potentially to 1,2-CAT, may explain the isomeric differences in developmental toxicity of 2-OHCHR.

Identification of a window of androgen sensitivity for somatic cell function in human fetal testis cultured ex vivo.

BACKGROUND: Reduced androgen action during early fetal development has been suggested as the origin of reproductive disorders comprised within the testicular dysgenesis syndrome (TDS). This hypothesis has been supported by studies in rats demonstrating that normal male development and adult reproductive function depend on sufficient androgen exposure during a sensitive fetal period, called the masculinization programming window (MPW). The main aim of this study was therefore to examine the effects of manipulating androgen production during different timepoints during early human fetal testis development to identify the existence and timing of a possible window of androgen sensitivity resembling the MPW in rats. METHODS: The effects of experimentally reduced androgen exposure during different periods of human fetal testis development and function were examined using an established and validated human ex vivo tissue culture model. The androgen production was reduced by treatment with ketoconazole and validated by treatment with flutamide which blocks the androgen receptor. Testicular hormone production ex vivo was measured by liquid chromatography-tandem mass spectrometry or ELISA assays, and selected protein markers were assessed by immunohistochemistry. RESULTS: Ketoconazole reduced androgen production in testes from gestational weeks (GW) 7-21, which were subsequently divided into four age groups: GW 7-10, 10-12, 12-16 and 16-21. Additionally, reduced secretion of testicular hormones INSL3, AMH and Inhibin B was observed, but only in the age groups GW 7-10 and 10-12, while a decrease in the total density of germ cells and OCT4+ gonocytes was found in the GW 7-10 age group. Flutamide treatment in specimens aged GW 7-12 did not alter androgen production, but the secretion of INSL3, AMH and Inhibin B was reduced, and a reduced number of pre-spermatogonia was observed. CONCLUSIONS: This study showed that reduced androgen action during early development affects the function and density of several cell types in the human fetal testis, with similar effects observed after ketoconazole and flutamide treatment. The effects were only observed within the GW 7-14 period-thereby indicating the presence of a window of androgen sensitivity in the human fetal testis.

Evidence for Metabolic Activation of Omeprazole In Vitro and In Vivo.

Omeprazole (OPZ) is a proton pump inhibitor commonly used for the treatment of gastric acid hypersecretion. Studies have revealed that use of OPZ can induce hepatotoxicity, but the mechanisms by which it induces liver injury are unclear. This study aimed to identify reactive metabolites of OPZ, determine the pathways of the metabolic activation, and define the correlation of the bioactivation with OPZ cytotoxicity. Quinone imine-derived glutathione (GSH), N-acetylcysteine (NAC), and cysteine (Cys) conjugates were detected in OPZ-fortified rat and human liver microsomal incubations captured with GSH, NAC, or Cys. The same GSH conjugates were detected in bile of rats and cultured liver primary cells after exposure to OPZ. Similarly, the same NAC conjugates were detected in urine of OPZ-treated rats. The resulting quinone imine was found to react with Cys residues of hepatic protein. CYP3A4 dominated the metabolic activation of OPZ. Exposure to OPZ resulted in decreased cell survival in cultured primary hepatocytes. Pretreatment with ketoconazole attenuated the susceptibility of hepatocytes to the cytotoxicity of OPZ.

Permeabilized Cryopreserved Human Hepatocytes as an Exogenous Metabolic System in a Novel Metabolism-Dependent Cytotoxicity Assay for the Evaluation of Metabolic Activation and Detoxification of Drugs Associated with Drug-Induced Liver Injuries: Results with Acetaminophen, Amiodarone, Cyclophosphamide, Ketoconazole, Nefazodone, and Troglitazone.

We report here a novel in vitro experimental system, the metabolism-dependent cytotoxicity assay (MDCA), for the definition of the roles of hepatic drug metabolism in toxicity. MDCA employs permeabilized cofactor-supplemented cryopreserved human hepatocytes (MetMax Human Hepatocytes, MMHH), as an exogenous metabolic activating system, and human embryonic kidney 293 (HEK293) cells, a cell line devoid of drug-metabolizing enzyme activity, as target cells for the quantification of drug toxicity. The assay was performed in the presence and absence of cofactors for key drug metabolism pathways known to play key roles in drug toxicity: NADPH/NAD+ for phase 1 oxidation, uridine 5'-diphosphoglucuronic acid (UDPGA) for uridine 5'-diphospho-glucuronosyltransferase (UGT) mediated glucuronidation, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) for cytosolic sulfotransferase (SULT) mediated sulfation, and glutathione (GSH) for glutathione S-transferase (GST) mediated GSH conjugation. Six drugs with clinically significant hepatoxicity, resulting in liver failure or a need for liver transplantation: acetaminophen, amiodarone, cyclophosphamide, ketoconazole, nefazodone, and troglitazone were evaluated. All six drugs exhibited cytotoxicity enhancement by NADPH/NAD+, suggesting metabolic activation via phase 1 oxidation. Attenuation of cytotoxicity by UDPGA was observed for acetaminophen, ketoconazole, and troglitazone, by PAPS for acetaminophen, ketoconazole, and troglitazone, and by GSH for all six drugs. Our results suggest that MDCA can be applied toward the elucidation of metabolic activation and detoxification pathways, providing information that can be applied in drug development to guide structure optimization to reduce toxicity and to aid the assessment of metabolism-based risk factors for drug toxicity. GSH detoxification represents an endpoint for the identification of drugs forming cytotoxic reactive metabolites, a key property of drugs with idiosyncratic hepatotoxicity. SIGNIFICANCE STATEMENT: Application of the metabolism-dependent cytotoxicity assay (MDCA) for the elucidation of the roles of metabolic activation and detoxification pathways in drug toxicity may provide information to guide structure optimization in drug development to reduce hepatotoxic potential and to aid the assessment of metabolism-based risk factors. Glutathione (GSH) detoxification represents an endpoint for the identification of drugs forming cytotoxic reactive metabolites that may be applied toward the evaluation of idiosyncratic hepatotoxicity.

Arylacetamide deacetylase knockout mice are sensitive to ketoconazole-induced hepatotoxicity and adrenal insufficiency.

Orally administered ketoconazole may rarely induce liver injury and adrenal insufficiency. A metabolite formed by arylacetamide deacetylase (AADAC)-mediated hydrolysis has been observed in cellulo studies, and it is relevant to ketoconazole-induced cytotoxicity. This study tried to examine the significance of AADAC in ketoconazole-induced toxicity in vivo using Aadac knockout mice. Oral administration of 150 mg/kg ketoconazole resulted in the area under the plasma concentration-time curve values of ketoconazole and N-deacetylketoconazole, a hydrolyzed metabolite of ketoconazole, in Aadac knockout mice being significantly higher and lower than those in wild-type mice, respectively. With the administration of ketoconazole (300 mg/kg/day) for 7 days, Aadac knockout mice showed higher mortality (100%) than wild-type mice (42.9%), and they also showed significantly higher plasma alanine transaminase and lower corticosterone levels, thus representing liver injury and steroidogenesis inhibition, respectively. It was suggested that a higher plasma ketoconazole concentration likely accounts for the inhibition of the synthesis of corticosterone, which has anti-inflammatory effects, in the adrenal gland in Aadac KO mice. In Aadac knockout mice, hepatic mRNA levels of immune- and inflammation-related factors were increased by the administration of 300 mg/kg ketoconazole, and the increase was restored by the replenishment of corticosterone (40 mg/kg, s.c.) along with recoveries of plasma alanine transaminase levels. In conclusion, Aadac defects exacerbate ketoconazole-induced liver injury by inhibiting glucocorticoid synthesis and enhancing the inflammatory response. This in vivo study revealed that the hydrolysis of ketoconazole by AADAC can mitigate ketoconazole-induced toxicities.

In Vitro Evaluation of P-gp-Mediated Drug-Drug Interactions Using the RPTEC/TERT1 Human Renal Cell Model.

BACKGROUND AND OBJECTIVES: In vitro evaluation of the P-glycoprotein (P-gp) inhibitory potential is an important issue when predicting clinically relevant drug-drug interactions (DDIs). Located within all physiological barriers, including intestine, liver, and kidneys, P-gp plays a major role in the pharmacokinetics of various therapeutic classes. However, few data are available about DDIs involving renal transporters during the active tubular secretion of drugs. In this context, the present study was designed to investigate the application of the human renal cell line RPTEC/TERT1 to study drug interactions mediated by P-gp. METHODS: The P-gp inhibitory potentials of a panel of drugs were first determined by measuring the intracellular accumulation of rhodamine 123 in RPTEC/TERT1 cells. Then four drugs were selected to assess the half-maximal inhibitor concentration (IC50) values by measuring the intracellular accumulation of two P-gp-substrate drugs, apixaban and rivaroxaban. Finally, according to the FDA guidelines, the [I1]/IC50 ratio was calculated for each combination of drugs to assess the clinical relevance of the DDIs. RESULTS: The data showed that drugs which are known P-gp inhibitors, including cyclosporin A, ketoconazole, and verapamil, caused great increases in rhodamine 123 retention, whereas noninhibitors did not affect the intracellular accumulation of the P-gp substrate. The determined IC50 values were in accordance with the inhibition profiles observed in the rhodamine 123 accumulation assays, confirming the reliability of the RPTEC/TERT1 model. CONCLUSIONS: Taken together, the data demonstrate the feasibility of the application of the RPTEC/TERT1 model for evaluating the P-gp inhibitory potentials of drugs and consequently predicting renal drug interactions.

Analyzing Potential Intestinal Transporter Drug-Drug Interactions: Reevaluating Ticagrelor Interaction Studies.

PURPOSE: Previous studies evaluating ticagrelor drug-drug interactions have not differentiated intestinal versus systemic mechanisms, which we do here. METHODS: Using recently published methodologies from our laboratory to differentiate metabolic- from transporter-mediated drug-drug interactions, a critical evaluation of five published ticagrelor drug-drug interactions was carried out to investigate the purported clinical significance of enzymes and transporters in ticagrelor disposition. RESULTS: The suggested CYP3A4 inhibitors, ketoconazole and diltiazem, displayed unchanged mean absorption time (MAT) and time of maximum concentration (Tmax) values as was expected, i.e., the interactions were mainly mediated by metabolic enzymes. The potential CYP3A4/P-gp inhibitor cyclosporine also showed an unchanged MAT value. Further analysis assuming there was no P-gp effect suggested that the increased AUC and unchanged t1/2 for ticagrelor after cyclosporine administration were attributed to the inhibition of intestinal CYP3A4 rather than P-gp. Rifampin, an inducer of CYP3As after multiple dosing, unexpectedly showed decreased MAT and Tmax values, which cannot be completely explained. In contrast, grapefruit juice, an intestinal CYP3A/P-gp/OATP inhibitor, significantly increased MAT and Tmax values for ticagrelor, which may be due to activation of P-gp or inhibition of OATPs expressed in intestine. CONCLUSIONS: This study provides new insight into the role of transporter pathways in ticagrelor intestinal absorption by examining potential MAT and Tmax changes mediated by drug-drug interactions.

Effects of nanoplastic on toxicity of azole fungicides (ketoconazole and fluconazole) in zebrafish embryos.

The ubiquity of nanoplastics (NPs) raises concerns about their interactions and combined toxicity with other common contaminants. Although azoles are present throughout the natural environment, their interactions with NP are not well known. We investigated the effects of polystyrene (PS) NP on the toxicity of ketoconazole (KCZ) and fluconazole (FCZ) in zebrafish embryos using the developmental toxicity, oxidative-stress-related biochemical parameters, and expression of genes related to neurotoxicity (ache), cardiotoxicity (gata4, bmp4), inflammation (il1b), oxidative stress (sod1, sod2, cyp1a), and apoptosis (bax, bcl2). Co-exposure to NP (1 mg/L) and KCZ/FCZ (1 mg/L) for 96 h reduced the hatching rate, survival rate, and heart rate and increased the malformation rate and catalase activity. The bax/bcl2 ratio, an apoptosis indicator, was higher after NP, KCZ, or FCZ treatment. However, the bax/bcl2 ratio after exposure to NP + KCZ or NP + FCZ was much higher than that after single exposure. Overall, the results indicated that NP aggravated the toxicity of azole by significantly increasing the reactive oxygen species, lipid peroxidation and altering the expression of oxidative-stress- and apoptosis-related genes. The interactive toxicity of PS NP with KCZ/FCZ reported in this study emphasises the need for caution in the release of azole fungicides in the environment.

Ketoconazole-p aminobenzoic cocrystal, an improved antimycotic drug formulation, does not induce skin sensitization on the skin of BALBc mice.

Fungal infections are a growing global health problem. Therefore, our group has synthetized and characterized an improved antimycotic by co-crystallization of ketoconazole and para-amino benzoic acid, named KET-PABA. The aim was to increase bioavailability, biocompatibility, and efficiency of the parent drug-ketoconazole. Based on our previous results showing the cocrystal improved physical properties, such as stability in suspension, solubility, as well as antimycotic efficiency compared to ketoconazole, the current study investigated the local possible side effects induced on the skin of BALBc mice by the application of KET-PABA cocrystal, in view of a further use as a topically applied antimycotic drug. A specific test (mouse ear-swelling test) was used, combined with the histopathological examination and the measurement of pro and anti-inflammatory cytokines and inflammation mediators. KET-PABA application was safe, without signs of skin sensitization shown by the mouse ear sensitization test, or histopathology. KET-PABA strongly inhibited proinflammatory cytokines such as IL1 α, IL1 ß, IL6 and TNF α, and other proinflammatory inducers such as NRF2, compared to vehicle. KET-PABA had no effect on the levels of the anti-inflammatory cytokine IL10, or proinflammatory enzyme COX2 and had minimal effects on the activation of the NF-κB pathway. Overall, KET-PABA application induced no sensitization, moreover, it decreased the skin levels of proinflammatory molecules. The lack of skin sensitization effects on BALBc mice skin along with the inhibition of the proinflammatory markers show a good safety profile for topical applications of KET-PABA and show promise for a further clinical use in the treatment of cutaneous mycosis.

Tertiary Oxidation of Deoxycholate Is Predictive of CYP3A Activity in Dogs.

Deoxycholic acid (DCA, 3α, 12α-dihydroxy-5ß-cholan-24-oic acid) is the major circulating secondary bile acid, which is synthesized by gut flora in the lower gut and selectively oxidized by CYP3A into tertiary metabolites, including 1ß,3α,12α-trihydroxy-5ß-cholan-24-oic acid (DCA-1ß-ol) and 3α,5ß,12α-trihydroxy-5ß-cholan-24-oic acid (DCA-5ß-ol) in humans. Since DCA has the similar exogenous nature and disposition mechanisms as xenobiotics, this work aimed to investigate whether the tertiary oxidations of DCA are predictive of in vivo CYP3A activities in beagle dogs. In vitro metabolism of midazolam (MDZ) and DCA in recombinant canine CYP1A1, 1A2, 2B11, 2C21, 2C41, 2D15, 3A12, and 3A26 enzymes clarified that CYP3A12 was primarily responsible for either the oxidation elimination of MDZ or the regioselective oxidation metabolism of DCA into DCA-1ß-ol and DCA-5ß-ol in dog liver microsomes. Six male dogs completed the CYP3A intervention studies including phases of baseline, inhibition (ketoconazole treatments), recovery, and induction (rifampicin treatments). The oral MDZ clearance after a single dose was determined on the last day of the baseline, inhibition, and induction phases, and subjected to correlation analysis with the tertiary oxidation ratios of DCA detected in serum and urine samples. The results confirmed that the predosing serum ratios of DCA oxidation, DCA-5ß-ol/DCA, and DCA-1ß-ol/DCA were significantly and positively correlated both intraindividually and interindividually with oral MDZ clearance. It was therefore concluded that the tertiary oxidation of DCA is predictive of CYP3A activity in beagle dogs. Clinical transitional studies following the preclinical evidence are promising to provide novel biomarkers of the enterohepatic CYP3A activities. SIGNIFICANCE STATEMENT: Drug development, clinical pharmacology, and therapeutics are under insistent demands of endogenous CYP3A biomarkers that avoid unnecessary drug exposure and invasive sampling. This work has provided the first proof-of-concept preclinical evidence that the CYP3A catalyzed tertiary oxidation of deoxycholate, the major circulating secondary bile acid synthesized in the lower gut by bacteria, may be developed as novel in vivo biomarkers of the enterohepatic CYP3A activities.

Development and Validation of Sample Preparation and an HPLC Analytical Method for Dissolution Testing in Fed-State Simulated Gastric Fluid-Illustrating Its Application for Ibuprofen and Ketoconazole Immediate Release Tablets.

Dissolution testing and solubility determinations in different biorelevant media have gained considerable interest in the pharmaceutical industry from early-stage development of new products to forecasting bioequivalence. Among all biorelevant fluids, the preparation of fed-state simulated gastric fluid (FeSSGF) and handling of samples from dissolution/solubility testing in FeSSGF is considered to be relatively challenging. Challenges include maintaining the stability of FeSSGF medium upon sampling, filtration, and mitigating analytical interference of excipients and milk components. To overcome these challenges, standard and uniform working practices are required that are not only helpful in preparation of stable FeSSGF but also serve as a harmonizing guide for the collection of dissolution/solubility samples and their subsequent processing (i.e., handling and assay). The optimization of sample preparation methodology is crucial to reduce method-related variance by ensuring specificity, robustness, and reproducibility with acceptable recovery of the analytes. The sample preparation methodology includes a combination of techniques including filtration, solvent treatment, and centrifugation to remove the interfering media-related components and excipients from the analyte. The analytes of interest were chromatographically separated from the interfering analytes to quantify the drug concentration using the new high-performance liquid chromatography methods with ultraviolet detection. The methods developed allow rapid sample preparation, acceptable specificity, reproducible recoveries (greater than 95% of label claim), and quantification of study drugs (ibuprofen and ketoconazole). The sample preparation technique and method considerations provided here for ibuprofen and ketoconazole can serve as a starting point for solubility and dissolution testing of other small molecules in FeSSGF.

Topical application of nanoparticles integrated supramolecular hydrogels for the potential treatment of seborrhoeic dermatitis.

The current application was aimed to evaluate the therapeutic potential of selenium and ketoconazole nanoparticles loaded hyaluronic acid gel against seborrhoeic dermatitis (SD). Amalgamation of ketoconazole (antifungal medication) and selenium (pro-oxidant) in an optimized formulation setting may help in the treatment of SD. In this study, selenium and ketoconazole nanoparticles loaded hyaluronic acid (HA) hydrogel was prepared by mechanical mixing followed by sonication. Results of the optimized batch showed a mean particle size of 121 ± 12 nm for ketoconazole and 51 ± 7 nm for selenium. SEM and TEM study revealed the prepared nanoparticles are of nanoscale dimension, with smooth spherical outline. Finally, the optimized nanoparticles were incorporated into HA hydrogel. Hydrogel exhibits desirable physical, mechanical and rheological characteristics appropriate for topical application. Optimized gel formulation exhibited an enhanced permeability with better antifungal, and anti-inflammatory activities, compared with the plain drug suspension. The optimized hydrogel with ketoconazole and selenium in nanotemplate could offer a potential strategy for the treatment of SD.

Nano-elastic liposomes as multidrug carrier of sodium stibogluconate and ketoconazole: A potential new approach for the topical treatment of cutaneous Leishmaniasis.

The present study evaluates the efficacy of sodium stibogluconate (SSG) co-loaded with ketoconazole (KTZ) in nano-elastic liposomes (NELs) for the topical treatment of cutaneous leishmaniasis (CL). SSG-KTZ co-loaded NELs were developed and assessed for various physicochemical properties and anti-leishmanial potential. The optimized nano-vesicles have an average size of 212.8 ± 3.1 nm and entrapment efficiency of 61.2 ± 2.9%. SSG-KTZ co-loaded NELs displayed 5.37-fold higher skin permeation of SSG as compared to drug solution. SSG and KTZ displayed a synergistic interaction and flow cytometry revealed enhanced killing of DsRed Leishmania mexicana in infected macrophages. In-vitro and in-vivo anti-leishmanial studies indicated a 10.67-fold lower IC50 value and a 35.33-fold reduced parasitic burden as compared with plain SSG solution, respectively. SSG-KTZ co-loaded NELs were found to be a promising approach for the topical treatment of CL.

Piperine Is a Mechanism-Based Inactivator of CYP3A.

Piperine (PPR) is the representative alkaloid component of the piper species (family: Piperaceae). Our rapid screening study found PPR caused time-dependent inhibition of cytochrome P450s (CYP) 3A and 2D6, and CYP3A was inactivated the most. Further study demonstrated that PPR is a time-, concentration-, and NADPH-dependent inhibitor of CYP3A, and significant loss (49.5% ± 3.9%) of CYP3A activity was observed after 20minute incubations with 80 µM PPR at 37°C. The values of K I and k inact were 30.7 µM and 0.041 minutes-1, respectively. CYP3A competitive inhibitor ketoconazole showed protective effect against the enzyme inactivation. Superoxide dismutase/catalase and GSH displayed minor protection against the PPR-caused enzyme inactivation. Ferricyanide partially reduced the enzyme inhibition by PPR. Additionally, NADPH-dependent formation of reactive metabolites from PPR were found in human liver microsomal incubation mixtures. An ortho-quinone intermediate was trapped by NAC in microsomal incubations with PPR. DM-PPR, demethylene metabolite of PPR, showed weak enzyme inactivation relative to that caused by PPR. It appears that both carbene and ortho-quinone intermediates were involved in the inactivation of CYP3A caused by PPR. SIGNIFICANCE STATEMENT: CYP3A subfamily members (mainly CYP3A4 and CYP3A5) play a critical role in drug metabolism. Piperine (PPR), a methylenedioxyphenyl derivative combined with an unsaturated ketone, is the major active ingredient of pepper. PPR revealed time-, concentration-, and NADPH-dependent inhibitory effect on CYP3A. Carbene and quinone metabolites were both involved in the observed CYP3A inactivation by PPR. Apparently, the unsaturated ketone moiety did not participate in the enzyme inactivation. The present study sounds an alert of potential risk for food-drug interactions.

Conformational Response of N-Terminally Truncated Cytochrome P450 3A4 to Ligand Binding in Solution.

Human cytochrome P450 3A4 (CYP3A4) is a membrane-associated monooxygenase that is responsible for metabolizing >50% of the pharmaceuticals in the current market, so studying its chemical mechanism and structural changes upon ligand binding will help provide deeper insights into drug metabolism and further drug development. The best-characterized cytochrome P450 is a bacterial form, P450cam, which undergoes significant conformational changes upon binding substrate and its redox partner, putidaredoxin. In contrast, most crystal structures of CYP3A4 with or without ligands have shown few changes, although allosteric effects and multiple-substrate binding in solution are well-documented. In this study, we use double electron-electron resonance (DEER) to measure distances between spatially separated spin-labels on CYP3A4 and molecular dynamics to interpret the DEER data. These methods were applied to a soluble N-terminally truncated CYP3A4 form, and the results show that there are few changes in the average structure upon binding ketoconazole, ritonavir, or midazolam. However, binding of midazolam, but not ketoconazole or ritonavir, resulted in a significant change in the motion and/or disorder in the F/G helix region near the substrate binding pocket. These results suggest that soluble CYP3A4 behaves in a unique way in response to inhibitor and substrate binding.

Characterization of Genetic Variation in CYP3A4 on the Metabolism of Cabozantinib in Vitro.

Cabozantinib is a multityrosine kinase inhibitor and has a wide range of applications in the clinic, whose metabolism is predominately dependent on CYP3A4. This study was performed to characterize the enzymatic properties of 29 CYP3A4 alleles toward cabozantinib and the functional changes of five selected alleles (the wild-type, CYP3A4.2.8.14 and .15) toward cabozantinib in the presence of ketoconazole. Cabozantinib, 1-100 µM, with/without the presence of ketoconazole and CYP3A4 enzymes in the incubation system went through 30 min incubation at 37 °C, and the concentrations of cabozantinib N-oxide were quantified by UPLC-MS/MS to calculate the corresponding kinetic parameters of each variant. Collectively, without the presence of ketoconazole, most variants displayed defective enzymatic activities in different degrees, and only CYP3A4.14 and .15 showed significantly augmented enzymatic activities. With the presence of ketoconazole, five tested CYP3A4 alleles, even CYP3A4.14 and .15, exhibited obvious reductions in intrinsic clearance. Besides, we compared cabozantinib with regorafenib in relative clearance to confirm that CYP3A4 has the property of substrate specificity. As the first study of CYP3A4 genetic polymorphisms toward cabozantinib, our observations can provide prediction of an individual's capability in response to cabozantinib and guidance for medication and treatment of cabozantinib.

Drug-drug interaction (DDI) assessments of ruxolitinib, a dual substrate of CYP3A4 and CYP2C9, using a verified physiologically based pharmacokinetic (PBPK) model to support regulatory submissions.

Ruxolitinib is mainly metabolized by cytochrome P450 (CYP) enzymes CYP3A4 and CYP2C9 followed by minor contributions of other hepatic CYP enzymes in vitro. A physiologically based pharmacokinetic (PBPK) model was established to evaluate the changes in the ruxolitinib systemic exposures with co-administration of CYP3A4 and CYP2C9 perpetrators. The fractions metabolized in the liver via oxidation by CYP enzymes (fm,CYP3A4 = 0.75, fm,CYP2C9 = 0.19, and fm,CYPothers = 0.06) for an initial ruxolitinib model based on in vitro data were optimized (0.43, 0.56, and 0.01, respectively) using the observed exposure changes of ruxolitinib (10 mg) with co-administered ketoconazole (200 mg). The reduced amount of fm,CYP3A4 was distributed to fm,CYP2C9. For the initial ruxolitinib model with co-administration of ketoconazole, the area under the curve (AUC) increase of 2.60-fold was over-estimated compared with the respective observation (1.91-fold). With the optimized fm values, the predicted AUC ratio was 1.82. The estimated AUC ratios of ruxolitinib by co-administration of the moderate CYP3A4 inhibitor erythromycin (500 mg) and the strong CYP3A4 inducer rifampicin (600 mg) were within a 20% error compared with the clinically observed values. The PBPK modeling results may provide information on the labeling, i.e. supporting a dose reduction by half for co-administration of strong CYP3A4 inhibitors. Furthermore, an AUC increase of ruxolitinib in the absence or presence of the dual CYP3A4 and CYP2C9 inhibitor fluconazole (100-400 mg) was prospectively estimated to be 1.94- to 4.31-fold. Fluconazole simulation results were used as a basis for ruxolitinib dose adjustment when co-administering perpetrator drugs. A ruxolitinib PBPK model with optimized fm,CYP3A4 and fm,CYP2C9 was established to evaluate victim DDI risks. The previous minimal PBPK model was supported by the FDA for the dose reduction strategy, halving the dose with the concomitant use of strong CYP3A4 inhibitors and dual inhibitors on CYP2C9 and CYP3A4, such as fluconazole at ≤200 mg. Fluconazole simulation results were used as supportive evidence in discussions with the FDA and EMA about ruxolitinib dose adjustment when co-administering perpetrator drugs. Thus, this study demonstrated that PBPK modeling can support characterizing DDI liabilities to inform the drug label and might help reduce the number of clinical DDI studies by simulations of untested scenarios, when a robust PBPK model is established.