The discovery of fused oxadiazepines as gamma secretase modulators for treatment of Alzheimer's disease.

In an attempt to further improve overall profiles of the oxadiazine series of GSMs, in particular the hERG activity, conformational modifications of the core structure resulted in the identification of fused oxadiazepines such as 7i which had an improved hERG inhibition profile and was a highly efficacious GSM in vitro and in vivo in rats. These SAR explorations offer opportunities to identify potential drugs to treat Alzheimer's disease.

Rapid Evolution of a Fragment-like Molecule to Pan-Metallo-Beta-Lactamase Inhibitors: Initial Leads toward Clinical Candidates.

With the emergence and rapid spreading of NDM-1 and existence of clinically relevant VIM-1 and IMP-1, discovery of pan inhibitors targeting metallo-beta-lactamases (MBLs) became critical in our battle against bacterial infection. Concurrent with our fragment and high-throughput screenings, we performed a knowledge-based search of known metallo-beta-lactamase inhibitors (MBLIs) to identify starting points for early engagement of medicinal chemistry. A class of compounds exemplified by 11, discovered earlier as B. fragilis metallo-beta-lactamase inhibitors, was selected for in silico virtual screening. From these efforts, compound 12 was identified with activity against NDM-1 only. Initial exploration on metal binding design followed by structure-guided optimization led to the discovery of a series of compounds represented by 23 with a pan MBL inhibition profile. In in vivo studies, compound 23 in combination with imipenem (IPM) robustly lowered the bacterial burden in a murine infection model and became the lead for the invention of MBLI clinical candidates.

Protein binding-dependent decreases in hERG channel blocker potency assessed by whole-cell voltage clamp in serum.

In vitro hERG blocking potency is measured in drug discovery as part of an integrated cardiovascular risk assessment. Typically, the concentrations producing 50% inhibition are measured in protein-free saline solutions and compared with calculated free therapeutic in vivo Cmax values to estimate a hERG safety multiple. The free/unbound fraction is believed responsible for activity. We tested the validity of this approach with 12 compounds by determining potencies in voltage clamp studies conducted in the absence and presence of 100% dialyzed fetal bovine serum (FBS). Bath drug concentrations in saline solutions were measured to account for loss of compounds due to solubility, stability, and/or adsorption. Protein binding in dialyzed FBS was measured to enable predictions of serum IC50s based on the unbound fraction and the saline IC50. For 11 of 12 compounds, the measured potency in the presence of dialyzed FBS was within 2-fold of the predicted potency. The predicted IC50 in dialyzed FBS for one highly bound compound, amiodarone, was 9-fold higher than the measured serum IC50. These data suggest that for highly bound compounds, direct measurement of IC50s in the presence of 100% serum may provide a more accurate estimate of in vivo potencies than the approach based on calculated serum shifts.

Iminoheterocycles as gamma-secretase modulators.

The synthesis of a novel series of iminoheterocycles and their structure-activity relationship (SAR) as modulators of gamma-secretase activity will be detailed. Encouraging SAR generated from a monocyclic core led to a structurally unique bicyclic core. Selected compounds exhibit good potency as gamma-secretase modulators, excellent rat pharmacokinetics, and lowering of Abeta42 levels in various in vivo models.

Discovery of amide and heteroaryl isosteres as carbamate replacements in a series of orally active gamma-secretase inhibitors.

The design of amide and heteroaryl amide isosteres as replacements for the carbamate substructure in previously disclosed 2,6-disubstituted piperidine N-arylsulfonamides is described. In several cases, amides lessened CYP liabilities in this class of gamma-secretase inhibitors. Selected compounds showed significant reduction of Abeta levels upon oral dosing in a transgenic murine model of Alzheimer's disease.

Temporal evaluation of CYP mRNA in mice administered with prototypical P450 inducers: comparison with conventional protein/enzyme methods.

Assessment of cytochrome P450 (CYP) induction at the mRNA level in preclinical rodent studies has gained interest in recent years, but there are still concerns regarding correlations between the mRNA and the enzyme activity levels, especially in mice. The purpose of the present study was to systematically evaluate patterns of temporal changes of CYPs 1a1, 1a2, 2b10, 3a11, and 4a10 at mRNA, protein, and activity levels in order to determine to what extent mRNA levels could be used either qualitatively or quantitatively for the assessment of CYP enzyme induction. In this study, livers from male CD-1 mice treated daily with beta-naphthoflavone, phenobarbital, dexamethasone, clofibrate, and control vehicles were collected for RNA and microsomal analysis after 0.5, 1, 2, 4, and 8 days of daily dose. The results revealed a good correlation among mRNA, protein, and enzyme activity levels, with the best correlation at the time points between Days 2 and 8, suggesting that the appropriate time to monitor CYP mRNA may be beyond Day 2 of chemical treatments. Based on these results, we concluded that the mRNA approach is a useful tool to monitor CYP induction in mice, particularly when treatment duration is beyond 2 days.

Utility of mass spectrometry for in-vitro ADME assays.

A balance between pharmacological activity, safety and drug metabolism and pharmacokinetics (DMPK) attributes determines the fate of a new chemical entity (NCE) in drug discovery. Because of the increased number of NCEs requiring DMPK evaluation, several in vitro higher-throughput screens and counter screens designed to evaluate DMPK attributes have been introduced in drug discovery. The DMPK screens evaluate NCEs for potential absorption, metabolism, drug-drug interactions, brain penetration, protein binding and pharmacokinetics. Higher-throughput analytical methodologies for the determination of either a common end product of a screen or the parent compound (and/or possible metabolites) are essential for successful DMPK screens. Because of its speed, sensitivity and specificity, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the technology of choice for sample analysis. In this review, several in vitro screening assays that we employ in drug discovery are discussed with an emphasis on LC-MS/MS role in accelerating them.

Synthesis and SAR Studies of Fused Oxadiazines as γ-Secretase Modulators for Treatment of Alzheimer's Disease.

Fused oxadiazines (3) were discovered as selective and orally bioavailable γ-secretase modulators (GSMs) based on the structural framework of oxadiazoline GSMs. Although structurally related, initial modifications showed that structure-activity relationships (SARs) did not translate from the oxadiazoline to the oxadiazine series. Subsequent SAR studies on modifications at the C3 and C4 positions of the fused oxadiazine core helped to identify GSMs such as compounds 8r and 8s that were highly efficacious in vitro and in vivo in a number of animal models with highly desirable physical and pharmacological properties. Further improvements of in vitro activity and selectivity were achieved by the preparation of fused morpholine oxadiazines. The shift in specificity of APP cleavage rather than a reduction in overall γ-secretase activity and the lack of changes in substrate accumulation and Notch processing as observed in the animal studies of compound 8s confirm that the oxadiazine series of compounds are potent GSMs.

Cyclic hydroxyamidines as amide isosteres: discovery of oxadiazolines and oxadiazines as potent and highly efficacious γ-secretase modulators in vivo.

Cyclic hydroxyamidines were designed and validated as isosteric replacements of the amide functionality. Compounds with these structural motifs were found to be metabolically stable and to possess highly desirable pharmacokinetic profiles. These designs were applied in the identification of γ-secretase modulators leading to highly efficacious agents for reduction of central nervous system Aß(42) in various animal models.

The Discovery of Pyridone and Pyridazone Heterocycles as γ-Secretase Modulators.

A series of novel pyridazone and pyridone compounds as γ-secretase modulators were discovered. Starting from the initial lead, structure-activity relationship studies were carried out in which an internal hydrogen bond was introduced to conformationally fix the side chain, and compounds with improved in vitro Aß42 inhibition activity and good Aßtotal/Aß42 selectivity were quickly discovered. Compound 35 displayed very good in vitro activity and excellent selectivity with good in vivo efficacy in both CRND8 mouse and nontransgenic rat models. This compound displayed a good overall profile in terms of rat pharmacokinetics and ancillary profile. No abnormal behavior and side effects were observed in all of the studies.

The impact of protein on Caco-2 permeability of low mass balance compounds for absorption projection and efflux substrate identification.

This study was to evaluate the mechanistic effect of protein to help better interpret the permeability results for compounds with low mass balance in Caco-2 permeability assay. The absorptive or bi-directional permeability of lipophilic compounds with mass balance were measured across Caco-2 cell monolayers as well as the empty transport devices with or without protein (4% bovine serum albumin, BSA) added to the receiver side. The results from empty transport device study indicated that the filter membrane is a permeability barrier for the low mass balance compounds and protein increases permeability by improving the compound diffusivity through the filter membrane. Caco-2 permeability measured with protein provided better absorption projection. Assuming the amount of compound associated with cells as transported did not correlate with absorption. For efflux substrate identification using Caco-2 bi-directional permeability assay, protein at the receiver side had no significant effect on the conclusion regarding the tested compounds as efflux substrate but increased the permeability measurement from both transport directions. In conclusions, Caco-2 permeability results measured using protein-containing buffer at the receiver side for low mass balance compound seems to provide better correlation with in vivo absorption. The fact that protein at receiver side has minimal effect on efflux substrate identification provides scientific basis for further specific transporter characterization (such as P-gp or BCRP) using specific inhibitors, in which same concentration of inhibitor is used in both sides of the Caco-2 cell system and protein for optimal permeability assessment has to be avoided.

Blocking ion channel KCNN4 alleviates the symptoms of experimental autoimmune encephalomyelitis in mice.

The KCNN4 potassium-ion channel has been reported to play an important role in regulating antigen-induced T cell effector functions in vitro. This study presents the first evidence that a selective KCNN4 blocker, TRAM-34, confers protection against experimental autoimmune encephalomyelitis (EAE) in the mouse model. Treatment with the KCNN4 blocker did not prevent infiltration of T cells in the spinal cord, but resulted in the reduction of both the protein and the message levels of TNF-alpha and IFN-gamma as well as the message levels of several other pro-inflammatory molecules in the spinal cord. Plasma concentrations of TRAM-34 within a 24-h period were between the in vitro IC(50) and IC(90) values for the KCNN4 channel. The effect of TRAM-34 was reversible, as indicated by the development of clinical EAE symptoms within 48 h after withdrawal of treatment. In summary, our data support the idea that KCNN4 channels play a critical role in the immune response during the development of MOG-induced EAE in C57BL/6 mice.

A computer program for automated data evaluation to support in vitro higher-throughput screening for drug metabolism and pharmacokinetics attributes.

With the advances in analytical techniques, higher-throughput screening for drug metabolism and pharmacokinetics (DMPK) attributes has become an integral part of drug discovery. However, as the number of compounds increases, the volume of data that needs to be processed and evaluated increases exponentially. As a result, a major challenge for the analytical chemist is how to quickly process the vast amount of data so as to keep up with the throughput of the screening assay. We have developed a customized computer program for automated evaluation of the liquid chromatography/tandem mass spectrometric (LC/MS/MS) data generated from the in vitro DMPK screening assays. This program performs automatic data processing and quality control. It identifies analytical anomalies, such as low internal standard intensity and poor reproducibility of replicates. All analytical anomalies for individual compounds are summarized into an 'E-Log' in a color-coded format for reviewing. With the use of this program and other supporting software, data processing and evaluation for up to 100 compounds are accomplished in several minutes.

Generic fast gradient liquid chromatography/tandem mass spectrometry techniques for the assessment of the in vitro permeability across the blood-brain barrier in drug discovery.

Rapid, generic gradient liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays, designed to accelerate sample analyses, have been developed to keep pace with the productivity of advanced synthetic procedures. In this study, LC/MS/MS was combined with an in vitro, cell-based, blood-brain barrier (BBB) model to evaluate the potential of new chemical entities (NCEs) to cross the BBB. This in vitro assay provides the permeability of discovery compounds across a monolayer of a primary culture of bovine brain microvessel endothelial cells in a fraction of the time that is required for in vivo studies (brain/plasma concentrations), using only 2 mg of the compound. The results are consistent with in vivo brain/plasma concentration ratio data.

Higher-throughput screening for Caco-2 permeability utilizing a multiple sprayer liquid chromatography/tandem mass spectrometry system.

In the current drug discovery environment, higher-throughput analytical assays have become essential to keep pace with the screening demands for drug metabolism and pharmacokinetics (DMPK) attributes. This has been dictated by advances primarily in chemical procedures, notably combinatorial and parallel syntheses, which has resulted in many-fold increases in the number of compounds requiring DMPK evaluation. Because of its speed and specificity, liquid chromatography/tandem mass spectrometry (LC/MS/MS) has become the dominant technology for sample analysis in the DMPK screening assays. For higher-throughput assays, analytical speed as well as other factors such as method development, data processing, quality control, and report generation, must be optimized. The four-way multiplexed electrospray interface (MUX), which allows for the analysis of four LC eluents simultaneously, has been adopted to maximize the rate of sample introduction into the mass spectrometer. Generic fast-gradient HPLC methods that are suitable for approximately 80% of the new chemical entities encountered have been developed. In-house-written software programs have been used to streamline information flow within the system, and for quality control by automatically identifying analytical anomalies. By integrating these components together with automated method development and data processing, a system capable of screening 100 compounds per week for Caco-2 permeability has been established.

A fully automated nanoelectrospray tandem mass spectrometric method for analysis of Caco-2 samples.

Caco-2 cells offer a means to rapidly screen permeability of drug candidates, allowing pharmaceutical companies to eliminate candidates unable to cross the intestinal barrier early in the discovery process. This screening process is typically performed by conventional liquid chromatography/tandem mass spectrometry (LC/MS/MS), which can require time-consuming method development. An alternative to LC/MS/MS, automated nanoelectrospray tandem mass spectrometry (nanoESI-MS/MS), is introduced. This novel approach requires an off-line ZipTip desalting step followed by automated nanoESI-MS/MS, using the NanoMate 100 and ESI Chip. In addition to reduced method development time, automated nanoESI-MS/MS also offers no carry-over between samples, low sample consumption, and ease-of-use as compared with conventional pulled-capillary nanoelectrospray. Furthermore, the infusion system described has the potential to be high-throughput. A comparison of Caco-2 samples analyzed both by LC/MS/MS and by automated nanoESI-MS/MS is presented. The permeability and recovery data of the two compounds analyzed in this study obtained from conventional LC/MS/MS and by automated nanoESI-MS/MS were in excellent agreement.

The use of in vitro metabolic stability for rapid selection of compounds in early discovery based on their expected hepatic extraction ratios.

PURPOSE: The in vivo hepatic extraction ratio of cynomolgus monkeys was correlated with the corresponding in vitro extraction ratios that were determined in monkey microsomal incubations. METHOD: For compounds that are eliminated mainly through liver phase I metabolism, the extraction ratio calculated from liver microsomal stability studies should correlate with their in vivo hepatic extraction ratios and also with their oral bioavailability in monkey. We used both well-stirred and parallel tube models of intrinsic clearance for the correlation. We also calculated extraction ratios for compounds within a given therapeutic area from fraction absorbed values that were estimated from the Caco-2 absorption model. RESULT: The present data show that in vitro extraction ratios in monkey microsomes are predictive of the in vivo hepatic extraction ratios in monkeys. All compounds with high extraction ratio (>70%) in vivo were successfully classified as high-extraction-ratio compounds based on the in vitro monkey microsomal stability data. From the results of this study, it appears that the parallel tube model provided a slightly better classification than the well-stirred model. CONCULUSIONS: The present method appears to be a valuable tool to rapidly screen and prioritize compounds with respect to liver first-pass metabolism in monkeys at an early phase of drug discovery.

Assessment of temporal biochemical and gene transcription changes in rat liver cytochrome P450: utility of real-time quantitative RT-PCR.

PURPOSE: A conventional approach to assess cytochrome P450 (CYP) induction in preclinical animal models involves daily dosing for a least a week followed by Western blot and/or enzyme activity analysis. To evaluate the potential benefit of a third more specific and sensitive assay, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), with the objective of reducing the duration of the conventional 1-week study, we simultaneously assessed gene expression by qRT-PCR along with Western blots and enzyme activity assays as a time course in an in vivo model. METHODS: Rats were dosed daily for 8 days with model inducers of CYP1A, CYP2B, CYP3A, or CYP4A. Liver P450 levels were measured after 0.5, 1, 2, 4, and 8 days of dosing by qRT-PCR, Western blot, and enzyme activity. RESULTS: CYP1A, CYP3A, and CYP4A genes were maximally induced very rapidly (0.5-1 day), whereas the CYP2B gene was maximally induced after a lag time of 4 days. In all cases, fold changes in induction detected by qRT-PCR were greater than fold changes in protein levels and enzyme activities. CONCLUSIONS: Maximal persistent and larger fold changes observed by qRT-PCR either preceded or occurred simultaneously with maximal sustained fold changes in protein levels as measured by Western blots and enzyme activity assays. Our data show that qRT-PCR provides increased sensitivity and specificity over conventional assays and may be key information for reliable assessment of drug-related changes in CYP induction during the transition from discovery to toxicology studies.