Challenges and Opportunities for In Vitro-In Vivo Extrapolation of Aldehyde Oxidase-Mediated Clearance: Toward a Roadmap for Quantitative Translation.

Underestimation of aldehyde oxidase (AO)-mediated clearance by current in vitro assays leads to uncertainty in human dose projections, thereby reducing the likelihood of success in drug development. In the present study we first evaluated the current drug development practices for AO substrates. Next, the overall predictive performance of in vitro-in vivo extrapolation of unbound hepatic intrinsic clearance (CLint,u) and unbound hepatic intrinsic clearance by AO (CLint,u,AO) was assessed using a comprehensive literature database of in vitro (human cytosol/S9/hepatocytes) and in vivo (intravenous/oral) data collated for 22 AO substrates (total of 100 datapoints from multiple studies). Correction for unbound fraction in the incubation was done by experimental data or in silico predictions. The fraction metabolized by AO (fmAO) determined via in vitro/in vivo approaches was found to be highly variable. The geometric mean fold errors (gmfe) for scaled CLint,u (mL/min/kg) were 10.4 for human hepatocytes, 5.6 for human liver cytosols, and 5.0 for human liver S9, respectively. Application of these gmfe's as empirical scaling factors improved predictions (45%-57% within twofold of observed) compared with no correction (11%-27% within twofold), with the scaling factors qualified by leave-one-out cross-validation. A road map for quantitative translation was then proposed following a critical evaluation on the in vitro and clinical methodology to estimate in vivo fmAO In conclusion, the study provides the most robust system-specific empirical scaling factors to date as a pragmatic approach for the prediction of in vivo CLint,u,AO in the early stages of drug development. SIGNIFICANCE STATEMENT: Confidence remains low when predicting in vivo clearance of AO substrates using in vitro systems, leading to de-prioritization of AO substrates from the drug development pipeline to mitigate risk of unexpected and costly in vivo impact. The current study establishes a set of empirical scaling factors as a pragmatic tool to improve predictability of in vivo AO clearance. Developing clinical pharmacology strategies for AO substrates by utilizing mass balance/clinical drug-drug interaction data will help build confidence in fmAO.

Prostate-Specific Membrane Antigen-Targeted Turn-on Probe for Imaging Cargo Release in Prostate Cancer Cells.

The tunable nature of phosphoramidate linkers enables broad applicability as pH-triggered controlled-release platforms, particularly in the context of antibody- and small-molecule-drug conjugates (ADCs and SMDCs), where there remains a need for new linker technology. Herein, we explored in-depth the release of turn-on fluorogenic payloads from a homoserinyl-based phosphoramidate acid-cleavable linker. Kinetics of payload release from the scaffold was observed in buffers representing the pH conditions of systemic circulation, early and late endosomes, and lysosomes. It was found that payload release takes place in two key consecutive steps: (1) P-N bond hydrolysis and (2) spacer immolation. These two steps were found to follow pseudo-first-order kinetics and had opposite dependencies on pH. P-N bond hydrolysis increased with decreasing pH, while spacer immolation was most rapid at physiological pH. Despite the contrasting release kinetics of these two steps, maximal payload release was observed at the mildly acidic pH (5.0-5.5), while minimal payload release occurred at physiological pH. We integrated this phosphoramidate-payload linker system into a PSMA-targeted fluorescent turn-on probe to study the intracellular trafficking and release of a fluorescent payload in PSMA-expressing prostate cancer cells. Results showed excellent turn-on and accumulation of the coumarin payload in the late endosomal and lysosomal compartments of these cells. The release properties of this linker mark it as an attractive alternative in the modular design of ADCs and SMDCs, which demand selective intracellular payload release triggered by the pH changes that accompany intracellular trafficking.

Case Study 12: Roadmap to Quantifying Ago2-Mediated siRNA Metabolic Activation Kinetics.

Therapeutic siRNA is a prodrug that requires Ago2-mediated site-specific hydrolysis of the sense strand before RNA interference can occur. Although this metabolic activation step was first described 15 years ago, the kinetics of this reaction, and its relationship to in vivo siRNA efficacy, remains unexplored in the literature. To provide a roadmap to address these gaps, we describe a liquid chromatography-mass spectrometry method to monitor formation of the cleaved sense-strand metabolites in a reconstituted system. In the absence of metabolite standards for quantitation, we apply an ionization efficiency correction across a panel of siRNA molecules and find that it improves in vitro-in vivo correlation in a transgenic mouse model. Finally, we lay out a case for why Michaelis-Menten kinetics will likely be inadequate for describing Ago2-mediated metabolic activation kinetics, and propose several alternative models that can be solved numerically and applied to quantitated kinetic data when it becomes available.

Effects of alcohol-induced increase in CYP2E1 content in human liver microsomes on the activity and cooperativity of CYP3A4.

We investigate the effect of the alcohol-induced increase in the content of CYP2E1 in human liver microsomes (HLM) on the function of CYP3A4. Membrane incorporation of the purified CYP2E1 into HLM considerably increases the rate of metabolism of 7-benzyloxyquinoline (BQ) and attenuates the homotropic cooperativity observed with this CYP3A4-specific substrate. It also eliminates the activating effect of α-naphthoflavone (ANF) seen in some HLM samples. To probe the physiological relevance of these effects, we compared three pooled preparations of HLM from normal donors (HLM-N) with a pooled preparation from ten heavy alcohol consumers (HLM-A). The composition of the P450 pool in all samples was characterized by the mass-spectrometric determination of 11 cytochrome P450 species. The fractional content of CYP2E1 in HLM-A was from 2.0 to 3.4 times higher than in HLM-N. In contrast, the content of CYP3A4 in HLM-A was the lowest among all samples. Despite that, HLM-A exhibited a much higher metabolism rate and a lower homotropic cooperativity with BQ, similar to CYP2E1-enriched HLM-N. To substantiate the involvement of interactions between CYP2E1 and CYP3A4 in these effects, we probed hetero-association of these proteins in CYP3A4-containing Supersomes™ with a technique employing CYP2E1 labeled with BODIPY-618 maleimide. These experiments evinced the interactions between the two enzymes and revealed an inhibitory effect of ANF on their association. Our results demonstrate that the functional properties of CYP3A4 are fundamentally dependent on the composition of the cytochrome P450 ensemble and suggest a possible impact of chronic alcohol exposure on the pharmacokinetics of drugs metabolized by CYP3A4.

Site-Directed Mutagenesis at the Molybdenum Pterin Cofactor Site of the Human Aldehyde Oxidase: Interrogating the Kinetic Differences Between Human and Cynomolgus Monkey.

The estimation of the drug clearance by aldehyde oxidase (AO) has been complicated because of this enzyme's atypical kinetics and species and substrate specificity. Since human AO (hAO) and cynomolgus monkey AO (mAO) have a 95.1% sequence identity, cynomolgus monkeys may be the best species for estimating AO clearance in humans. Here, O6-benzylguanine (O6BG) and dantrolene were used under anaerobic conditions, as oxidative and reductive substrates of AO, respectively, to compare and contrast the kinetics of these two species through numerical modeling. Whereas dantrolene reduction followed the same linear kinetics in both species, the oxidation rate of O6BG was also linear in mAO and did not follow the already established biphasic kinetics of hAO. In an attempt to determine why hAO and mAO are kinetically distinct, we have altered the hAO V811 and F885 amino acids at the oxidation site adjacent to the molybdenum pterin cofactor to the corresponding alanine and leucine in mAO, respectively. Although some shift to a more monkey-like kinetics was observed for the V811A mutant, five more mutations around the AO cofactors still need to be investigated for this purpose. In comparing the oxidative and reductive rates of metabolism under anaerobic conditions, we have come to the conclusion that despite having similar rates of reduction (4-fold difference), the oxidation rate in mAO is more than 50-fold slower than hAO. This finding implies that the presence of nonlinearity in AO kinetics is dependent upon the degree of imbalance between the rates of oxidation and reduction in this enzyme. SIGNIFICANCE STATEMENT: Although they have as much as 95.1% sequence identity, human and cynomolgus monkey aldehyde oxidase are kinetically distinct. Therefore, monkeys may not be good estimators of drug clearance in humans.

Comparing the immunogenicity of glycosidase-directed resiquimod prodrugs mediated by cancer cell metabolism.

We have recently developed an enzyme-directed immunostimulant (EDI) prodrug motif, which is metabolized to active immunostimulant by cancer cells and, following drug efflux, activates nearby immune cells, resulting in immunogenicity. In this study, we synthesized several EDI prodrugs featuring an imidazoquinoline immunostimulant resiquimod (a Toll-like receptor 7/8 agonist) covalently modified with glycosidase enzyme-directing groups selected from substrates of ß-glucuronidase, α-mannosidase, or ß-galactosidase. We compared the glycosidase-dependent immunogenicity elicited by each EDI in RAW-Blue macrophages following conversion to active immunostimulant by complementary glycosidase. At a cellular level, we examined EDI metabolism across three cancer cell lines (B16 melanoma, TC2 prostate, and 4T1 breast cancer). Comparing the relative immunogenicity elicited by each EDI/cancer cell combination, we found that B16 cells produced the highest EDI prodrug immunogenicity, achieving >95% of that elicited by unmodified resiquimod, followed by TC2 and 4T1 cells (40% and 30%, respectively). Immunogenicity elicited was comparable for a given cell type and independent of the glycosidase substrate in the EDIs or differences in functional glycosidase activity between cell lines. Measuring drug efflux of the immunostimulant payload and efflux protein expression revealed that EDI/cancer cell-mediated immunogenicity was governed by efflux potential of the cancer cells. We determined that, following EDI conversion, immunostimulant efflux occurred through both P-glycoprotein-dependent and P-glycoprotein-independent transport mechanisms. Overall, this study highlights the broad ability of EDIs to couple immunogenicity to the metabolism of many cancers that exhibit drug efflux and suggests that designing future generations of EDIs with immunostimulant payloads that are optimized for drug efflux could be particularly beneficial.

Time Course of Aldehyde Oxidase and Why It Is Nonlinear.

Many promising drug candidates metabolized by aldehyde oxidase (AOX) fail during clinical trial owing to underestimation of their clearance. AOX is species-specific, which makes traditional allometric studies a poor choice for estimating human clearance. Other studies have suggested using half-life calculated by measuring substrate depletion to measure clearance. In this study, we proposed using numerical fitting to enzymatic pathways other than Michaelis-Menten (MM) to avoid missing the initial high turnover rate of product formation. Here, product formation over a 240-minute time course of six AOX substrates-O6-benzylguanine, N-(2-dimethylamino)ethyl)acridine-4-carboxamide, zaleplon, phthalazine, BIBX1382 [N8-(3-Chloro-4-fluorophenyl)-N2-(1-methyl-4-piperidinyl)-pyrimido[5,4-d]pyrimidine-2,8-diamine dihydrochloride], and zoniporide-have been provided to illustrate enzyme deactivation over time to help better understand why MM kinetics sometimes leads to underestimation of rate constants. Based on the data provided in this article, the total velocity for substrates becomes slower than the initial velocity by 3.1-, 6.5-, 2.9-, 32.2-, 2.7-, and 0.2-fold, respectively, in human expressed purified enzyme, whereas the K m remains constant. Also, our studies on the role of reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, show that ROS did not significantly alter the change in enzyme activity over time. Providing a new electron acceptor, 5-nitroquinoline, did, however, alter the change in rate over time for mumerous compounds. The data also illustrate the difficulties in using substrate disappearance to estimate intrinsic clearance.