Effect of chronic treatment with the inducible nitric oxide synthase inhibitor N-iminoethyl-L-lysine or with L-arginine on progression of coronary and aortic atherosclerosis in hypercholesterolemic rabbits.

BACKGROUND: We examined the implications of iNOS in atherosclerosis progression using the selective inducible NO synthase (iNOS) inhibitor N:-iminoethyl-L-lysine (L-NIL) in hypercholesterolemic rabbits. METHODS AND RESULTS: Nine rabbits were fed a 0.3% cholesterol diet for 24 weeks (Baseline group); 25 animals were maintained on the diet and treated for 12 extra weeks with L-NIL (5 mg x kg(-1) x d(-1), L-NIL group, n=8), vehicle (Saline group, n=9), or L-arginine (2.25%, L-Arg group, n=8). In abdominal aortas of Saline rabbits, the lesions (53.7+/-5.7%, Baseline) increased to 75.0+/-5.0% (P:<0.05) but remained unaltered in the L-NIL group (63. 4+/-6.6%). Similar results were obtained for the intima/media ratio in thoracic aortas. In coronary arteries, the intima/media ratio was comparable in Baseline (0.68+/-0.18) and Saline (0.96+/-0.19) rabbits but decreased to 0.34+/-0.19 (P:<0.05) in L-NIL rabbits. L-Arginine had beneficial effects only in abdominal aortas. An increased thoracic aorta collagen content was found in Saline and L-Arg but not in L-NIL rabbits. In thoracic aortas of the Saline group, acetylcholine caused modest relaxations that slightly increased by L-arginine but not by L-NIL. Relaxations to nitroglycerin were ameliorated by L-NIL. CONCLUSIONS: This is the first study showing that chronic treatment with an iNOS inhibitor, L-NIL, limits progression of preexisting atherosclerosis in hypercholesterolemic rabbits. Increased intimal collagen accumulation may participate in iNOS-induced atherosclerosis progression.

Combinatorial peptide libraries: robotic synthesis and analysis by nuclear magnetic resonance, mass spectrometry, tandem mass spectrometry, and high-performance capillary electrophoresis techniques.

Combinatorial peptide libraries are a new source of compounds from which a large number of pharmacological leads will emerge in the next few years. A large body of literature shows that this approach is of considerable interest in many areas of biological sciences for the search of enzyme substrates and inhibitors, of receptor agonists or antagonists, or of antigen sequences. Nevertheless, the analytical investigation of such complex mixtures as libraries which contain up to millions of individual molecules is still poorly documented. In this work, we present analytical solutions for their characterization. NMR and tandem mass spectrometry (MS/MS) can provide an in deep description of any type of combinatorial libraries, while MS and high-performance capillary electrophoresis can bring a rapid and overall information at the routine level, sufficient to ensure a first assessment of their composition. MS in the fast atom bombardment mode was used to describe the libraries O1X2X3X4X5 or O1X2X3X4 (Oi and Xi are fixed and random residue in position i, respectively). Advantage was taken of the high proton affinity of arginine and of its induction of charge remote fragmentation to interpret the MS spectra of whole libraries and neutral losses (MS/MS) in the model sublibraries ArgGlyX3X4 and NipValX3X4 (Nip,4-nitrophenylalanine). Two-dimensional NMR allowed the incorporation of the individual residues during synthesis to be tested in 24 sublibraries O1X2X3X4. While from the pharmacological point of view, impressive discoveries made with combinatorial peptide libraries have already been reported, our results show that they should be complemented by appropriate analytical tools, crucial for the proper characterization and exploitation of these libraries.

S 16118 (p-guanidobenzoyl-[Hyp3,Thi5,D-Tic7,Oic8]bradykinin) is a potent and long-acting bradykinin B2 receptor antagonist, in vitro and in vivo.

The in vitro and in vivo effects of S 16118 [p-guanidobenzoyl-[Hyp3, Thi5,D-Tic7,Oic8]bradykinin (BK)], a new BK receptor antagonist, were studied. S 16118 inhibited the contraction produced by BK in the rabbit jugular vein, but was ineffective in the rabbit aorta, indicating the BK B2 receptor specificity of the compound. In isolated organs from various species including humans, S 16118 was a potent antagonist (Ki, pA2 or pKB value from 9.58-7.37). The effect of S 16118 was specific as it did not show any affinity for a number of other receptors or channels and did not possess residual agonistic properties in most of the tissues studied. Furthermore, S 16118 is a poor secretagogue agent either in the rat or human mast cells and is resistant to degradation with an in vitro half-life in blood from different species, including humans, of more than 24 hr. In vivo, in the rabbit, i.v. injection of S 16118 inhibited the hypotension induced by BK up to 4 hr after administration. In the guinea pig, it was also effective in inhibiting the bronchoconstriction induced by BK, although when administered i.v. it had a shorter duration than in the rabbit. However, in the same species, when aerosolized, S 16118 was effective and long-acting against BK-induced bronchoconstriction. Changes in permeability induced by BK injection in the guinea pig trachea and bronchus, and by BK superfusion in the hamster cheek pouch, were abolished by i.v. pretreatment with S 16118.(ABSTRACT TRUNCATED AT 250 WORDS)

Estimation of blood levels of endothelin and neurokinin receptor antagonists at the rat portal and jugular veins after oral administration as a tool in peptide drug design.

The blood concentration of three representative endothelin and four neurokinin receptor antagonists were monitored both at the portal and jugular vein of rats 30, 60 and 90 min after oral administration. Peptide-derived structures, in the size range tetra-pentapeptides, were shown to be absorbed in the reverse order of their log P values, to be weakly metabolized in the first hepatic transit and to maintain high blood levels during the observation time. These interesting results obtained by a simple and convenient UV assay, stress once again the importance of monitoring oral absorption early in the process of peptide drug design.

Evaluation and comparison of urinary pyridinium crosslinks in two rat models of bone loss--ovariectomy and adjuvant polyarthritis--using a new automated HPLC method.

A specific HPLC system was developed to assess urinary excretion of collagen crosslinks (pyridinoline (Pyr) and deoxypyridinoline (D.Pyr)) in two models of osteopenia in rats, ovariectomy and adjuvant polyarthritis. The sensitivity of this method was in the picomolar range. In ovariectomized rats, a specific model of bone resorption, Pyr and D.Pyr levels rose early, reaching a peak 2 weeks after surgery. Both levels remained raised during the whole observation period (6 weeks) with no change in the Pyr/D.Pyr ratio. So, in this high bone turnover model, hyperresorption is reflected by the parallel increase of both crosslinks resulting in a significant decrease of bone mineral density (BMD) at 6 weeks (-7.3% vs. control). In polyarthritic rats, in the 2 post-adjuvant weeks, Pyr levels increased in parallel with inflammatory parameters, whereas D.Pyr levels remained unchanged. This is in agreement with our previous report that at the end of the 2nd week after adjuvant there is no change in bone resorption. From the 3rd week, both Pyr and D.Pyr increased. The Pyr/D.Pyr ratio was always significantly higher in polyarthritic rats. These results suggest that the early increase of Pyr level reflects non-osseous collagen breakdown and that bone resorption occurs at a later stage when D.Pyr rises, leading to a dramatic decrease of BMD at 4 weeks (-17.7% vs. control). Taken together, our results suggest that in rat as in human, urinary Pyr is a marker of bone and cartilage breakdown, whereas D.Pyr is a specific marker of bone loss. This automated method described may constitute a very useful tool to evaluate bone and/or cartilage breakdown in rats and for the assessment of protective treatments.

In vivo and in vitro glucuronidation of the flavonoid diosmetin in rats.

Flavonoids form an important family of compounds widely present in plants and therefore in food, sometimes as substitutes for synthetic antioxidants. Although the excretion routes of flavonoids in animals has been explored, little is known about the details of their conjugation in the xenobiotic metabolism pathway. In this study, we investigate the metabolism of diosmetin as a model compound in the rat, particularly its level in blood after treatment (100 mg/kg, po) and the presence of its glucuronide(s) in both blood and urine. We demonstrate that after po treatment of the rat, a rapid glucuronidation takes place and that diosmetin circulates as glucuronides, whereas no free diosmetin is present in blood and urine. The glucuronides formed are present in the blood plasma at a high level (approximately 10 micrograms/ml), for at least 6 hr after the treatment and the conjugates are excreted in urine. We have detected four different glucuronides in blood and characterized the two major ones after purification by a combination of MS, NMR, and UV spectroscopy: diosmetin-7,3'-diglucuronide and diosmetin-3'-glucuronide. A brief characterization of the in vitro glucuronidation of some flavonoid compounds using liver microsomal preparations suggests that this important class of natural compounds might be conjugated by a specific isoform of UDP-glucuronosyltransferase. Thus, this work brings evidence that diosmetin is rapidly glucuronoconjugated in the rat and provides an explanation for the low po bioavailability of the unchanged compound that can be generalized to this important class of natural compounds.

Kinetics of photoperoxidation of arachidonic acid: molecular mechanisms and effects of antioxidants.

The kinetics of photoperoxidation of [1-14C]arachidonic acid (20:4n-6) at 1.32 mM was studied either with the unsaturated fatty acid alone or in the presence of 10 microM of antioxidants and/or inhibitors of eicosanoid metabolism. The photosensitizer used was meso-tetraphenylporphine. The time-course of the reactions was followed by ultraviolet spectral analysis, thiobarbituric acid reactivity and high-performance liquid chromatographic analysis of aliquots sampled every 15 min during the 4 h of irradiation. The kinetics of photoperoxidation of 20:4n-6 can be divided into three main successive steps: (i) monohydroperoxidation, characterized by the appearance of conjugated diene patterns and monohydroperoxidized 20:4n-6; (ii) secondary oxidation characterized by polyoxygenated products such as dihydroperoxidized 20:4n-6 possessing conjugated triene patterns; and (iii) the disappearance of conjugated patterns and the oxidative cleavage of the products of the two first steps into aldehydic molecules reacting with thiobarbituric acid. During the first 90 min of irradiation, the mechanism of monohydroperoxidation (step one) is purely or predominantly type II photoperoxidation involving only singlet oxygen. This step was inhibited by beta-carotene and by BW755C (3-amino-1-[3-trifluoromethylphenyl]2-pyrazoline). In contrast, the reactions involved in the second and third steps were predominantly type I photoperoxidation involving radical mechanisms. These latter steps were inhibited by beta-carotene, BW755C, vitamin E and probucol. Indomethacin and 5,8,11,14-eicosatetraynoic acid did not alter 20:4n-6 photoperoxidation. This in vitro model of lipid photoperoxidation allows the screening of antioxidants in accordance with their singlet oxygen quenching and/or free radical scavenging properties.