Francine Liard

Tissue Specificity of Endothelin Binding Sites

A measurement was made of the binding of 125I-labeled endothelin (125I-ET) to crude membrane fractions prepared from rat aorta, atrium, ventricle, portal vein, trachea, lung parenchyma, vas deferens, ileum, bladder, and guinea-pig taenia coli and lung parenchyma. Scatchard analysis of 125I-ET binding in all tissues indicated binding to a single class of saturable sites. The affinity and density of 125I-ET binding sites varied between tissues. The Kd of 125I-ET binding was ≤0.5 nM for rat aorta, trachea, lung parenchyma, ventricle, bladder, and vas deferens, and guinea-pig taenia coli and lung parenchyma, 1.8 nM for rat portal vein and atrium, and 3.3 nM for ileum. The Bmax of 125I-ET binding had the following rank order of density in rat tissues: trachea > lung parenchyma = vas deferens ± aorta = portal vein = atrium > bladder > ventricle = ileum. The properties of 125I-ET endothelin binding were characterized in rat ventricular membranes. 125I-ET binding was time dependent, reaching a maximum within 45–60 min at 25°C. The calculated microassociation constant was 9.67 ± 105 s−1 M−1. Only 15–20% of 125I-ET dissociated from its binding site even when dissociation was studied as long as 3 h. Preincubation of ventricular membranes with ET prevented binding of 125I-ET. 125I-ET binding was destroyed by boiling of ventricular membranes and was temperature, pH, and cation (Ca2+, Mg2+, and Na+) dependent. F125I-ET binding was not affected by specific calcium channel ligands, N-methyl-D-aspartate (NMDA) receptor-gated ionophore antagonists, peripheral benzodiazepines, opiates, atrial natriuretic factor, angiotensin II, and sodium nitroprusside. There was no correlation between the density of ET binding sites and the contractile efficacy of ET in rat and guinea pig tissues. Exposure of rat atrium tissues to ET (10−7 M) in vitro resulted in a significant (25%) inhibition of ET binding to atrium membranes isolated from these tissues. These results indicate that specific high-affinity 125I-ET binding sites with varying properties exist in a number of tissues. ET binding does not display simple reversible bimolecular kinetics but instead demonstrates kinetics characteristic of irreversible binding.

Oral Bioavailability and In Vivo Efficacy of the Helicase-Primase Inhibitor BILS 45 BS against Acyclovir-Resistant Herpes Simplex Virus Type 1

ABSTRACT This study investigated the oral bioavailability and efficacy of BILS 45 BS, a selective herpes simplex virus (HSV) helicase-primase inhibitor, against acyclovir (ACV)-resistant (ACVr) infections mediated by the HSV type 1 (HSV-1) dlsptk and PAAr5 mutant strains. In vitro, the compound was more potent than ACV against wild-type clinical and laboratory HSV-1 strains and ACVr HSV isolates, as determined by a standard plaque reduction assay, with a mean 50% effective concentration of about 0.15 μM. The oral bioavailability of BILS 45 BS in hairless mice was 49%, with a peak concentration in plasma of 31.5 μM after administration of a single dose of 25 mg/kg. Following cutaneous infection of nude mice, both the HSV-1 dlsptk and PAAr5 mutant strains induced significant, reproducible, and persistent cutaneous lesions that lasted for more than 2 weeks. Oral treatment with ACV (100 or 125 mg/kg/day, three times a day by gavage) did not affect either mutant-induced infection. In contrast, BILS 45 BS at an oral dose of 100 mg/kg/day almost completely abolished cutaneous lesions mediated by both ACVr HSV-1 mutants. The 50% effective doses of BILS 45 BS were 56.7 and 61 mg/kg/day against dlsptk- and PAAr5-induced infections, respectively. Taken together, our results demonstrate very effective oral therapy of experimental ACVr HSV-1 infections in nude mice and support the potential use of HSV helicase-primase inhibitors for the treatment of nucleoside-resistant HSV disease in humans.

Tissue selectivity and calcium dependence of contractile responses to endothelin

The tissue selectivity and calcium dependence of endothelin, a potent vasoconstrictor peptide obtained from endothelial cells, were investigated in a number of rat and guinea pig tissues. Endothelin produced a significant dose-dependent contraction of rat tissues with the following rank order of efficacy: aorta > portal vein > trachea >> vas deferens = lung parenchyma >> bladder. In the rat atrium and ventricle, endothelin possessed positive inotropic effects at low doses and arrhythmogenic effects at high doses. Endothelin also produced a significant contraction of guinea pig lung parenchyma and induced relaxations of guinea pig taenia coli precontracted with potassium chloride. The aortic ring contractile response to endothelin was characterized by both rapid and slow phases of contraction. The rapid phase of contraction was abolished by removal of extracellular calcium or preincubation of tissues with the dihydropyridine calcium channel antagonist, nifedipine. Removal of the endothelium from aortic rings resulted in an increase of approximately 10-fold in the contractile potency of endothelin. In comparison, the potency of the dihydropyridine calcium channel activator, (-)-S-BAY K 8644, was increased approximately 15-fold by the same treatment. These findings suggest that endothelin, while displaying some selectivity as a vascular smooth muscle spasmogen, is a general smooth and cardiac muscle spasmogen which utilizes both intra- and extracellular sources of calcium to support its contractions.

Cutaneously applied acyclovir acts systemically in the treatment of herpetic infection in the hairless mouse

Using the SKH-1 hairless mouse (HM) we have addressed the issue as to whether topically applied acyclovir (ACV) may mediate some of its antiviral actions by a systemic effect. When topically applied in a formulation consisting of polyvinyl alcohol (25% w/v):DMSO:cremophor EL:linoleic acid (63:16:16:5, v/v/v/v), ACV penetrated hairless mouse skin in a concentration-dependent manner and dose-dependently reduced cutaneous herpes simplex virus 1 (HSV-1) KOS infection. Topically applied ACV also effectively reduced the mortality associated with disseminated HSV-2 HG-52 infection. At 1 h following topical application of 1.7% w/v ACV the plasma and skin concentrations of ACV were 5.5 nmoles/ml and 120 nmoles/g. At 1 h following an oral dose of ACV with antiviral efficacy comparable to topically applied ACV (1.7% w/v) the plasma and skin concentrations of ACV were 21.3 nmoles/ml and 51 nmoles/g. These findings imply that when applied topically to the HM, ACV can mediate a portion of its antiviral activity through a systemic mode of action.

Determination of the HIV protease inhibitor BILA 2185 BS in rat plasma by liquid-liquid extraction and high performance liquid chromatography photodiode array detector

A novel series of hydroxyethylamine-based inhibitors of HIV protease which contain a substituted pipecolinic amide were developed. After preliminary screening, a representative of this series, compound BILA 2185 BS, demonstrated an IC50 value of 3.3 nM in the enzymatic assay and an EC50 value of 2.0 nM in cell culture. The plasma profile and bioavailability values for BILA 2185 BS in the rat will be presented. The analyte was isolated from rat plasma using a liquid-liquid extraction procedure. The analytical technique used utilizes a high performance liquid chromatography system with photodiode array detector. The range of the standard curve was from 10 to 5000 nM. Recovery values averaged 72.4 +/- 8.6% (mean +/- S.D.). The limit of detection for BILA 2185 BS was 6-12 nM.

A Novel Oral Vehicle for Poorly Soluble HSV-Helicase Inhibitors: PK/PD Validations

AbstractPurpose. The current study describes the design and validation of a novel oral vehicle for delivering poorly water-soluble herpes simplex virus (HSV)-helicase inhibitors in preclinical pharmacokinetic (PK) and pharmacodynamic (PD) evaluations. Methods. Poorly water-soluble compounds were used in solubility and drinking compliance tests in mice. A preferred vehicle containing 0.1% bovine serum albumin (BSA), 3% dextrose, 5% polyethylene glycol (PEG) 400, and 2% peanut oil, pH 2.8 with HCL (BDPP) was selected. This vehicle was further validated with oral PK and in vivo antiviral PD studies using BILS 45 BS. Results. Solubility screen and drinking compliance tests revealed that the BDPP vehicle could solubilize BILS compounds at 0.5-3 mg/ml concentration range and could be administered to mice without reducing water consumption. Comparative oral PK of BILS 45 BS in HCL or BDPP by gavage at 40 mg/kg showed overlapping PK profiles. In vivo antiviral efficacy and potency of BILS 45 BS in BDPP by oral gavages or in drinking water were confirmed to be comparable as that achieved by gavage in HCL solution. Conclusions. These results provide a protein-enriched novel oral vehicle for delivering poorly water-soluble antiviral compounds in a continuous administration mode. Similar approaches may be applicable to other poorly soluble compounds by gavage or in drinking solution.