Biomedical subjects

Li Di

Publications and source records attributed to Li Di.

At least 19 recordsLinked to original sources

PubMed2006

Comparison of cytochrome P450 inhibition assays for drug discovery using human liver microsomes with LC-MS, rhCYP450 isozymes with fluorescence, and double cocktail with LC-MS.

The disparity of IC(50)s from CYP450 inhibition assays used to assess drug-drug interaction potential was investigated, in order to have evidence for selecting a reliable in vitro CYP450 inhibition assay to support drug discovery. Three assays were studied: individual rhCYP isozymes and corresponding coumarin derivative-probe substrates with fluorescent detection, human liver microsomes (HLM) and cocktail drug-probe substrates with LC-MS detection, and double cocktail rhCYP isozymes mix and drug-probe mix with LC-MS detection. Data comparisons showed that the rhCYP-fluorescent assay and the cocktail assay with HLM-LC-MS had weak correlation. Detection method and probe substrates were shown to not be the major cause of the disparity in IC(50)s. However, the enzyme source and composition (HLM versus, rhCYP) caused disparity in IC(50)s. Specifically, the high concentrations of CYP isozymes often used with HLM-based assays produced high probe substrate conversion and test compound metabolism, which should both contribute to artificially higher IC(50)s. Non-specific binding of substrate to higher concentration proteins and lipids in the HLM-based assays should also contribute to higher IC(50)s. The modified double cocktail assay was found to overcome limitations of the other two assays. It uses an rhCYP isozymes mix, drug-probe substrate mix, low protein concentration, and LC-MS detection. The double cocktail assay is sensitive, selective, and high throughout for use in drug discovery to provide an early alert to potential toxicity with regard to drug-drug interaction, prioritize chemical series, and guide structural modification to circumvent CYP450 inhibition.

Aryl Hydrocarbon Hydroxylases↗

PubMed2006

5-Alkylated thiazolidinones as follicle-stimulating hormone (FSH) receptor agonists.

We prepared analogs of potent thiazolidinone-based follicle-stimulating hormone (FSH) agonists 1, that is, 3 that contained an additional 5-alkyl substituent. This extra substituent was added to reduce synthetic problems that arose during preparation of analogs of 1. These compounds (3) were evaluated in a Chinese hamster ovary (CHO) cell line that expressed recombinant human FSH receptor (FSHR) and a luciferase reporter gene regulated by a cAMP response element (CRE). Selected compounds were also tested on a CHO-cell line that over expressed the FSHR for the ability to induce cAMP production. When the 5-alkyl substituent was a methyl group as in analog 16a, similar FSH activity (i.e., EC(50) = 51 nM, 100% efficacy relative to hFSH) to the analogous 5-hydrogen series compound (e.g., 2) was observed; thus, proving that a small 5-alkyl substituent was well tolerated. New derivatives of 3, in which the potentially hydrolytically labile secondary amide function of 1 (-CONH-) was modified to other moieties (e.g., -CH(2)NH-, -CH(2)S-, and -CH(2)OCONH-), were also prepared and evaluated. These congeners (namely 21, 22, and 24) also displayed good potency in the CRE-luciferase assay.

Alkylation↗

PubMed2006

High throughput microsomal stability assay for insoluble compounds.

High throughput metabolic stability assays are widely implemented in drug discovery to guide structural modification, predict in vivo performance, develop structure-metabolic stability relationships, and triage compounds for in vivo animal studies. However, these methods are often developed and validated using commercial drugs. Many drug discovery compounds differ from commercial drugs, with many having high lipophilicity, high molecular weight and low solubility. The impact of very low solubility on metabolic stability assay results was explored. Two metabolic stability assays, the 'aqueous dilution method' and the 'cosolvent method, were compared. For commercial drugs and most discovery compounds having reasonable drug-like properties, the two methods gave comparable results. For highly lipophilic, insoluble drug discovery compounds, the 'aqueous dilution method' gave artificially higher stability results. The cosolvent method performs compound dilutions in solutions with higher organic solvent content and adds solutions directly to microsomes to assist with solubilization, minimize precipitation and reduce non-specific binding to plastics. This method is more applicable in drug discovery where compounds of a wide range of solubility are studied.

Acetonitriles↗

PubMed2006

Biological assay challenges from compound solubility: strategies for bioassay optimization.

Compound solubility in buffers and dimethyl sulfoxide (DMSO) has emerged as an important issue. Many discovery compounds have low solubility but are potentially valuable as leads. Unfortunately, low solubility affects bioassays by causing underestimated activity, reduced HTS-hit rates, variable data, inaccurate SAR, discrepancies between enzyme and cell assays and inaccurate in vitro ADME-Tox testing. Strategies for optimizing bioassays include: considering solubility in HTS-library design; early screening for solubility; improving storage and handling of DMSO stocks; optimizing dilution protocols; and ensuring that low-solubility compounds are fully solubilized in bioassays. These approaches allow for adequate assessments of valuable pharmacophores for which solubility can be chemically optimized at a later date.

Biological Assay↗

PubMed2006

Utility of mass spectrometry for pharmaceutical profiling applications.

MS has great utility for pharmaceutical profiling, the measurement of physicochemical and metabolic properties that are crucial to the discovery and development of new drug candidates. An evaluation of the capabilities of MS to improve the speed, specificity, sensitivity and cost per compound of method in development indicates when MS technologies have utility compared to other analytical techniques. MS has been used successfully for methods that profile the critical properties: permeability, lipophilicity, plasma and solution stability, solubility, plasma protein binding and integrity. In general, MS has utility in these methods using analytical strategies involving unique MS technologies (e.g., parallel multiplexed interfaces, trap-and-elute), orthogonal detection to UV, high sensitivity for low LOQs, low concentration studies, highly specific MS/MS SRM, combinatorial analysis, use of internal standards, providing initial structural data in addition to quantitative and facile integration with HPLC autosamplers and other hardware that allow enhanced on-line experiments. Ultimately, it is important to evaluate the appropriateness of any technique that is being considered for use in a method, to insure that it best meets all of the criteria for the organization's needs.

Caco-2 Cells↗

PubMed2005

Development and application of an automated solution stability assay for drug discovery.

Screening of solution stability provides an early alert on potential liabilities of drug candidates so that strategies can be developed to overcome the challenges. A fully automated solution stability assay has been developed to accelerate traditional manual operation. The assay uses the advanced capabilities of a high-performance liquid chromatography instrument that is present in many pharmaceutical research laboratories. The samples are prepared automatically by a temperature-controlled autosampler. The samples are delivered to the stability matrices, mixed, incubated, and injected at selected time points during the reaction time course. This automated process occurs without operator intervention, thus allowing 96 experiments to be run with 0.5 h of a scientist's time compared to 8 h for the same study when performed manually. Automation not only eliminates the manual operation but also improves accuracy and throughput. The assay protocol has been optimized to achieve homogenous mixing and eliminate carryover. The assay is robust, flexible, and high throughput. It can be used to study stability for a large number of samples under multiple incubation conditions and has a wide range of applications in drug discovery and development, such as screening compound stability in biological assay media, obtaining a stability-pH profile, surveying compound stability in physiological fluids, and performing development forced degradation and excipient compatibility.

Automation↗

PubMed2005

Synthesis and SAR of 2-carboxylic acid indoles as inhibitors of plasminogen activator inhibitor-1.

We synthesized and evaluated a novel series of 2-carboxylic acid indole-based inhibitors of plasminogen activator inhibitor-1 (PAI-1). Systematic modification of the N-1 position and the 5-position of the indole scaffold resulted in the identification of several compounds that showed good potency against PAI-1 in the spectrophotometric assay. This potency did not always translate to the antibody assay. Solubility and serum protein binding studies on selected analogs revealed that protein binding might be a factor in the poor correlation between the two assays.

Binding Sites↗

PubMed2005

Development and application of high throughput plasma stability assay for drug discovery.

Plasma stability plays an important role in drug discovery and development. Unstable compounds tend to have rapid clearance and short half-life, resulting in poor in vivo performance. This paper examines the variables that affect the plasma stability assay results, including substrate concentration, %DMSO, plasma concentration, enzyme activity upon incubation and batch variation. The results show that plasma stability can accommodate a wide range of experimental conditions. Relatively minor differences in results are produced with major differences in conditions. Significant batch-to-batch variations were observed for rat plasma. We selected the following conditions: 1 microM substrate concentration, 2.5% DMSO, and 50% dilution of plasma in pH 7.4 buffer. Plasma stability can be used as a diagnostic assay when compounds are unexpectedly rapidly cleared, as a special assay when structural classes contain groups that may be susceptible to plasma enzyme hydrolysis, or as general screen for compounds if resources are available. Plasma stability assay has many applications in drug discovery: to alert teams to labile structural motifs, to prioritize compounds for in vivo studies and to screen prodrugs and antedrugs.

Animals↗

PubMed2005

Integrity profiling of high throughput screening hits using LC-MS and related techniques.

Integrity profiling of HTS hits is valuable for verification of the hit identity and purity. This provides early discovery researchers with more confident SAR theories. Methodology for integrity profiling of HTS hits must be high throughput, consume little material, and selectively provide structure-based data. Analytical techniques that can be utilized for integrity profiling methods are reviewed for their appropriateness in sample preparation, component separation, detection, purity quantitation, identity confirmation, and follow-up.

Chromatography, Liquid↗

PubMed2005

Application of pharmaceutical profiling assays for optimization of drug-like properties.

Pharmaceutical profiling assays provide an early assessment of drug-like properties, such as solubility, permeability, metabolism, stability and drug-drug interactions. This information can be used to alert project teams to potential property issues, predict and diagnose in vivo assay results, guide structure-property relationships, provide insight into structure modification, and help drug discovery teams to make informed decisions. Successful drugs can be developed when biological activities of interest and pharmaceutical properties are optimized in parallel.

Animals↗

PubMed2004

Rapid, automated screening method for enzymatic transformations using a robotic system and supercritical fluid chromatography.

An automated screening method was developed for enzymatic transformations using a robotic system and rapid chiral supercritical fluid chromatography (SFC) analysis with a run time of 1.5 min. The method accelerates the enzyme selection process for screening biocatalysts, where a large number of enzymes are evaluated for activity and enantioselectivity. Kinetic resolution of secondary alcohols by enzymatic transesterification was used as a prototype for method development. The rapid automated method can be used effectively for screening enzymes and optimizing reaction conditions in biocatalysis.

Automation↗

PubMed2004

Integrated high capacity solid phase extraction-MS/MS system for pharmaceutical profiling in drug discovery.

A method is described for use in analysis of samples from pharmaceutical profiling of early drug discovery compounds. The method consists of a high capacity autosampler which injects samples into one of two solid phase extraction columns operated in parallel for alternating trapping, washing and elution into a tandem quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) MS/MS mode. A primary method, which is useful for 80-90% of compounds, and a secondary method, which is useful for a majority of the remaining compounds, are described. No analytical HPLC column is used and the analysis rate is approximately 50 samples/h. Specificity is obtained using MRM analysis. Application of the method for high capacity analysis of metabolic stability samples is described.

Drug Design↗

PubMed2004

Combined application of parallel artificial membrane permeability assay and Caco-2 permeability assays in drug discovery.

Data from permeability profiling using the parallel artificial membrane permeability assay (PAMPA) and cell monolayer (Caco-2 and MDR1-MDCKII) methods were compared for two published compound sets and one in-house set. A majority of compounds in each set correlated (R(2) = 0.76-0.92), indicating the predominance of passive diffusion in the permeation of these compounds. Compounds that did not correlate grouped into two subsets. One subset had higher PAMPA permeability than cell monolayer permeability and consisted of compounds that are subject to secretory mechanisms: efflux or reduced passive diffusion of bases under Caco-2 when run under a pH gradient. The other subset had higher cell monolayer permeability than PAMPA permeability and consisted of compounds that are subject to absorptive mechanisms: paracellular, active transport, or increased passive diffusion of acids under Caco-2 when run under a pH gradient. Given the characteristics of the two methods, these studies suggest how PAMPA and Caco-2 can be synergistically applied for efficient and rapid investigation of permeation mechanisms in drug discovery. During early discovery, all compounds can be rapidly screened using PAMPA at low pH and neutral pH to assess passive diffusion permeability to indicate potential for gastrointestinal and cell assay permeation. During intermediate discovery, selected compounds can be additionally assayed by apical-to-basolateral Caco-2, which, in combination with PAMPA data, indicates susceptibility to additional permeation mechanisms (secretory and absorptive). During mid-to-late discovery, selected candidates can be examined in detail via multiple directional Caco-2 experiments and with transporter inhibitors for complete characterization of permeation mechanisms.

Caco-2 Cells↗

PubMed2004

Experimental design on single-time-point high-throughput microsomal stability assay.

An experimental design for a single-time-point microsomal stability assay was evaluated as compared with multiple-time-point studies. Results obtained from single-time-point experiments are in excellent agreement with those from multiple time points. First-order reaction kinetics revealed rapid changes of predicted half-life from percent remaining of the parent compound at the inflection points, suggesting a maximum predictive limit for half-life. Selection of the incubation time in single-time-point assays is important to obtain balanced information for stable and unstable compounds. A short incubation time (e.g., 5 min) is most useful for differentiating between unstable compounds, which is beneficial to direct the synthetic efforts in projects with poor metabolic stability. A long incubation time (e.g., 30 min) is more applicable to a compound series with high metabolic stability. For screening purposes, a moderate incubation time (e.g., 15 min) is recommended to achieve good resolution and a sufficiently high maximum predictive limit for half-life. This study suggests that a single-time-point assay is sufficient for ranking compounds in early drug discovery. It increases throughput and reduces turnaround time and cost.

Animals↗

PubMed2003

Pharmaceutical profiling method for lipophilicity and integrity using liquid chromatography-mass spectrometry.

A method is described for the simultaneous profiling of sample lipophilicity, integrity, and purity. The method is rapid and is applicable to high throughput profiling of pharmaceutical properties in drug discovery. A short Polaris C(18) column is used with a rapid, wide-polarity mobile phase gradient, UV detection, and MS analysis. The lipophilicity of each component is estimated from a calibration curve using six drug or organic compounds and plotting their respective measured retention time versus LogD(7.4) (literature). The correlation of LogD(7.4) (literature) to LogD(7.4) (HPLC) for 60 structurally diverse drugs has a correlation coefficient r(2) of 0.89. The method is applicable to compounds with MW>200 and retention time>1.5 min for rapid, initial pharmaceutical profiling in drug discovery.

Chromatography, High Pressure Liquid↗

PubMed2003

Pharmaceutical profiling in drug discovery.

Drug discovery research organizations are building capability for measuring an ensemble of crucial 'drug-like' properties. These structure-property relationship (SPR) data complement current SAR information. This pharmaceutical profiling strategy enables research teams to better plan and interpret discovery experiments, be alerted to potential 'show stoppers', improve property liabilities, and select the best candidates for advancement. High throughput property assays for physicochemical properties--solubility, permeability, lipophilicity, stability, and pK(a)--in vitro ADME--metabolism, transporters, protein binding and CYP inhibition--and in vivo PK/exposure provide a wealth of data for teams to make informed decisions.

Animals↗

PubMed2003

High throughput artificial membrane permeability assay for blood-brain barrier.

The recent advances in high throughput screening for biological activities and combinatorial chemistry have greatly expanded the number of drug candidates. Rapid screening for BBB penetration potential early in drug discovery programs provides important information for compound selection and guidance of synthesis for desirable CNS properties. In this paper, we discuss a modification of the parallel artificial membrane permeation assay (PAMPA) for the prediction of blood-brain barrier penetration (PAMPA-BBB). The assay was developed with 30 structurally diverse commercial drugs and validated with 14 Wyeth Research compounds. The PAMPA-BBB assay has the advantages of: predicting passive blood-brain barrier penetration with high success, high throughput, low cost, and reproducibility.

Animals↗

PubMed2003

Profiling drug-like properties in discovery research.

Measurement and application of compound properties for candidate selection and optimization is an emerging trend. Property-based design supplements successful activity-based strategies to produce drug-like candidates. High-throughput screening hits are evaluated for integrity and aggregation to ensure quality leads. Solubility data assures accurate activity assays and predicts absorbance. Cellular and artificial membrane permeability assays indicate compound penetration through membranes in cells, intestine and blood-brain barrier. Lipophilicity and pK(a) provide fundamental structure design elements. Stability in liver, plasma and buffer evaluates compound lifetime. Drug-drug interaction is predicted using CYP inhibition assays. Drug-like properties are vital to successful drug candidates and enhance drug discovery.

Animals↗