Tetrahydro-naphthols as orally available TRPV1 inhibitors.

Starting from a naphthol-based lead series with low oral bioavailability, we have identified potent TRPV1 antagonists with oral bioavailability in rats. These compounds emerged from SAR studies aimed at replacing the lead's phenol structure whilst maintaining potency. Compound rac-6a is an orally available TRPV1 antagonist with single-digit nanomolar activity. The enantiomers show a low eudismic ratio at the receptor level.

Naphthol derivatives as TRPV1 inhibitors for the treatment of urinary incontinence.

We have identified naphthol derivatives as inhibitors of the vanilloid receptor TRPV1 by high throughput screening. The initial lead showed high clearance in rats and has been optimized by enhancing the acidity of the phenol group. Compound 6b has reduced clearance, improved potency and is active in rat cystometry models of urinary incontinence after intravenous administration.

Selective indole-based ECE inhibitors: synthesis and pharmacological evaluation.

Inhibition of the metalloprotease ECE-1 may be beneficial for the treatment of coronary heart disease, cancer, renal failure, and urological disorders. A novel class of indole-based ECE inhibitors was identified by high throughput screening. Optimization of the original screening lead structure 6 led to highly potent inhibitors such as 11, which bears a bisaryl amide moiety linked to the indole C2 position through an amide group. Docking of 11 into a model structure of ECE revealed a unique binding mode in which the Zn center of the enzyme is not directly addressed by the inhibitor, but key interactions are suggested for the central amide group. Testing of the lead compound 6 in hypertensive Dahl S rats resulted in a decrease in blood pressure after an initial period in which the blood pressure remained unchanged, most probably the result of ET-1 already present. Indole derivative 6 also displays a cardio-protective effect in a mouse model of acute myocardial infarction after oral administration. The more potent chloropyridine derivative 9 antagonizes big-ET-1-induced increase in blood pressure in rats at intravenous administration of 3 mg kg-1. All ECE inhibitors of the indole class showed high selectivity for ECE over related metalloproteases such as NEP and ACE. Therefore, these compounds might have further potential as drugs for the treatment of coronary heart diseases.

[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.

A recirculatory model for local absorption and disposition of ciprofloxacin by measuring portal and systemic blood concentration difference.

A recirculatory model for the portal-systemic blood concentration difference (P-S difference) was developed to separately evaluate the rate and extent of absorption from the gastrointestinal tract into the portal system and disposition of a drug in the body. To apply this model to pharmacokinetic analysis, ciprofloxacin was selected as a model drug possessing a moderate intestinal absorption, and renal and hepatic elimination. The portal and systemic blood samples were simultaneously taken from rats at appropriate time points after intravenous and oral administration of ciprofloxacin at a dose of 5 mg/kg. After intravenous administration, little or no difference in the concentrations between the portal and systemic blood was observed, whereas after oral administration the concentrations of ciprofloxacin in the portal blood were consistently higher than those in the systemic blood over the time studied. This difference observed after oral administration is attributed to the absorption of ciprofloxacin from the gastrointestinal tract into the portal system. On the basis of the moment analysis deduced from the recirculatory model, the portal blood flow rate (Q(p)), the local absorption ratio from the gastrointestinal tract into the portal system (F(a)), the hepatic recovery ratio (F(h)), and bioavailability (BA) were then estimated. The obtained Q(p) of 2.81 L/h/kg, F(a) of 32.6, F(h) of 68.1, and BA of 22.2% were found to be in good agreement with the reported values. Furthermore, the mean local absorption time from the gastrointestinal tract into the portal system (t(a)) calculated by a nonlinear least-squares program [MULTI (FILT)] was almost identical with that by the global moments. These results suggest that the model proposed in this study would be useful for evaluating both in vivo absorption and disposition of drugs.

Species difference in nisoldipine oxidation activity in the small intestine.

Species difference in nisoldipine oxidation activities was investigated using small intestinal microsomes of rats, guinea pigs, dogs, monkeys and humans. The oxidation activities were estimated by measuring metabolites formation (BAY o 3199 and BAY r 9425) of nisoldipine. For the preparation of small intestinal microsomes of various animal species, the effect of protease inhibitors was preliminarily investigated. The formation of BAY o 3199 significantly increased in the rat small intestinal microsomes prepared with trypsin inhibitor. Using the trypsin inhibitor-treated small intestinal microsomes of various animals, metabolic intrinsic clearances (CL(int, in vitro)) for BAY o 3199 and BAY r 9425 formations were estimated based on an Eadie-Hofstee plot. The total CL(int,in vitro) estimated by the sum of CL(int, in vitro) for both formations in the small intestines of all species was much lower than that in the liver. There was a marked species difference in the nisoldipine oxidation activities in the small intestines, with the rank order being humans=monkeys>dogs>rats>guinea pigs, versus the following order in the liver: rats>monkeys=guinea pigs>humans>dogs. The formations of both BAY o 3199 and BAY r 9425 in the human intestinal microsomes were inhibited by pretreatment with troleandomycin (TAO) and antiserum against CYP3A4. Similar inhibition profile by TAO was obtained from the monkey intestinal microsomes. These results implied that monkeys would be a good predictor of human small intestinal metabolism for CYP3A4 substrates.