Design and Synthesis of Selective Phosphodiesterase 4D (PDE4D) Allosteric Inhibitors for the Treatment of Fragile X Syndrome and Other Brain Disorders.

Novel pyridine- and pyrimidine-based allosteric inhibitors are reported that achieve PDE4D subtype selectivity through recognition of a single amino acid difference on a key regulatory domain, known as UCR2, that opens and closes over the catalytic site for cAMP hydrolysis. The design and optimization of lead compounds was based on iterative analysis of X-ray crystal structures combined with metabolite identification. Selectivity for the activated, dimeric form of PDE4D provided potent memory enhancing effects in a mouse model of novel object recognition with improved tolerability and reduced vascular toxicity over earlier PDE4 inhibitors that lack subtype selectivity. The lead compound, 28 (BPN14770), has entered midstage, human phase 2 clinical trials for the treatment of Fragile X Syndrome.

Meeting report: metabolites in safety testing (MIST) symposium-safety assessment of human metabolites: what's REALLY necessary to ascertain exposure coverage in safety tests?

In the 2012 AAPS metabolites in safety testing (MIST) symposium held in Chicago, IL, USA, on October 15, 2012, regulatory experts and industrial scientists joined together to discuss their perspectives and strategies in addressing contemporary MIST recommendations (FDA 2008, International Conference on Harmonization (ICH) M3(R2), ICH M(R2) Q&A). Overall, these regulatory guidances indicate that metabolites identified in human plasma should circulate at similar or greater concentrations in at least one of the animal species used in nonclinical safety assessment of the parent drug. However, synthetic standards for the metabolites often do not exist or they are intractable to synthesize, thus introducing multiple challenges in drug development for the quantitative comparison of metabolites between human and animals. A tiered bioanalytical strategy for metabolite analysis is a prevalent approach to demonstrate coverage in animals. Recent developments in bioanalytical methodology have yielded several time- and resource-sparing strategies to provide fit-for-purpose approaches that can enable critical decisions related to metabolite quantification and monitoring in plasma. This report summarizes the presentations and panel discussions at the symposium.

Radiolabeled mass-balance excretion and metabolism studies in laboratory animals: a commentary on why they are still necessary.

The necessity of conducting traditional radiolabeled absorption, distribution, metabolism and excretion (ADME) studies in animals during development of new investigative agents has been questioned in a recent review. We present a compilation of the benefits of such studies in the understanding of the in vivo pharmacological activity and disposition of new drug candidates, including interpretation of preclinical toxicology findings, characterization of circulating metabolites, and determination of principal pathways of clearance. This understanding is valuable in anticipating the human disposition of the drugs and the planning of the clinical development program. Because of new technologies, evolving regulatory expectations, and increased scientific understanding of the disposition of drugs, the traditional design and timing of both animal and human ADME studies should be reviewed. Innovative "fit-for-purpose" studies may well be a better choice in a particular drug development program than a standard animal ADME "package". However, we submit that, at this time, radiolabeled animal ADME studies still provide a definitive and irreplaceable component of our understanding of the in vivo actions and behaviors of drugs and should continue to be performed prior to the exposure of large numbers of human subjects to investigative drugs.

The role of exploratory drug metabolism and pharmacokinetics in new drug research: case study-selection of a thrombin receptor antagonist for development.

The rising costs and time associated with bringing new medicines to the market have created a need for a new paradigm for reducing the attrition rates of drug candidates in both preclinical and clinical development stages. Early appraisal of drug metabolism and pharmacokinetic (DMPK) parameters is now possible due to several higher throughput in vitro and in vivo screens. This knowledge of DMPK properties should not only shorten the timelines for the selection of drug candidates but also enhance the probability of their success for development. The role of DMPK researchers in the drug research paradigm should not be limited to screening a large array of compounds during the lead optimization process but should include a strive for an understanding of the absorption, distribution, metabolism, excretion, and potential drug-related toxicities of a chemical series. As an example, in this article we present a specific DMPK research screening paradigm and describe a case study using the Thrombin Receptor Antagonist program. This screening paradigm followed by the extensive lead optimization process culminated in the selection of SCH 530348, a potent, selective and orally active thrombin receptor antagonist for the treatment of thrombosis.

Application and interpretation of hPXR screening data: Validation of reporter signal requirements for prediction of clinically relevant CYP3A4 inducers.

A human pregnane X receptor (PXR) reporter-gene assay was established and validated using 19 therapeutic agents known to be clinical CYP3A4 inducers, 5 clinical non-inducers, and 6 known inducers in human hepatocytes. The extent of CYP3A4 induction (measured as RIF ratio in comparison to rifampicin) and EC50 was obtained from the dose-response curve. All of the clinical inducers (19/19) and human hepatocyte inducers (6/6) showed positive responses in the PXR assay. One out of five clinical non-inducers, pioglitazone, also showed a positive response. An additional series of 18 commonly used drugs with no reports of clinical induction was also evaluated as putative negative controls. Sixteen of these were negative (89%), whereas two of these, flutamide and haloperidol showed 16-fold (RIF ratio 0.79) and 10-fold (RIF ratio 0.48) maximal induction, respectively in the reporter-gene system. Flutamide and haloperidol were further demonstrated to cause CYP3A4 induction in human cryopreserved hepatocytes based on testosterone 6beta-hydroxylation activity. The induction potential index calculated based on the maximum RIF ratio, EC50, and in vivo maximum plasma concentration was used to predict the likelihood of CYP3A4 induction in humans. When the induction potential index is greater than 0.08, the compound is likely to cause induction in humans. A high-throughput screening strategy was developed based on the validation results at 1microM and 10microM for the same set of drugs. A RIF ratio of 0.4 was set as more practical screening cut-off to minimize the possibility of generating false positives. Thus, a tiered approach was implemented to use the human PXR reporter-gene assay from early lead optimization to late lead characterization in drug discovery.

Evolution of thiazolidine-based blockers of human Kv1.5 for the treatment of atrial arrhythmias.

Blockade of the Kv1.5 ion channel is a potentially atrial-selective avenue for the treatment of atrial fibrillation and atrial flutter. The development and biological evaluation of a series of thiazolidine-based blockers of Kv1.5 is described.

Lead Optimization in Discovery Drug Metabolism and Pharmacokinetics/Case study: The Hepatitis C Virus (HCV) Protease Inhibitor SCH 503034.

Lead optimization using drug metabolism and pharmacokinetics (DMPK) parameters has become one of the primary focuses of research organizations involved in drug discovery in the last decade. Using a combination of rapid in vivo and in vitro DMPK screening procedures on a large array of compounds during the lead optimization process has resulted in development of compounds that have acceptable DMPK properties. In this review, we present a general screening paradigm that is currently being used as part of drug discovery at Schering-Plough and we describe a case study using the Hepatitis C Virus (HCV) protease inhibitor program as an example. By using the DMPK optimization tools, a potent HCV protease inhibitor, SCH 503034, was selected for development as a candidate drug.

Development of a high-throughput in vitro assay using a novel Caco-2/rat hepatocyte system for the prediction of oral plasma area under the concentration versus time curve (AUC) in rats.

INTRODUCTION: Previously, we have shown that a novel Caco-2/human hepatocyte system is a useful model for the prediction of oral bioavailability in humans. In this study, we attempted to use a similar system in a high-throughput screening mode for the selection of new compound entities (NCE) in drug discovery. METHODS: A total of 72 compounds randomly selected from three different chemotypes were dosed orally in rats. In vivo plasma area under the concentration versus time curve (AUC) from 0-6 h of the parent compound was determined. The same compounds were also tested in the Caco-2/rat hepatocyte system. In vitro AUC from 0-3 h in the Caco-2 rat hepatocyte system was determined. RESULTS: The predictive usefulness of the Caco-2/rat hepatocyte system was evaluated by comparing the in vivo plasma AUC and the in vitro AUC. Linear regression analysis showed a reasonable correlation (R2 = 0.5) between the in vivo AUC and the in vitro AUC. Using 0.4 microM h in vivo AUC as a cut-off, compounds were categorized as either low or high AUC. The in vitro AUC successfully matched the corresponding in vivo category for sixty-three out of seventy-two compounds. DISCUSSION: The results presented in this study suggest that the Caco-2/rat hepatocyte system may be used as a high-throughput screen in drug discovery for pharmacokinetic behaviors of compounds in rats.

Rat PXR reporter-gene activity correlates with the induction of CYP3A in rat precision-cut liver slices.

Recent studies have suggested that both constitutive androstane receptor (CAR) and pregnane X-receptor (PXR) are involved in the induction of rat liver microsomal cytochrome P-450 (CYP) 2B and 3A through a mechanism called cross-talk. In this study we intend to determine if a PXR-reporter gene assay could be used for the prediction of CYP3A and/or CYP2B induction in rats. The induction of rat CYP2B and CYP3A by nineteen structurally diverse compounds was evaluated by using rat precision-cut liver slices and a rat PXR reporter-gene system. Induction of CYP2B and CYP3A mRNAs in rat liver slices was quantified by real-time polymerase chain reaction. Rat PXR activation was measured by induction of luciferase activity in rat PXR reporter-gene system. Linear regression analysis of the fold of induction of mRNA in liver slices and the fold of luciferase activity in rat PXR reporter-gene system shows that a reasonable correlation (r2 = 0.6) exists between the CYP3A induction and the rat PXR activation. A much lower correlation was observed between CYP2B induction and the rat PXR activation (r2 = 0.1). The results from this study suggest that the PXR may play a major role in the induction of rat CYP3A, but not CYP2B. Therefore, the PXR-reporter gene assay may be useful in a high-throughput screening to predict CYP3A induction in rats.

Quantitative PCR assay for cytochromes P450 2B and 3A induction in rat precision-cut liver slices: correlation study with induction in vivo.

INTRODUCTION: In drug development, new chemical entities that cause cytochrome P450 induction are considered to be undesirable since P450 induction is linked to tumor formation and may compromise the evaluation of drug safety when autoinduction results in poor drug exposure. METHODS: We evaluated the use of the precision-cut liver slice as a model for measuring induction of cytochrome P450 in rats. Quantitative real-time reverse-transcription polymerase chain reaction was used to analyze the induction of selected forms of cytochrome P450 at the mRNA level. Firstly, the system was validated against known inducers of CYP2B and 3A. Subsequently, 26 proprietary compounds were tested in rat liver slices and rats in vivo for CYP2B and 3A induction. RESULTS: Exposure of liver slices to the known CYP2B inducers phenobarbital, benzoyl-pyridine, cabarmazepine, metyrapone, RU486 and dexamethasone caused elevation of CYP2B1/2 expression 10- to 40-fold compared to the control values. The CYP3A inducers PCN, dexamethasone, nicardipine, nifedipine, clotrimazole and RU486 induced a 4- to 50-fold expression of CYP3A14. For 26 proprietary compounds, a correlation with an R(2) value of 0.74 was established between the induction of CYP2B expression in liver slices and that in rats in vivo. When liver slice results were used to predict the induction of CYP2B in rats in vivo, the success rate was 91%. The induction of CYP3A in rats in vivo was analyzed by Western blot, then quantified by densitometry. There was a good correlation between CYP3A induction in liver slices and induction in vivo as assessed by Western blot, with an 86% positive prediction rate. DISCUSSION: The use of liver slices in combination with TaqMan technology provides a good model for predicting CYP induction in the rat. This method is useful for identifying compounds with CYP2B and 3A induction liability in the early phase of drug discovery.

Evaluation of a novel in vitro Caco-2 hepatocyte hybrid system for predicting in vivo oral bioavailability.

A novel in vitro Caco-2 hepatocyte hybrid system was set up and tested for its ability to predict the oral bioavailability (F) in humans of 24 randomly chosen marketed drugs. Caco-2 cells were cultured on the transwell filters to form tight junctions. Pooled cryopreserved human hepatocytes were placed in the basolateral receiver compartment. To evaluate the permeability and hepatic first pass in one experiment, compounds were dissolved in medium and added to the apical donor compartment of the transwell apparatus, and the amount of the parent compound appearing in the basolateral receiver compartment was determined over a 3-h time course. The area under the concentration versus time curve (AUC) of the parent compound was determined. The predictive usefulness of this Caco-2 hepatocyte model was tested by comparing the AUC with the in vivo oral bioavailability reported in the literature. Linear regression analysis shows a reasonable correlation (R(2) = 0.86) between the in vitro AUC and oral bioavailability reported in the literature. Based on the literature data, the compounds were classified into low (F < 20%), medium (20 < F < 50%), and high (F > 50%) bioavailability categories. The oral bioavailability predicted from the experimental Caco-2 hepatocyte system successfully matches the appropriate literature-based bioavailability category for 22 of 24 of the compounds. The results presented in this study suggest that it may be feasible to combine Caco-2 cells and hepatocytes into one system for the prediction of oral absorption and first-pass effect in humans.

Whatever happened to cassette-dosing pharmacokinetics?

Cassette dosing is a procedure that is used for rapidly assessing the pharmacokinetics of a series of discovery drug candidates by dosing a mixture of compounds rather than a single compound. Cassette dosing has advantages and disadvantages associated with its use, which leads to controversy about how and if it should be used. To assess the current practices of the pharmaceutical industry regarding cassette dosing, a survey of several pharmaceutical companies was conducted. Analysis of the survey revealed that opinion on this subject is divided within the pharmaceutical industry. In addition, it was determined that approximately only a half of those companies that perform in vivo pharmacokinetic screening use cassette dosing for this purpose.

Interactions between penicillin-binding proteins (PBPs) and two novel classes of PBP inhibitors, arylalkylidene rhodanines and arylalkylidene iminothiazolidin-4-ones.

Several non-beta-lactam compounds were active against various gram-positive and gram-negative bacterial strains. The MICs of arylalkylidene rhodanines and arylalkylidene iminothiazolidin-4-ones were lower than those of ampicillin and cefotaxime for methicillin-resistant Staphylococcus aureus MI339 and vancomycin-resistant Enterococcus faecium EF12. Several compounds were found to inhibit the cell wall synthesis of S. aureus and the last two steps of peptidoglycan biosynthesis catalyzed by ether-treated cells of Escherichia coli or cell wall membrane preparations of Bacillus megaterium. The effects of the arylalkylidene rhodanines and arylalkylidene iminothiazolidin-4-one derivatives on E. coli PBP 3 and PBP 5, Streptococcus pneumoniae PBP 2xS (PBP 2x from a penicillin-sensitive strain) and PBP 2xR (PBP 2x from a penicillin-resistant strain), low-affinity PBP 2a of S. aureus, and the Actinomadura sp. strain R39 and Streptomyces sp. strain R61 DD-peptidases were studied. Some of the compounds exhibited inhibitory activities in the 10 to 100 microM concentration range. The inhibition of PBP 2xS by several of them appeared to be noncompetitive. The dissociation constant for the best inhibitor (Ki = 10 microM) was not influenced by the presence of the substrate.

Development of a screening assay to measure the loss of antibacterial activity in the presence of proteins: its use in optimizing compound structure.

An assay quantifying the loss of antibacterial potency of compounds, originally identified via target-based screening, in the presence of increasing albumin concentration was developed and used as a technique to measure potential association of compounds with proteins unrelated to their molecular target. Minimum inhibitory concentrations (MICs) of test compounds were measured against Staphylococcus aureus strain ATCC 6538 in the presence of 0-12 muM bovine serum albumin (BSA). The linear regression coefficient r(2) for the correlation between MIC and BSA concentration was >/= 0.9 for 49 and > 0.5 for 62 out of a total of 69 compounds tested. The slope of these correlations varied widely from < 1 to 99, suggesting that the loss of potency due to a given concentration of BSA could vary from compound to compound due to wide variation in the apparent stoichiometry for protein-ligand association. Follow-up experiments using additional proteins and a fatty acid, oleic acid, showed that this compound:BSA association was not protein specific, but was likely driven by hydrophobicity. The method described in this report can be used to optimize compound design and minimize this undesirable effect.

Development of a novel in vitro model to predict hepatic clearance using fresh, cryopreserved, and sandwich-cultured hepatocytes.

Sixty-four compounds with diverse structures were used in evaluation of intrinsic clearance by various hepatocyte preparations from rats, dogs, monkeys, and humans. Intrinsic clearance (CL(int)) was calculated from the ratio of the initial amount of the test compound minus the amount remaining after 2 h of incubation and the corresponding area under the concentration versus time curve. The predictive potential of this in vitro model was tested by comparing the intrinsic clearance with in vivo clearance using linear regression analysis. In addition, the intrinsic clearance values obtained from three different types of hepatocytes (cryopreserved, fresh, and sandwich-cultured) from the same species were compared to determine the influence of preservation and culture conditions. It seems that intrinsic clearance determined from human cryopreserved hepatocyte (R(2) = 0.87) was the best predictor for the corresponding human in vivo clearance. Dog and rat hepatocyte clearances were also demonstrated to be reasonable predictors (R(2) ranged 0.68-0.85 in dogs and 0.65-0.72 in rats) for their corresponding in vivo clearances. Monkey hepatocyte clearance seems to be the worst predictor for the corresponding in vivo clearance (R(2) = 0.35-0.67). Comparison of intrinsic clearance generated from cryopreserved and fresh hepatocytes demonstrated a very good correlation in dog (R(2) = 0.82) followed by rat (R(2) = 0.77), and then by monkey (R(2) = 0.70). A similar correlation profile was shown between cryopreserved hepatocytes and sandwich-cultured hepatocytes. These results demonstrated the predictive potential of intrinsic clearance for rat, dog, and human in vivo clearance, but also showed some limitation of the approach for monkey.

Semi-automated determination of plasma stability of drug discovery compounds using liquid chromatography-tandem mass spectrometry.

A simple procedure for the measurement of stability of drug candidates in plasma was developed to eliminate the traditional labor-intensive and time-consuming sample preparation procedures that are typically used for these studies. The procedure makes use of a thermostatic autosampler as an incubator combined with the direct plasma injection method based on high-performance liquid chromatography (HPLC) coupled to tandem mass spectrometry (MS-MS). Untreated human, monkey, mouse and rat plasma containing the test compound was directly injected into a mixed-function column for on-line protein removal and chromatography. The test compound and its biotransformation product were separated via HPLC and monitored using the tandem mass spectrometer. The need for adequate chromatographic separation of the test compound (M) from its carboxylic acid metabolite (M+1) is demonstrated. Plasma samples from four different species at specified incubation temperatures were sequentially assayed in one analytical procedure. The injection-to-injection time was about 6 min. The peak responses of the test compound in individual plasma samples were repeatedly determined every 24 min. The retention times and peak shape of all analytes were found to be consistent throughout the experiments. The stability of the test compound in plasma was found to be a function of animal species, incubation time and temperature. The test compound was rapidly degraded in rat plasma at 37 degrees C, but it could be stabilized by adding sodium thiosulfate.

Insights from a three-dimensional model into ligand binding to constitutive active receptor.

Two orphan nuclear receptors, constitutive active (or androstane) receptor (CAR) and pregnane X receptor (PXR), are among the most important mediators of ligand-activated transcriptional induction of liver microsomal cytochrome P450 drug-metabolizing enzymes. CAR and PXR belong to the same NR1I receptor subfamily and show high sequence homology to each other. The vitamin D receptor (VDR) also belongs to the NR1I subfamily and has the second highest homology to CAR in the ligand binding domain. A 3D model of the ligand binding domain of human CAR (hCAR) was constructed based on the available X-ray structures of human PXR (hPXR) and VDR (hVDR). The model shows that the size of the ligand binding cavities of hCAR and hPXR are similar, but larger than that of hVDR. hPXR's capability of binding to extremely large ligands, such as rifampicin, implies that its binding cavity may be able to expand further through the flexibility of a surface loop. In contrast, hCAR does not have this loop so that its cavity cannot expand, suggesting that hCAR would not bind to the largest hPXR ligands. Docking calculations of selected ligands to hCAR, based on the structural model, are consistent with previously reported receptor binding data. The results from this study indicate that structural modeling will be a useful tool for understanding ligand binding to hCAR and for design of drugs free of hCAR-mediated enzyme induction.

Rapid determination of pharmacokinetic properties of new chemical entities: in vivo approaches.

There is a continuing need for increased throughput in the evaluation of new chemical entities in terms of their pharmacokinetic (PK) parameters as part of new drug discovery. This review summarizes various approaches that have been used to increase throughput in this area. The article divides the approaches into two areas: assay enhancement and sample reduction.

A high-throughput cell-based reporter gene system for measurement of CYP1A1 induction.

INTRODUCTION: Enzyme induction is undesirable in new drug discovery process, with consequences spanning from auto-induction to toxicity. Cytochrome P450 (CYP) 1A1 has long been known to be one of the metabolic enzymes involved in activating many procarcinogens, the first step toward tumor formation during chemical carcinogenesis. Induction of CYP1A1 during drug treatment may predispose the patients to some risk of chemical carcinogenesis. METHODS: Based on the signal-transduction mechanism of CYP1A1 induction, a high-throughput reporter-gene system was established by stable transformation of H4IIE cells to incorporate the luciferase gene under control of CYP1A1 promoter. This stable cell line was validated with known CYP1A1 inducers, such as 3-methylcholanthrene (3-MC), beta-naphthoflavone (beta-NF), alpha-naphthoflavone (alpha-NF) and 3-indocarbinol. Thirty in-house new chemical entities (NCEs) were then screened with this reporter-gene system, and also administered to rats to evaluate in vivo CYP1A1 induction. RESULTS: CYP1A1 reporter gene system can be used to identify strong inducers, such as 3-MC, beta-NF and alpha-NF, and weak inducers, such as 3-indocarbinol. In vitro induction of 30 in-house compounds in reporter gene system did not correlate with in vivo induction in rat liver microsome measured by ethoxyresorufin-O-dealkylation (EROD) activity, but had a reasonable correlation with Western blot signals. DISCUSSION: This reporter-gene system may be useful in eliminating compounds that can cause CYP1A1 induction at an early stage of drug discovery.