The Critical Role of Passive Permeability in Designing Successful Drugs.

Passive permeability is a key property in drug disposition and delivery. It is critical for gastrointestinal absorption, brain penetration, renal reabsorption, defining clearance mechanisms and drug-drug interactions. Passive diffusion rate is translatable across tissues and animal species, while the extent of absorption is dependent on drug properties, as well as in vivo physiology/pathophysiology. Design principles have been developed to guide medicinal chemistry to enhance absorption, which combine the balance of aqueous solubility, permeability and the sometimes unfavorable compound characteristic demanded by the target. Permeability assays have been implemented that enable rapid development of structure-permeability relationships for absorption improvement. Future advances in assay development to reduce nonspecific binding and improve mass balance will enable more accurately measurement of passive permeability. Design principles that integrate potency, selectivity, passive permeability and other ADMET properties facilitate rapid advancement of successful drug candidates to patients.

Quantification of cyclocreatine in mouse and rat plasma using hydrophilic-interaction ultra-performance liquid chromatography-tandem mass spectrometry.

An accurate, rapid and selective method was developed to quantify cyclocreatine in mouse and rat plasma using hydrophilic interaction (HILIC) ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The plasma samples were prepared by protein precipitation with acetonitrile:methanol (70:30). Chromatographic separation was performed on a HILIC BEH amide column (2.1mm×50mm, 1.7µm) with a 3min gradient elution at a flow rate of 0.5mL/min. For mass spectrometric detection, selected reaction monitoring (SRM) was used; the SRM transitions were m/z 144→98 and m/z 144→56 for cyclocreatine and m/z 148→102 for the internal standard (D4-cyclocreatine) in the positive ionization mode. No endogenous components interfered with the analysis of cyclocreatine and the internal standard in mouse and rat plasma. Plasma calibration curves were constructed in the range of 0.01-25µM. The correlation coefficient of the calibration curves was greater than 0.99. The mean intraday assay accuracy for all quality control (QC) replicates was between 93 and 105%. The mean intraday assay precision (CV%) was 1.9-11% for all QC levels. The HILIC-UPLC-MS/MS method was successfully applied in pharmacokinetic (PK) studies of cyclocreatine in mice and rats for the first time. After a single 30mg/kg oral administration in mice and rats, the AUC0-∞ (area under the curve) was 84.1µgh/mL and 91.7±18.0µgh/mL, respectively.

Synthesis and structure-activity relationship studies of N-benzyl-2-phenylpyrimidin-4-amine derivatives as potent USP1/UAF1 deubiquitinase inhibitors with anticancer activity against nonsmall cell lung cancer.

Deregulation of ubiquitin conjugation or deconjugation has been implicated in the pathogenesis of many human diseases including cancer. The deubiquitinating enzyme USP1 (ubiquitin-specific protease 1), in association with UAF1 (USP1-associated factor 1), is a known regulator of DNA damage response and has been shown as a promising anticancer target. To further evaluate USP1/UAF1 as a therapeutic target, we conducted a quantitative high throughput screen of >400000 compounds and subsequent medicinal chemistry optimization of small molecules that inhibit the deubiquitinating activity of USP1/UAF1. Ultimately, these efforts led to the identification of ML323 (70) and related N-benzyl-2-phenylpyrimidin-4-amine derivatives, which possess nanomolar USP1/UAF1 inhibitory potency. Moreover, we demonstrate a strong correlation between compound IC50 values for USP1/UAF1 inhibition and activity in nonsmall cell lung cancer cells, specifically increased monoubiquitinated PCNA (Ub-PCNA) levels and decreased cell survival. Our results establish the druggability of the USP1/UAF1 deubiquitinase complex and its potential as a molecular target for anticancer therapies.

Passive lipoidal diffusion and carrier-mediated cell uptake are both important mechanisms of membrane permeation in drug disposition.

Recently, it has been proposed that drug permeation is essentially carrier-mediated only and that passive lipoidal diffusion is negligible. This opposes the prevailing hypothesis of drug permeation through biological membranes, which integrates the contribution of multiple permeation mechanisms, including both carrier-mediated and passive lipoidal diffusion, depending on the compound's properties, membrane properties, and solution properties. The prevailing hypothesis of drug permeation continues to be successful for application and prediction in drug development. Proponents of the carrier-mediated only concept argue against passive lipoidal diffusion. However, the arguments are not supported by broad pharmaceutics literature. The carrier-mediated only concept lacks substantial supporting evidence and successful applications in drug development.

Synthesis and structure-activity relationship studies of 4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide derivatives as potent and selective inhibitors of 12-lipoxygenase.

Human lipoxygenases (LOXs) are a family of iron-containing enzymes which catalyze the oxidation of polyunsaturated fatty acids to provide the corresponding bioactive hydroxyeicosatetraenoic acid (HETE) metabolites. These eicosanoid signaling molecules are involved in a number of physiologic responses such as platelet aggregation, inflammation, and cell proliferation. Our group has taken a particular interest in platelet-type 12-(S)-LOX (12-LOX) because of its demonstrated role in skin diseases, diabetes, platelet hemostasis, thrombosis, and cancer. Herein, we report the identification and medicinal chemistry optimization of a 4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide-based scaffold. Top compounds, exemplified by 35 and 36, display nM potency against 12-LOX, excellent selectivity over related lipoxygenases and cyclooxygenases, and possess favorable ADME properties. In addition, both compounds inhibit PAR-4 induced aggregation and calcium mobilization in human platelets and reduce 12-HETE in ß-cells.

Evidence-based approach to assess passive diffusion and carrier-mediated drug transport.

Evidence supporting the action of passive diffusion and carrier-mediated (CM) transport in drug bioavailability and disposition is discussed to refute the recently proposed theory that drug transport is CM-only and that new transporters will be discovered that possess transport characteristics ascribed to passive diffusion. Misconceptions and faulty speculations are addressed to provide reliable guidance on choosing appropriate tools for drug design and optimization.

Identification of 3-sulfonylindazole derivatives as potent and selective 5-HT(6) antagonists.

As part of our efforts to develop agents for cognitive enhancement, we have been focused on the 5-HT(6) receptor in order to identify potent and selective ligands for this purpose. Herein we report the identification of a novel series of 3-sulfonylindazole derivatives with acyclic amino side chains as potent and selective 5-HT(6) antagonists. The synthesis and detailed SAR of this class of compounds are reported.

The effect of plasma protein binding on in vivo efficacy: misconceptions in drug discovery.

Data from in vitro plasma protein binding experiments that determine the fraction of protein-bound drug are frequently used in drug discovery to guide structure design and to prioritize compounds for in vivo studies. However, we consider that these practices are usually misleading, because in vivo efficacy is determined by the free (unbound) drug concentration surrounding the therapeutic target, not by the free drug fraction. These practices yield no enhancement of the in vivo free drug concentration. So, decisions based on free drug fraction could result in the wrong compounds being advanced through drug discovery programmes. This Perspective provides guidance on the application of plasma protein binding information in drug discovery.

Insights for predicting blood-brain barrier penetration of CNS targeted molecules using QSPR approaches.

Due to the high attrition rate of central nervous system drug candidates during clinical trials, the assessment of blood-brain barrier (BBB) penetration in early research is particularly important. A genetic approximation (GA)-based regression model was developed for predicting in vivo blood-brain partitioning data, expressed as logBB (log[brain]/[blood]). The model was built using an in-house data set of 193 compounds assembled from 22 different therapeutic projects. The final model (cross-validated r(2) = 0.72) with five molecular descriptors was selected based on validation using several large internal and external test sets. We demonstrate the potential utility of the model by applying it to a set of literature reported secretase inhibitors. In addition, we describe a rule-based approach for rapid assessment of brain penetration with several simple molecular descriptors.

Stability challenges in drug discovery.

Stability is one of the most important properties of drug candidates. Instable compounds can lead to false positive high-throughput screening (HTS) hits, incorrect bioassay results, erroneous structure-activity relationships (SAR), low oral bioavailability, drug withdrawal, toxic reactions from degradation products, and difficult formulation development. Screening of stability has been implemented early in drug discovery to identify labile chemotypes and guide structural modification. The most commonly applied stability studies in drug discovery are stability-pH profile, stability in gastrointestinal fluids, stability in bioassay media, excipient compatibility, and prodrug screening. The strategy enhances the quality of drug development candidates and reduces the risks.

Drug-like property concepts in pharmaceutical design.

The pharmaceutical industry is facing an ever increasing challenge to deliver safer and more effective medicines. Traditionally, drug discovery programs were driven solely by potency, regardless of the properties. As a result, the development of non-drug-like molecules was costly, had high risk and low success rate. To meet the challenges, the bar has been rising higher for drug candidates. They not only need to be active, but also drug-like to be advanced to clinical development. Drug-like properties, such as solubility, permeability, metabolic stability and transporter effects are of critical importance for the success of drug candidates. They affect oral bioavailability, metabolism, clearance, toxicity, as well as in vitro pharmacology. Insoluble and impermeable compounds can result in erroneous biological data and unreliable SAR in enzyme and cell-based assays. Rapid metabolism by enzymes and high efflux by transporters can lead to high clearance, short half-life, low systemic exposure and inadequate efficacy. Early property information helps teams make informed decisions and avoids wasting precious resources. Structure-property relationships are essential to guide structural modification to improve properties. High throughput ADME/TOX assays have been implemented and are being widely used to drive drug discovery projects in parallel with activity screening. Property design has become an integrated and inseparable part of the modern drug discovery paradigm. The approach has been proven to be a winning strategy.

Identification of a novel series of 3-piperidinyl-5-sulfonylindazoles as potent 5-HT6 ligands.

Cognitive dysfunction is a characteristic of various forms of dementia such as Alzheimer's disease (AD) and a core feature of schizophrenia. As part of our continuing efforts to develop agents for cognitive enhancement, we have been focused on the 5-HT(6) receptor-one of the emerging therapeutic targets in this area. Herein, we report the identification of a novel series of 3-piperidinyl-5-sulfonylindazole derivatives as potent 5-HT(6) antagonists. The synthesis and SAR of this class of compounds are reported.

1-Sulfonylindazoles as potent and selective 5-HT6 ligands.

As part of our continuing efforts to identify therapeutics for CNS diseases, such as schizophrenia and Alzheimer's disease (AD), we have been focused on the 5-HT(6) receptor in an attempt to identify ligands as a potential treatment for cognitive dysfunction. Herein we report the identification of a novel series of 1-sulfonylindazole derivatives as potent and selective 5-HT(6) antagonists. The synthesis and SAR of this class of compounds are reported. Several potent compounds in both binding and cyclase functional assays also display good selectivity, microsomal stability, solubility, and brain penetration as well as low cytochrome P450 inhibition. One compound exemplified in this series showed 24% oral bioavailability and in vivo efficacy in a NOR cognition model at 10mg/kg following an oral administration in rats.

Identification of a series of benzoxazoles as potent 5-HT6 ligands.

As part of our continuing efforts to identify therapeutics for CNS diseases such as schizophrenia and Alzheimer's disease (AD), we have been focused on the 5-HT(6) receptor in order to identify potent and selective ligands as a potential treatment for cognitive dysfunction. Herein we report the identification of a novel series of benzoxazole derivatives as potent 5-HT(6) ligands. The synthesis and detailed SAR of this class of compounds are reported. The compounds have been shown to be full antagonists in a cyclic AMP functional assay.

Comparison of blood-brain barrier permeability assays: in situ brain perfusion, MDR1-MDCKII and PAMPA-BBB.

Permeability data from MDR1-MDCKII and PAMPA-BBB assays were compared to data from in situ brain perfusion to evaluate the accuracy of in vitro assays in predicting in vivo blood-brain barrier (BBB) permeability. PAMPA-BBB significantly correlated to in situ brain perfusion, however, MDR1-MDCKII had no correlation with in situ brain perfusion. PAMPA-BBB also significantly correlated with MDR1-MDCKII. The differential correlation of PAMPA-BBB and MDR1-MDCKII to in situ brain perfusion appears to be mainly due to the difference in membrane characteristics rather than binding to brain tissue. The MDR1-MDCKII cell membrane has lower ratios of: phospholipid to cholesterol, unsaturated to saturated acyl chains, and phosphatidyl-choline (PC) to sphingomyelin (SM) than brain endothelial cells, making it a poor passive permeability model for BBB. The BBB is more hydrophobic, rigid, and less fluidic than MDR1-MDCKII cell membrane. PAMPA-BBB more closely matches the BBB membrane in these characteristics and is a more accurate passive diffusion permeability model for BBB than MDR1-MDCKII. PAMPA-BBB is high throughput, low cost and has good prediction of in vivo BBB permeability, and therefore, it is a valuable tool in drug discovery to screen compounds for the rate of brain penetration.

Solution stability--plasma, gastrointestinal, bioassay.

Solution stability of drug candidates in plasma, gastrointestinal fluids and bioassays is important in order to achieve low clearance, good oral bioavailability and have robust SAR. Screening of solution stability early in drug discovery can avoid pursuing hits with high risk of instability, prioritize chemical series, guide structural modification, and enhance the chance of project success. The conditions of solution stability methods are critical in generating relevant data and include: test compound concentration, enzyme source and preparation, limits of solubility, cosolvent, plasma protein binding effect, detection techniques (LC-UV vs. LC-MS), and what to detect (disappearance of parent vs. formation of degradants). Details of methodologies, applications, structure-stability relationships and case studies are discussed.

In vitro solubility assays in drug discovery.

The solubility of a compound depends on its structure and solution conditions. Structure determines the lipophilicity, hydrogen bonding, molecular volume, crystal energy and ionizability, which determine solubility. Solution conditions are affected by pH, co-solvents, additives, ionic strength, time and temperature. Many drug discovery experiments are conducted under "kinetic" solubility conditions. In drug discovery, solubility has a major impact on bioassays, formulation for in vivo dosing, and intestinal absorption. A good goal for the solubility of drug discovery compounds is >60 ug/mL. Equilibrium solubility assays can be conducted in moderate throughput, by incubating excess solid with buffer and agitating for several days, prior to filtration and HPLC quantitation. Kinetic solubility assays are performed in high throughput with shorter incubation times and high throughput analyses using plate readers. The most frequently used of these are the nephelometric assay and direct UV assay, which begin by adding a small volume of DMSO stock solution of each test compound to buffer. In nephelometry, this solution is serially diluted across a microtitre plate and undissolved particles are detected via light scattering. In direct UV, undissolved particles are separated by filtration, after which the dissolved material is quantitated using UV absorption. Equilibrium solubility is useful for preformulation. Kinetic solubility is useful for rapid compound assessment, guiding optimization via structure modification, and diagnosing bioassays. It is often useful to customize solubility experiments using conditions that answer specific research questions of drug discovery teams, such as compound selection and vehicle development for pharmacology and PK studies.

Applications of high throughput microsomal stability assay in drug discovery.

High throughput in vitro microsomal stability assays are widely used in drug discovery as an indicator for in vivo stability, which affects pharmacokinetics. This is based on in-depth research involving a limited number of model drug-like compounds that are cleared predominantly by cytochrome P450 metabolism. However, drug discovery compounds are often not drug-like, are assessed with high throughput assays, and have many potential uncharacterized in vivo clearance mechanisms. Therefore, it is important to determine the correlation between high throughput in vitro microsomal stability data and abbreviated discovery in vivo pharmacokinetics study data for a set of drug discovery compounds in order to have evidence for how the in vitro assay can be reliably applied by discovery teams for making critical decisions. In this study the relationship between in vitro single time point high throughput microsomal stability and in vivo clearance from abbreviated drug discovery pharmacokinetics studies was examined using 306 real world drug discovery compounds. The results showed that in vitro Phase I microsomal stability t(1/2) is significantly correlated to in vivo clearance with a p-value<0.001. For compounds with low in vitro rat microsomal stability (t(1/2)<15 min), 87% showed high clearance in vivo (CL>25 mL/min/kg). This demonstrates that high throughput microsomal stability data are very effective in identifying compounds with significant clearance liabilities in vivo. For compounds with high in vitro rat microsomal stability (t(1/2)>15 min), no significant differentiation was observed between high and low clearance compounds. This is likely owing to other clearance pathways, in addition to cytochrome P450 metabolism that enhances in vivo clearance. This finding supports the strategy used by medicinal chemists and drug discovery teams of applying the in vitro data to triage compounds for in vivo PK and efficacy studies and guide structural modification to improve metabolic stability. When in vitro and in vivo data are both available for a compound, potential in vivo clearance pathways can be diagnosed to guide further discovery studies.