Evaluating variations in bilirubin glucuronidation activity by protease inhibitors in canine and human primary hepatocytes cultured in a 3D culture system.

Bilirubin is excreted into the bile from hepatocytes, mainly as monoglucuronosyl and bisglucuronosyl conjugates, reflecting bilirubin glucuronidation activity. However, there is limited information on the in vitro evaluation of liver cell lines or primary hepatocytes. This study aimed to investigate variations in the bilirubin metabolic function of canine and human hepatocyte spheroids formed in a three-dimensional (3D) culture system indicated by the formation of bilirubin glucuronides when protease inhibitors such as atazanavir, indinavir, ritonavir, and nelfinavir were treated with bilirubin. The culture supernatant was collected for bilirubin glucuronidation assessment and the cells were used to evaluate viability. On day 8 of culture, both canine and human hepatocyte spheroids showed high albumin secretion and distinct spheroid formation, and their bilirubin glucuronidation activities were evaluated considering cell viability. Treatment with atazanavir and ritonavir remarkably inhibited bilirubin glucuronide formation, wherein atazanavir showed the highest inhibition, particularly in human hepatocyte spheroids. These results may reflect the effects on cellular uptake of bilirubin and its intracellular metabolic function. Thus, primary hepatocytes cultured in a 3D culture system may be a useful in vitro system for the comprehensive evaluation of bilirubin metabolic function and risk assessment in bilirubin metabolic disorders for drug development.

Mechanistic in vitro studies indicate that the clinical drug-drug interactions between protease inhibitors and rosuvastatin are driven by inhibition of intestinal BCRP and hepatic OATP1B1 with minimal contribution from OATP1B3, NTCP and OAT3.

Previous use of a mechanistic static model to accurately quantify the increased rosuvastatin exposure due to drug-drug interaction (DDI) with coadministered atazanavir underpredicted the magnitude of area under the plasma concentration-time curve ratio (AUCR) based on inhibition of breast cancer resistance protein (BCRP) and organic anion transporting polypeptide (OATP) 1B1. To reconcile the disconnect between predicted and clinical AUCR, atazanavir and other protease inhibitors (darunavir, lopinavir and ritonavir) were evaluated as inhibitors of BCRP, OATP1B1, OATP1B3, sodium taurocholate cotransporting polypeptide (NTCP) and organic anion transporter (OAT) 3. None of the drugs inhibited OAT3, nor did darunavir and ritonavir inhibit OATP1B3 or NTCP. All drugs inhibited BCRP-mediated estrone 3-sulfate transport or OATP1B1-mediated estradiol 17ß-D-glucuronide transport with the same rank order of inhibitory potency (lopinavir>ritonavir>atazanavir>>darunavir) and mean IC50 values ranging from 15.5 ± 2.80 µM to 143 ± 14.7 µM or 0.220 ± 0.0655 µM to 9.53 ± 2.50 µM, respectively. Atazanavir and lopinavir also inhibited OATP1B3- or NTCP-mediated transport with a mean IC50 of 1.86 ± 0.500 µM or 65.6 ± 10.7 µM and 5.04 ± 0.0950 µM or 20.3 ± 2.13 µM, respectively. Following integration of a combined hepatic transport component into the previous mechanistic static model using the in vitro inhibitory kinetic parameters determined above for atazanavir, the newly predicted rosuvastatin AUCR reconciled with the clinically observed AUCR confirming additional minor involvement of OATP1B3 and NTCP inhibition in its DDI. The predictions for the other protease inhibitors confirmed inhibition of intestinal BCRP and hepatic OATP1B1 as the principal pathways involved in their clinical DDI with rosuvastatin.

Decoding molecular mechanism underlying binding of drugs to HIV-1 protease with molecular dynamics simulations and MM-GBSA calculations.

HIV-1 protease (PR) is thought to be efficient targets of anti-AIDS drug design. Molecular dynamics (MD) simulations and multiple post-processing analysis technologies were applied to decipher molecular mechanism underlying binding of three drugs Lopinavir (LPV), Nelfinavir (NFV) and Atazanavir (ATV) to the PR. Binding free energies calculated by molecular mechanics generalized Born surface area (MM-GBSA) suggest that compensation between binding enthalpy and entropy plays a vital role in binding of drugs to PR. Dynamics analyses show that binding of LPV, NFV and ATV highly affects structural flexibility, motion modes and dynamics behaviour of the PR, especially for two flaps. Computational alanine scanning and interaction network analysis verify that although three drugs have structural difference, they share similar binding modes to the PR and common interaction clusters with the PR. The current findings also confirm that residues located interaction clusters, such as Asp25/Asp25', Gly27/Gly27', Ala28/Ala28', Asp29, Ile47/Ile47', Gly49/Gly49', Ile50/Ile50', Val82/Val82' and Ile84/Ile84, can be used as efficient targets of clinically available inhibitors towards the PR.

Quantum simulations of SARS-CoV-2 main protease Mpro enable high-quality scoring of diverse ligands.

The COVID-19 pandemic has led to unprecedented efforts to identify drugs that can reduce its associated morbidity/mortality rate. Computational chemistry approaches hold the potential for triaging potential candidates far more quickly than their experimental counterparts. These methods have been widely used to search for small molecules that can inhibit critical proteins involved in the SARS-CoV-2 replication cycle. An important target is the SARS-CoV-2 main protease Mpro, an enzyme that cleaves the viral polyproteins into individual proteins required for viral replication and transcription. Unfortunately, standard computational screening methods face difficulties in ranking diverse ligands to a receptor due to disparate ligand scaffolds and varying charge states. Here, we describe full density functional quantum mechanical (DFT) simulations of Mpro in complex with various ligands to obtain absolute ligand binding energies. Our calculations are enabled by a new cloud-native parallel DFT implementation running on computational resources from Amazon Web Services (AWS). The results we obtain are promising: the approach is quite capable of scoring a very diverse set of existing drug compounds for their affinities to M pro and suggest the DFT approach is potentially more broadly applicable to repurpose screening against this target. In addition, each DFT simulation required only ~ 1 h (wall clock time) per ligand. The fast turnaround time raises the practical possibility of a broad application of large-scale quantum mechanics in the drug discovery pipeline at stages where ligand diversity is essential.

(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.

Coupling of an Acyl Migration Prodrug Strategy with Bio-activation To Improve Oral Delivery of the HIV-1 Protease Inhibitor Atazanavir.

HIV-1 protease inhibitors (PIs), which include atazanavir (ATV, 1), remain important medicines to treat HIV-1 infection. However, they are characterized by poor oral bioavailability and a need for boosting with a pharmacokinetic enhancer, which results in additional drug-drug interactions that are sometimes difficult to manage. We investigated a chemo-activated, acyl migration-based prodrug design approach to improve the pharmacokinetic profile of 1 but failed to obtain improved oral bioavailability over dosing the parent drug in rats. This strategy was refined by conjugating the amine with a promoiety designed to undergo bio-activation, as a means of modulating the subsequent chemo-activation. This culminated in a lead prodrug that (1) yielded substantially better oral drug delivery of 1 when compared to the parent itself, the simple acyl migration-based prodrug, and the corresponding simple l-Val prodrug, (2) acted as a depot which resulted in a sustained release of the parent drug in vivo, and (3) offered the benefit of mitigating the pH-dependent absorption associated with 1, thereby potentially reducing the risk of decreased bioavailability with concurrent use of stomach-acid-reducing drugs.

A Genome-Wide Association Study Identifies a Candidate Gene Associated With Atazanavir Exposure Measured in Hair.

Hair provides a direct measure of long-term exposure of atazanavir (ATV). We report the results of the first genome-wide association study (GWAS) of ATV exposure measured in hair in an observational cohort representative of US women living with HIV; the Women's Interagency HIV Study. Approximately 14.1 million single nucleotide polymorphisms (SNPs) were analyzed in linear regression-based GWAS, with replication, adjusted for nongenetic predictors collected under conditions of actual use of ATV in 398 participants. Lastly, the PharmGKB database was used to identify pharmacogene associations with ATV exposure. The rs73208473, within intron 1 of SORCS2, resulted in a 0.46-fold decrease in ATV exposure, with the strongest association (P = 1.71×10-8 ) in GWAS. A priori pharmacogene screening did not identify additional variants statistically significantly associated with ATV exposure, including those previously published in ATV plasma candidate pharmacogene studies. The findings demonstrate the potential value of pharmacogenomic GWAS in ethnically diverse populations under conditions of actual use.

A novel humanized mouse lacking murine P450 oxidoreductase for studying human drug metabolism.

Only one out of 10 drugs in development passes clinical trials. Many fail because experimental animal models poorly predict human xenobiotic metabolism. Human liver chimeric mice are a step forward in this regard, as the human hepatocytes in chimeric livers generate human metabolites, but the remaining murine hepatocytes contain an expanded set of P450 cytochromes that form the major class of drug-metabolizing enzymes. We therefore generated a conditional knock-out of the NADPH-P450 oxidoreductase (Por) gene combined with Il2rg - /- /Rag2 - /- /Fah - /- (PIRF) mice. Here we show that homozygous PIRF mouse livers are readily repopulated with human hepatocytes, and when the murine Por gene is deleted (<5%), they predominantly use human cytochrome metabolism. When given the anticancer drug gefitinib or the retroviral drug atazanavir, the Por-deleted humanized PIRF mice develop higher levels of the major human metabolites than current models. Humanized, murine Por-deficient PIRF mice can thus predict human drug metabolism and should be useful for preclinical drug development.Human liver chimeric mice are increasingly used for drug testing in preclinical development, but express residual murine p450 cytochromes. Here the authors generate mice lacking the Por gene in the liver, and show that human cytochrome metabolism is used following repopulation with human hepatocytes.

Effect of Methamphetamine on Spectral Binding, Ligand Docking and Metabolism of Anti-HIV Drugs with CYP3A4.

Cytochrome P450 3A4 (CYP3A4) is the major drug metabolic enzyme, and is involved in the metabolism of antiretroviral drugs, especially protease inhibitors (PIs). This study was undertaken to examine the effect of methamphetamine on the binding and metabolism of PIs with CYP3A4. We showed that methamphetamine exhibits a type I spectral change upon binding to CYP3A4 with δAmax and KD of 0.016±0.001 and 204±18 µM, respectively. Methamphetamine-CYP3A4 docking showed that methamphetamine binds to the heme of CYP3A4 in two modes, both leading to N-demethylation. We then studied the effect of methamphetamine binding on PIs with CYP3A4. Our results showed that methamphetamine alters spectral binding of nelfinavir but not the other type I PIs (lopinavir, atazanavir, tipranavir). The change in spectral binding for nelfinavir was observed at both δAmax (0.004±0.0003 vs. 0.0068±0.0001) and KD (1.42±0.36 vs.2.93±0.08 µM) levels. We further tested effect of methamphetamine on binding of 2 type II PIs; ritonavir and indinavir. Our results showed that methamphetamine alters the ritonavir binding to CYP3A4 by decreasing both the δAmax (0.0038±0.0003 vs. 0.0055±0.0003) and KD (0.043±0.0001 vs. 0.065±0.001 nM), while indinavir showed only reduced KD in presence of methamphetamine (0.086±0.01 vs. 0.174±0.03 nM). Furthermore, LC-MS/MS studies in high CYP3A4 human liver microsomes showed a decrease in the formation of hydroxy ritonavir in the presence of methamphetamine. Finally, CYP3A4 docking with lopinavir and ritonavir in the absence and presence of methamphetamine showed that methamphetamine alters the docking of ritonavir, which is consistent with the results obtained from spectral binding and metabolism studies. Overall, our results demonstrated differential effects of methamphetamine on the binding and metabolism of PIs with CYP3A4. These findings have clinical implication in terms of drug dose adjustment of antiretroviral medication, especially with ritonavir-boosted antiretroviral therapy, in HIV-1-infected individuals who abuse methamphetamine.

Transport-Metabolism Interplay of Atazanavir in Rat Hepatocytes.

The aim of this study was to explore the mechanisms governing the intra- to extracellular unbound concentration ratio (Kpu,u) for the HIV protease inhibitor atazanavir (ATV) in rat hepatocytes. We had previously proposed a new method to determine Kpu,u by using the unbound Km values from metabolism studies with suspended rat hepatocytes and rat liver microsomes. Following that method, we determined that the value of ATV Kpu,u was 0.32, indicating that ATV hepatocellular clearance is uptake rate-limited. This hypothesis was supported by the linear correlation between Kpu,u and active uptake clearance (P = 0.04; R(2)=0.82) in the presence of increasing concentrations of the uptake transport inhibitor losartan. Moreover, in contrast to an expected increase of Kpu,u upon inhibition of ATV metabolism, a decrease of Kpu,u was observed, suggesting an increased impact of sinusoidal efflux. In summary, involvement of active uptake transport does not guarantee high intracellular accumulation; however, it has a key role in regulating intracellular drug concentrations and drug metabolism. These findings will help improve future in vitro-to-in vivo extrapolations and likewise physiologically based pharmacokinetic models.