Relationships between structure and high-throughput screening permeability of diverse drugs with artificial membranes: application to prediction of Caco-2 cell permeability.

To evaluate the absorption of drugs with diverse structures across a membrane via the transcellular route, their permeability was measured using the parallel artificial membrane permeation assay (PAMPA). The permeability coefficients obtained by PAMPA were analyzed using a classical quantitative structure-activity relationship (QSAR) approach with simple physicochemical parameters and 3D-QSAR, VolSurf. We formulated correlation equations for diverse drugs similar to the equation obtained for peptide-related compounds in our previous study. The hydrogen-bonding ability of molecules, not only the hydrogen-accepting ability but also the hydrogen-donating ability, in addition to hydrophobicity at a particular pH, was significant in determining variations in PAMPA permeability coefficients. Based on this result, an in silico good prediction model for the passive transcellular permeability of diverse structural compounds was obtained. The artificial lipid-membrane permeability coefficients of the drugs, except salicylic acid, were well correlated with the Caco-2 permeability in a previous report suggesting the importance of absorption by the transcellular mechanism for these drugs.

Metabolic flux analysis of the phenylpropanoid pathway in elicitor-treated potato tuber tissue.

The effects of beta-1,3-oligosaccharide elicitor on the metabolism of phenylpropanoids in potato tuber were analyzed quantitatively, by monitoring the time-dependent changes in the levels of seven compounds. The elicitor treatment caused an increase in the pool size of octopamine and tyramine amides (N-p-coumaroyloctopamine, N-feruloyloctopamine, N-p-coumaroyltyramine and N-feruloyltyramine), as well as a decrease in that of chlorogenic acid and putrescine amides (caffeoylputrescine and feruloylputrescine). An analysis of metabolic flux using stable isotope labeling and liquid chromatography-spectrometry (LC-MS) detection clearly demonstrated that the changes in the pool size of these compounds were correlated with the changes in their flux for biosynthesis (Jin) upon elicitor treatment. The increase in Jin in the cases of octopamine and tyramine amides was accompanied by an increase in flux for the transformation (Jout), indicating a rapid turnover of these compounds in the elicitor-treated tuber tissue. The result of the flux analysis indicated that the actual activation of the biosynthesis of octopamine and tyramine amides after the elicitor treatment was greater than that estimated from the changes in their levels in the potato tissue. These findings suggest that these amide compounds and their metabolic derivatives play an important role in the defense-related metabolism of phenylpropanoids in potato.

[High throughput screening of pharmacokinetics and metabolism in drug discovery (I)--establishment of assessment system for absorption to compounds with a wide diversity of physical properties].

The application of combinatorial chemistry and high-throughput screening to biological targets has led to efficient identification of lead compounds in wide therapeutic areas. However, the physicochemical properties of some lead compounds are lipophilic with low water soluble. Since 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 structure-pharmacokinetic parameter relationship (SPR). Of test compounds with different core structures, turbidimetric solubility and apparent solubility as determined by HPLC-UV analysis after dilution of aqueous 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 the traditional method was developed using 96-well plates for equilibrium dialysis. The throughput of the method was the almost the same as that using the apparent solubility method. In a conventional Caco-2 assay, membrane permeability (P(app)) of some lipophilic compounds was underestimated due to low solubility in the apical site and adhesion to the device, resulting in a poor relationship between the in vivo absorption fraction and the P(app) values. The addition of 0.1% Gelucire 44/14 into the apical site and 4% bovine serum albumin into the basolateral site improved the relationship. These newly developed methods are therefore useful to optimize lead compounds with less water solubility and high lipophilicity on the basis of SPR.

[High throughput screening of pharmacokinetics and metabolism in drug discovery (II)--investigation on in vitro and in vivo correlation in drug metabolism screening].

Metabolic screening using liver microsomes of rats and humans is an indispensable tool to optimize a lead structure and to select compounds for in vivo study. Elucidating the relationship between in vitro intrinsic clearance (CL(int, app)) and in vivo clearance (CL(b)) is a prerequisite for screening. We investigated the relationship between CL(int, app) in rat liver microsomes and CL(b) after intravenous administration in rats in eight projects. No relationship between these two parameters was found across all of the projects examined. However, there was a certain relationship in the same core structure of six projects, but not in the other two projects. The poor correlation in the projects was improved by considering serum protein binding or microsomal binding in the estimation of in vitro clearances. Although the binding assay was labor intensive, unlike metabolic screening, the introduction of the equilibrium dialysis method using a 96-well format increased the throughput. Optimization of metabolic stability was conducted on the basis of the structure-metabolic stability relationship (SMR) in one of the projects, showing a good correlation without the binding factors. The replacement of the piperazine with a homopiperazine moiety improved metabolic stability in the rat and human liver microsomes. The compound also showed a desirable in vivo pharmacokinetic profile in rats, suggesting that the SMR study on the confirmed in vitro and in vivo correlation is essential to the optimization.

[High throughput screening of pharmacokinetics and metabolism in drug discovery (III)--investigation on in- silico model for membrane permeability and CYP1A2 inhibition].

Pharmacokinetic and metabolic screening plays an important role in the optimization of a lead compound in drug discovery. Since those 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 cytochrome P450 (CYP) inhibition using molecular weight, lipophilicity (clog 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 clog 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 number of RB, PSA, and clog 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 gastrointestine and CYP1A2 inhibition, respectively.