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.

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.

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.

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.

Novel antibody-antibiotic conjugate eliminates intracellular S. aureus.

Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S. aureus within host cells may provide a reservoir relatively protected from antibiotics, thus enabling long-term colonization of the host and explaining clinical failures and relapses after antibiotic therapy. Here we confirm that intracellular reservoirs of S. aureus in mice comprise a virulent subset of bacteria that can establish infection even in the presence of vancomycin, and we introduce a novel therapeutic that effectively kills intracellular S. aureus. This antibody-antibiotic conjugate consists of an anti-S. aureus antibody conjugated to a highly efficacious antibiotic that is activated only after it is released in the proteolytic environment of the phagolysosome. The antibody-antibiotic conjugate is superior to vancomycin for treatment of bacteraemia and provides direct evidence that intracellular S. aureus represents an important component of invasive infections.

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.

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.

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.

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.

Differential effects of Rifampin and Ketoconazole on the blood and liver concentration of atorvastatin in wild-type and Cyp3a and Oatp1a/b knockout mice.

Atorvastatin is eliminated by CYP3A4 which follows carrier-mediated uptake into hepatocytes by OATP1B1, OATP1B3, and OATP2B1. Multiple clinical studies demonstrated that OATP inhibition by rifampin had a greater impact on atorvastatin systemic concentration than itraconazole-mediated CYP3A4 inhibition. If it is assumed that the blood and hepatocyte compartments are differentiated by the concentration gradient that is established by OATPs, and if the rate of uptake into the hepatocyte is rate-determining to the elimination of atorvastatin from the body, then it is hypothesized that blood concentrations may not necessarily reflect liver concentrations. In wild-type mice, rifampin had a greater effect on systemic exposure of atorvastatin than ketoconazole, as the blood area under the blood concentration-time curve increased 7- and 2-fold, respectively. In contrast, liver concentrations were affected more by ketoconazole than by rifampin, as liver levels increased 21- and 4-fold, respectively. Similarly, in Cyp3a knockout animals, 39-fold increases in liver concentrations were observed despite insignificant changes in the blood area under the blood concentration-time curve. Interestingly, blood and liver levels in Oatp1a/b knockout animals were similar to wild types, suggesting that Oatp1a/b knockout may be necessary but not sufficient to completely describe atorvastatin uptake in mice. Data presented in this work indicate that there is a substantial drug interaction when blocking atorvastatin metabolism, but the effects of this interaction are predominantly manifested in the liver and may not be captured when monitoring changes in the systemic circulation. Consequently, there may be a disconnect when trying to relate blood exposure to instances of hepatotoxicity because a pharmacokinetic-toxicity relationship may not be obvious from blood concentrations.

Role of P-glycoprotein on the brain penetration and brain pharmacodynamic activity of the MEK inhibitor cobimetinib.

Cobimetinib is a MEK inhibitor currently in clinical trials as an anticancer agent. The objectives of this study were to determine in vitro and in vivo if cobimetinib is a substrate of P-glycoprotein (P-gp) and/or breast cancer resistance protein (Bcrp1) and to assess the implications of efflux on cobimetinib pharmacokinetics (PK), brain penetration, and target modulation. Cell lines transfected with P-gp or Bcrp1 established that cobimetinib was a substrate of P-gp but not a substrate of Bcrp1. In vivo, after intravenous and oral administration of cobimetinib to FVB (wild-type; WT), Mdr1a/b(-/-), Bcrp1 (-/-), and Mdr1a/b(-/-)/Bcrp(-/-) knockout (KO) mice, clearance was similar in WT (35.5 ± 16.7 mL/min/kg) and KO animals (22.0 ± 3.6 to 27.6 ± 5.2 mL/min/kg); oral exposure was also similar between WT and KO animals. After an oral 10 mg/kg dose of cobimetinib, the mean total brain to plasma ratio (Kp) at 6 h postdose was 0.3 and 0.2 in WT and Bcrp1(-/-) mice, respectively. In Mdr1a/b(-/-) and Mdr1a/1b/Bcrp1(-/-) KO mice and WT mice treated with elacridar (a P-gp and BCRP inhibitor), Kp increased to 11, 6, and 7, respectively. Increased brain exposure in Mdr1a/b(-/-) and Mdr1a/1b/Bcrp1(-/-) KO and elacridar treated mice was accompanied by up to ∼65% suppression of the target (pErk) in brain tissue, compared to WT mice. By MALDI imaging, the cobimetinib signal intensity was relatively high and was dispersed throughout the brain of Mdr1a/1b/Bcrp1(-/-) KO mice compared to low/undetectable signal intensity in WT mice. The efflux of cobimetinib by P-gp may have implications for the treatment of patients with brain tumors/metastases.

Variability in P-glycoprotein inhibitory potency (IC50) using various in vitro experimental systems: implications for universal digoxin drug-drug interaction risk assessment decision criteria.

A P-glycoprotein (P-gp) IC50 working group was established with 23 participating pharmaceutical and contract research laboratories and one academic institution to assess interlaboratory variability in P-gp IC50 determinations. Each laboratory followed its in-house protocol to determine in vitro IC50 values for 16 inhibitors using four different test systems: human colon adenocarcinoma cells (Caco-2; eleven laboratories), Madin-Darby canine kidney cells transfected with MDR1 cDNA (MDCKII-MDR1; six laboratories), and Lilly Laboratories Cells--Porcine Kidney Nr. 1 cells transfected with MDR1 cDNA (LLC-PK1-MDR1; four laboratories), and membrane vesicles containing human P-glycoprotein (P-gp; five laboratories). For cell models, various equations to calculate remaining transport activity (e.g., efflux ratio, unidirectional flux, net-secretory-flux) were also evaluated. The difference in IC50 values for each of the inhibitors across all test systems and equations ranged from a minimum of 20- and 24-fold between lowest and highest IC50 values for sertraline and isradipine, to a maximum of 407- and 796-fold for telmisartan and verapamil, respectively. For telmisartan and verapamil, variability was greatly influenced by data from one laboratory in each case. Excluding these two data sets brings the range in IC50 values for telmisartan and verapamil down to 69- and 159-fold. The efflux ratio-based equation generally resulted in severalfold lower IC50 values compared with unidirectional or net-secretory-flux equations. Statistical analysis indicated that variability in IC50 values was mainly due to interlaboratory variability, rather than an implicit systematic difference between test systems. Potential reasons for variability are discussed and the simplest, most robust experimental design for P-gp IC50 determination proposed. The impact of these findings on drug-drug interaction risk assessment is discussed in the companion article (Ellens et al., 2013) and recommendations are provided.

Evaluating the in vitro inhibition of UGT1A1, OATP1B1, OATP1B3, MRP2, and BSEP in predicting drug-induced hyperbilirubinemia.

Hyperbilirubinemia may arise due to inadequate clearance of bilirubin from the body. Bilirubin elimination is a multifaceted process consisting of uptake of bilirubin into the hepatocytes facilitated by OATP1B1 and OATP1B3. Once in the hepatocytes, it is extensively glucuronidated by UGT1A1. Eventually, the glucuronide metabolite is excreted into the bile via MRP2. UGT1A1 inhibition has been previously shown to be linked with hyperbilirubinemia. However, because drug transporters also contribute to bilirubin elimination, the purpose of this work was to investigate the in vitro inhibition of OATP1B1, OATP1B3, MRP2, and BSEP of select test drugs known to elicit hyperbilirubinemia. Test drugs investigated in this study were atazanavir and indinavir, which are associated with hyperbilirubinemia and elevations in serum transaminase; ritonavir and nelfinavir, which are not associated with hyperbilirubinemia; and bromfenac, troglitazone, and trovafloxacin, which are associated with severe idiosyncratic hepatotoxicity exhibiting elevations in serum bilirubin and transaminase. Due to limited solubility and poor ionization of bilirubin and its glucuronide, the formation of estradiol 3-glucuronide was used as a surrogate to assess UGT1A1 activity, while the transport of pitavastatin, CDCF, and taurocholate were used as surrogate probe substrates to monitor the function of OATP1B1/OATP1B3, MRP2, and BSEP, respectively. It was assumed that any inhibition of the surrogate probe substrates by test drugs is indicative of the potential impact of test drugs to modulate the function of proteins involved in bilirubin disposition. In vitro inhibition was determined by calculating IC50. Moreover, Cmax and Cmax,free were integrated with IC50 values to calculate R and Rfree, respectively, which represents the ratio of probe drug glucuronidation/transport in the absence and presence of test drugs. Analysis of the data showed that Rfree demonstrated the best correlation to hyperbilirubinemia. Specifically, Rfree was above the 1.1 target threshold against UGT1A1, OATP1B1, and BSEP for atazanavir and indinavir. In contrast, Rfree was below this threshold for ritonavir and nelfinavir as well as for bromfenac, troglitazone, and trovafloxacin. For all test drugs examined, only minor inhibition against OATP1B3 and MRP2 were observed. These data suggest that the proposed surrogate probe substrates to evaluate the in vitro inhibition of UGT1A1, OATP1B1, and BSEP may be suitable to assess bilirubin disposition. For protease inhibitors, inclusion of OATP1B1 and BSEP inhibition may improve the predictability of hyperbilirubinemia.

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.

Preclinical assessment of the absorption and disposition of the phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor GDC-0980 and prediction of its pharmacokinetics and efficacy in human.

(S)-1-{4-[2-(2-Amino-pyrimidin-5-yl)-7-methyl-4-morpholin-4-yl-thieno[3,2-d]pyrimidin-6-ylmethyl]-piperazin-1-yl}-2-hydroxy-propan-1-one (GDC-0980) is a potent and selective inhibitor of phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin, two key components of the PI3K pathway, the deregulation of which is associated with the development of many cancers. The objectives of these studies were to characterize the absorption and disposition of GDC-0980 and assess its efficacy in an MCF7-neo/HER2 human breast cancer xenograft model in immunocompromised mice. Studies in parental Madin-Darby canine kidney cells indicated that GDC-0980 had high permeability (P(app) = 18 × 10⁻6 cm/s), suggesting good absorption potential. However, it was found to be a P-glycoprotein and breast cancer resistance protein substrate in transfected cells and in knockout mice studies. Plasma protein binding was low, with the fraction unbound ranging from 29 to 52% across species. GDC-0980 hepatic clearance (CL) was predicted to be low in all of the species tested from hepatocyte incubations. The plasma CL of GDC-0980 was low in mouse (6.30 ml · min⁻¹ · kg⁻¹), rat (15.4 ml · min⁻¹ · kg⁻¹), and dog (6.37 ml · min⁻¹ · kg⁻¹) and moderate in cynomolgus monkey (18.9 ml · min⁻¹ · kg⁻¹). Oral bioavailability ranged from 14.4% in monkey to 125% in dog. Predicted human plasma CL and volume of distribution using allometry were 5.1 ml · min⁻¹ · kg⁻¹ and 1.8 l/kg, respectively. Parameters estimated from the pharmacokinetic/pharmacodynamic modeling of the MCF7-neo/HER2 xenograft data indicated that the GDC-0980 plasma concentration required for tumor stasis was approximately 0.5 µM. These parameters, combined with the predicted human pharmacokinetic profile, suggested that 55 mg once daily may be a clinically efficacious dose. GDC-0980 preclinical characterization and the predictions of its human properties supported its clinical development; it is currently in Phase II clinical trials.

Preclinical disposition of GDC-0973 and prospective and retrospective analysis of human dose and efficacy predictions.

[3,4-Difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyl]-((S)-3-hydroxy-3-piperidin-2-yl-azetidin-1-yl)-methanone (GDC-0973) is a potent and highly selective inhibitor of mitogen-activated protein kinase(MAPK)/extracellular signal-regulated kinase (ERK) 1/2 (MEK1/2), a MAPK kinase that activates ERK1/2. The objectives of these studies were to characterize the disposition of GDC-0973 in preclinical species and to determine the relationship of GDC-0973 plasma concentrations to efficacy in Colo205 mouse xenograft models. The clearance (CL) of GDC-0973 was moderate in mouse (33.5 ml · min(-1) · kg(-1)), rat (37.9 ± 7.2 ml · min(-1) · kg(-1)), and monkey (29.6 ± 8.5 ml · min(-1) · kg(-1)). CL in dog was low (5.5 ± 0.3 ml · min(-1) · kg(-1)). The volume of distribution across species was large, 6-fold to 15-fold body water; half-lives ranged from 4 to 13 h. Protein binding in mouse, rat, dog, monkey, and human was high, with percentage unbound, 1 to 6%. GDC-0973-related radioactivity was rapidly and extensively distributed to tissues; however, low concentrations were observed in the brain. In rats and dogs, [(14)C]GDC-0973 was well absorbed (fraction absorbed, 70-80%). The majority of [(14)C]GDC-0973-related radioactivity was recovered in the bile of rat (74-81%) and dog (65%). The CL and volume of distribution of GDC-0973 in human, predicted by allometry, was 2.9 ml · min(-1) · kg(-1) and 9.9 l/kg, respectively. The predicted half-life was 39 h. To characterize the relationship between plasma concentration of GDC-0973 and tumor growth inhibition, pharmacokinetic-pharmacodynamic modeling was applied using an indirect response model. The KC(50) value for tumor growth inhibition in Colo205 xenografts was estimated to be 0.389 µM, and the predicted clinical efficacious dose was ∼10 mg. Taken together, these data are useful in assessing the disposition of GDC-0973, and where available, comparisons with human data were made.

Preclinical stereoselective disposition and toxicokinetics of two novel MET inhibitors.

The R- and S-enantiomer of N-(4-(3-(1-ethyl-3,3-difluoropiperidin-4-ylamino)-1H-pyrazolo[3,4-b]pyridin-4-yloxy)-3-fluorophenyl)-2-(4-fluorophenyl)-3-oxo-2,3-dihydropyridazine-4-carboxamide are novel MET kinase inhibitors that have been investigated as potential anticancer agents. The effect of the chirality of these compounds on preclinical in vivo pharmacokinetics and toxicity was studied. The plasma clearance for the S-enantiomer was low in mice and monkeys (23.7 and 7.8 mL min(-1) kg(-1), respectively) and high in rats (79.2 mL min(-1) kg(-1)). The R/S enantiomer clearance ratio was 1.5 except in rats (0.49). After oral single-dose administration at 5 mg kg(-1) the R/S enantiomer ratio of AUC(inf) was 0.95, 1.9 and 0.41 in mice, rats and monkeys, respectively. In an oral single-dose dose-ranging study at 200 and 500 mg kg(-1) and multi-dose toxicity study in mice plasma AUC exposure was approximately 2- to 3-fold higher for the R-enantiomer compared to the S-enantiomer. Greater toxicity of the S-enantiomer was observed which appeared to be due to high plasma C(min) values and tissue concentrations approximately 24 h after the final dose. Both enantiomers showed low to moderate permeability in MDCKI cells with no significant efflux, no preferential distribution into red blood cells and similar plasma protein binding in vitro. Overall, the differences between the enantiomers with respect to low dose pharmacokinetics and in vitro properties were relatively modest. However, toxicity results warrant further development of the R-enantiomer over the S-enantiomer.

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.