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.

Alpha-Smooth Muscle Actin Expression and Parafoveal Blood Flow Pathways Are Altered in Preclinical Diabetic Retinopathy.

Purpose: To investigate differences in alpha smooth muscle actin (αSMA) expression and parafoveal blood flow pathways in diabetic retinopathy (DR). Methods: Human donor eyes from healthy subjects (n = 8), patients with diabetes but no DR (DR-; n = 7), and patients with clinical DR (DR+; n = 13) were perfusion labeled with antibodies targeting αSMA, lectin, collagen IV, and filamentous actin. High-resolution confocal scanning laser microscopy was used to quantify αSMA staining and capillary density in the parafoveal circulation. Quantitative analyses of connections between retinal arteries and veins within the superficial vascular plexus (SVP), intermediate capillary plexus (ICP) and deep capillary plexus (DCP) were performed. Results: Mean age between the groups was not different (P = 0.979). αSMA staining was seen in the SVP and ICP of all groups. The DCP was predominantly devoid of αSMA staining in control eyes but increased in a disease stage-specific manner in the DR- and DR+ groups. The increase in αSMA staining was localized to pericytes and endothelia of terminal arterioles and adjacent capillary segments. Capillary density was less in the DCP in the DR+ group (P < 0.001). ICP of the DR- and DR+ groups received more direct arteriole supplies than the control group (P < 0.001). Venous outflow pathways were not altered (all P > 0.284). Conclusions: Alterations in αSMA and vascular inflow pathways in preclinical DR suggest that perfusion abnormalities precede structural vascular changes such as capillary loss. Preclinical DR may be characterized by a "steal" phenomenon where blood flow is preferentially diverted from the SVP to the ICP and DCP.

Effect of magnesium stearate surface coating method on the aerosol performance and permeability of micronized fluticasone propionate.

In this study, we evaluated the aerodynamic performance, dissolution, and permeation behavior of micronized fluticasone propionate (FP) and magnesium stearate (MgSt) binary mixtures. Micronized FP was dry mixed with 2% w/w MgSt using a tumble mixer and a resonant acoustic mixer (RAM) with and without heating. The mixing efficacy was determined by X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC) analysis. Additional techniques were used to determine powder properties such as the dynamic vapor sorption (DVS), particle size distribution (PSD) by laser diffraction light scattering, and particle surface properties by scanning electron microscope (SEM). The aerodynamic performance was studied by the next-generation impactor (NGI) using drug-loaded capsules in a PlastiApi® device. Physiochemical properties such as porosity, particle size distribution, and surface area of the formulations were studied with adsorption and desorption curves fitted to several well-known models including Brunauer-Emmett-Teller (BET), Barret Joyner Halenda (BJH), and the density functional theory (DFT). The dissolution behavior of the formulations collected on the transwell inserts incorporated into stages 3, 5, and 7 of the NGI with a membrane providing an air interface was evaluated. Drug permeability of formulations was assessed by directly depositing particles on Calu-3 cells at the air-liquid interface (ALI). Drug concentration was determined by LC-MS/MS. A better MgSt mixing on micronized FP particles was achieved by mixing with a RAM with and without heating than with a tumble mixer. A significant concomitant increase in the % of emitted dose and powder aerosol performance was observed after MgSt mixing. Formulation 4 (RAM mixing at room temperature) showed the highest rate of permeability and correlation with dissolution profile. The results show that the surface enrichment of hydrophobic MgSt improved aerosolization properties and the dissolution and permeability rate of micronized FP by reducing powder agglomerations. A simple low-shear acoustic dry powder mixing method was found to be efficient and substantially improved the powder aerosolization properties and enhanced dissolution and permeability rate.

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.

Improving the Translation of Organic Anion Transporting Polypeptide Substrates using HEK293 Cell Data in the Presence and Absence of Human Plasma via Physiologically Based Pharmacokinetic Modeling.

Accurately predicting the pharmacokinetics of compounds that are transporter substrates has been notoriously challenging using traditional in vitro systems and physiologically based pharmacokinetic (PBPK) modeling. The objective of this study was to use PBPK modeling to understand the translational accuracy of data generated with human embryonic kidney 293 (HEK293) cells overexpressing the hepatic uptake transporters organic anion transporting polypeptide (OATP) 1B1/3 with and without plasma while accounting for transporter expression. Models of four OATP substrates, two with low protein binding (pravastatin and rosuvastatin) and two with high protein binding (repaglinide and pitavastatin) were explored, and the OATP in vitro data generated in plasma incubations were used for a plasma model, and in buffer incubations for a buffer model. The pharmacokinetic parameters and concentration-time profiles of pravastatin and rosuvastatin were similar and well predicted (within 2-fold of observed values) using the plasma and buffer models without needing an empirical scaling factor, whereas the dispositions of the highly protein bound repaglinide and pitavastatin were more accurately simulated with the plasma models than the buffer models. This work suggests that data from HEK293 overexpressing transporter cells corrected for transporter expression represent a valid approach to improve bottom-up PBPK modeling for highly protein bound OATP substrates with plasma incubations and low protein binding OATP substrates with or without plasma incubations. SIGNIFICANCE STATEMENT: This work demonstrates the bottom-up approach of using in vitro data directly without employing empirical scaling factors to predict the intravenous pharmacokinetic (PK) profiles reasonably well for four organic anion transporting polypeptide (OATP) substrates. Based on these results, using HEK293 overexpressing cells, examining the impact of plasma for highly bound compounds, and incorporating transporter quantitation for the lot in which the in vitro data were generated represents a valid approach to achieve more accurate prospective PK predictions for OATP substrates.

Optimization of globomycin analogs as novel gram-negative antibiotics.

Discovery of novel classes of Gram-negative antibiotics with activity against multi-drug resistant infections is a critical unmet need. As an essential member of the lipoprotein biosynthetic pathway, lipoprotein signal peptidase II (LspA) is an attractive target for antibacterial drug discovery, with the natural product inhibitor globomycin offering a modestly-active starting point. Informed by structure-based design, the globomycin depsipeptide was optimized to improve activity against E. coli. Backbone modifications, together with adjustment of physicochemical properties, afforded potent compounds with good in vivo pharmacokinetic profiles. Optimized compounds such as 51 (E. coli MIC 3.1 µM) and 61 (E. coli MIC 0.78 µM) demonstrate broad spectrum activity against gram-negative pathogens and may provide opportunities for future antibiotic discovery.

Functional similarity of modified cascade impactor to deposit drug particles on cells.

Pulmonary drug delivery is a non-invasive and effective route for local or systemic drug administration. Despite several products in the market, the mechanism of drug absorption from the lungs is not well understood. An in vitro model for aerosol deposition and transport across epithelia that uses particle deposition may be a good predictor of and help understand in vivo drug disposition. The objective of this study was to examine the uptake of HFA fluticasone (Flovent HFA) particles at various stages of the Next Generation Impactor (NGI) by human Calu-3 cell line derived from human bronchial respiratory epithelial cell monolayer. Particles were directly deposited on Calu-3 cells incorporated onto stages 3, 5, and 7 of the NGI at the air-liquid interface (ALI). We modified the NGI apparatus to allow particle deposition directly on cells and determined the in vitro deposition characteristics using modified NGI. Particles of different size ranges showed different in vitro epithelial transport rates. This study highlights the need to develop in vitro test systems to determine the deposition of aerosol particles on cell monolayers by simultaneously considering aerodynamic properties.

Investigating the Impact of Albumin on the Liver Uptake of Pitavastatin and Warfarin in Nagase Analbuminemic Rats.

Albumin has been suggested to enhance the hepatic uptake of organic anion-transporting polypeptide (Oatp) substrates in various in vitro as well as liver perfusion models. However, it is not known whether the interplay between albumin and Oatp substrates is an experimental artifact or if this interaction occurs in vivo. The objective of this work was to investigate the hepatic uptake of warfarin and pitavastatin, which are both extensively bound to albumin but only pitavastatin being an Oatp substrate. Experiments were conducted in Nagase analbuminemic rats (NAR) which exhibit reduced albumin levels compared with F344 (wild type, WT). The fraction unbound (f u) was 140- and 10-fold greater in NAR plasma for warfarin and pitavastatin, respectively, whereas no meaningful differences were observed with tissue binding. In vitro, pitavastatin uptake into hepatocytes reconstituted in WT plasma was 17- and 3-fold greater than when reconstituted in buffer or NAR plasma, respectively. In vivo, the free tissue-to-free plasma ratios (K p,u,u) from brain and liver in intact WT and NAR were not significantly different for warfarin. Contrarily, liver K p,u,u of pitavastatin was 6-fold higher in WT animals, which corresponded to a 2.3-fold reduction in free plasma and 2.6-fold increase in free liver exposure. These results suggest that the enhanced hepatic uptake by albumin is not necessarily an experimental artifact but is also a relevant phenomenon in vivo. This work raises the possibility that other plasma proteins may also effect the function of additional drug transporters, and that modulating plasma protein binding may exhibit meaningful clinical relevance in the disposition of drugs. SIGNIFICANCE STATEMENT: The interplay between albumin and Oatp substrates has been reported in hepatocytes and in liver perfusion studies, but the in vivo relevance of this interaction has yet to be elucidated. Using NAR and its corresponding WT animal, this study demonstrates that albumin may indeed enhance the hepatic uptake of pitavastatin in intact animals. In vivo demonstration of this interplay not only provides further justification for continued investigation into this particular mechanism but also raises the possibility that other plasma proteins may affect additional drug transporters and that modulating plasma protein binding may exhibit meaningful clinical relevance in the disposition of drugs.

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.

Unremarkable impact of Oatp inhibition on the liver concentration of fluvastatin, lovastatin and pitavastatin in wild-type and Oatp1a/1b knockout mouse.

1. Oatp inhibitors have been shown to significantly increase the plasma exposure of statins. However, understanding alterations of liver concentration is also important. While modeling has simulated liver concentration changes, availability of experimental data is limited, especially when concerning drug-drug interactions (DDI). The objective of this work was to determine blood and liver concentrations of fluvastatin, lovastatin and pitavastatin, when blocking uptake transporters. 2. In wild-type mouse, rifampin pre-treatment decreased the unbound liver-to-plasma ratio (Kp,uu) of fluvastatin by 4.2-fold to 2.2, lovastatin by 4.9-fold to 0.81 and pitavastatin by 10-fold to 0.21. Changes in Kp,uu were driven by increases in systemic exposures as liver concentrations were not greatly altered. 3. In Oatp1a/1b knockout mouse (KO), rifampin exerted no additional effect on fluvastatin and lovastatin. Contrarily, rifampin further decreased pitavastatin Kp,uu by 3.4-fold, suggesting that the KO is inadequate to completely block liver uptake of pitavastatin as there are additional rifampin-sensitive uptake mechanism(s) not captured in the KO model. 4. This work provides experimental data showing that the plasma compartment is more sensitive to Oatp modulation than the liver compartment, even for rifampin-mediated DDI. Consistent with previous simulations, inhibiting or targeting Oatps may change Kp,uu, but exhibit only a minimal effect on absolute liver concentrations.

Preclinical assessment of the ADME, efficacy and drug-drug interaction potential of a novel NAMPT inhibitor.

GNE-617 (N-(4-((3,5-difluorophenyl)sulfonyl)benzyl)imidazo[1,2-a]pyridine-6-carboxamide) is a potent, selective nicotinamide phosphoribosyltransferase (NAMPT) inhibitor being explored as a potential treatment for human cancers. Plasma clearance was low in monkeys and dogs (9.14 mL min-1 kg-1 and 4.62 mL min-1 kg-1, respectively) and moderate in mice and rats (36.4 mL min-1 kg-1 and 19.3 mL min-1 kg-1, respectively). Oral bioavailability in mice, rats, monkeys and dogs was 29.7, 33.9, 29.4 and 65.2%, respectively. Allometric scaling predicted a low clearance of 3.3 mL min-1 kg-1 and a volume of distribution of 1.3 L kg-1 in human. Efficacy (57% tumor growth inhibition) in Colo-205 CRC tumor xenograft mice was observed at an oral dose of 15 mg/kg BID (AUC = 10.4 µM h). Plasma protein binding was moderately high. GNE-617 was stable to moderately stable in vitro. Main human metabolites identified in human hepatocytes were formed primarily by CYP3A4/5. Transporter studies suggested that GNE-617 is likely a substrate for MDR1 but not for BCRP. Simcyp® simulations suggested a low (CYP2C9 and CYP2C8) or moderate (CYP3A4/5) potential for drug-drug interactions. The potential for autoinhibition was low. Overall, GNE-617 exhibited acceptable preclinical properties and projected human PK and dose estimates.

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.

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.

Discovery of Clinical Development Candidate GDC-0084, a Brain Penetrant Inhibitor of PI3K and mTOR.

Inhibition of phosphoinositide 3-kinase (PI3K) signaling is an appealing approach to treat brain tumors, especially glioblastoma multiforme (GBM). We previously disclosed our successful approach to prospectively design potent and blood-brain barrier (BBB) penetrating PI3K inhibitors. The previously disclosed molecules were ultimately deemed not suitable for clinical development due to projected poor metabolic stability in humans. We, therefore, extended our studies to identify a BBB penetrating inhibitor of PI3K that was also projected to be metabolically stable in human. These efforts required identification of a distinct scaffold for PI3K inhibitors relative to our previous efforts and ultimately resulted in the identification of GDC-0084 (16). The discovery and preclinical characterization of this molecule are described within.

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.

Structure-Guided Design of Group I Selective p21-Activated Kinase Inhibitors.

The p21-activated kinases (PAKs) play important roles in cytoskeletal organization, cellular morphogenesis, and survival and have generated significant attention as potential therapeutic targets for cancer. Following a high-throughput screen, we identified an aminopyrazole scaffold-based series that was optimized to yield group I selective PAK inhibitors. A structure-based design effort aimed at targeting the ribose pocket for both potency and selectivity led to much-improved group I vs II selectivity. Early lead compounds contained a basic primary amine, which was found to be a major metabolic soft spot with in vivo clearance proceeding predominantly via N-acetylation. We succeeded in identifying replacements with improved metabolic stability, leading to compounds with lower in vivo rodent clearance and excellent group I PAK selectivity.

Practical permeability-based hepatic clearance classification system (HepCCS) in drug discovery.

BACKGROUND: The use of liver microsomes and hepatocytes to predict total in vivo clearance is standard practice in the pharmaceutical industry; however, metabolic stability data alone cannot always predict in vivo clearance accurately. RESULTS: Apparent permeability generated from Mardin-Darby canine kidney cells and rat hepatocyte uptake for 33 discovery compounds were obtained. CONCLUSION: When there is underprediction of in vivo clearance, compounds with low apparent permeability (less than 3 × 10(-6) cm/s) all exhibited hepatic uptake. A systematic approach in the form of a classification system (hepatic clearance classification system) and decision tree that will help drug discovery scientists understand in vitro-in vivo clearance prediction disconnect early is proposed.

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.

Use of cassette dosing approach to examine the effects of P-glycoprotein on the brain and cerebrospinal fluid concentrations in wild-type and P-glycoprotein knockout rats.

The study objectives were 1) to test the hypothesis that the lack of P-glycoprotein (P-gp) and the inhibition of breast cancer resistance protein (Bcrp) at the blood-brain barrier after cassette dosing of potent P-gp and Bcrp inhibitors were due to low plasma concentrations of those inhibitors and 2) to examine the effects of P-gp on the unbound brain (C(u,brain)) and cerebrospinal fluid (CSF) concentrations (C(u,CSF)) of P-gp substrates in rats. In vitro inhibition of 11 compounds (amprenavir, citalopram, digoxin, elacridar, imatinib, Ko143 [(3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino[1',2':1,6]pyrido[3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester], loperamide, prazosin, quinidine, sulfasalazine, and verapamil) on P-gp and Bcrp was examined in P-gp- and Bcrp-expressing Madin-Darby canine kidney cells, respectively. An in vivo study was conducted in wild-type and Mdr1a(-/-) rats after subcutaneous cassette dosing of the 11 compounds at 1-3 mg/kg, and the brain, CSF, and plasma concentrations of these compounds were determined. At the maximal unbound concentrations observed in rats at 1-3 mg/kg, P-gp and Bcrp were not inhibited by a cassette of the 11 compounds. For non-P-gp/Bcrp substrates, similar C(u,brain), C(u,CSF), and unbound plasma concentrations (C(u,plasma)) were observed in wild-type and P-gp knockout rats. For P-gp/Bcrp substrates, C(u,brain) ≤ C(u,CSF) ≤ C(u,plasma) in wild-type rats, but C(u,brain) and C(u,CSF) increased in the P-gp knockout rats and were within 3-fold of C(u,plasma) for six of the seven P-gp substrates. These results indicate that P-gp and Bcrp inhibition at the blood-brain barrier is unlikely in cassette dosing and also suggest that P-gp and Bcrp activity at the blood-CSF barrier is functionally not important in determination of the CSF concentration for their substrates.