Membrane Permeability in a Large Macrocyclic Peptide Driven by a Saddle-Shaped Conformation.

The effort to modulate challenging protein targets has stimulated interest in ligands that are larger and more complex than typical small-molecule drugs. While combinatorial techniques such as mRNA display routinely produce high-affinity macrocyclic peptides against classically undruggable targets, poor membrane permeability has limited their use toward primarily extracellular targets. Understanding the passive membrane permeability of macrocyclic peptides would, in principle, improve our ability to design libraries whose leads can be more readily optimized against intracellular targets. Here, we investigate the permeabilities of over 200 macrocyclic 10-mers using the thioether cyclization motif commonly found in mRNA display macrocycle libraries. We identified the optimal lipophilicity range for achieving permeability in thioether-cyclized 10-mer cyclic peptide-peptoid hybrid scaffolds and showed that permeability could be maintained upon extensive permutation in the backbone. In one case, changing a single amino acid from d-Pro to d-NMe-Ala, representing the loss of a single methylene group in the side chain, resulted in a highly permeable scaffold in which the low-dielectric conformation shifted from the canonical cross-beta geometry of the parent compounds into a novel saddle-shaped fold in which all four backbone NH groups were sequestered from the solvent. This work provides an example by which pre-existing physicochemical knowledge of a scaffold can benefit the design of macrocyclic peptide mRNA display libraries, pointing toward an approach for biasing libraries toward permeability by design. Moreover, the compounds described herein are a further demonstration that geometrically diverse, highly permeable scaffolds exist well beyond conventional drug-like chemical space.

Preclinical characterization of the absorption and disposition of the brain penetrant PI3K/mTOR inhibitor paxalisib and prediction of its pharmacokinetics and efficacy in human.

Glioblastoma multiforme (GBM) is the most common primary brain tumour in adults. Available treatments have not markedly improved patient survival in the last twenty years. However, genomic investigations have showed that the PI3K pathway is frequently altered in this glioma, making it a potential therapeutic target.Paxalisib is a brain penetrant PI3K/mTOR inhibitor (mouse Kp,uu 0.31) specifically developed for the treatment of GBM. We characterised the preclinical pharmacokinetics and efficacy of paxalisib and predicted its pharmacokinetics and efficacious dose in humans.Plasma protein binding of paxalisib was low, with the fraction unbound ranging from 0.25 to 0.43 across species. The hepatic clearance of paxalisib was predicted to be low in mice, rats, dogs and humans, and high in monkeys, from hepatocytes incubations. The plasma clearance was low in mice, moderate in rats and high in dogs and monkeys. Oral bioavailability ranged from 6% in monkeys to 76% in rats.The parameters estimated from the pharmacokinetic/pharmacodynamic modelling of the efficacy in the subcutaneous U87 xenograft model combined with the human pharmacokinetics profile predicted by PBPK modelling suggested that a dose of 56 mg may be efficacious in humans. Paxalisib is currently tested in Phase III clinical trials.

Ternary complex dissociation kinetics contribute to mutant-selective EGFR degradation.

Targeted degradation of proteins by chimeric heterobifunctional degraders has emerged as a major drug discovery paradigm. Despite the increased interest in this approach, the criteria dictating target protein degradation by a degrader remain poorly understood, and potent target engagement by a degrader does not strongly correlate with target degradation. In this study, we present the biochemical characterization of an epidermal growth factor receptor (EGFR) degrader that potently binds both wild-type and mutant EGFR, but only degrades EGFR mutant variants. Mechanistic studies reveal that ternary complex half-life strongly correlates with processive ubiquitination with purified components and mutant-selective degradation in cells. We present cryoelectron microscopy and hydrogen-deuterium exchange mass spectroscopy data on wild-type and mutant EGFR ternary complexes, which demonstrate that potent target degradation can be achieved in the absence of stable compound-induced protein-protein interactions. These results highlight the importance of considering target conformation during degrader development as well as leveraging heterobifunctional ligand binding kinetics to achieve robust target degradation.

Evaluation of bottom-up modeling of the blood-brain barrier to improve brain penetration prediction via physiologically based pharmacokinetic modeling.

Predicting the brain penetration of drugs has been notoriously difficult; however, recently, permeability-limited brain models have been constructed. Lead optimization for central nervous system compounds often focuses on compounds that have low transporter efflux, where passive permeability could be a main driver in determining cerebrospinal fluid (CSF)/brain concentrations. The main objective of this study was to evaluate the translatability of passive permeability data generated from different in vitro systems and its impact on the prediction of human CSF/brain concentrations using physiologically-based pharmacokinetic (PBPK) modeling. In vitro data were generated using gMDCK and parallel artificial membrane permeability assay-blood-brain barrier for comparison and predictions using a quantitative structure-activity relationship model were also evaluated. PBPK modeling was then performed for seven compounds with moderate-high permeability and a range of efflux in vitro, and the CSF/brain mass concentrations and Kpuu were reasonably predicted. This work provides the first step of a promising approach using bottom-up PBPK modeling for CSF/brain penetration prediction to support lead optimization and clinical candidate selection.

Elucidating a Potential Mechanism of Permeability Enhancer Sodium N-[8-(2-hydroxybenzoyl) amino] Caprylate in Rats: Evidence of Lymphatic Absorption of Cyanocobalamin using the Mesenteric Lymph Duct Cannulated Rat.

Oral administration is the most popular and convenient route for drug delivery, yet the success of oral drug delivery is dependent on the ADME properties of the drug. Among those ADME properties, permeability is considered one of the key attributes for successful oral drug absorption. Hence, the utilization of permeability enhancers to improve drug oral absorption is an important area of research in drug delivery. A multitude of data suggests that sodium N-[8-(2-hydroxybenzoyl) amino] caprylate (SNAC) is an effective permeability enhancer. Despite its success, the mechanism of how SNAC works to enhance the oral absorption of compounds is poorly understood. To better understand how SNAC worked, we investigated the hypothesis of SNAC promotes lymphatic absorption of target compounds. In this study, cyanocobalamin was used as the model compound and mesenteric lymph duct cannulated rats were used to investigate its absorption with or without SNAC. The present study demonstrated that SNAC enhanced the lymphatic absorption of cyanocobalamin when the two were co-dosed in rats. Furthermore, levels of SNAC in lymph fluid and the systemic circulation were higher when co-dosed with cyanocobalamin.

Turning the Other Cheek: Influence of the cis-Tetrafluorocyclohexyl Motif on Physicochemical and Metabolic Properties.

The targeted introduction of substituents in order to tailor a molecule's pharmacologic, physicochemical, and metabolic properties has long been of interest to medicinal chemists. The all-cis tetrafluorocyclohexyl motif-dubbed Janus face, due to its electrostatically polarized cyclohexyl ring-represents one such example where chemists might incorporate a metabolically stable, polar, lipocompatible motif. To better understand its potential utility, we have synthesized three series of matched molecular pairs (MMPs) where each MMP differs only in the cyclohexane unit, i.e., with a tetrafluorocyclohexyl or a standard cyclohexyl motif. With the introduction of the facially polarized all-cis tetrafluorocyclohexyl ring, the resulting compounds have significantly modified physicochemical properties (e.g., kinetic solubility, lipophilicity and permeability) and metabolic stabilities. These results further speak to the promise of this substituent as a tactic to improve the drug-like properties of molecules.

An Integrated Strategy for Assessing the Metabolic Stability and Biotransformation of Macrocyclic Peptides in Drug Discovery toward Oral Delivery.

Macrocyclic peptides (MCPs) are an emerging class of promising drug modalities that can be used to interrogate hard-to-drug ("undruggable") targets. However, their poor intestinal stability is one of the major liabilities or obstacles for oral drug delivery. We therefore investigated the metabolic stability and biotransformation of MCPs via a systematic approach and established an integrated in vitro assay strategy to facilitate MCP drug discovery, with a focus on oral delivery liabilities. A group of diverse MCPs were incubated with representative matrices, including simulated intestinal fluid with pancreatin (SIFP), human enterocytes, liver S9 fractions, liver lysosomes, plasma, and recombinant enzymes. The results revealed that the stability and biotransformation of MCPs varied, with the major metabolic pathways identified in different matrices. Under the given conditions, the selected MCPs generally showed better stability in plasma compared to that in SIFP. Our data suggest that pancreatic enzymes act as the primary metabolic barrier for the oral delivery of MCPs, mainly through hydrolysis of their backbone amide bonds. Whereas in enterocytes, multiple metabolic pathways appeared to be involved and resulted in metabolic reactions such as oxidation and reduction in addition to hydrolysis. Further studies suggested that lysosomal peptidase cathepsin B could be a major enzyme responsible for the cleavage of side-chain amide bonds in lysosomes. Collectively, we developed and implemented an integrated assay for assessing the metabolic stability and biotransformation of MCPs for compound screening in the discovery stage toward oral delivery. The proposed question-driven assay cascade can provide biotransformation insights that help to guide and facilitate lead candidate selection and optimization.

RTK-Dependent Inducible Degradation of Mutant PI3Kα Drives GDC-0077 (Inavolisib) Efficacy.

PIK3CA is one of the most frequently mutated oncogenes; the p110a protein it encodes plays a central role in tumor cell proliferation. Small-molecule inhibitors targeting the PI3K p110a catalytic subunit have entered clinical trials, with early-phase GDC-0077 studies showing antitumor activity and a manageable safety profile in patients with PIK3CA-mutant breast cancer. However, preclinical studies have shown that PI3K pathway inhibition releases negative feedback and activates receptor tyrosine kinase signaling, reengaging the pathway and attenuating drug activity. Here we discover that GDC-0077 and taselisib more potently inhibit mutant PI3K pathway signaling and cell viability through unique HER2-dependent mutant p110a degradation. Both are more effective than other PI3K inhibitors at maintaining prolonged pathway suppression. This study establishes a new strategy for identifying inhibitors that specifically target mutant tumors by selective degradation of the mutant oncoprotein and provide a strong rationale for pursuing PI3Kα degraders in patients with HER2-positive breast cancer. SIGNIFICANCE: The PI3K inhibitors GDC-0077 and taselisib have a unique mechanism of action; both inhibitors lead to degradation of mutant p110a protein. The inhibitors that have the ability to trigger specific degradation of mutant p110a without significant change in wild-type p110a protein may result in improved therapeutic index in PIK3CA-mutant tumors.See related commentary by Vanhaesebroeck et al., p. 20.This article is highlighted in the In This Issue feature, p. 1.

Evaluation of Ipatasertib Interactions with Itraconazole and Coproporphyrin I and III in a Single Drug Interaction Study in Healthy Subjects.

Ipatasertib is a pan-AKT inhibitor in development for the treatment of cancer. Ipatasertib was metabolized by CYP3A4 to its major metabolite, M1 (G-037720), and was a P-gp substrate and OATP1B1/1B3 inhibitor in vitro. A phase I drug-drug interaction (DDI) study (n = 15) was conducted in healthy subjects to evaluate the effect of itraconazole (200-mg solution QD, 4 days), a strong CYP3A4 and P-gp inhibitor, on pharmacokinetics of ipatasertib (100-mg single dose). Itraconazole increased the Cmax and AUC0 -∞ of ipatasertib by 2.3- and 5.5-fold, respectively, increased the half-life by 53%, and delayed the tmax by 1 hour. The Cmax and AUC0-72h of its metabolite M1 (G-037720) reduced by 91% and 68%, respectively. This study confirmed that CYP3A4 plays a major role in ipatasertib clearance. Furthermore, the interaction of ipatasertib with coproporphyrin (CP) I and CPIII, the two endogenous substrates of OATP1B1/1B3, was evaluated in this study. CPI and CPIII plasma levels were unchanged in the presence of ipatasertib, both at exposures of 100 mg and at higher exposures in combination with itraconazole. This indicated no in vivo inhibition of OATP1B1/1B3 by ipatasertib. Additionally, it was shown that CPI and CPIII were not P-gp substrates in vitro, and itraconazole had no effect on CPI and CPIII concentrations in vivo. The latter is an important finding because it will simplify interpretation of future DDI studies using CPI/CPIII as OATP1B1/1B3 biomarkers. SIGNIFICANCE STATEMENT: This drug-drug interaction study in healthy volunteers demonstrated that CYP3A4 plays a major role in ipatasertib clearance, and that ipatasertib is not an organic anion transporting polypeptide 1B1/1B3 inhibitor. Furthermore, it was demonstrated that itraconazole, an inhibitor of CYP3A4 and several transporters, did not affect CPI/CPIII levels in vivo. This increases the understanding and application of these endogenous substrates as well as itraconazole in complex drug interaction studies.

Calibrating the In Vitro-In Vivo Correlation for OATP-Mediated Drug-Drug Interactions with Rosuvastatin Using Static and PBPK Models.

Organic anion-transporting polypeptide (OATP) 1B1/3-mediated drug-drug interaction (DDI) potential is evaluated in vivo with rosuvastatin (RST) as a probe substrate in clinical studies. We calibrated our assay with RST and estradiol 17-ß-D-glucuronide (E217ßG)/cholecystokinin-8 (CCK8) as in vitro probes for qualitative and quantitative prediction of OATP1B-mediated DDI potential for RST. In vitro OATP1B1/1B3 inhibition using E217ßG and CCK8 yielded higher area under the curve (AUC) ratio (AUCR) values numerically with the static model, but all probes performed similarly from a qualitative cutoff-based prediction, as described in regulatory guidances. However, the magnitudes of DDI were not captured satisfactorily. Considering that clearance of RST is also mediated by gut breast cancer resistance protein (BCRP), inhibition of BCRP was also incorporated in the DDI prediction if the gut inhibitor concentrations were 10 × IC50 for BCRP inhibition. This combined static model closely predicted the magnitude of RST DDI with root-mean-square error values of 0.767-0.812 and 1.24-1.31 with and without BCRP inhibition, respectively, for in vitro-in vivo correlation of DDI. Physiologically based pharmacokinetic (PBPK) modeling was also used to simulate DDI between RST and rifampicin, asunaprevir, and velpatasvir. Predicted AUCR for rifampicin and asunaprevir was within 1.5-fold of that observed, whereas that for velpatasvir showed a 2-fold underprediction. Overall, the combined static model incorporating both OATP1B and BCRP inhibition provides a quick and simple mathematical approach to quantitatively predict the magnitude of transporter-mediated DDI for RST for routine application. PBPK complements the static model and provides a framework for studying molecules when a dynamic model is needed. SIGNIFICANCE STATEMENT: Using 22 drugs, we show that a static model for organic anion-transporting polypeptide (OATP) 1B1/1B3 inhibition can qualitatively predict potential for drug-drug interaction (DDI) using a cutoff-based approach, as in regulatory guidances. However, consideration of both OATP1B1/3 and gut breast cancer resistance protein inhibition provided a better prediction of the magnitude of the transporter-mediated DDI of these inhibitors with rosuvastatin. Based on these results, we have proposed an empirical mechanistic-static approach for a more reliable prediction of transporter-mediated DDI liability with rosuvastatin that drug development teams can leverage.

Understanding the Effect of Hydroxypropyl-ß-Cyclodextrin on Fenebrutinib Absorption in an Itraconazole-Fenebrutinib Drug-Drug Interaction Study.

Cyclodextrins are widely used pharmaceutical excipients, particularly for insoluble compounds dosed orally, such as the oral solution of itraconazole, which is frequently used in clinical drug-drug interaction studies to inhibit cytochrome P450 3A. Since cyclodextrins act by forming inclusion complexes with their coformulated drug, they could have an unintended consequence of affecting absorption if they form a strong complex with the potential victim drug in an itraconazole drug-drug interaction study. This observation was made in a drug-drug interaction study with the Bruton's tyrosine kinase (BTK) inhibitor fenebrutinib and itraconazole, in which, relative to the control group, the expected increase in fenebrutinib maximum plasma concentration (Cmax ) was not observed in the itraconazole group, and a delay in time to reach maximum plasma concentration (Tmax ) was observed in the itraconazole group. The in vitro binding constant between fenebrutinib and hydroxypropyl-ß-cyclodextrin was determined to be 2 × 105  M-1 , and the apparent permeability of fenebrutinib across a Madin-Darby canine kidney cell monolayer decreased in a cyclodextrin concentration-dependent manner. This observation was confirmed in vivo, in a pentagastrin-pretreated dog model, in which fenebrutinib was administered with or without cyclodextrin; a reduction in Cmax , a prolonged Tmax , and increased fenebrutinib recovery in feces replicated the previous observation in healthy volunteers and supported the hypothesis that complexation with cyclodextrin decreased rate and extent of fenebrutinib absorption. Physiologically-based pharmacokinetic modeling was used to translate the in vitro effect of cyclodextrin on fenebrutinib apparent permeability to the in vivo effect on absorption, which was then confirmed using the in vivo dog pharmacokinetic data.

Evaluating the Pharmacokinetics and Systemic Effects of a Permeability Enhancer Sodium N-[8-(2-hydroxybenzoyl)amino] Caprylate in Rats.

Oral administration is the preferred route for drug delivery and its success is highly dependent on a compound's ADME properties, of which, permeability plays a major role. Therefore, permeability enhancers are an attractive area of research in the pharmaceutical industry. Recent data suggest that sodium N-[8-(2-hydroxybenzoyl) amino] caprylate (SNAC) is an effective permeability enhancer, yet the pharmacokinetic (PK) and systemic effects of SNAC are poorly understood, specifically its oral bioavailability and systemic effects on distribution, which could influence the safety of certain drugs. To answer these questions, both in vitro and in vivo studies were conducted. Of 3 permeability enhancers (SNAC, 4-CNAB, and 5-CNAC), SNAC was found to have the greatest effect on the absorption of cyanocobalamin in rats. It was also found that SNAC is orally bioavailable (almost 40%) when dosed to rats. Based on these findings, tool compounds were co-dosed in rats to further evaluate the systemic effects of SNAC. Oral co-dosing of SNAC with an intravenous infusion of 2 poorly brain penetrant compounds, quinidine, and gabapentin, did not increase brain ISF: plasma ratio or total brain:plasma ratio for either of these compounds, implying that SNAC is effective only in the intestine at pharmacologically relevant concentrations.

Comprehensive Evaluation of Bile Acid Homeostasis in Human Hepatocyte Co-Culture in the Presence of Troglitazone, Pioglitazone, and Acetylsalicylic Acid.

Interruption of bile acid (BA) homeostasis has been hypothesized for a variety of liver diseases and for drug-induced liver injury (DILI). Consequently, BA is gaining increasing prominence as a potential biomarker. The objective of this work was to evaluate the effect of troglitazone (TZN, associated with severe DILI), pioglitazone (PZN, rarely associated with DILI), and acetylsalicylic acid (ASA, or aspirin, not associated with DILI) on the in vitro BA homeostasis in hepatocytes co-cultured with nonparenchymal cells by monitoring the disposition of 36 BAs. The cells were supplemented with 2.5 µM d4-cholic acid, d4-chenodeoxycholic acid, d4-lithocholic acid, d4-deoxycholic acid, d4-ursodeoxycholic acid, and hyodeoxycholic acid. Concentration-time profiles of BAs were used to determine the area under the curve from the supernatant, lysate, or bile compartments, in the presence or absence of TZN, PZN, or ASA. When applicable, IC50 describing depletion of individual BAs was calculated, or accumulation greater than 200% of dimethyl sulfoxide control was noted. Thiazolidinediones significantly altered the concentration of glycine and sulfate conjugates; however, more BAs were impacted by TZN than with PZN. For commonly shared BAs, TZN exhibited 3- to 13-fold stronger inhibition than PZN. In contrast, no changes were observed with ASA. Modulation of BA disposition by thiazolidinediones and ASA was appropriately differentiated. Particularly for thiazolidinediones, TZN was more potent in interrupting BA homeostasis, and, when also considering its higher dose, may explain differences in their clinical instances of DILI. This is one of the first works which comprehensively evaluated the disposition of primary and secondary BAs along with their metabolites in an in vitro system. Differing degrees of BA homeostasis modulation was observed with various perpetrators associated with varying clinical instances of DILI. These data indicate that in vitro systems such as hepatocyte co-cultures may be a promising tool to gain a detailed insight into how drugs affect BA handling to further probe into the mechanism of DILI related to BA homeostasis.

Design of a brain-penetrant CDK4/6 inhibitor for glioblastoma.

CDK4 and CDK6 are kinases with similar sequences that regulate cell cycle progression and are validated targets in the treatment of cancer. Glioblastoma is characterized by a high frequency of CDKN2A/CCND2/CDK4/CDK6 pathway dysregulation, making dual inhibition of CDK4 and CDK6 an attractive therapeutic approach for this disease. Abemaciclib, ribociclib, and palbociclib are approved CDK4/6 inhibitors for the treatment of HR+/HER2- breast cancer, but these drugs are not expected to show strong activity in brain tumors due to poor blood brain barrier penetration. Herein, we report the identification of a brain-penetrant CDK4/6 inhibitor derived from a literature molecule with low molecular weight and topological polar surface area (MW = 285 and TPSA = 66 Å2), but lacking the CDK2/1 selectivity profile due to the absence of a basic amine. Removal of a hydrogen bond donor via cyclization of the pyrazole allowed for the introduction of basic and semi-basic amines, while maintaining in many cases efflux ratios reasonable for a CNS program. Ultimately, a basic spiroazetidine (cpKa = 8.8) was identified that afforded acceptable selectivity over anti-target CDK1 while maintaining brain-penetration in vivo (mouse Kp,uu = 0.20-0.59). To probe the potency and selectivity, our lead compound was evaluated in a panel of glioblastoma cell lines. Potency comparable to abemaciclib was observed in Rb-wild type lines U87MG, DBTRG-05MG, A172, and T98G, while Rb-deficient cell lines SF539 and M059J exhibited a lack of sensitivity.

Targeting the PI3K/Akt/mTOR pathway with the pan-Akt inhibitor GDC-0068 in PIK3CA-mutant breast cancer brain metastases.

BACKGROUND: Activating mutations in the pathway of phosphatidylinositol-3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) occur in 43-70% of breast cancer brain metastasis patients. To date, the treatment of these patients presents an ongoing challenge, mainly because of the lack of targeted agents that are able to sufficiently penetrate the blood-brain barrier. GDC-0068 is a pan-Akt inhibitor that has shown to be effective in various preclinical tumor models as well as in clinical trials. The purpose of this study was to analyze the efficacy of GDC-0068 in a breast cancer brain metastases model. METHODS: In in vitro studies, antitumor activity of GDC-0068 was assessed in breast cancer cells of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA)-mutant and PIK3CA-wildtype breast cancer cell lines using cell viability and apoptosis assays, cell cycle analysis, and western blots. In vivo, the efficacy of GDC-0068 was analyzed in a PIK3CA-mutant breast cancer brain metastasis orthotopic xenograft mouse model and evaluated by repeated bioluminescent imaging and immunohistochemistry. RESULTS: GDC-0068 decreased cell viability, induced apoptosis, and inhibited phosphorylation of proline rich Akt substrate 40 kDa and p70 S6 kinase in a dose-dependent manner in PIK3CA-mutant breast cancer brain metastatic cell lines compared with PIK3CA-wildtype cell lines. In vivo, treatment with GDC-0068 notably inhibited the growth of PIK3CA-mutant tumors and resulted in a significant survival benefit compared with sham, whereas no effect was detected in a PIK3CA-wildtype model. CONCLUSIONS: This study suggests that the Akt inhibitor GDC-0068 may be an encouraging targeted treatment strategy for breast cancer brain metastasis patients with activating mutations in the PI3K pathway. These data provide a rationale to further evaluate the efficacy of GDC-0068 in patients with brain metastases.

An Integrated Analysis of Solid Form Change Impact on Solubility and Permeability: Case Study of Oral Exposure in Rats of an RAR Related Orphan Receptor C Inhibitor.

It is well acknowledged that the oral absorption of a drug can be influenced by its solubility, which is usually associated with its solid form properties. G1032 is a retinoic acid-related orphan receptor inverse agonist. Crystalline solid (form A) was identified with an aqueous solubility of 130 µg/mL. This form was used in an oral dose escalation study in rodents up to 300 mg/kg and achieved good exposures. Later on, a more stable crystalline hydrate (form B) was identified and the aqueous solubility was reduced to 55 µg/mL. A modeling exercise suggested that this solubility change would cause a 2-fold decrease in exposure at tested doses; however, the actual reduction was far larger than the model predicted. At high dose, exposure was found to be reduced by almost 10-fold. A parameter sensitivity analysis suggested that such a drop in exposure could be associated with permeability reduction as well. More in vitro permeability experiments were performed, indicating G1032 was an efflux transporter substrate. This finding was integrated into the modeling and the design for in vivo studies. Data obtained from those studies allowed us to better understand the causes of the higher-than-expected exposure change and enabled decision-making.

Evaluating the Utility of Canine Mdr1 Knockout Madin-Darby Canine Kidney I Cells in Permeability Screening and Efflux Substrate Determination.

Permeability assays are commonly conducted with Madin-Darby canine kidney (MDCK) cells to predict the intestinal absorption of small-molecule drug candidates. In addition, MDCK cells transfected to overexpress efflux transporters are often used to identify substrates. However, MDCK cells exhibit endogenous efflux activity for a significant proportion of experimental compounds, potentially leading to the underestimation of permeability and confounded findings in transport studies. The goal of this study was to evaluate canine Mdr1 knockout MDCK (gMDCKI) cells in permeability screening and human MDR1 substrate determination in a drug discovery setting. The gMDCKI cells were established by CRISPR-Cas9-mediated knockout of the canine Mdr1 gene in MDCKI wildtype (wt) cells. A comparison of efflux ratios (ER) between MDCKI wt and gMDCKI showed that out of 135 compounds tested, 38% showed efflux activity in MDCKI wt, while no significant efflux was observed in gMDCKI cells. Apparent permeability (Papp) from apical-to-basolateral (A-to-B) and basolateral-to-apical were near unity in gMDCKI cells, which approximated passive permeability, and 17% of compounds demonstrated increases in their Papp A-to-B values. Overexpression of human MDR1 in gMDCKI (gMDCKI-MDR1) cells enabled substrate determination without the contribution of endogenous efflux, and the assay was able to deconvolute ambiguous results from MDCKI-MDR1 and identify species differences in substrate specificity. An analysis of 395 and 474 compounds in gMDCKI and gMDCKI-MDR1, respectively, suggested physicochemical properties that were associated with low permeability correlated with MDR1 recognition. Poorly permeable compounds and MDR1 substrates were more likely to be large, flexible, and more capable of forming external hydrogen bonds. On the basis of our evaluation, we concluded that gMDCKI is a better cell line for permeability screening and efflux substrate determination than the MDCK wt cell line.

The Effects of Drug Metabolizing Enzyme Inhibitors on Hepatic Efflux and Uptake Transporters.

BACKGROUND: Non-selective chemical inhibitors of phase I and phase II enzymes are commonly used in in vitro metabolic studies to elucidate the biotransformation pathways of drugs. However, the inhibition of the inhibitors on efflux and uptake transporters is not well investigated, potentially leading to unexpected and ambiguous results in these studies. OBJECTIVE: The commonly used metabolizing enzyme inhibitors, 1-aminobenzotriazole (ABT), SKF- 525A, pargyline, allopurinol, menadione, methimazole, piperine and raloxifene, were examined for their potential inhibition of the major hepatic ABC (ATP binding cassette) and SLC (solute carrier) transporters. METHODS: Different concentrations of the metabolizing enzyme inhibitors were used to study their effects on ABC and SLC transporters expressed in MDR1-MDCKI, Bcrp1-MDCKII, OATP1B1-HEK, OATP1B3-HEK, OCT1-HEK, OCT3-HEK cells and MRP2 vesicles. RESULTS: ABT, allopurinol and methimazole had no inhibitory effects on MDR1, Bcrp1, MRP2 or on OATP1B1, OATP1B3, OCT1 or OCT3. Pargyline did not inhibit OATP1B1 or OATP1B3, but weakly inhibited OCT1 and OCT3. In contrast, SKF-525A showed inhibition of not only MDR1, Bcrp1 and MRP2 but also OATP1B1, OATP1B3 and OCT1. Menadione and raloxifene weakly inhibited Bcrp1, but the inhibition of raloxifene on MDR1 was as potent as on the xanthine oxidase pterin oxidation. Piperine showed inhibition of MDR1, Bcrp1, OATP1B1, OCT1 and OCT3. CONCLUSION: ABT, pargyline, allopurinol and methimazole have no inhibitory effects on the studied ABC and SLC transporters, suggesting the inhibitors are unlikely to cause confounding inhibition of transporters when used in metabolism studies. However, SKF525A, menadione, raloxifene and piperine can inhibit the activities of ABC and/or SLC transporters.

GluN2A-Selective Pyridopyrimidinone Series of NMDAR Positive Allosteric Modulators with an Improved in Vivo Profile.

The N-methyl-d-aspartate receptor (NMDAR) is an ionotropic glutamate receptor, gated by the endogenous coagonists glutamate and glycine, permeable to Ca2+ and Na+. NMDAR dysfunction is associated with numerous neurological and psychiatric disorders, including schizophrenia, depression, and Alzheimer's disease. Recently, we have disclosed GNE-0723 (1), a GluN2A subunit-selective and brain-penetrant positive allosteric modulator (PAM) of NMDARs. This work highlights the discovery of a related pyridopyrimidinone core with distinct structure-activity relationships, despite the structural similarity to GNE-0723. GNE-5729 (13), a pyridopyrimidinone-based NMDAR PAM, was identified with both an improved pharmacokinetic profile and increased selectivity against AMPARs. We also include X-ray structure analysis and modeling to propose hypotheses for the activity and selectivity differences.

Brain Distribution and Efficacy of the Brain Penetrant PI3K Inhibitor GDC-0084 in Orthotopic Mouse Models of Human Glioblastoma.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. Limited treatment options have only marginally impacted patient survival over the past decades. The phophatidylinositol 3-kinase (PI3K) pathway, frequently altered in GBM, represents a potential target for the treatment of this glioma. 5-(6,6-Dimethyl-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[4,3-e]purin-2-yl)pyrimidin-2-amine (GDC-0084) is a PI3K inhibitor that was specifically optimized to cross the blood-brain barrier. The goals of our studies were to characterize the brain distribution, pharmacodynamic (PD) effect, and efficacy of GDC-0084 in orthotopic xenograft models of GBM. GDC-0084 was tested in vitro to assess its sensitivity to the efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) and in vivo in mice to evaluate its effects on the PI3K pathway in intact brain. Mice bearing U87 or GS2 intracranial tumors were treated with GDC-0084 to assess its brain distribution by matrix-assisted laser desorption ionization (MALDI) imaging and measure its PD effects and efficacy in GBM orthotopic models. Studies in transfected cells indicated that GDC-0084 was not a substrate of P-gp or BCRP. GDC-0084 markedly inhibited the PI3K pathway in mouse brain, causing up to 90% suppression of the pAkt signal. MALDI imaging showed GDC-0084 distributed evenly in brain and intracranial U87 and GS2 tumors. GDC-0084 achieved significant tumor growth inhibition of 70% and 40% against the U87 and GS2 orthotopic models, respectively. GDC-0084 distribution throughout the brain and intracranial tumors led to potent inhibition of the PI3K pathway. Its efficacy in orthotopic models of GBM suggests that it could be effective in the treatment of GBM. GDC-0084 is currently in phase I clinical trials.