Drug Metabolism and Disposition最新文献

Compromised hepatic drug metabolism in response to proinflammatory cytokine release is primarily attributed to downregulation of cytochrome P450 (CYP) enzymes. However, whether inflammation also affects other phase I and phase II drug metabolizing enzymes (DMEs), such as the flavin monooxygenases (FMOs), carboxylesterases (CESs), and UDP glucuronosyltransferases (UGTs), remains unclear. This study aimed to decipher the impact of physiologically relevant concentrations of proinflammatory cytokines on expression and activity of phase I and phase II enzymes, to establish a hierarchy of their sensitivity as compared with the CYPs. Hereto, HepaRG cells were exposed to interleukin-6 and interleukin-1β to measure alterations in DME gene expression (24 h) and activity (72 h). Sensitivity of DMEs toward proinflammatory cytokines was evaluated by determining IC₅₀ (potency) and I_max (maximal inhibition) values from the concentration-response curves. Proinflammatory cytokine treatment led to nearly complete downregulation of CYP3A4 (∼98%) but was generally less efficacious at reducing gene expression of the non-CYP DME families. Importantly, FMO, CES, and UGT family members were less sensitive toward interleukin-6 induced inhibition in terms of potency, with IC₅₀ values that were 4.3- to 7.4-fold higher than CYP3A4. Similarly, 18- to 31-fold more interleukin-1β was required to achieve 50% of the maximal downregulation of FMO3, FMO4, CES1, UGT2B4, and UGT2B7 expression. The differential sensitivity persisted at enzyme activity level, highlighting that alterations in DME gene expression during inflammation are predictive for subsequent alterations in enzyme activity. In conclusion, this study has shown that FMOs, CESs, and UGTs enzymes are less impacted by IL-6 and IL-1β treatment as compared with CYP enzymes. SIGNIFICANCE STATEMENT: While the impact of proinflammatory cytokines on CYP expression is well established, their effects on non-CYP phase I and phase II drug metabolism remains underexplored, particularly regarding alterations in drug metabolizing enzyme (DME) activity. This study provides a quantitative understanding of the sensitivity differences to inflammation between DME family members, suggesting that non-CYP DMEs may become more important for the metabolism of drugs during inflammatory conditions due to their lower sensitivity as compared with the CYPs.

Evaluation of the CYP3A induction risk is important in early drug development stages. This study focused on 4β-hydroxycholesterol (4β-HC) as an endogenous biomarker of drug-drug interactions (DDIs) caused by CYP3A induction. We investigated a new approach using 4β-HC for quantitative prediction of DDIs caused by CYP3A induction based on the mechanistic static pharmacokinetic (MSPK) model. The induction ratio, i.e., the ratio of plasma 4β-HC or 4β-HC/cholesterol (4β-HC/C) with and without a coadministered CYP3A inducer, and the ratio of the area under the plasma concentration-time curve (AUCR), i.e., the ratio of the AUC of plasma CYP3A substrate drugs with and without a coadministered CYP3A inducer, were collected. The scaling factor (d) in the MSPK model was calculated from the induction ratio of 4β-HC or 4β-HC/C based on the systemic term in the MSPK model. The AUCR of 18 CYP3A substrates with and without coadministration of seven CYP3A inducers were then predicted by substituting the calculated d value into the MSPK model. This approach showed that approximately 84% of the predicted AUCR values were within a twofold range of the observed values, showing that this approach can be a good tool to quantitatively predict DDIs caused by CYP3A induction. SIGNIFICANCE STATEMENT: A concise approach to predict drug interactions with adequate accuracy is preferable in the early drug development stage. In this study, a new approach using 4β-hydroxycholesterol for quantitative prediction of drug-drug interactions caused by CYP3A induction was investigated. The predictability was verified using seven CYP3A inducers and 18 substrates.

Eteplirsen, golodirsen, and casimersen are phosphorodiamidate morpholino oligomers (PMOs) that are approved in the United States for the treatment of patients with Duchenne muscular dystrophy (DMD) with mutations in the DMD gene that are amenable to exon 51, 53, and 45 skipping, respectively. Here we report a series of in vivo and in vitro studies characterizing the drug metabolism and pharmacokinetic (DMPK) properties of these three PMOs. Following a single intravenous dose, plasma exposure was consistent for all three PMOs in mouse, rat, and nonhuman primate (NHP), and plasma half-lives were similar for eteplirsen (2.0-4.1 h) and golodirsen (2.1-8.7 h) across species and more variable for casimersen (3.2-18.1 h). Plasma protein binding was low (<40%) for all three PMOs in mouse, rat, NHP, and human and was largely concentration independent. In the mdx mouse model of DMD, following a single intravenous injection, extensive biodistribution was observed in the target skeletal muscle tissues and the kidney for all three PMOs; consistent with the latter finding, the predominant route of elimination was renal. In vitro studies using liver microsomes showed no evidence of hepatic metabolism, and none of the PMOs were identified as inhibitors or inducers of the human cytochrome P450 enzymes or membrane drug transporters tested at clinically relevant concentrations. These findings suggest that key DMPK features are consistent for eteplirsen, golodirsen, and casimersen and provide evidence for the concept of a PMO drug class with potential application to novel exon-skipping drug candidates. SIGNIFICANCE STATEMENT: The PMOs eteplirsen, golodirsen, and casimersen share similar absorption, distribution, metabolism, and excretion and DMPK properties, which provides evidence for the concept of a PMO treatment class. A PMO drug class may support a platform approach to enhance understanding of the pharmacokinetic and pharmacodynamic behavior of these molecules. The grouping of novel agent series into platforms could be beneficial in the development of drug candidates for populations in which traditional clinical trials are not feasible.

Icenticaftor (QBW251) is a potentiator of the cystic fibrosis transmembrane conductance regulator protein and is currently in clinical development for the treatment of chronic obstructive pulmonary disease and chronic bronchitis. An absorption, distribution, metabolism, and excretion study was performed at steady state to determine the pharmacokinetics, mass balance, and metabolite profiles of icenticaftor in humans. In this open-label study, six healthy men were treated with unlabeled oral icenticaftor (400 mg b.i.d.) for 4 days. A single oral dose of [¹⁴C]icenticaftor was administered on Day 5, and unlabeled icenticaftor was administered twice daily from the evening of Day 5 to Day 12. Unchanged icenticaftor accounted for 18.5% of plasma radioactivity. Moderate to rapid absorption of icenticaftor was observed (median time to reach peak or maximum concentration: 4 hours), with 93.4% of the dose absorbed. It exhibited moderate distribution (Vz/F: 335 L) and was extensively metabolized, principally through N-glucuronidation, O-glucuronidation, and/or O-demethylation. The metabolites M8 and M9, formed by N-glucuronidation and O-glucuronidation of icenticaftor, respectively, represented the main entities detected in plasma (35.3% and 14.5%, respectively) in addition to unchanged icenticaftor (18.5%). The apparent mean terminal half-life of icenticaftor was 15.4 hours in blood and 20.6 hours in plasma. Icenticaftor was eliminated from the body mainly through metabolism followed by renal excretion, and excretion of radioactivity was complete after 9 days. In vitro phenotyping of icenticaftor showed that cytochrome P450 and uridine diphosphate glucuronosyltransferase were responsible for 31% and 69% of the total icenticaftor metabolism in human liver microsomes, respectively. This study provided invaluable insights into the disposition of icenticaftor. SIGNIFICANCE STATEMENT: The absorption, distribution, metabolism, and excretion of a single radioactive oral dose of icenticaftor was evaluated at steady state to investigate the nonlinear pharmacokinetics observed previously with icenticaftor. [¹⁴C]Icenticaftor demonstrated good systemic availability after oral administration and was extensively metabolized and moderately distributed to peripheral tissues. The most abundant metabolites, M8 and M9, were formed by N-glucuronidation and O-glucuronidation of icenticaftor, respectively. Phenotyping demonstrated that [¹⁴C]icenticaftor was metabolized predominantly by UGT1A9 with a remarkably low K_m value.

In vitro evidence shows that the acyl-β-D-glucuronide metabolite of candesartan inhibits cytochrome P450 (CYP) 2C8 with an inhibition constant of 7.12 μM. We investigated the effect of candesartan on the plasma concentrations and glucose-lowering effect of repaglinide, a sensitive clinical CYP2C8 index substrate. In a randomized crossover study, ten healthy volunteers ingested 8 mg of candesartan or placebo daily for three days, and on day 3, they also ingested 0.25 mg of repaglinide one hour after candesartan or placebo. We measured the plasma concentrations of repaglinide, candesartan, and candesartan acyl-β-D-glucuronide, and blood glucose concentrations for up to nine hours after repaglinide intake. Candesartan had no effect on the area under the plasma concentration-time curve and peak plasma concentration of repaglinide compared with placebo, with ratios of geometric means of 1.02 [P = 0.809; 90% confidence interval (CI) 0.90-1.15] and 1.13 (P = 0.346; 90% CI 0.90-1.43), respectively. Other pharmacokinetic variables and blood glucose concentrations were neither affected. Candesartan acyl-β-D-glucuronide was detectable in seven subjects, in whom the peak concentration of repaglinide was 1.32-fold higher in the candesartan phase than in the placebo phase (P = 0.041; 90% CI 1.07-1.62). Systemic concentrations of candesartan acyl-β-D-glucuronide were very low compared with its CYP2C8 inhibition constant (ratio ≪ 0.1). Furthermore, in a cohort of 93 cancer patients, no indication of decreased paclitaxel clearance was found in four patients using candesartan concomitantly. In conclusion, candesartan therapy is unlikely to inhibit CYP2C8-mediated metabolism of other drugs to any clinically significant extent. SIGNIFICANCE STATEMENT: The findings of this study suggest that candesartan is unlikely to cause drug-drug interactions via inhibition of cytochrome P450 (CYP) 2C8. Although candesartan acyl-β-D-glucuronide has been shown to inhibit CYP2C8 in vitro, it shows no clinically relevant CYP2C8 inhibition in humans due to low systemic concentrations.

Millions of people globally are exposed to the proven human carcinogen arsenic at unacceptable levels in drinking water. In contrast, arsenic is a poor rodent carcinogen, requiring >100-fold higher doses for tumor induction, which may be explained by toxicokinetic differences between humans and mice. The human ATP-binding cassette subfamily C (ABCC) transporter hABCC4 mediates the cellular efflux of a diverse array of metabolites, including the glutathione (GSH) conjugate of the highly toxic monomethylarsonous acid (MMA^III), monomethylarsenic diglutathione [MMA(GS)₂], and the major human urinary arsenic metabolite dimethylarsinic acid (DMA^V). Our objective was to determine if mouse Abcc4 (mAbcc4) protected against and/or transported the same arsenic species as hABCC4. The anti-ABCC4 antibody M₄I-10 epitope was first mapped to an octapeptide (⁴¹¹HVQDFTA⁴¹⁸F) present in both hABCC4 and mAbcc4, enabling quantification of relative amounts of hABCC4/mAbcc4. mAbcc4 expressed in human embryonic kidney (HEK)293 cells did not protect against any of the six arsenic species tested [arsenite, arsenate, MMA^III, monomethylarsonic acid, dimethylarsinous acid, or DMA^V], despite displaying remarkable resistance against the antimetabolite 6-mercaptopurine (>9-fold higher than hABCC4). Furthermore, mAbcc4-enriched membrane vesicles prepared from transfected HEK293 cells did not transport MMA(GS)₂ or DMA^V despite a >3-fold higher transport activity than hABCC4-enriched vesicles for the prototypic substrate 17β-estradiol-17-(β-D-glucuronide). Abcc4^(+/+) mouse embryonic fibroblasts (MEFs) were ∼3-fold more resistant to arsenate than Abcc4^(-/-) MEFs; however, further characterization indicated that this was not mAbcc4 mediated. Thus, under the conditions tested, arsenicals are not transported by mAbcc4, and differences between the substrate selectivity of hABCC4 and mAbcc4 seem likely to contribute to arsenic toxicokinetic differences between human and mouse. SIGNIFICANCE STATEMENT: Toxicokinetics of the carcinogen arsenic differ among animal species. Arsenic methylation is known to contribute to this, whereas arsenic transporters have not been considered. Human ATP-binding cassette subfamily C member 4 (hABCC4) is a high-affinity transporter of toxicologically important arsenic metabolites. Here we used multiple approaches to demonstrate that mouse Abcc4 does not protect cells against or transport any arsenic species tested. Thus, differences between hABCC4 and mAbcc4 substrate selectivity likely contribute to differences in human and mouse arsenic toxicokinetics.

Remimazolam (Byfavo^®), a recent FDA-approved ester-linked benzodiazepine, offers advantages in sedation, such as rapid onset and predictable duration, making it suitable for broad anesthesia applications. Its favorable pharmacological profile is primarily attributed to rapid hydrolysis, the primary metabolism pathway for its deactivation. Thus, understanding remimazolam hydrolysis determinants is essential for optimizing its clinical use. This study aimed to identify the enzyme(s) and tissue(s) responsible for remimazolam hydrolysis and to evaluate the influence of genetic polymorphisms and drug-drug interactions (DDIs) on its hydrolysis in the human liver. An initial incubation study with remimazolam and phosphate buffer saline (PBS), human serum, and the S9 fractions of human liver and intestine demonstrated that remimazolam was exclusively hydrolyzed by human liver S9 fractions. Subsequent incubation studies utilizing a Carboxylesterase inhibitor (Bis-para-nitrophenylphosphate, BNPP), recombinant human Carboxylesterase1 (CES1) and Carboxylesterase 2 (CES2) confirmed that remimazolam is specifically hydrolyzed by CES1 in human liver. Furthermore, in vitro studies with wild-type CES1 and CES1 variants transfected cells revealed that certain genetic polymorphisms significantly impair remimazolam deactivation. Notably, the impact of CES1 G143E was verified using individual human liver samples. Moreover, our evaluation of the DDIs between remimazolam and several other substrates/inhibitors of CES1-including simvastatin, enalapril, clopidogrel and sacubitril- found that clopidogrel significantly inhibited remimazolam hydrolysis at clinically relevant concentrations, with CES1 genetic variants potentially influencing the interactions. In summary, CES1 genetic variants and its interacting drugs are crucial factors contributing to interindividual variability in remimazolam hepatic hydrolysis, holding the potential to serve as biomarkers for optimizing remimazolam use. Significance Statement This investigation demonstrates that remimazolam is deactivated by CES1 in the human liver, with CES1 genetic variants and DDIs significantly influencing its metabolism. These findings emphasize the need to consider CES1 genetic variability and potential DDIs in remimazolam use, especially in personalized pharmacotherapy to achieve optimal anesthetic outcomes.

Predictions of drug-drug interactions resulting from time-dependent inhibition (TDI) of CYP3A4 have consistently overestimated or mis-predicted (i.e. false positives) the interaction that is observed in vivo. Recent findings demonstrated that the presence of the allosteric modulator progesterone (PGS) in the in vitro assay could alter the in vitro kinetics of CYP3A4 TDI with inhibitors that interact with the heme moiety, such as metabolic-intermediate complex (MIC) forming inhibitors. The impact of the presence of 100 µM PGS on the TDI of molecules in the class of macrolides typically associated with MIC formation was investigated. Presence of PGS resulted in varied responses across the inhibitors tested. The TDI signal was eliminated for five inhibitors, and unaltered in the case of one, fidaxomicin. The remaining molecules erythromycin, clarithromycin, and troleandomycin, were observed to have a decrease in both potency and maximum inactivation rate ranging from 1.7-fold to 6.7-fold. These changes in TDI kinetics led to a >90% decrease in inactivation efficiency. In order to determine in vitro conditions that could reproduce in vivo inhibition, varied concentrations of PGS were incubated with clarithromycin and erythromycin. Resulting in vitro TDI kinetics were incorporated into dynamic physiologically-based pharmacokinetic (PBPK) models to predict clinically observed interactions. The results suggested that a concentration of ~45 µM PGS would result in TDI kinetic values that could reproduce in vivo observations and could potentially improve predictions for CYP3A4 TDI. Significance Statement The impact of the allosteric heterotropic modulator progesterone on the CYP3A4 time-dependent inhibition kinetics was quantified for a set of metabolic-intermediate complex forming mechanism-based inhibitors. We identify the in vitro conditions that optimally predict time-dependent inhibition for in vivo drug-drug interactions through dynamic physiologically-based pharmacokinetic modeling. The optimized assay conditions improve in vitro to in vivo translation and prediction of time-dependent inhibition.

Several clinical studies have shown that COVID-19 increases the systemic concentration of drugs in hospitalized COVID-19 patients. However, it is unclear how COVID-19-mediated bidirectional dysregulation of hepatic and pulmonary CYP3A4 impacts drug concentrations, especially in the lung tissue which is most affected by the disease. Herein, PBPK modeling was used to demonstrate the differences in systemic and pulmonary concentrations of four respiratory infectious disease drugs when CYP3A4 is concurrently downregulated in the liver and upregulated in the lung based on existing clinical data on COVID-19 - CYP3A4 interactions at varying severity levels including outpatients, non-ICU, and ICU patients. The study showed that hepatic metabolism is the primary determinant of both systemic and pulmonary drug concentrations despite the concurrent bidirectional dysregulation of liver and lung CYP3A4. ICU patients had the most systemic and pulmonary drug exposure with a percentage increase in AUC_plasma of approximately 44%, 56%, 114%, and 196% for clarithromycin, nirmatrelvir, dexamethasone, and itraconazole, respectively, relative to the healthy group. Within the ICU cohort, clarithromycin exhibited its highest exposure in lung tissue mass with a fold change of 1189, while nirmatrelvir and dexamethasone showed their highest exposure in the plasma compartment, with fold changes of about 126 and 5, respectively, compared to the maximum therapeutic concentrations for their target pathogens. Itraconazole was significantly under-exposed in the lung fluid compartment potentially explaining its limited efficacy for the treatment of COVID-19. These findings underscore the importance of optimizing dosing regimens in at risk ICU patients to enhance both efficacy and safety profiles. Significance Statement This study investigated whether COVID-19-mediated concurrent hepatic downregulation and pulmonary upregulation of CYP3A4 leads to differences in the systemic and pulmonary concentrations of four respiratory medicines. The study demonstrated that intercompartmental differences in drug concentrations were driven by only hepatic CYP3A4 expression. This work suggests that ICU patients with significant COVID-19 - CYP3A4 interactions may be at risk of clinically relevant COVID-19-drug interactions, highlighting the need for optimizing dosing regimens in this patient group to improve safety and efficacy.