Utility of PBPK Modeling in Predicting and Characterizing Clinical Drug Interactions.

Physiologically-based pharmacokinetic (PBPK) modeling is a mechanistic dynamic modeling approach that can be used to predict or retrospectively describe changes in drug exposure due to drug-drug interactions. With advancements in commercially available PBPK software, PBPK DDI modeling has become a mainstream approach from early drug discovery through to late stage drug development and is often utilized to support regulatory packages for new drug applications. This minireview will briefly describe the approaches to predicting DDI utilizing PBPK and static modeling approaches, the basic model structures and features inherent to PBPK DDI models and key examples where PBPK DDI models have been used to describe complex DDI mechanisms. Future directions aimed at using PBPK models to characterize transporter-mediated DDI, predict DDI in special populations and assess the DDI potential of protein therapeutics will be discussed. A summary of the 209 PBPK DDI examples published to date in 2023 will be provided. Overall, current data and trends suggest a continued role for PBPK models in the characterization and prediction of DDI for therapeutic molecules. Significance Statement PBPK models have been a key tool in the characterization of various pharmacokinetic phenomenon, including drug-drug interactions. This minireview will highlight recent advancements and publications around PBPK DDI modeling, an important area of drug discovery and development research in light of the increasing prevalence of polypharmacology in clinical settings.

Cytochrome P450 and Other Drug-Metabolizing Enzymes As Therapeutic Targets.

Cytochrome P450 and other families of drug-metabolizing enzymes are commonly thought of and studied for their ability to metabolize xenobiotics and other foreign entities as they are eliminated from the body. Equally as important, however, is the homeostatic role that many of these enzymes play in maintaining the proper levels of endogenous signaling molecules such as lipids, steroids, and eicosanoids as well as their ability to modulate protein-protein interactions involved in downstream signaling cascades. Throughout the years, many of these endogenous ligands or protein partners of drug-metabolizing enzymes have been associated with a wide range of disease states from cancer to various cardiovascular, neurologic, or inflammatory diseases, prompting an interest in whether modulation of drug-metabolizing enzyme activity could have a subsequent pharmacological impact or lessening of disease severity. Beyond direct regulation of endogenous pathways, drug-metabolizing enzymes have also been proactively targeted for their ability to activate prodrugs with subsequent pharmacological activity or enhance the efficacy of a coadministered drug by inhibiting the metabolism of that drug through a rationally designed drug-drug interaction (i.e., ritonavir and human immunodeficiency virus antiretroviral therapy). The focus of this minireview will be to highlight research aimed at characterizing cytochrome P450 and other drug-metabolizing enzymes as therapeutic targets. Examples of successfully marketed drugs as well as early research efforts will be discussed. Finally, emerging areas of research utilizing typical drug-metabolizing enzymes to impact clinical outcomes will be discussed. SIGNIFICANCE STATEMENT: Although generally thought of for their drug-metabolizing capabilities, enzymes such as the cytochromes P450, glutathione S-transferases, soluble epoxide hydrolases, and others play a significant role in regulating key endogenous pathways, making them potential drug targets. This minireview will cover various efforts over the years to modulate drug-metabolizing enzyme activity toward pharmacological outcomes.

Use of Cryopreserved Hepatocytes as Part of an Integrated Strategy to Characterize In Vivo Clearance for Peptide-Antibody Conjugate Inhibitors of Nav1.7 in Preclinical Species.

The identification of nonopioid alternatives to treat chronic pain has received a great deal of interest in recent years. Recently, the engineering of a series of Nav1.7 inhibitory peptide-antibody conjugates has been reported, and herein, the preclinical efforts to identify novel approaches to characterize the pharmacokinetic properties of the peptide conjugates are described. A cryopreserved plated mouse hepatocyte assay was designed to measure the depletion of the peptide-antibody conjugates from the media, with a correlation being observed between percentage remaining in the media and in vivo clearance (Pearson r = -0.5525). Physicochemical (charge and hydrophobicity), receptor-binding [neonatal Fc receptor (FcRn)], and in vivo pharmacokinetic data were generated and compared with the results from our in vitro hepatocyte assay, which was hypothesized to encompass all of the aforementioned properties. Correlations were observed among hydrophobicity; FcRn binding; depletion rates from the hepatocyte assay; and ultimately, in vivo clearance. Subsequent studies identified potential roles for the low-density lipoprotein and mannose/galactose receptors in the association of the Nav1.7 peptide conjugates with mouse hepatocytes, although in vivo studies suggested that FcRn was still the primary receptor involved in determining the pharmacokinetics of the peptide conjugates. Ultimately, the use of the cryopreserved hepatocyte assay along with FcRn binding and hydrophobic interaction chromatography provided an efficient and integrated approach to rapidly triage molecules for advancement while reducing the number of in vivo pharmacokinetic studies. SIGNIFICANCE STATEMENT: Although multiple in vitro and in silico tools are available in small-molecule drug discovery, pharmacokinetic characterization of protein therapeutics is still highly dependent upon the use of in vivo studies in preclinical species. The current work demonstrates the combined use of cryopreserved hepatocytes, hydrophobic interaction chromatography, and neonatal Fc receptor binding to characterize a series of Nav1.7 peptide-antibody conjugates prior to conducting in vivo studies, thus providing a means to rapidly evaluate novel protein therapeutic platforms while concomitantly reducing the number of in vivo studies conducted in preclinical species.

Engineering NaV1.7 Inhibitory JzTx-V Peptides with a Potency and Basicity Profile Suitable for Antibody Conjugation To Enhance Pharmacokinetics.

Drug discovery research on new pain targets with human genetic validation, including the voltage-gated sodium channel NaV1.7, is being pursued to address the unmet medical need with respect to chronic pain and the rising opioid epidemic. As part of early research efforts on this front, we have previously developed NaV1.7 inhibitory peptide-antibody conjugates with tarantula venom-derived GpTx-1 toxin peptides with an extended half-life (80 h) in rodents but only moderate in vitro activity (hNaV1.7 IC50 = 250 nM) and without in vivo activity. We identified the more potent peptide JzTx-V from our natural peptide collection and improved its selectivity against other sodium channel isoforms through positional analogueing. Here we report utilization of the JzTx-V scaffold in a peptide-antibody conjugate and architectural variations in the linker, peptide loading, and antibody attachment site. We found conjugates with 100-fold improved in vitro potency relative to those of complementary GpTx-1 analogues, but pharmacokinetic and bioimaging analyses of these JzTx-V conjugates revealed a shorter than expected plasma half-life in vivo with accumulation in the liver. In an attempt to increase circulatory serum levels, we sought the reduction of the net +6 charge of the JzTx-V scaffold while retaining a desirable NaV in vitro activity profile. The conjugate of a JzTx-V peptide analogue with a +2 formal charge maintained NaV1.7 potency with 18-fold improved plasma exposure in rodents. Balancing the loss of peptide and conjugate potency associated with the reduction of net charge necessary for improved target exposure resulted in a compound with moderate activity in a NaV1.7-dependent pharmacodynamic model but requires further optimization to identify a conjugate that can fully engage NaV1.7 in vivo.

Discovery and in Vivo Evaluation of the Potent and Selective PI3Kδ Inhibitors 2-((1S)-1-((6-Amino-5-cyano-4-pyrimidinyl)amino)ethyl)-6-fluoro-N-methyl-3-(2-pyridinyl)-4-quinolinecarboxamide (AM-0687) and 2-((1S)-1-((6-Amino-5-cyano-4-pyrimidinyl)amino)ethyl)-5-fluoro-N-methyl-3-(2-pyridinyl)-4-quinolinecarboxamide (AM-1430).

Optimization of the potency and pharmacokinetic profile of 2,3,4-trisubstituted quinoline, 4, led to the discovery of two potent, selective, and orally bioavailable PI3Kδ inhibitors, 6a (AM-0687) and 7 (AM-1430). On the basis of their improved profile, these analogs were selected for in vivo pharmacodynamic (PD) and efficacy experiments in animal models of inflammation. The in vivo PD studies, which were carried out in a mouse pAKT inhibition animal model, confirmed the observed potency of 6a and 7 in biochemical and cellular assays. Efficacy experiments in a keyhole limpet hemocyanin model in rats demonstrated that administration of either 6a or 7 resulted in a strong dose-dependent reduction of IgG and IgM specific antibodies. The excellent in vitro and in vivo profiles of these analogs make them suitable for further development.

Induction of CYP26A1 by metabolites of retinoic acid: evidence that CYP26A1 is an important enzyme in the elimination of active retinoids.

All-trans-retinoic acid (atRA), the active metabolite of vitamin A, induces gene transcription via binding to nuclear retinoic acid receptors (RARs). The primary hydroxylated metabolites formed from atRA by CYP26A1, and the subsequent metabolite 4-oxo-atRA, bind to RARs and potentially have biologic activity. Hence, CYP26A1, the main atRA hydroxylase, may function either to deplete bioactive retinoids or to form active metabolites. This study aimed to determine the role of CYP26A1 in modulating RAR activation via formation and elimination of active retinoids. After treatment of HepG2 cells with atRA, (4S)-OH-atRA, (4R)-OH-atRA, 4-oxo-atRA, and 18-OH-atRA, mRNAs of CYP26A1 and RARß were increased 300- to 3000-fold, with 4-oxo-atRA and atRA being the most potent inducers. However, >60% of the 4-OH-atRA enantiomers were converted to 4-oxo-atRA in the first 12 hours of treatment, suggesting that the activity of the 4-OH-atRA was due to 4-oxo-atRA. In human hepatocytes, atRA, 4-OH-atRA, and 4-oxo-atRA induced CYP26A1 and 4-oxo-atRA formation was observed from 4-OH-atRA. In HepG2 cells, 4-oxo-atRA formation was observed even in the absence of CYP26A1 activity and this formation was not inhibited by ketoconazole. In human liver microsomes, 4-oxo-atRA formation was supported by NAD(+), suggesting that 4-oxo-atRA formation is mediated by a microsomal alcohol dehydrogenase. Although 4-oxo-atRA was not formed by CYP26A1, it was depleted by CYP26A1 (Km = 63 nM and intrinsic clearance = 90 µl/min per pmol). Similarly, CYP26A1 depleted 18-OH-atRA and the 4-OH-atRA enantiomers. These data support the role of CYP26A1 to clear bioactive retinoids, and suggest that the enzyme forming active 4-oxo-atRA may be important in modulating retinoid action.

Evaluation of UGT protein interactions in human hepatocytes: effect of siRNA down regulation of UGT1A9 and UGT2B7 on propofol glucuronidation in human hepatocytes.

Previous experiments performed in recombinant systems have suggested that protein-protein interactions occur between the UGTs and may play a significant role in modulating enzyme activity. However, evidence of UGT protein-protein interactions either in vivo or in more physiologically relevant in vitro systems has yet to be demonstrated. In this study, we examined oligomerization and its ability to affect glucuronidation in plated human hepatocytes. siRNA down regulation experiments and activity studies were used to examine changes in metabolite formation of one UGT isoform due to down regulation of a second UGT isoform. Selective siRNA directed towards UGT1A9 or UGT2B7 resulted in significant and selective decreases in their respective mRNA levels. As expected, the metabolism of the UGT1A9 substrate propofol decreased with UGT1A9 down regulation. Interestingly, UGT1A9 activity, but not UGT1A9 mRNA expression, was also diminished when UGT2B7 expression was selectively inhibited, implying potential interactions between the two isoforms. Minor changes to UGT1A4, UGT2B4 and UGT2B7 activity were also observed when UGT1A9 expression was selectively down regulated. To our knowledge, this represents the first piece of evidence that UGT protein-protein interactions occur in human hepatocytes and suggests that expression levels of UGT2B7 may directly impact the glucuronidation activity of selective UGT1A9 substrates.

Cytochrome p450 architecture and cysteine nucleophile placement impact raloxifene-mediated mechanism-based inactivation.

The propensity for cytochrome P450 (P450) enzymes to bioactivate xenobiotics is governed by the inherent chemistry of the xenobiotic itself and the active site architecture of the P450 enzyme(s). Accessible nucleophiles in the active site or egress channels of the P450 enzyme have the potential of sequestering reactive metabolites through covalent modification, thereby limiting their exposure to other proteins. Raloxifene, a drug known to undergo CYP3A-mediated reactive metabolite formation and time-dependent inhibition in vitro, was used to explore the potential for bioactivation and enzyme inactivation of additional P450 enzymes (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A5). Every P450 tested except CYP2E1 was capable of raloxifene bioactivation, based on glutathione adduct formation. However, raloxifene-mediated time-dependent inhibition only occurred in CYP2C8 and CYP3A4. Comparable inactivation kinetics were achieved with K(I) and k(inact) values of 0.26 µM and 0.10 min(-1) and 0.81 µM and 0.20 min(-1) for CYP2C8 and CYP3A4, respectively. Proteolytic digests of CYP2C8 and CYP3A4 Supersomes revealed adducts to Cys225 and Cys239 for CYP2C8 and CYP3A4, respectively. For each P450 enzyme, proposed substrate/metabolite access channels were mapped and active site cysteines were identified, which revealed that only CYP2C8 and CYP3A4 possess accessible cysteine residues near the active site cavities, a result consistent with the observed kinetics. The combined data suggest that the extent of bioactivation across P450 enzymes does not correlate with P450 inactivation. In addition, multiple factors contribute to the ability of reactive metabolites to form apo-adducts with P450 enzymes.

Predicting the drug interaction potential of AMG 853, a dual antagonist of the D-prostanoid and chemoattractant receptor-homologous molecule expressed on T helper 2 cells receptors.

2-(4-(4-(tert-Butylcarbamoyl)-2-(2-chloro-4-cyclopropylphenylsulfonamido)phenoxy)-5-chloro-2-fluorophenyl)acetic acid (AMG 853) is an orally bioavailable and potent dual antagonist of the D-prostanoid and chemoattractant receptor-homologous molecule expressed on T helper 2 cells receptors. The drug interaction potential of AMG 853, both as a victim and a perpetrator, was investigated using in vitro, in silico, and in vivo methodologies. Experiments in human liver microsomes (HLM) and recombinant enzymes identified CYP2C8, CYP2J2, and CYP3A as well as multiple UDP-glucuronosyltransferase isoforms as being responsible for the metabolic clearance of AMG 853. With use of HLM and selective probe substrates, both AMG 853 and its acyl glucuronide metabolite (M1) were shown to be inhibitors of CYP2C8. AMG 853 and M1 did not inhibit any of the other cytochrome P450 isoforms tested, and AMG 853 exhibited minimal enzyme induction properties in human hepatocytes cultures. In light of the in vitro findings, modeling and simulation approaches were used to examine the potential for ketoconazole (a CYP3A inhibitor) to inhibit the metabolism of AMG 853 as well as for AMG 853 to inhibit the metabolism of paclitaxel, rosiglitazone, and montelukast, commonly used substrates of CYP2C8. A weak and clinically insignificant drug interaction (area under the drug concentration-time curve (AUC)(i)/AUC <2) was predicted between ketoconazole and AMG 853. No drug interactions were predicted for AMG 853 and paclitaxel, rosiglitazone, or montelukast. Finally, administration of AMG 853 to healthy human subjects in clinical trials in the presence or absence of ketoconazole confirmed that AMG 853 is unlikely to be involved in clinically significant drug interactions.

Evaluation of CYP2C8 inhibition in vitro: utility of montelukast as a selective CYP2C8 probe substrate.

Understanding the potential for cytochrome P450 (P450)-mediated drug-drug interactions is a critical step in the drug discovery process. Although in vitro studies with CYP3A4, CYP2C9, and CYP2C19 have suggested the presence of multiple binding regions within the P450 active site based on probe substrate-dependent inhibition profiles, similar studies have not been performed with CYP2C8. The ability to understand CYP2C8 probe substrate sensitivity will enable appropriate in vitro and in vivo probe selection. To characterize the potential for probe substrate-dependent inhibition with CYP2C8, the inhibition potency of 22 known inhibitors of CYP2C8 were measured in vitro using four clinically relevant CYP2C8 probe substrates (montelukast, paclitaxel, repaglinide, and rosiglitazone) and amodiaquine. Repaglinide exhibited the highest sensitivity to inhibition in vitro. In vitro phenotyping indicated that montelukast is an appropriate probe for CYP2C8 inhibition studies. The in vivo sensitivities of the CYP2C8 probe substrates cerivastatin, fluvastatin, montelukast, pioglitazone, and rosiglitazone were determined in relation to repaglinide on the basis of clinical drug-drug interaction (DDI) data. Repaglinide exhibited the highest sensitivity in vivo, followed by cerivastatin, montelukast, and pioglitazone. Finally, the magnitude of in vivo CYP2C8 DDI caused by gemfibrozil-1-O-ß-glucuronide was predicted. Comparisons of the predictions with clinical data coupled with the potential liabilities of other CYP2C8 probes suggest that montelukast is an appropriate CYP2C8 probe substrate to use for the in vivo situation.

In vitro inhibition of multiple cytochrome P450 isoforms by xanthone derivatives from mangosteen extract.

Mangosteen is a xanthone-containing fruit found in Southeast Asia for which health claims include maintaining healthy immune and gastrointestinal systems to slowing the progression of tumor growth and neurodegenerative diseases. Previous studies have identified multiple xanthones in the pericarp of the mangosteen fruit. The aim of the current study was to assess the drug inhibition potential of mangosteen in vitro as well as the cytochrome P450 (P450) enzymes responsible for the metabolism of its individual components. The various xanthone derivatives were found to be both substrates and inhibitors for multiple P450 isoforms. Aqueous extracts of the mangosteen pericarp were analyzed for xanthone content as well as inhibition potency. Finally, in vivo plasma concentrations of alpha-mangostin, the most abundant xanthone derivative found in mangosteen, were predicted using Simcyp and found to be well above their respective in vitro K(i) values for CYP2C8 and CYP2C9.

Differential time-dependent inactivation of P450 3A4 and P450 3A5 by raloxifene: a key role for C239 in quenching reactive intermediates.

The role of C239 as the active-site residue responsible for forming the covalent linkage with raloxifene during P450 3A4 time-dependent inactivation (TDI) was recently identified. The corresponding residue in CYP3A5 is S239, and when the potential for TDI in P450 3A5 was investigated, only reversible inhibition was observed against midazolam and testosterone, with median inhibitory concentration (IC50) values of 2.4 and 2.9 microM, respectively. In a similar fashion, when C239 was replaced with alanine in P450 3A4, TDI was successfully engineered out, and the reversible inhibition was characterized by IC50 values of 3.7 and 3.5 microM against midazolam and testosterone, respectively. Metabolism studies confirmed that the reactive diquinone methide intermediate required for P450 3A4 inactivation formed in all of the P450 3A enzymes investigated. Furthermore, the absence of TDI in P450 3A5 led to an increase in the formation of GSH-related adducts of raloxifene compared with that for P450 3A4. Consequently, the absence of the nucleophilic cysteine leads to differential TDI and generation of reactive metabolites in the P450 3A enzyme, providing the foundation for pharmacogenetics that contributes to individual differences in susceptibility to adverse drug reactions.