In Vitro Permeability of Poorly Aqueous Soluble Compounds Using Different Solubilizers in the PAMPA Assay with Liquid Chromatography/Mass Spectrometry Detection

PURPOSE: This study compares the use of UV-VIS detection with liquid chromatography/mass spectrometry (LC/MS) detection for the PAMPA (Parallel Artificial Membrane Permeability Assay) permeability determination of compounds in the drug discovery stage. LC/MS detection offers a selective and sensitive method for the determination of the PAMPA permeability for compounds that do not contain a UV chromophore or possess a low UV extinction coefficient. To enhance the reliability of our permeability measurements for compounds with low aqueous solubility, we demonstrated the use of LC/MS detection as a means for facilitating the study of solubilizing agents to enhance aqueous solubility that normally would interfere with UV-VIS detection. In doing so, the PAMPA assay can be expanded to study the in vitro permeability of poorly water soluble compounds and evaluate the effects of solubilizers' on the membrane permeability of different compounds. This might be useful in selecting solubilizers for poorly water soluble compounds to be used for further in vivo studies. METHODS: A diverse set of 20 drugs using UV-VIS detection were compared with data using LC/MS detection. A PAMPA screening method was designed which used solubilizers (Brij 35, Cremophor EL, ethanol, and Tween 80) for compounds with low aqueous solubility. The stability of the artificial membrane was determined using various solubilizer concentrations (0.1-5% w/v) to ensure the phospholipid membrane was not disrupted. Two compounds, amiodarone and miconazole, with low aqueous solubility yielding an undetected response in the PAMPA assay using UV-VIS detection were subjected to the different solubilizing agents and their PAMPA permeability was measured using LC/MS detection. RESULTS: Most of the compounds showed similar PAMPA permeability using the two detection systems. However, for compounds lacking a UV chromophore or with a low UV extinction coefficient, LC/MS was the detection method of choice for determination of PAMPA permeability values. LC/MS also gave reliable quantification data for compounds containing impurities, as well as compounds that were not stable during the assay. Although many solubilizers were found to interfere with UV-VIS detection, the LC/MS approach was applicable to determine the permeability values of compounds with normally low aqueous solubility. CONCLUSIONS: LC/MS detection offered greater sensitivity and selectivity as compared with UV-VIS detection for the PAMPA assay. With this added versatility in detection, PAMPA can be used in both discovery and pre-formulation applications, which has not been described before.     

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.     

Application of LC/MS and related techniques to high-throughput drug discovery

The broad coordinated implementation of common platform technologies, such as LC/MS, can be a key factor in attaining increased throughput, sensitivity and data quality for pharmaceutical discovery. These platform technologies are the key tools that Medicinal Analytical Chemists rely upon to address the ever-changing needs of a drug discovery team. Despite the recent advances in key areas of sensitivity and speed for LC/MS, additional progress to address these never-ending analytical problems can be anticipated. This review will highlight current status and future advances for the applications of LC/MS and related techniques to high-throughput drug discovery.     

Advances in LC platforms for drug discovery

Importance of the field: The major application areas of liquid chromatography (LC) in modern drug discovery are the identification and structural characterization of new potential lead compounds from natural and/or synthetic sources and the determination of their physico-chemical and pharmacokinetic properties. Areas covered in this review: Significant advances in terms of LC platforms achieved in the last 5 years are highlighted in this review. Special attention is paid to novel LC strategies used in the discovery of new bioactive molecules and the determination of lipophilicity, pK(a) values, passive drug permeability and in vitro metabolism of new chemical entities. What the reader will gain: Many improvements were achieved in terms of LC instrumentation, columns technology and analytes detection to attain ultra-fast and/or high-resolution chromatographic separations. These advances are particularly beneficial to face the complexity and high number of samples studied in the early phases of the discovery process. Advantages and drawbacks of each strategy are discussed. Take home message: LC and ultra high pressure liquid chromatography coupled with mass spectrometry detection constitute the most promising strategies to achieve high-throughput and/or high-resolution analyses in a drug discovery environment.     

平行人工膜渗透模型及其应用进展

随着新药研发水平的提高,每类药物的候选化合物增多,研究工作量明显增加,对化合物进行快速筛选的方法已经成为研究的重点之一。平行人工膜渗透模型(PAMPA)作为药物筛选的有力工具,已用于药物研究中。该模型主要以人工磷脂作为生物膜来模拟药物跨膜的屏障,用于药物的膜渗透研究。本文介绍了平行人工膜渗透模型的建立、特点和研究进展,概述了利用PAMPA进行药物筛选等方面内容。通过调整人工磷脂膜的配比,PAMPA能够作为肠道、血脑屏障和皮肤吸收等体外模型。作为药物被动转运模型,PAMPA能够对药物进行高通量快速筛选,具有成本低、灵活、用药量少和重现性好等特点。     

Perspectives on bioanalytical mass spectrometry and automation in drug discovery

The use of high speed synthesis technologies has resulted in a steady increase in the number of new chemical entities active in the drug discovery research stream. Large organizations can have thousands of chemical entities in various stages of testing and evaluation across numerous projects on a weekly basis. Qualitative and quantitative measurements made using LC/MS are integrated throughout this process from early stage lead generation through candidate nomination. Nearly all analytical processes and procedures in modern research organizations are automated to some degree. This includes both hardware and software automation. In this review we discuss bioanalytical mass spectrometry and automation as components of the analytical chemistry infrastructure in pharma. Analytical chemists are presented as members of distinct groups with similar skillsets that build automated systems, manage test compounds, assays and reagents, and deliver data to project teams. The ADME-screening process in drug discovery is used as a model to highlight the relationships between analytical tasks in drug discovery. Emerging software and process automation tools are described that can potentially address gaps and link analytical chemistry related tasks. The role of analytical chemists and groups in modern 'industrialized' drug discovery is also discussed.     

Introduction to early in vitro identification of metabolites of new chemical entities in drug discovery and development

The introduction of combinatorial chemistry and robotics for high throughput screening has changed the way drugs are discovered today compared with 10-15 years ago when fewer compounds were tested in animal or organ models. The introduction of new analytical techniques, especially liquid chromatography/mass spectrometry (LC/MS) has made it possible to characterize the chemical properties, permeability, metabolic stability and metabolic fate of a large number of screening hits for further development in a funnel-like manner. The purpose of this contribution is to discuss principles and recent strategies for metabolite identification and to give an introduction to biotransformation studies. Metabolites are experimentally generated with the use of animal and human recombinant expressed enzymes, and different liver and other tissue fractions like microsomes and slices. For separation and identification of structurally diverse metabolites, LC/MS and tandem mass spectrometry (LC/MS/MS) techniques are commonly used. The LC/MS and LC/MS/MS techniques are rapid, sensitive, easy to automate and robust, and therefore, they are the methods of choice for these studies. The outcome of the metabolite identification studies is detection of metabolites that could be pharmacologically active and contribute to the efficacy of a new chemical entity (NCE), and elimination of compounds that form reactive intermediates and/or toxic metabolites that could cause adverse effects of NCE. If such information is available at an early stage during the drug discovery process, the chemical structure of the compound may be modified to reduce the risk of idiosyncratic and/or adverse drug reactions during clinical development.     

Recent development in liquid chromatography/mass spectrometry and emerging technologies for metabolite identification

Metabolism studies play a pivotal role in drug discovery and development since the active metabolites is critical to toxicological profile, efficacy and designing new drug candidates. From the instrumentation standpoint, liquid chromatography/mass spectrometry (LC/MS) has secured a central analytical technique for metabolite identification with the continuous developments and improvements in LC and MS technologies. Recently, a wide range of experimental strategies and post acquisition data processing and mining modes have emerged driven by the need to identify and characterize metabolites at ever increasing sensitivity and in ever more complex samples. In this article, the classical and practical mass spectrometry-based techniques, such as low resolution MS (quadruple, ion trap, linear ion trap, etc), high resolution MS (time-of-flight, hybrid time-of-flight instruments, Qrbitrap, Fourier transform ion cyclotron resonance MS, etc) and corresponding post acquisition data processing and mining modes (precursor ion filtering, neutral loss filtering, mass defect filter, isotope-pattern-filtering, etc) are described comprehensively. In addition, this review is also devote to discuss several novel MS technologies (ambient ionization techniques, ion mobility MS, imaging MS, LC/MNR/MS, etc) that hold additional promise for the advancement of metabolism studies.     

Future technology insight: mass spectrometry imaging as a tool in drug research and development

In pharmaceutical research, understanding the biodistribution, accumulation and metabolism of drugs in tissue plays a key role during drug discovery and development. In particular, information regarding pharmacokinetics, pharmacodynamics and transport properties of compounds in tissues is crucial during early screening. Historically, the abundance and distribution of drugs have been assessed by well-established techniques such as quantitative whole-body autoradiography (WBA) or tissue homogenization with LC/MS analysis. However, WBA does not distinguish active drug from its metabolites and LC/MS, while highly sensitive, does not report spatial distribution. Mass spectrometry imaging (MSI) can discriminate drug and its metabolites and endogenous compounds, while simultaneously reporting their distribution. MSI data are influencing drug development and currently used in investigational studies in areas such as compound toxicity. In in vivo studies MSI results may soon be used to support new drug regulatory applications, although clinical trial MSI data will take longer to be validated for incorporation into submissions. We review the current and future applications of MSI, focussing on applications for drug discovery and development, with examples to highlight the impact of this promising technique in early drug screening. Recent sample preparation and analysis methods that enable effective MSI, including quantitative analysis of drugs from tissue sections will be summarized and key aspects of methodological protocols to increase the effectiveness of MSI analysis for previously undetectable targets addressed. These examples highlight how MSI has become a powerful tool in drug research and development and offers great potential in streamlining the drug discovery process.     

Applications of mass spectrometry in drug metabolism: 50 years of progress

Abstract

Mass spectrometry plays a pivotal role in drug metabolism studies, which are an integral part of drug discovery and development nowadays. Metabolite identification has become critical to understanding the metabolic fate of drug candidates and to aid lead optimization with improved metabolic stability, toxicology and efficacy profiles. Ever since the introduction of atmospheric ionization techniques in the early 1990s, liquid chromatography coupled with mass spectrometry (LC/MS) has secured a central role as the predominant analytical platform for metabolite identification as LC and MS technologies continually advanced. In this review, we discuss the evolution of both MS technology and its applications over the past 50 years to meet the increasing demand of drug metabolism studies. These advances include ionization sources, mass analyzers, a wide range of MS acquisition strategies and data mining tools that have substantially accelerated the metabolite identification process and changed the overall drug metabolism landscape. Exemplary applications for characterization and identification of both small-molecule xenobiotics and biological macromolecules are described. In addition, this review discusses novel MS technologies and applications, including xenobiotic metabolomics that hold additional promise for advancing drug metabolism research, and offers thoughts on remaining challenges in studying the metabolism and disposition of drugs and other xenobiotics.     

Lipid composition effect on permeability across PAMPA.

The parallel artificial membrane permeability assay (PAMPA) system has promise to rapidly screen drug candidate passive permeability, but has been poorly described in terms of its lipid membrane structure and function. The objective was to investigate the role of PAMPA lipid composition on the permeability of five model compounds. PAMPA was used and employed individual phospholipids that varied in phosphate head group and acyl chain unsaturation. Transport of benzoic acid, taurocholic acid, metoprolol, sucrose, and mannitol was measured. Membrane fluidity was assessed by 1,3-diphenylhexatriene fluorescence anisotropy. Results indicate that compound permeability across PAMPA differed in their sensitivity to membrane lipid composition, where compounds with appreciable permeability (i.e. at least 0.2 x 10(-6)cm/s) were possibly sensitive to membrane fluidity and apparent ion pair effects. Benzoic acid permeability ranged 51-fold across membrane types, suggesting acyl chain effect on membrane fluidity. Mannitol, sucrose, and taurocholic acid permeabilities were low and independent of lipid composition. Metoprolol permeability ranged 17-fold and exhibited a markedly high permeability across 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] due to apparent ion pair-facilitated transport. Compound permeability was lowest across the phosphatidylcholines, which is consistent with phosphatidylcholine exhibiting relatively high membrane rigidity. In contrast to results from phosphatidylethanolamines and phosphatidylserines, acyl chain unsaturation had no effect on permeability across phosphatidylcholines. In conclusion, while much remains unknown about PAMPA structure and subsequent PAMPA permeability, results here from five solutes suggest that, for solutes with appreciable permeability, lipid composition modulated drug permeability through possible membrane fluidity and apparent ion pair influences.     

High-throughput techniques for compound characterization and purification

A new paradigm in drug discovery is the synthesis of structurally diverse collections of compounds, so-called libraries, followed by high-throughput biological screening. High-throughput characterization and purification techniques are required to provide high-quality compounds and reliable biological data, which has led to the development of faster methods, system automation and parallel approaches. This review summarizes recent advances in support of analytical characterization and preparative purification technologies. Notably, mass spectrometry (MS) and supercritical fluid chromatography (SFC) are among the areas where new developments have had a major impact on defining these high-throughput applications.     

PAMPA--critical factors for better predictions of absorption

PAMPA, log P(OCT), and Caco-2 are useful tools in drug discovery for the prediction of oral absorption, brain penetration and for the development of structure-permeability relationships. Each approach has its advantages and limitations. Selection criteria for methods are based on many different factors: predictability, throughput, cost and personal preferences (people factor). The PAMPA concerns raised by Galinis-Luciani et al. (Galinis-Luciani et al., 2007, J Pharm Sci, this issue) are answered by experienced PAMPA practitioners, inventors and developers from diverse research organizations. Guidelines on how to use PAMPA are discussed. PAMPA and PAMPA-BBB have much better predictivity for oral absorption and brain penetration than log P(OCT) for real-world drug discovery compounds. PAMPA and Caco-2 have similar predictivity for passive oral absorption. However, it is not advisable to use PAMPA to predict absorption involving transporter-mediated processes, such as active uptake or efflux. Measurement of PAMPA is much more rapid and cost effective than Caco-2 and log P(OCT). PAMPA assay conditions are critical in order to generate high quality and relevant data, including permeation time, assay pH, stirring, use of cosolvents and selection of detection techniques. The success of using PAMPA in drug discovery depends on careful data interpretation, use of optimal assay conditions, implementation and integration strategies, and education of users.     

Tools for GPCR drug discovery

G-protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic targets for a broad spectrum of diseases. The design and implementation of high-throughput GPCR assays that allow the cost-effective screening of large compound libraries to identify novel drug candidates are critical in early drug discovery. Early functional GPCR assays depend primarily on the measurement of G-protein-mediated 2nd messenger generation. Taking advantage of the continuously deepening understanding of GPCR signal transduction, many G-protein-independent pathways are utilized to detect the activity of GPCRs, and may provide additional information on functional selectivity of candidate compounds. With the combination of automated imaging systems and label-free detection systems, such assays are now suitable for high-throughput screening (HTS). In this review, we summarize the most widely used GPCR assays and recent advances in HTS technologies for GPCR drug discovery.     

Simple strategies for reducing sample loads in in vitro metabolic stability high-throughput screening experiments: a comparison between traditional, two-time-point and pooled sample analyses

Higher-throughput ADME programs in early drug discovery are becoming common throughout the pharmaceutical industry as companies strive to reduce their compound attrition in later-stage development. Many of the ADME assays developed into higher-throughput formats rely on LC/MS analyses. Since the biological aspects of the assay are amenable to parallel processes using dense plate formats, the number of samples generated from these assays produce a large analysis load for serial LC/MS. Presented in this report are two novel strategies, including a sample pooling method and a two time-point method, that could be used in drug discovery to reduce the number of samples generated during multiple time-point in-vitro ADME assays. One hundred and sixty-three compounds were subjected to human microsomal incubations with full time-point method samples taken at t = 0, 5, 15, 30, and 45 min. The ER data correlation (R(2)) between the full time-point method and the pooling method and two time-point methods were 0.98 and 0.97, respectively. Both methods have the potential to: 1. produce data of similar quality to traditional high throughput ADME assays, 2. be easily implemented, 3. shorten analytical run times, and 4. be reproducible and robust.     

Advances in high-throughput mass spectrometry

The evolution of high-throughput drug discovery is readily apparent as the pharmaceutical industry continues to stress the rapid progression of new chemical entities and biological agents through drug discovery and development pipelines. Mass spectrometry and high performance liquid chromatography-mass spectrometry have played an instrumental role in the support and advancement of all facets of high-throughput drug discovery. The introduction of new instrumentation has extended the breadth of mass spectrometric-based capabilities from the characterization of high-throughput organic synthesis products to early adsorption, distribution, metabolism and excretion profiling. Additionally, advances in the capacity and throughput of mass spectrometry systems have concurrently led to the introduction of data management tools to address automated data reduction, archival and mining, as well as analytical data integration to chemical and biological databases.     

Medicines from nature: are natural products still relevant to drug discovery?

Historically, most drugs have been derived from natural products, but there has been a shift away from their use with the increasing predominance of molecular approaches to drug discovery. Nevertheless, their structural diversity makes them a valuable source of novel lead compounds against newly discovered therapeutic targets. Technical advances in analytical techniques mean that the use of natural products is easier than before. However, there is a widening gap between natural-product researchers in countries rich in biodiversity and drug discovery scientists immersed in proteomics and high-throughput screening.     

PAMAM Dendrimers: A Review of Methodologies Employed in Biopharmaceutical Classification

The oral route is the preferred way of drug administration for most drugs, whose treatment success is directly related to the compound intestinal absorption. This absorption process, in its turn, is influenced by several factors impacting the drug bioavailability, which is extremely dependent on the maximum solubility and permeability. However, optimizing these last two factors, without chemical structural modification, is challenging. Although poly(amidoamine) dendrimers (PAMAM) are an innovative and promising strategy as drug delivery compounds, there are few studies that determine the permeability and solubility of PAMAM-drugs derivatives. Considering this scenario, this paper aimed to carry out a literature review of the last five years concerning biopharmaceutical characterizations of dendrimer delivery systems. In vitro methodologies, such as the Parallel artificial membrane permeability assay (PAMPA) (non-cellular based model) and Caco-2 cells (cellular based model), used for the permeability evaluation in the early stages of drug discovery proved to be the most promising methodologies. As a result, we discussed, for instance, that through the usage of PAMPA it was possible to evaluate the higher capacity for transdermal delivery of DNA of TAT-conjugated PAMAM, when in comparison with unmodified PAMAM dendrimer with a P<0.05. We also presented the importance of choosing the best methods of biopharmaceutical characterization, which will be essential to guarantee the efficacy and safety of the drug candidate.     

Antimycotic Drug Discovery in the Age of Genomics

Genomic-based methodologies are increasingly used at all stages of drug development. The most extensive applications have occurred in early drug discovery stages due to advances in technologies that allow for automated synthesis and characterization of organic compounds, and for high-throughput screening of these molecules against known drug targets. The adaptation of genomic-based methodologies in later stages of drug development presents a more difficult task. In this review we describe how genomics can be used to identify previously uncharacterized pharmacologic actions that provide a basis for the development of new classes of antimycotic agents or for adverse event aversion. Clinically, novel antimycotics are gravely needed. This review provides a perspective on new technologies that will bridge the gap between drug discovery and development that may enable more rapid access to new antimycotic agents.     

Applications of mass spectrometry in early stages of target based drug discovery

Mass spectrometry (MS) has been applied to drug discovery for many years. With the advent of new ionization techniques, MS has emerged as an important analytical tool in identification and characterization of protein targets, structure elucidation of synthetic compounds, and early drug metabolism and pharmacokinetics studies. Two MS-based strategies, function-based and affinity-based, have been employed in recent years for screening and evaluation of compounds. In the function-based approach, the effects of compounds on the biological activity of a target molecule are measured. In the affinity-based approach, compounds are screened based on their binding affinities to target molecules. The interaction between targets and compounds can be directly evaluated by monitoring the formation of non-covalent target-ligand complexes (direct detection) or indirectly evaluated by detecting the compounds after separating bound compounds from unbound (indirect detection). Various techniques including high performance liquid chromatography (HPLC)-MS, size exclusion chromatography (SEC)-MS, frontal affinity chromatography (FAC)-MS and desorption/ionization on silicon (DIOS)-MS can be applied. The recent advances, relative advantages, and limitations of each MS-based method as a tool in compound screening and compound evaluation in the early stages of drug discovery are discussed in this review.