Applications of high-throughput ADME in drug discovery

Assessment of physicochemical and pharmacological properties is now conducted at very early stages of drug discovery for the purpose of accelerating the conversion of hits and leads into qualified development candidates. In particular, in vitro absorption, distribution, metabolism and elimination (ADME) assays and in vivo drug metabolism pharmacokinetic (DMPK) studies are being conducted throughout the discovery process, from hit generation through to lead optimization, with the goal of reducing the attrition rate of these potential drug candidates as they progress through development. Because the continuing trend in drug discovery has been to access ADME information earlier and earlier in the discovery process, the need has arisen within the analytical community to introduce faster and better analytical methods to enhance the 'developability' of drug leads. Strategies for streamlined ADME assessment of drug candidates in discovery and pre-clinical development are presented within.     

Discovery of novel, highly potent and selective beta-hairpin mimetic CXCR4 inhibitors with excellent anti-HIV activity and pharmacokinetic profiles

Novel highly potent CXCR4 inhibitors with good pharmacokinetic properties were designed and optimized starting from the naturally occurring β-hairpin peptide polyphemusin II. The design involved incorporating important residues from polyphemusin II into a macrocyclic template-bound β-hairpin mimetic. Using a parallel synthesis approach, the potency and ADME properties of the mimetics were optimized in iterative cycles, resulting in the CXCR4 inhibitors POL2438 and POL3026. The inhibitory potencies of these compounds were confirmed in a series of HIV-1 invasion assays in vitro. POL3026 showed excellent plasma stability, high selectivity for CXCR4, favorable pharmacokinetic properties in the dog, and thus has the potential to become a therapeutic compound for application in the treatment of HIV infections (as an entry inhibitor), cancer (for angiogenesis suppression and inhibition of metastasis), inflammation, and in stem cell transplant therapy.     

Human hepatocytes: isolation, cryopreservation and applications in drug development

The recent developments in the isolation, culturing, and cryopreservation of human hepatocytes, and the application of the cells in drug development are reviewed. Recent advances include the improvement of cryopreservation procedures to allow cell attachment, thereby extending the use of the cells to assays that requires prolong culturing such as enzyme induction studies. Applications of human hepatocytes in drug development include the evaluation of metabolic stability, metabolite profiling and identification, drug-drug interaction potential, and hepatotoxic potential. The use of intact human hepatocytes, because of the complete, undisrupted metabolic pathways and cofactors, allows the development of data more relevant to humans in vivo than tissue fractions such as human liver microsomes. Incorporation of key in vivo factors with the intact hepatocytes in vitro may help predictive human in vivo drug properties. For instance, evaluation of drug metabolism and drug-drug interactions with intact human hepatocytes in 100% human serum may eliminate the need to determine in vivo intracellular concentrations for the extrapolation of in vitro data to in vivo. Co-culturing of hepatocytes and nonhepatic primary cells from other organs in the integrated discrete multiple organ co-culture (IdMOC) may allow the evaluation of multiple organ interactions in drug metabolism and drug toxicity. In conclusion, human hepatocytes represent a critical experimental model for drug development, allowing early evaluation of human drug properties to guide the design and selection of drug candidates with a high probability of clinical success.     

Drug screening of α-amylase inhibitors as candidates for treating diabetes

In the present study, the identification of potential α-amylase inhibitors is explored as a potential strategy for treating type-2 diabetes mellitus. A computationally driven approach using molecular docking was employed to search for new α-amylase inhibitors. The interactions of potential drugs with the enzyme's active site were investigated and compared with the contacts established by acarbose (a reference drug for α-amylase inhibition) in the crystallographic structure 1B2Y. For this active site characterization, both molecular docking and molecular dynamics simulations were performed, and the residues involved in the α-amylase–acarbose complex were considered to analyse the potential drug's interaction with the enzyme. Two potential α-amylase inhibitors (AN-153I105594 and AN-153I104845) have been selected following this computational strategy. Both compounds established a large number of interactions with key binding site α-amylase amino acids and obtained a comparable docking score concerning the reference drug (acarbose). Aiming to further analyse candidates' properties, their ADME (absorption, distribution, metabolism, excretion) parameters, druglikeness, organ toxicity, toxicological endpoints and median lethal dose (LD₅₀) were estimated. Overall estimations are promising for both candidates, and in silico toxicity predictions suggest that a low toxicity should be expected.     

Consensus hologram QSAR modeling for the prediction of human intestinal absorption

Consistent in silico models for ADME properties are useful tools in early drug discovery. Here, we report the hologram QSAR modeling of human intestinal absorption using a dataset of 638 compounds with experimental data associated. The final validated models are consistent and robust for the consensus prediction of this important pharmacokinetic property and are suitable for virtual screening applications.     

Application of a rapid and integrated analysis system (RIAS) as a high-throughput processing tool for in vitro ADME samples by liquid chromatography/tandem mass spectrometry

Over the past decade, drug discovery programs have started to address the optimization of key ADME properties already at an early stage of the process. Hence, analytical chemists have been confronted with tremendously rising sample numbers and have had to develop methodologies accelerating quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS). This article focuses on the application of a generic and fully automated LC/MS/MS, named Rapid and Integrated Analysis System (RIAS), as a high-throughput platform for the rapid quantification of drug-like compounds in various in vitro ADME assays. Previous efforts were dedicated to the setup and feasibility study of a workflow-integrated platform combining a modified high-throughput liquid handling LC/MS/MS system controlled by a customized software interface and a customized data-processing and reporting tool. Herein the authors present an extension of this previously developed basic application to a broad set of ADME screening campaigns, covering CYP inhibition, Caco-2, and PAMPA assays. The platform is capable of switching automatically between various ADME assays, performs MS compound optimization if required, and provides a speed of 8 s from sample to sample, independently of the type of ADME assay. Quantification and peak review are adopted to the high-throughput environment and tested against a standard HPLC-ESI technology.     

Preclinical in vitro screening of newly synthesised amidino substituted benzimidazoles and benzothiazoles

Newly synthesised benzimidazole/benzotiazole derivatives bearing amidino, namely 3,4,5,6-tetrahydropyrimidin-1-ium chloride, substituents have been evaluated for their potential antitumor activity in vitro. Compounds and standard drugs (doxorubicin, staurosporine and vandetanib) were tested on three human lung cancer cell lines A549, HCC827 and NCI-H358. We tested compounds in MTS citotoxicity assay and in BrdU proliferative assay performed on 2 D and 3 D assay format. Because benzmidazole scaffold is similar to natural purines, we tested the most active compounds for ability to induce cell apoptosis of A549 by binding to DNA in comparison with doxorubicin and saturosporine. Additionally, the ADME properties of the most active benzothiazole/benzimidazole and non-active compounds were determined to see if the different ADME properties are the cause of different activity in 2 D and 3 D assays, as well as to see if the tested active compounds have drug like properties and potency for further profilation. ADME characterisation included solubility, lipophilicity, permeability, metabolic stability and binding to plasma proteins. In general, the benzothiazole derivatives were more active in comparison to their benzimidazole analogues. The exception was 2-phenyl substituted benzimidazole 6a being active with very pronounced activity especially towards HCC827 cells. All active compounds have similar mode of action on A549 cell line as standard compound doxorubicin, which binds to nucleic acids with the DNA double helix. Tested active benzothiazole compounds were characterised by moderate to good solubility, good metabolic stability, low permeability and high binding to plasma proteins. One tested active benzimidazole derivative showed ADME properties, but lower lipophilicity resulted in low PPB and higher metabolic instability. In addition, no significant difference was observed in ADME profile between active and non-active compounds.     

Olomoucine II,but Not Purvalanol A,Is Transported by Breast Cancer Resistance Protein (ABCG2) and P-Glycoprotein (ABCB1)

Purine cyclin-dependent kinase inhibitors have been recognized as promising candidates for the treatment of various cancers; nevertheless, data regarding interaction of these substances with drug efflux transporters is still lacking. Recently, we have demonstrated inhibition of breast cancer resistance protein (ABCG2) by olomoucine II and purvalanol A and shown that these compounds are able to synergistically potentiate the antiproliferative effect of mitoxantrone, an ABCG2 substrate. In this follow up study, we investigated whether olomoucine II and purvalanol A are transported by ABCG2 and ABCB1 (P-glycoprotein). Using monolayers of MDCKII cells stably expressing human ABCB1 or ABCG2, we demonstrated that olomoucine II, but not purvalanol A, is a dual substrate of both ABCG2 and ABCB1. We, therefore, assume that pharmacokinetics of olomoucine II will be affected by both ABCB1 and ABCG2 transport proteins, which might potentially result in limited accumulation of the compound in tumor tissues or lead to drug-drug interactions. Pharmacokinetic behavior of purvalanol A, on the other hand, does not seem to be affected by either ABCG2 or ABCB1, theoretically favoring this drug in the potential treatment of efflux transporter-based multidrug resistant tumors. In addition, we observed intensive sulfatation of olomoucine II in MDCKII cell lines with subsequent active efflux of the metabolite out of the cells. Therefore, care should be taken when performing pharmacokinetic studies in MDCKII cells, especially if radiolabeled substrates are used; the generated sulfated conjugate may largely contaminate pharmacokinetic analysis and result in misleading interpretation. With regard to chemical structures of olomoucine II and purvalanol A, our data emphasize that even drugs with remarkable structure similarity may show different pharmacokinetic behavior such as interactions with ABC transporters or biotransformation enzymes.     

Novel triazines as potent and selective phosphodiesterase 10A inhibitors

The identification of highly potent and orally active triazines for the inhibition of PDE10A is reported. The new analogs exhibit low-nanomolar potency for PDE10A, demonstrate high selectivity against all other members of the PDE family, and show desired drug-like properties. Employing structure-based drug design approaches, we investigated the selectivity of PDE10A inhibitors against other known PDE isoforms, by methodically exploring the various sub-regions of the PDE10A ligand binding pocket. A systematic assessment of the ADME and pharmacokinetic properties of the newly synthesized compounds has led to the design of drug-like candidates with good brain permeability and desirable drug kinetics (t(1/2), bioavailability, clearance). Compound 66 was highly potent for PDE10A (IC(50)=1.4nM), demonstrated high selectivity (>200×) for the other PDEs, and was efficacious in animal models of psychoses; reversal of MK-801 induced hyperactivity (MED=0.1mg/kg) and conditioned avoidance responding (CAR; ID(50)=0.2mg/kg).     

Fluorescence-based assay for the interaction of small molecules with the human renal organic anion transporter 1

Secretion of small molecules from the systemic blood circulation into urine is one of the physiologically essential functions of the kidney. The human organic anion transporter (hOAT1) is a key component in the renal tubular secretion of negatively charged molecules including a variety of important therapeutics. In some cases, compounds interacting with hOAT1 may induce pharmacokinetic drug-drug interactions or cause nephrotoxicity. We developed a fluorescence-based, 96-well format assay using CHO cells stably expressing hOAT1, which allows for the evaluation of interactions between small molecules and hOAT1. The assay is based on the inhibition of the transport of 6-carboxyfluorescein, a high-affinity hOAT1 substrate (Km = 3.9 microM), which was identified as one of several fluorescent organic anions. The relative inhibition potency of various known hOAT1 substrates determined using the 6-carboxyfluorescein-based inhibition assay correlated well with their Km values, indicating that the fluorescent assay exhibits a proper specificity. This in vitro assay can be employed to evaluate the mechanism of renal clearance of organic anions, to assess potential drug-drug interactions and/or nephrotoxic effects of various therapeutics, and to screen for novel hOAT1 inhibitors that could serve as efficient nephroprotectants.     

Fluorescence-based assays for the assessment of drug interaction with the human transporters OATP1B1 and OATP1B3

Hepatic disposition plays a significant role in the pharmacokinetics and pharmacodynamics of a variety of drugs. Sinusoidal membrane transporters have been shown to participate in the hepatic disposition of many pharmaceuticals. Two sinusoidal membrane transporters with an established role in hepatic disposition are OATP1B1 and OATP1B3 (organic anion-transporting polypeptides 1B1 and 1B3, respectively). OATP1B1 and OATP1B3 have been implicated in the hepatic uptake of statin drugs, and polymorphisms linked to OATP1B1 have been associated with deleterious patient endpoints. As a result, OATP1B1 and OATP1B3 represent sites for potential drug-drug interactions. Numerous methods exist for identifying potential drug-drug interactions with transporters. However, relatively few offer the convenience and speed of fluorescence-based assays. Here a fluorescence-based assay was developed for measuring the OATP1B1- and OATP1B3-mediated transport of 8-fluorescein-cAMP (8-FcA). The OATP1B1- and OATP1B3-mediated transport of 8-FcA was time dependent and saturable (K_m = 2.9 and 1.8 μM, V_max = 0.20 and 0.33 pmol/min/cm², respectively). Molecules known to interact with OATPs, including cyclosporin A, rifampicin, and glibenclamide, each demonstrated concentration-dependent inhibition of 8-FcA transport by OATP1B1 and OATP1B3. The in vitro fluorescence-based assays described here using 8-FcA as the substrate are convenient and rapid and have utility in screening drug candidates for potential drug-drug interactions with OATP1B1 and OATP1B3.     

PK/DB: database for pharmacokinetic properties and predictive in silico ADME models

The study of pharmacokinetic properties (PK) is of great importance in drug discovery and development. In the present work, PK/DB (a new freely available database for PK) was designed with the aim of creating robust databases for pharmacokinetic studies and in silico absorption, distribution, metabolism and excretion (ADME) prediction. Comprehensive, web-based and easy to access, PK/DB manages 1203 compounds which represent 2973 pharmacokinetic measurements, including five models for in silico ADME prediction (human intestinal absorption, human oral bioavailability, plasma protein binding, blood-brain barrier and water solubility). AVAILABILITY: http://www.pkdb.ifsc.usp.br     

Some distinctions between flavin-containing and cytochrome P450 monooxygenases

This minireview summarizes information concerning the differences and similarities of the human flavin-containing- (FMO, E.C. 1.14.13.8) and the cytochrome P450-monooxygenases (CYP, E.C. 1.14.14.1). Human FMO oxygenates soft nucleophiles. CYP mainly catalyzes C-H abstraction but also oxidizes nitrogen- and sulfur-containing compounds. Both FMO and CYP generally convert lipophilic compounds into more hydrophilic metabolites. The mechanism by which each monooxygenase operates is quite distinct. Sometimes, CYP or FMO bioactivate chemicals to reactive metabolites but to date, drug toxicity thus far observed in the clinic is mainly the result of CYP-dependent oxidation. Both FMO and CYP possess genetic variability that may contribute to inter-individual variability observed for drug metabolism. In contrast to CYP, FMO is not induced or readily inhibited and potential adverse drug-drug interactions are minimized for drugs prominently metabolized by FMO. These properties may provide advantages in drug design, and by incorporating FMO detoxication pathways into drug candidates, more drug-like materials may emerge.     

QSAR and ADME

The prediction from structure of ADME (absorption, distribution, metabolism, elimination) of drug candidates is an important goal to achieve since it can considerably reduce the cost of drug development. Using our database of 10,700 QSAR, we are now reaching the point where we can make many useful comparisons that illustrate how ADME is a practical way to describe the way organic compounds react with living systems. We also show that Caco-2 cells are useful to model absorption, but the most generally useful parameter is the octanol/water partition coefficient. It should be noted, however, that in our opinion, an in silico prediction of ADME is still a long way in the future.     

Indolinone based phosphoinositide-dependent kinase-1 (PDK1) inhibitors. Part 2: optimization of BX-517

Based on the lead compound BX-517, a series of C-4' substituted indolinones have been synthesized and evaluated for PDK1 inhibition. Modification at C-4' of the pyrrole afforded potent compounds (7b and 7d) with improved solubility and ADME properties. In this letter, we describe the synthesis, selectivity profile, and pharmacokinetic data of selected compounds.     

Use of Optical Biosensor Technology to Study Immunological Cross-Reactivity between Different Sulfonamide Drugs

Adverse reactions to medications account for a substantial number of hospitalizations and in some cases fatalities. The nature of the many drug-drug interactions caused by the inhibition of drug-metabolizing enzymes can now be predicted and examined with a greater deal of accuracy due to research developments in the understanding of the drug-metabolizing enzymes. However, the more troubling aspects of drug-drug interactions are the idiosyncratic reactions that are unpredictable and quite often life-threatening. These reactions are often caused by a prior sensitization of a person's immune system to a given drug or class of drugs. The following work offers a technique to examine in a medium-throughput system the cross-reactivity of drugs to antibodies in order to predict if structures share the same antigenic potential toward a sensitized individual. Two commercially important sulfonamide drugs, sulfamethazine and furosemide, were taken and their binding to their respective antibodies were tested in the presence of other structurally related sulfonamide drugs. The BIACORE 3000 biosensor was used for the study and the solution-phase equilibrium assay principle was employed. The data obtained help us determine which drugs can react, and to what extent, with sulfamethazine and furosemide, giving rise to possible allergic or hypersensitivity reactions. Though sulfamethazine and furosemide were used in this study; this principle and methodology can be applied to study any drug molecule-antibody pair.     

秀丽隐杆线虫（Caenorhabditis elegans）在药物筛选中的应用

基于靶点的体外药物筛选操作相对简单,成本较低,但是由于药物在体内的作用并不仅仅取决于其与靶点的作用程度,吸收、分布、代谢、排泄特征和毒性均会对早期先导物能否进入临床使用产生极大的影响,因此,药物的体内筛选受到重视。本文重点综述了秀丽隐杆线虫（C.elegans）在抗衰老、抗感染药物筛选中的应用情况。秀丽隐杆线虫结构简单、易于培养和可实现高通量筛选,在未来的药物筛选中必将发挥更重要的作用。     

Evaluation of fluorescence- and mass spectrometry-based CYP inhibition assays for use in drug discovery

The potential for metabolism-related drug-drug interactions by new chemical entities is assessed by monitoring the impact of these compounds on cytochrome P450 (CYP) activity using well-characterized CYP substrates. The conventional gold standard approach for in vitro evaluation of CYP inhibitory potential uses pooled human liver microsomes (HLM) in conjunction with prototypical drug substrates, often quantified by LC-MS/MS. However, fluorescent CYP inhibition assays, which use recombinantly expressed CYPs and fluorogenic probe substrates, have been employed in early drug discovery to provide low-cost, high-throughput assessment of new chemical entities. Despite its greatly enhanced throughput, this approach has been met with mixed success in predicting the data obtained with the conventional gold standard approach (HLM+LC-MS). The authors find that the predictivity of fluorogenic assays for the major CYP isoforms 3A4 and 2D6 may depend on the quality of the test compounds. Although the structurally more optimized marketed drugs yielded acceptable correlations between the fluorogenic and HLM+LC-MS/MS assays for CYPs 3A4, 2D6, and 2C9 (r2 = 0.5-0.7; p < 0.005), preoptimization, early discovery compounds yielded poorer correlations (r2 < or = 0.2) for 2 of these major isoforms, CYPs 3A4 and 2D6. Potential reasons for the observed differences are discussed.     

Combinatorial design of nonsymmetrical cyclic urea inhibitors of aspartic protease of HIV-1

The aspartic protease (PR) of the human immunodeficiency virus type 1 (HIV-1) is an important target for the design of specific antiviral agents dedicated to treatment of HIV-1 infection. We have employed computer-assisted combinatorial chemistry methods to design a small focused virtual library of nonsymmetrically substituted cyclic urea inhibitors of the PR. Nonsymmetrical compounds with decreased peptidic character were namely found to inhibit the PR with comparable inhibition potencies as their C2-pseudosymmetric counterparts and to possess superior pharmacokinetic properties. To generate the virtual library of fully nonsymmetrical cyclic urea analogs, diverse reagents were selected from databases of available chemicals with characteristics similar to those of the building blocks of known potent PR inhibitors. The X-ray structure of the protease-inhibitor complex PR-XV-638 was used as the receptor model in the structure-based focusing and in silico screening of the virtual library. A target-specific LUDI-type scoring function, parameterized for a QSAR training set of known cyclic urea inhibitors and validated on a set of compounds not included into the training set, was used to predict the inhibition constants (Ki) of the generated analogs toward the HIV-1 PR. The fragments most frequently occurring in the analogs with the highest predicted inhibition potencies (Ki*<10 pM) were then selected to constitute a highly focused library subset containing novel nonsymmetrical cyclic ureas with predicted Ki*s 1 order of magnitude lower than the most potent known cyclic urea inhibitors. ADME properties calculated for the most promising analogs suggested that the cyclic ureas are endowed with a wide range of favorable pharmacokinetic properties, which may favor the discovery of a potent orally administrable antiviral drug.