Using the Dynamic Well-stirred Model to Extrapolate Hepatic Clearance of OATP Substrates without Assuming Albumin-Mediated Hepatic Drug Uptake.

Extrapolating in vivo hepatic clearance from in vitro uptake data is a known challenge, especially for OATP substrates, and the well-stirred model (WSM) commonly yields systematic under-predictions for those anionic drugs hypothetically due to "albumin-mediated hepatic drug uptake". In the present study, we demonstrate that the WSM incorporating the dynamic free fraction (f D), a measure of drug protein binding affinity, performs reasonably well in predicting hepatic clearance of OATP substrates. For a selection of anionic drugs including atorvastatin, fluvastatin, pravastatin, rosuvastatin, pitavastatin, cerivastatin, and repaglinide, this dynamic well-stirred model (dWSM) correctly predicts hepatic plasma clearance within 2-fold error for six out of seven OATP substrates examined. The geometric mean of clearance ratios between the predicted and the observed values falls in the range of 1.21-1.38. As expected, the WSM with unbound fraction (f u) systematically under-predicts hepatic clearance with greater than 2-fold error for five out of seven drugs, and the geometric mean of clearance ratios between the predicted and the observed values is in the range of 0.20-0.29. The results suggest that, despite its simplicity, the dWSM operates well for transporter-mediated uptake clearance, and that clearance under-prediction of OATP substrates may not necessarily be associated with the chemical class of the anionic drugs, nor is it a result of albumin-mediated hepatic drug uptake as currently hypothesized. Instead, the superior prediction power of the dWSM confirms the utility of the dynamic free fraction in clearance prediction and the importance of drug plasma binding kinetics in hepatic uptake clearance. Significance Statement The traditional well-stirred model (WSM) consistently under predicts OATP-mediated hepatic uptake clearance, hypothetically due to the albumin-mediated hepatic drug uptake. In this manuscript, we apply the dynamic well-stirred model (dWSM) to extrapolate hepatic clearance of the OATP substrates, and our results show significant improvements in clearance prediction without assuming "albumin-mediated hepatic drug uptake".

Introducing the Dynamic Well-Stirred Model for Predicting Hepatic Clearance and Extraction Ratio.

The well-stirred model (WSM) incorporating the fraction of unbound drug (fu) to account for the effect of plasma binding on intrinsic clearance has been widely used for predicting hepatic clearance under the assumption that drug protein binding reaches equilibrium instantaneously. Our theoretical analysis reveals that the effect of protein binding on intrinsic clearance is better accounted for with the dynamic free fraction (fD), a measure of drug protein binding affinity, which leads to a putative dynamic well-stirred model (dWSM) without the instantaneous equilibrium assumption. Using recombinant CYP3A4 as the in vitro clearance system, we demonstrate that the binding effect of albumin on the intrinsic clearance of both highly bound midazolam and highly free verapamil is fully corrected by their corresponding fD values, respectively. On the other hand, fu only corrects the binding effect of albumin on the intrinsic clearance of verapamil, and yields severe over-correction of the intrinsic clearance of midazolam. The results suggest that the traditional WSM is suitable for highly free drugs like verapamil but not necessarily for highly bound drugs such as midazolam due to the violation of the instantaneous equilibrium assumption or under-estimating the true free drug concentration. In comparison, the dWSM incorporating fD holds true as long as drug elimination follows steady-state kinetics, and hence, it is more broadly applicable to drugs with different protein binding characteristics. Here we demonstrate with 36 diverse drugs, that the dWSM significantly improves the accuracy of predicting human hepatic clearance and liver extraction ratio from in vitro microsomal clearance data, highlighting the importance of drug plasma protein binding kinetics in addressing the under-prediction of hepatic clearance by the WSM.

Identification of a Discrete Diglucuronide of GDC-0810 in Human Plasma after Oral Administration.

GDC-0810 is a small molecule therapeutic agent having potential to treat breast cancer. In plasma of the first-in-human study, metabolite M2, accounting for 20.7% of total drug-related materials, was identified as a discrete diglucuronide that was absent in rats. Acyl glucuronide M6 and N-glucuronide M4 were also identified as prominent metabolites in human plasma. Several in vitro studies were conducted in incubations of [14C]GDC-0810, synthetic M6 and M4 with liver microsomes, intestinal microsomes, and hepatocytes of different species as well as recombinant UDP-glucuronosyltransferase (UGT) enzymes to further understand the formation of M2. The results suggested that 1) M2 was more efficiently formed from M6 than from M4, and 2) acyl glucuronidation was mainly catalyzed by UGT1A8/7/1 that is highly expressed in the intestines whereas N-glucuronidation was mainly catalyzed by UGT1A4 that is expressed in the human liver. This complicated mechanism presented challenges in predicting M2 formation using human in vitro systems. The absence of M2 and M4 in rats can be explained by low to no expression of UGT1A4 in rodents. M2 could be the first discrete diglucuronide that was formed from both acyl- and N-glucuronidation on a molecule identified in human plasma. SIGNIFICANCE STATEMENT: A discrete diglucuronidation metabolite of GDC-0810, a breast cancer drug candidate, was characterized as a unique circulating metabolite in humans that was not observed in rats or little formed in human in vitro system.

New Methodology for Determining Plasma Protein Binding Kinetics Using an Enzyme Reporter Assay Coupling with High-Resolution Mass Spectrometry.

Determination of drug binding kinetics in plasma is important yet extremely challenging. Accordingly, we introduce "dynamic free fraction" as a new binding parameter describing drug-protein binding kinetics. We demonstrate theoretically and experimentally that the dynamic free fraction can be determined by coupling the drug binding assay with a reporter enzyme in combination with high-resolution mass spectrometry measuring the relative initial steady-state rates of enzymatic reactions in the absence and presence of matrix proteins. This novel and simple methodology circumvents a long-standing challenge inherent in existing methods for determining binding kinetics constants, such as kon and koff, and enables assessment of the impact of protein binding kinetics on pharmaceutical properties of drugs. As demonstrated with nine model drugs, the predicted liver extraction ratio, a measure of efficiency of drug removal by the liver, correlates significantly better to the observed extraction ratio when using the dynamic free fraction (fD) in place of the unbound fraction (fu) of the drug in plasma. Similarly, the in vivo hepatic clearance of these drugs, a measure of liver drug elimination, is highly comparable to the clearance values calculated with the dynamic free fraction (fD), which is markedly better than those calculated with the unbound fraction (fu). In contrast to the prevailing view, these results indicate that protein binding kinetics is an important pharmacokinetic property of a drug. As plasma protein binding is one of the most important drug properties, this new methodology may represent a breakthrough and could have a real impact on the field.

Isotretinoin and its Metabolites Alter mRNA of Multiple Enzyme and Transporter Genes In Vitro, but Downregulation of Organic Anion Transporting Polypeptide Does Not Translate to the Clinic.

Isotretinoin [13-cis-retinoic acid (13cisRA)] is widely used for the treatment of neuroblastoma and acne. It acts via regulating gene transcription through binding to retinoic acid receptors. Yet, the potential for isotretinoin to cause transcriptionally mediated drug-drug interactions (DDIs) has not been fully explored. We hypothesized that isotretinoin and its active metabolites all-trans-retinoic acid (atRA) and 4-oxo-13cisRA would alter the transcription of enzymes and transporters in the human liver via binding to nuclear receptors. The goal of this study was to define the DDI potential of isotretinoin and its metabolites resulting from transcriptional regulation of cytochrome P450 and transporter mRNAs. In human hepatocytes (n = 3), 13cisRA, atRA, and 4-oxo-13cisRA decreased OATP1B1, CYP1A2, CYP2C9, and CYP2D6 mRNA and increased CYP2B6 and CYP3A4 mRNA in a concentration-dependent manner. The EC50 values for OATP1B1 mRNA downregulation ranged from 2 to 110 nM, with maximum effect (Emax ) ranging from 0.17- to 0.54-fold. Based on the EC50 and Emax values and the known circulating concentrations of 13cisRA and its metabolites after isotretinoin dosing, a 55% decrease in OATP1B1 activity was predicted in vivo. In vivo DDI potential was evaluated clinically in participants dosed with isotretinoin for up to 32 weeks using coproporphyrin-I (CP-I) as an OATP1B1 biomarker. CP-I steady-state serum concentrations were unaltered following 2, 8, or 16 weeks of isotretinoin treatment. These data show that isotretinoin and its metabolites alter transcription of multiple enzymes and transporters in vitro, but translation of these changes to in vivo drug-drug interactions requires clinical evaluation for each enzyme. SIGNIFICANCE STATEMENT: Isotretinoin and its metabolites alter the mRNA expression of multiple cytochrome P450s (CYPs) and transporters in human hepatocytes, suggesting that isotretinoin may cause clinically significant drug-drug interactions (DDIs). Despite the observed changes in organic anion transporting polypeptide 1B1 (OATP1B1) mRNA in human hepatocytes, no clinical DDI was observed when measuring a biomarker, coproporphyrin-I. Further work is needed to determine whether these findings can be extrapolated to a lack of a DDI with CYP1A2, CYP2B6, and CYP2C9 substrates.

Application of empirical scalars to enable early prediction of human hepatic clearance using IVIVE in drug discovery: an evaluation of 173 drugs.

The utilization of in vitro data to predict drug pharmacokinetics (PK) in vivo has been a consistent practice in early drug discovery for decades. However, its success is hampered by mispredictions attributed to uncharacterized biological phenomena/experimental artifacts. Predicted drug clearance (CL) from experimental data (i.e. hepatocyte intrinsic clearance: CLint, fraction unbound in plasma: fu,p) is often systematically underpredicted using the well-stirred model (WSM). The objective of this study was to evaluate using empirical scalars in the WSM to correct for CL mispredictions. Drugs (N=28) were used to generate numerical scalars on CLint (α), and fu,p (ß) to minimize the error (AAFE) for CL predictions. These scalars were validated using an additional dataset (N=28 drugs) and applied to a non-redundant AstraZeneca (AZ) dataset available in the literature (N=117 drugs) for a total of 173 compounds. CL predictions using the WSM were improved for most compounds using an α value of 3.66 (~64%<2-fold) compared to no scaling (~46%<2-fold). Similarly, using a ß value of 0.55 or combination of α and ß scalars (values of 1.74 and 0.66, respectively) resulted in a similar improvement in predictions (~64%<2-fold and ~65%<2-fold, respectively). For highly bound compounds (fu,p{less than or equal to}0.01), AAFE was substantially reduced across all scaling methods. Using the ß scalar alone or a combination of α and ß appeared optimal; and produce larger magnitude corrections for highly-bound compounds. Some drugs are still disproportionally mispredicted, however the improvements in prediction error and simplicity of applying these scalars suggests its utility for early-stage CL predictions. Significance Statement In early drug discovery, prediction of human clearance using in vitro experimental data plays an essential role in triaging compounds prior to in vivo studies. These predictions have been systematically underestimated. Here we introduce empirical scalars calibrated on the extent of plasma protein binding that appear to improve clearance prediction across multiple datasets. This approach can be used in early phases of drug discovery prior to the availability of pre-clinical data for early quantitative predictions of human clearance.

Phase I and scintigraphy studies to evaluate safety, tolerability, pharmacokinetics, and lung deposition of inhaled GDC-0214 in healthy volunteers.

Several inflammatory cytokines that promote inflammation and pathogenesis in asthma signal through the Janus kinase 1 (JAK1) pathway. This phase I, randomized, placebo-controlled trial assessed the pharmacokinetics and safety of single and multiple ascending doses up to 15 mg twice daily for 14 days of a JAK1 inhibitor, GDC-0214, in healthy volunteers (HVs; n = 66). Doses were administered with a dry powder, capsule-based inhaler. An accompanying open-label gamma scintigraphy study in HVs examined the lung deposition of a single dose of inhaled Technetium-99m (99m Tc)-radiolabeled GDC-0214. GDC-0214 plasma concentrations were linear and approximately dose-proportional after both single and multiple doses. Peak plasma concentrations occurred at 15-30 min after dosing. The mean apparent elimination half-life ranged from 32 to 56 h across all single and multiple dose cohorts. After single and multiple doses, all adverse events were mild or moderate, and none led to treatment withdrawal. There was no clear evidence of systemic toxicity due to JAK1 inhibition, and systemic exposure was low, with plasma concentrations at least 15-fold less than the plasma protein binding-corrected IC50 of JAK1 at the highest dose. Scintigraphy showed that approximately 50% of the emitted dose of radiolabeled GDC-0214 was deposited in the lungs and was distributed well to the peripheral airways. 99m Tc-radiolabeled GDC-0214 (1 mg) exhibited a mean plasma Cmax similar to that observed in phase I at the same dose level. Overall, inhaled GDC-0214 exhibited pharmacokinetic properties favorable for inhaled administration.

Inhaled JAK inhibitor GDC-0214 reduces exhaled nitric oxide in patients with mild asthma: A randomized, controlled, proof-of-activity trial.

BACKGROUND: The Janus kinase (JAK) pathway mediates the activity of many asthma-relevant cytokines, including IL-4 and IL-13. GDC-0214 is a potent, inhaled, small-molecule JAK inhibitor being developed for the treatment of asthma. OBJECTIVE: We sought to determine whether GDC-0214 reduces fractional exhaled nitric oxide (Feno), a JAK1-dependent biomarker of airway inflammation, in patients with mild asthma. METHODS: We conducted a double-blind, randomized, placebo-controlled, phase 1 proof-of-activity study in adults with mild asthma and Feno higher than 40 parts per billion (ppb). Subjects were randomized 2:1 (GDC-0214:placebo) into 4 sequential ascending-dose cohorts (1 mg once daily [QD], 4 mg QD, 15 mg QD, or 15 mg twice daily). All subjects received 4 days of blinded placebo, then 10 days of either active drug or placebo. The primary outcome was placebo-corrected percent reduction in Feno from baseline to day 14. Baseline was defined as the average Feno during the blinded placebo period. Pharmacokinetics, safety, and tolerability were also assessed. RESULTS: Thirty-six subjects (mean age, 28 years; 54% females) were enrolled. Mean Feno at baseline across all subjects was 93 ± 43 ppb. At day 14, placebo-corrected difference in Feno was -23% (95% CI, -37.3 to -9) for 15 mg QD and -42% (95% CI, -57 to -27.4) for 15 mg twice daily. Higher plasma exposure was associated with greater Feno reduction. No dose-limiting adverse events, serious adverse events, or treatment discontinuations occurred. There were no major imbalances in adverse events or laboratory findings, or evidence of systemic JAK inhibition. CONCLUSIONS: GDC-0214, an inhaled JAK inhibitor, caused dose-dependent reductions in Feno in mild asthma and was well tolerated without evidence of systemic toxicity.

Inter-individual and inter-regional variations in enteric drug metabolizing enzyme activities: Results with cryopreserved human intestinal mucosal epithelia (CHIM) from the small intestines of 14 donors.

We have previously reported successful isolation and cryopreservation of human intestinal mucosa (CHIM) with retention of viability and drug metabolizing enzyme activities. Here we report the results of the quantification of drug metabolizing enzyme activities in CHIM from different regions of the small intestines from 14 individual donors. CHIM were isolated from the duodenum, jejunum, and ileum of 10 individuals, and from 10 consecutive 12-inch segments starting from the pyloric sphincter of human small intestines from four additional individuals. P450 and non-P450 drug metabolizing enzyme activities (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A, UGT, SULT, FMO, MAO, AO, NAT1, and NAT2) were quantified via incubation with pathway-selective substrates. Quantifiable activities were observed for all pathways except for CYP2A6. Comparison of the duodenum, jejunum, and ileum in 10 donors shows jejunum had higher activities for CYP2C9, CYP3A, UGT, SULT, MAO, and NAT1. Further definition of regional variations with CHIM from ten 12-inch segments of the proximal small intestine shows that the segments immediately after the first 12-inch segment (duodenum) had the highest activity for most of the drug metabolizing enzymes but with substantial differences among the four donors. Our overall results demonstrate that there are substantial individual differences in drug metabolizing enzymes and that jejunum, especially the regions immediately after the duodenum, had the highest drug metabolizing enzyme activities.

Strategy for Determining the Free Fraction of Labile Covalent Modulators in Plasma Using Equilibrium Dialysis.

Determination of free drug fraction (fu) in plasma can be challenging for labile covalent modulators due to the off-target reactivity of chemical warheads to matrix proteins. The resulting poor drug recovery yields low confidence in fu. Two approaches using diluted plasma and low temperature (4 & 20 °C) for equilibrium dialysis (ED) have been investigated using covalent modulators including osimertinib, ibrutinib, rociletinib, afatinib, neratinib and acalabrutinib. Our data indicate that stability of covalent modulators in plasma varies in different species, and drug depletion may lead to overestimation of fu if true equilibrium is not reached. Additionally, although ED at low temperature improves the recovery of covalent modulators, the impact of low temperature may lead to underestimate of fu. Overall, ED using diluted plasma is a preferred method because of its faster equilibrium, improved recovery and free of temperature effect on fu. If low temperature ED must be used for extremely labile compounds, precaution must be taken to ensure no temperature dependence of fu in plasma. Nevertheless, an orthogonal ED approach is recommended for labile covalent modulators to confirm the true equilibrium and impact of temperature on fu. Additionally, this strategy can be used for determining fu of other liable compounds.

Strategies to Mitigate the Bioactivation of Aryl Amines.

The bioactivation of xenobiotics to yield reactive metabolites can lead to tolerability and toxicity concerns within a drug discovery program. Development of strategies for mitigating the metabolic liability of commonly encountered toxicophores, such as anilines, relies on an understanding of the relative tendency of these functionalities to undergo bioactivation. In this report, we present the first systematic study of the structure-activity relationships of the bioactivation of aryl amine fragments (molecular weight < 250 Da) using a glutathione (GSH) trapping assay in the presence of human liver microsomes and the reduced form of nicotinamide adenine dinucleotide phosphate. This study demonstrates that conversion of anilines to nitrogen-containing heteroarylamines results in a lower abundance of GSH conjugates in the order phenyl > pyrimidine ≈ pyridine > pyridazine. Introduction of electron-withdrawing functionality on the aromatic ring had a less pronounced effect on the extent of GSH conjugation. Examination of more drug-like compounds sourced from in-house drug discovery programs revealed similar trends in bioactivation between matched pairs containing (hetero)aryl amines. This study provides medicinal chemists with insights and qualitative guidance for the minimization of risks related to aryl amine metabolism.

Microphysiological systems for ADME-related applications: current status and recommendations for system development and characterization.

Over the last decade, progress has been made on the development of microphysiological systems (MPS) for absorption, distribution, metabolism, and excretion (ADME) applications. Central to this progress has been proof of concept data generated by academic and industrial institutions followed by broader characterization studies, which provide evidence for scalability and applicability to drug discovery and development. In this review, we describe some of the advances made for specific tissue MPS and outline the desired functionality for such systems, which are likely to make them applicable for practical use in the pharmaceutical industry. Single organ MPS platforms will be valuable for modelling tissue-specific functions. However, dynamic organ crosstalk, especially in the context of disease or toxicity, can only be obtained with the use of inter-linked MPS models which will enable scientists to address questions at the intersection of pharmacokinetics (PK) and efficacy, or PK and toxicity. In the future, successful application of MPS platforms that closely mimic human physiology may ultimately reduce the need for animal models to predict ADME outcomes and decrease the overall risk and cost associated with drug development.

Complex DDI by Fenebrutinib and the Use of Transporter Endogenous Biomarkers to Elucidate the Mechanism of DDI.

Mechanistic understanding of complex clinical drug-drug interactions (DDIs) with potential involvement of multiple elimination pathways has been challenging, especially given the general lack of specific probe substrates for transporters. Here, we conducted a clinical DDI study to evaluate the interaction potential of fenebrutinib using midazolam (MDZ; CYP3A), simvastatin (CYP3A and OATP1B), and rosuvastatin (BCRP and OATP1B) as probe substrates. Fenebrutinib (200 mg) increased the area under the curve (AUC) of these probe substrates twofold to threefold. To evaluate the mechanism of the observed DDIs, we measured the concentration of coproporphyrin I (CP-I) and coproporphyrin III (CP-III), endogenous biomarkers of OATP1B. There was no change in CP-I or CP-III levels with fenebrutinib, suggesting that the observed DDIs were caused by inhibition of CYP3A and BCRP rather than OATP1B, likely due to increased bioavailability. This is the first published account using an endogenous transporter biomarker to understand the mechanism of complex DDIs involving multiple elimination pathways.

Perspectives from the Innovation and Quality Consortium Induction Working Group on Factors Impacting Clinical Drug-Drug Interactions Resulting from Induction: Focus on Cytochrome 3A Substrates.

A recent publication from the Innovation and Quality Consortium Induction Working Group collated a large clinical data set with the goal of evaluating the accuracy of drug-drug interaction (DDI) prediction from in vitro data. Somewhat surprisingly, comparison across studies of the mean- or median-reported area under the curve ratio showed appreciable variability in the magnitude of outcome. This commentary explores the possible drivers of this range of outcomes observed in clinical induction studies. While recommendations on clinical study design are not being proposed, some key observations were informative during the aggregate analysis of clinical data. Although DDI data are often presented using median data, individual data would enable evaluation of how differences in study design, baseline expression, and the number of subjects contribute. Since variability in perpetrator pharmacokinetics (PK) could impact the overall DDI interpretation, should this be routinely captured? Maximal induction was typically observed after 5-7 days of dosing. Thus, when the half-life of the inducer is less than 30 hours, are there benefits to a more standardized study design? A large proportion of CYP3A4 inducers were also CYP3A4 inhibitors and/or inactivators based on in vitro data. In these cases, using CYP3A selective substrates has limitations. More intensive monitoring of changes in area under the curve over time is warranted. With selective CYP3A substrates, the net effect was often inhibition, whereas less selective substrates could discern induction through mechanisms not susceptible to inhibition. The latter included oral contraceptives, which raise concerns of reduced efficacy following induction. Alternative approaches for modeling induction, such as applying biomarkers and physiologically based pharmacokinetic modeling (PBPK), are also considered. SIGNIFICANCE STATEMENT: The goal of this commentary is to stimulate discussion on whether there are opportunities to optimize clinical drug-drug interaction study design. The overall aim is to reduce, understand and contextualize the variability observed in the magnitude of induction across reported clinical studies. A large clinical CYP3A induction dataset was collected and further analyzed to identify trends and gaps. Reporting individual victim PK data, characterizing perpetrator PK and including additional PK assessments for mixed-mechanism perpetrators may provide insights into how these factors impact differences observed in clinical outcomes. The potential utility of biomarkers and PBPK modeling are discussed in considering future directions.

Drug Concentration Asymmetry in Tissues and Plasma for Small Molecule-Related Therapeutic Modalities.

The well accepted "free drug hypothesis" for small-molecule drugs assumes that only the free (unbound) drug concentration at the therapeutic target can elicit a pharmacologic effect. Unbound (free) drug concentrations in plasma are readily measurable and are often used as surrogates for the drug concentrations at the site of pharmacologic action in pharmacokinetic-pharmacodynamic analysis and clinical dose projection in drug discovery. Furthermore, for permeable compounds at pharmacokinetic steady state, the free drug concentration in tissue is likely a close approximation of that in plasma; however, several factors can create and maintain disequilibrium between the free drug concentration in plasma and tissue, leading to free drug concentration asymmetry. These factors include drug uptake and extrusion mechanisms involving the uptake and efflux drug transporters, intracellular biotransformation of prodrugs, membrane receptor-mediated uptake of antibody-drug conjugates, pH gradients, unique distribution properties (covalent binders, nanoparticles), and local drug delivery (e.g., inhalation). The impact of these factors on the free drug concentrations in tissues can be represented by K p,uu, the ratio of free drug concentration between tissue and plasma at steady state. This review focuses on situations in which free drug concentrations in tissues may differ from those in plasma (e.g., K p,uu > or <1) and discusses the limitations of the surrogate approach of using plasma-free drug concentration to predict free drug concentrations in tissue. This is an important consideration for novel therapeutic modalities since systemic exposure as a driver of pharmacologic effects may provide limited value in guiding compound optimization, selection, and advancement. Ultimately, a deeper understanding of the relationship between free drug concentrations in plasma and tissues is needed.

Discovery of a class of highly potent Janus Kinase 1/2 (JAK1/2) inhibitors demonstrating effective cell-based blockade of IL-13 signaling.

Disruption of interleukin-13 (IL-13) signaling with large molecule antibody therapies has shown promise in diseases of allergic inflammation. Given that IL-13 recruits several members of the Janus Kinase family (JAK1, JAK2, and TYK2) to its receptor complex, JAK inhibition may offer an alternate small molecule approach to disrupting IL-13 signaling. Herein we demonstrate that JAK1 is likely the isoform most important to IL-13 signaling. Structure-based design was then used to improve the JAK1 potency of a series of previously reported JAK2 inhibitors. The ability to impede IL-13 signaling was thereby significantly improved, with the best compounds exhibiting single digit nM IC50's in cell-based assays dependent upon IL-13 signaling. Appropriate substitution was further found to influence inhibition of a key off-target, LRRK2. Finally, the most potent compounds were found to be metabolically labile, which makes them ideal scaffolds for further development as topical agents for IL-13 mediated diseases of the lungs and skin (for example asthma and atopic dermatitis, respectively).

Does In Vitro Cytochrome P450 Downregulation Translate to In Vivo Drug-Drug Interactions? Preclinical and Clinical Studies With 13-cis-Retinoic Acid.

All-trans-retinoic acid (atRA) downregulates cytochrome P450 (CYP)2D6 in several model systems. The aim of this study was to determine whether all active retinoids downregulate CYP2D6 and whether in vitro downregulation translates to in vivo drug-drug interactions (DDIs). The retinoids atRA, 13cisRA, and 4-oxo-13cisRA all decreased CYP2D6 mRNA in human hepatocytes in a concentration-dependent manner. The in vitro data predicted ~ 50% decrease in CYP2D6 activity in humans after dosing with 13cisRA. However, the geometric mean area under plasma concentration-time curve (AUC) ratio for dextromethorphan between treatment and control was 0.822, indicating a weak induction of dextromethorphan clearance following 13cisRA treatment. Similarly, in mice treatment with 4-oxo-13cisRA-induced mRNA expression of multiple mouse Cyp2d genes. In comparison, a weak induction of CYP3A4 in human hepatocytes translated to a weak in vivo induction of CYP3A4. These data suggest that in vitro CYP downregulation may not translate to in vivo DDIs, and better understanding of the mechanisms of CYP downregulation is needed.

Improving Confidence in the Determination of Free Fraction for Highly Bound Drugs Using Bidirectional Equilibrium Dialysis.

Equilibrium dialysis has been widely used for the measurement of the fraction of unbound drug (fu) in plasma, but it suffers from the accuracy and reliability for low fu values. To address this concern, an orthogonal approach, called the bidirectional equilibrium dialysis, is described to simultaneously measure a pair of fu values for each drug based on equilibration in 2 opposite dialysis directions: from plasma to buffer (fu,p/b) and from buffer to plasma (fu,b/p). Hypothetically, if true equilibrium is attained in both dialysis directions, the measured fu,b/p and fu,p/b values for a given drug should converge, and thus, the ratio of fu,b/p to fu,p/b becomes unity (1.0). Thus, the ratio can be used as a tangible readout for data reliability. This methodology has been extensively tested in the present study using various drugs with distinct plasma binding characteristics. Our results clearly showed that low fu values (<0.01) could be reliably determined and verified using either the standard or dilution bidirectional equilibrium dialysis method for some known highly bound drugs; for extensively bound drugs with high logD7.4, such as montelukast, bedaquiline, and venetoclax, only a range of fu can be reported with confidence because of uncertainty in the true equilibrium.

Lung-restricted inhibition of Janus kinase 1 is effective in rodent models of asthma.

Preclinical and clinical evidence indicates that a subset of asthma is driven by type 2 cytokines such as interleukin-4 (IL-4), IL-5, IL-9, and IL-13. Additional evidence predicts pathogenic roles for IL-6 and type I and type II interferons. Because each of these cytokines depends on Janus kinase 1 (JAK1) for signal transduction, and because many of the asthma-related effects of these cytokines manifest in the lung, we hypothesized that lung-restricted JAK1 inhibition may confer therapeutic benefit. To test this idea, we synthesized iJak-381, an inhalable small molecule specifically designed for local JAK1 inhibition in the lung. In pharmacodynamic models, iJak-381 suppressed signal transducer and activator of transcription 6 activation by IL-13. Furthermore, iJak-381 suppressed ovalbumin-induced lung inflammation in both murine and guinea pig asthma models and improved allergen-induced airway hyperresponsiveness in mice. In a model driven by human allergens, iJak-381 had a more potent suppressive effect on neutrophil-driven inflammation compared to systemic corticosteroid administration. The inhibitor iJak-381 reduced lung pathology, without affecting systemic Jak1 activity in rodents. Our data show that local inhibition of Jak1 in the lung can suppress lung inflammation without systemic Jak inhibition in rodents, suggesting that this strategy might be effective for treating asthma.

Considerations from the Innovation and Quality Induction Working Group in Response to Drug-Drug Interaction Guidances from Regulatory Agencies: Focus on CYP3A4 mRNA In Vitro Response Thresholds, Variability, and Clinical Relevance.

The Innovation and Quality Induction Working Group presents an assessment of best practice for data interpretation of in vitro induction, specifically, response thresholds, variability, application of controls, and translation to clinical risk assessment with focus on CYP3A4 mRNA. Single concentration control data and Emax/EC50 data for prototypical CYP3A4 inducers were compiled from many human hepatocyte donors in different laboratories. Clinical CYP3A induction and in vitro data were gathered for 51 compounds, 16 of which were proprietary. A large degree of variability was observed in both the clinical and in vitro induction responses; however, analysis confirmed in vitro data are able to predict clinical induction risk. Following extensive examination of this large data set, the following recommendations are proposed. a) Cytochrome P450 induction should continue to be evaluated in three separate human donors in vitro. b) In light of empirically divergent responses in rifampicin control and most test inducers, normalization of data to percent positive control appears to be of limited benefit. c) With concentration dependence, 2-fold induction is an acceptable threshold for positive identification of in vitro CYP3A4 mRNA induction. d) To reduce the risk of false positives, in the absence of a concentration-dependent response, induction ≥ 2-fold should be observed in more than one donor to classify a compound as an in vitro inducer. e) If qualifying a compound as negative for CYP3A4 mRNA induction, the magnitude of maximal rifampicin response in that donor should be ≥ 10-fold. f) Inclusion of a negative control adds no value beyond that of the vehicle control.