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sa predict direction-dependent drug fluxes in Caco-2 monolayers by determining the affinity of a compound to the exsorptive transporter Pgp and its passive membrane permeability. It was shown that a combination of high Pgp affinity with good passive membrane permeability, for example, in the case of verapamil, will readily compensate for the Pgp-mediated reduction of intestinal permeability, resulting in a narrow range in which the permeability depends on the apical drug concentration. On the other hand, the permeability of compounds with low passive membrane permeability (e.g., talinolol) might be affected over a wide concentration range despite low affinity to Pgp.

HT-microtiter-radiometric-fluorometric-cytochrome P450 (CYP): Development of predictive in vitro surrogate methods for Kariv et al. (2002) traditional approaches assessing bioavailability and pharmacokinetics of lead compounds must be made to both keep pace with HT lead identification and to mitigate the high costs associated with progression of compounds with poor chances of developmental success. Indeed, opportunities for improvement still exist in the lead optimization (LO) phase versus the lead identification phase, where HT methodologies have been nearly optimized. Review of examples, limitations, and development of HT microtiterplate-based assays for evaluating metabolic liabilities, such as in vitro radiometric and fluorometric assays for inhibition of CYP450 activity, determination of stability of a compound in liver microsomes, or cloned CYPs coupled to reconstituting systems are described. Parallel approaches to improve speed, resolution, sample preparation, as well as data analysis using LC/MS and LC/MS/MS approaches and technologies to assess compound integrity and biotransformation by automation and multiplexing are also discussed. Realization of the benefits in automation of cell-based models for determining drug permeability to predict drug absorption are still hampered by bottlenecks in analytical analysis of compounds. The implementation and limitations of surrogate physiochemical methods for passive adsorption, such as immobilized artificial membranes (IAM) and PAMPA, and compound solubility by laser nephelometry are reviewed as well. Additionally, data from a HT 96-well equilibrium dialysis device, showing good correlation to classical methods, is presented. Finally, the impact of improvements in these downstream bottlenecks in LO and preclinical drug discovery are discussed in this review.

HT-pharmacokinetics-CYP1A2 inhibition: Pharmacokinetic and metabolic screening plays an important role in the Komura et al. (2005)

optimization of a lead compound in drug discovery. Since these screening methods are time-consuming and labor intensive, in silico models would be effective to select compounds and guide derivatization prior to the screening. We investigated in silico models for permeability in Caco-2 cells, brain distribution and CYP450 inhibition using molecular weight, lipophilicity [c log D(7.4)], polar surface area (PSA), and number of rotatable bonds (RB). A variety of test compounds was selected from different Caco-2 assay projects. The permeability determined exhibited a good correlation with a combination of PSA and c log D(7.4) rather than with PSA alone. In the brain distribution, PSA, in addition to lipophilicity, was one of the determinant parameters, and compounds were significantly distributed to the brain in rats with the decrease in the PSA value. When this approach was adapted to CYP1A2 inhibition in the fluorometric assay, the inhibitory potential for two plane core structures was successfully predicted by utilizing the number of RB, PSA, and c log D(7.4). In particular, an increase in the number of RB weakened the inhibitory potential due to a loss of the plane structures. These results suggest that the PSA and RB are key parameters to design chemical structures in terms of the improvement of both membrane permeability in the brain and Gl and CYP1A2 inhibitions, respectively.

HT-Caco-2-structure-pharmacokinetic parameter relationship (SPR): The application of combinatorial chemistry and HTS Komura et al. (2005) 3)

to biological targets has led to efficient identification of lead compounds in wide therapeutic areas. However, the physico- S'

chemical properties of some lead compounds are lipophilic with low water solubility. As these parameters determine in vivo «

absorption, we established robust screening methods for solubility and Caco-2 membrane permeability, which are applicable to our screening strategy based on the SPR. Of test compounds with different core structures, turbidimetric solubility and ov apparent solubility as determined by high-performance liquid chromatography (HPLC)-UV analysis after dilution of aqueous o media from DMSO stock solution was overestimated in comparison with the corresponding thermodynamic solubility obtained §■.

using a traditional shake-flask method. A new powder-dissolution method providing thermodynamic solubility similar to that in a the traditional method was developed using 96-well plates for equilibrium dialysis. The throughput of the method was almost §

the same as that using the apparent solubility method. In a conventional Caco-2 assay, membrane permeability (Papp) of some Q-

lipophilic compounds was underestimated due to low solubility in the apical site and adhesion to the device, resulting in a poor c?

relationship between the in vivo absorption fraction and the Papp values. The addition of 0.1 % Gelucire 44/14 into the apical site 3

and 4% bovine serum albumin into the basolateral site improved the relationship. These newly developed methods are 2

therefore useful to optimize lead compounds with less water solubility and high lipophilicity on the basis of SPR. §

HT-drug metabolism and pharmacokinetics (DMPK)-LC/MS/MS: In the current drug discovery environment, HT analytical Fung et al. (2003) assays have become essential to keep pace with the screening demands for 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, LC/MS/MS has become the dominant technology for sample analysis in the DMPK screening assays. For HT assays, analytical speed and 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.

HT-LC/UV/chemiluminescent nitrogen detection (CLND)/evaporative light scattering detection (ELSD)/MS: As part of Popa-Burke et al. an overall systems approach to generating highly accurate screening data across large numbers of compounds and biological (2004) targets, we have developed and implemented streamlined methods for purifying and quantifying compounds at various stages of the screening process, coupled with automated "traditional" storage methods (DMSO, -20°C). Specifically, all of the compounds in our drug-like library are purified by LC/MS/UV and are then controlled for identity and concentration in their respective DMSO stock solutions by CLND/ELSD and MS/UV. In addition, the compound-buffer solutions used in the various biological assays are quantified by LC/UV/CLND to determine the concentration of the compound actually present during screening. Our results show that LC/UV/CLND/ELSD/MS is a widely applicable method that can be used to purify, quantify, and identify most small organic molecules from compound libraries. The LC/UV/CLND technique is a simple and sensitive

Hidalgo (2001)

method that can be easily and cost-effectively employed to rapidly determine the concentrations of even small amounts of any N-containing compound in aqueous solution. We present data to establish error limits for concentration determination that are well within the overall variability of the screening process. This study demonstrates that there is a significant difference between the predicted amount of soluble compound from stock DMSO solutions following dilution into assay buffer and the actual amount present in assay buffer solutions, even at the low concentrations employed for the assays. We also demonstrate that knowledge of the concentrations of compounds to which the biological target is exposed is critical for accurate potency determinations. Accurate potency values are in turn particularly important for drug discovery, for understanding structure-activity relationships, and for building useful empirical models of protein-ligand interactions. Our new understanding of relative solubility demonstrates that most, if not all, decisions that are made in early discovery are based upon missing or inaccurate information. Finally, we demonstrate that careful control of compound handling and concentration, coupled with accurate assay methods, allows the use of both positive and negative data in analyzing screening data sets for structure-activity relationships that determine potency and selectivity.

HT-models-absorption: Compounds with good biological activity may fail to become drugs due to insufficient oral absorption. Selection of drug development candidates with adequate absorption characteristics should increase the probability of success in the development phase. To assess the absorption potential of new chemical entities, numerous in vitro and in vivo model systems have been used. Many laboratories rely on cell culture models of intestinal permeability, such as Caco-2, HT-29, and MDCK. To attempt to increase the throughput of permeability measurements, several physico-chemical methods, such as IAM columns and PAMPA have been used. More recently, much attention has been given to the development of computational methods to predict drug absorption. However, it is clear that no single method is sufficient for studying drug absorption, but most likely a combination of systems will be needed. HT, less reliable methods could be used to discover "loser" compounds, whereas lower throughput, more accurate methods could be used to optimize the absorption properties of lead compounds. Finally, accurate methods are needed to understand absorption mechanisms (efflux-limited absorption, carrier-mediated, intestinal metabolism) that may limit intestinal drug absorption. This information could be extremely valuable to medicinal chemists in the selection of favorable chemo-types. This review describes different techniques used for evaluating drug absorption and indicates their advantages and disadvantages.

HT-permeability-microtiter plate: This study reports on a novel, HT assay, designed to predict passive, transcellular permeability in early drug discovery. The assay is carried out in 96-well microtiter plates and measures the ability of compounds to diffuse from a donorto an acceptor compartment, which are separated by a 9-10-|j,m hexadecane liquid layer. A set of 32 well-characterized, chemically diverse drugs was used to validate the method. The permeability values derived from the flux factors between donor and acceptor compartments show a good correlation with Gl absorption in humans. For comparison, correlations based on experimental or calculated octanol/water distribution coefficients [log D(o/w, 6.8)] were significantly lower. In addition, this simple and robust assay allows determination of pH permeability profiles, critical information to predict Gl absorption of ionizable drugs, and is difficult to obtain from cell culture experiments. Correction for the unstirred water layer effect allows to differentiate between effective and intrinsic membrane permeability and opens up the dynamic range of the method. In addition, alkane/water partition coefficients can be derived from intrinsic membrane permeabilities, making this assay the first HT method able to measure alkane/water log P in the microtiter plate format.

Co o

Wohnsland and Faller (2001)

Wexler et al. (2005)

HT-profiling: Measurement and application of compound properties for candidate selection and optimization is an emerging Di et al. (2003) trend. Property-based design supplements successful activity-based strategies to produce drug-like candidates. HTS 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, intestines, and BBB. Lipophilicity and pKa provide fundamental structure design elements. Stability in liver, plasma, and buffer evaluates compound's lifetime. Drug-drug interaction is predicted using CYP inhibition assays. Drug-like properties are vital to successful drug candidates and enhance drug discovery.

HT-solubility/permeability: First, solubility is determined at four pH values by comparing the concentration of a saturated compound solution with its dilute, known as the concentration. The filtered, saturated solution from the solubility assay is then used as input material for the membrane permeability determination. The permeability assay is a parallel artificial membrane technique whereby a membrane is created on a solid support, PAMPA. The two artificial membranes presented here model the GIT and the BBB. Data are presented for control compounds, which are well documented in the literature and exemplify a range of solubility and membrane permeability. The advantages of the combination method are: (/) reduction of sample usage and preparation time, (/'/) elimination of interference from compound precipitation in membrane permeability determination, (/'//) maximization of input concentration to permeability assay for improved reproducibility, and (iv) optimization of sample tracking by streamlining data entry and calculations. BBB permeability ranking of compounds correlates well with literature CNS activity.

1AM chromatography-HT-absorption: An 1AM chromatographic method was developed and validated. Absorption profiles of Chan et al. (2005) 32 structurally diverse compounds (acidic, basic, neutral, and amphoteric) were then evaluated based on their 1AM retention factor (log /<iam)> molecular weight (MW), calculated log P (C log P), PSA, hydrogen bonding capacity (HBD and HBA), and calculated Caco-2 permeability (QPCaco). Using regression and stepwise regression analysis, experimental Caco-2 permeability was correlated against log /<iAM and a combination of various physico-chemical variables for quantitative structural-permeability relationship (QSPR) study. For the 32 structurally diverse compounds, log /(¡am correlated poorly with Caco-2 permeability values (R2 = 0.227). Stepwise regression analysis confirmed that C log, PSA, HBD, and HBA parameters are not statistically significant and can be eliminated. Correlation between Caco-2 cell uptake and log /(¡am was enhanced when molecular size factor (MW) was included (R2 = 0.555). The exclusion of 11 compounds (paracellularly and actively transported, Pgp substrates and blocker, and molecules with MW lesser than 200 and greater than 800) improved the correlation between Caco-2 permeability, IAM, and MW factors to R2 value of 0.84. The results showed that IAM chromatography can only profile the passive absorption of drug molecules. Finally, it was confirmed in this study that the IAM model can accurately identify the Caco-2 permeability of nontransported Pgp substrates, such as verapamil and ketoconazole, through passive permeation because of their high permeability. IAM chromatography, combined with molecular size factor (MW), is useful for elucidating biopartitioning mechanism of drugs.

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