Pharmacokinetics of the enantiomers of felodipine in the dog after oral and intravenous administration of a pseudoracemic mixture.

1. A pseudoracemic mixture of deuterated (S)-felodipine and unlabelled (R)-felodipine was administered as single i.v. or oral doses to four dogs. Plasma concentrations of the enantiomers and their corresponding pyridine metabolites were determined by g.l.c.-mass spectrometry. 2. No isotope effects were observed after oral administration of equimolar amounts of deuterated and unlabelled (S)-felodipine. 3. The pharmacokinetic parameters of the enantiomers were similar after i.v. administration, indicating that the disposition of felodipine was not stereoselective. 4. After oral administration the bioavailability of (R)-felodipine was slightly higher than that of (S)-felodipine in two of the dogs, presumably due to a lower first-pass extraction of the (R)-enantiomer, while no difference was observed in the other two dogs. 5. No substantial differences in Cmax or AUC were observed between the deuterated and unlabelled pyridine metabolites, indicating that the oxidative clearances of the felodipine enantiomers were similar.

Identification of two main urinary metabolites of [14C]omeprazole in humans.

The excretion and metabolism of [14C]omeprazole given orally as a suspension was studied in 10 healthy male subjects. An average of 79% of the dose was recovered in the urine in 96 hr, with most of the radioactivity (76% of dose) being eliminated in the first 24 hr. Pooled urine (0-2 hr) from five subjects, containing about 47% of the dose, was analyzed by reverse phase gradient elution LC with radioisotope detection. Omeprazole was completely metabolized to at least six metabolites. The two major metabolites were extensively purified by LC and their structures were determined by MS with derivatization and use of stable isotopes, 1H NMR, and comparison with synthetic references. They were formed by hydroxylation of a methyl group in the pyridine ring, followed by further oxidation of the alcohol to the corresponding carboxylic acid. Both metabolites retained the sulfoxide group of omeprazole, rendering them as unstable as the parent compound at pH less than 7. They accounted for approximately 28% (hydroxyomeprazole) and 23% (omeprazole acid) of the amount excreted in the 0-2-hr collection interval. Based on in vitro studies with the synthetic metabolites in isolated gastric glands, it is unlikely that M1 and M2 will contribute to the pharmacological effect of omeprazole in humans.

Synthesis of 4-methoxy-2,3,5-trimethylpyridine: a specific building block for compounds with gastric-acid inhibiting activity.

A new synthesis of 4-methoxy-2,3,5-trimethylpyridine (2), an important building block for the preparation of gastric-acid inhibiting compounds, is described. Condensation of ethyl 3-amino-2-methyl-2-butenoate (3) and diethyl 2-methylmalonate (4) gives 4-hydroxy-3,5,6-trimethyl-2(1H)-pyridone 5. Reaction of 5 with phosphoryl chloride affords 2,4-dichloro-3,5,6-trimethylpyridine (9a), which, upon hydrogenolysis with palladium on charcoal, gives 2,3,5-trimethylpyridine (10). However, selective hydrogenolysis in acidic solution yields 4-chloro-2-3-5-trimethylpyridine (11). Substitution of the chlorine in 11 with methoxide ion gives 4-methoxy-2,3,5-trimethylpyridine (2), which can be oxidized to the corresponding N-oxide (13). This constitutes a new and efficient route to compound 2 in an overall yield of 43%.

Photoaffinity labeling of felodipine-binding proteins in vascular smooth muscle.

[3H]-Felodipine and high-intensity ultraviolet irradiation were used in the photoaffinity labeling of soluble proteins prepared from porcine mesenteric vascular smooth muscle. Irradiation of the soluble proteins in the presence of [3H]-felodipine resulted in the labeling of a protein with an apparent molecular weight of 62 kDa when analyzed by SDS-polyacrylamide gel electrophoresis. Labeling of the protein did not occur without ultraviolet irradiation. An [3H]-azido analog of felodipine was found to show less specificity than felodipine in its protein labeling when irradiated, since proteins with apparent molecular weights of 44, 29, and 14, as well as 62 kDa, were labeled. The photoaffinity labeling of the proteins were inhibited by excess of unlabeled felodipine.

Study of the specificity of thrombin with tripeptidyl-p-nitroanilide substrates.

The kinetic behaviour of human thrombin has been studied with 26 tripeptidyl-p-nitroanilide substrates protected at the N terminus and with 9 unprotected ones. By the regression analysis of experimentally determined 1/Km, kcat and kcat/Km values the individual contribution of each side chain of the various substrates to the kinetic parameters was calculated. The contributions to the kinetic parameters of the best substrates provide information about the structure of the binding site. The interaction of subsites S1 and P1, which determines primary specificity, proved to be marginal on the basis of contribution values, though it depends upon this contact whether the substrate is hydrolyzed at all. At subsite S2 proline appeared to be favourable. Subsite S3 plays an important role in efficiency. The best parameters were obtained here with the D configurations of bulky amino acid residues. The aromatic protecting groups applied did not improve the properties of substrates. BZDPhe-Pro-Arg-Nan was predicted by calculation to be better than the protected substrates assayed. The compound was synthesized and tested. Its experimentally determined 1/Km, 55.1 mM-1, was in good agreement with 50.9 mM-1 found by calculation.

Investigation of the substrate-binding site of trypsin by the aid of tripeptidyl-p-nitroanilide substrates.

The kinetic parameters of the tryptic hydrolysis of tripeptidyl-p-nitroanilide substrates were determined and the data were studied by regression analysis. The sequence of substrates optimal from the viewpoint of kinetic constants 1/Km, kcat and kcat/Km was established and the influence of amino acid side chains on the binding and reactivity of substrates was calculated. At subsite P3 [notation of Schechter and Berger (1967) Biochem. Biophys, Res. Commun. 27, 157] polar side chains (Asn, D-Arg) are favourable as regards 1/Km, whereas hydrophobic side chains are preferred definitely from the viewpoint of catalytic efficiency, just as at subsite P2. In the side chain contributions, calculated for the kinetic parameters, the P3-S3 interaction predominates, in spite of the fact that the properties of the residue at subsite P1 decide whether hydrolysis occurs at all. The ZAsn-Ile-Arg-Nan sequence was predicted as a better substrate than those tested experimentally. The compound was synthesized, and the calculated value of its 1/Km (116.4 mM-1) was in a good agreement with the measured value (100.2 mM-1). Comparing the data obtained with trypsin with those observed with thrombin, elastase and subtilisin, we can establish that the homology of these enzymes can be characterized at each binding subsite by the aid of tripeptidyl-p-nitroanilide substrates. The quantities derived allow one to envisage a novel type of comparison of the proteases.

Inhibition of the contractile action of bradykinin on isolated smooth muscle preparations by derivatives of low molecular weight peptides.

The carbonyl terminal tripeptide sequence of bradykinin (Pro-Phe-Arg) is molecularly manipulated to obtain agents with potent antagonistic activity towards the smooth muscle contractile activity of bradykinin. Screening of various peptide derivatives revealed that heptyl amides or esters of H-D-Pro-Phe-Arg, and H-D-Phe-Phe-Arg possessed relatively stronger antibradykinin activity on the isolated smooth muscle preparation. The parent tripeptides, H-D-Pro-Phe-Arg-OH, and H-D-Phe-Phe-Arg-OH, and their amino acid components, i.e. D-Proline, D-Phenylalanine, L-Phenylalanine and Arginine, did not possess any antibradykinin activity in concentrations of up to 10(-4) M. When the heptyl derivatives of these peptides were incubated with either heparinized or citrated whole blood or plasma, the antibradykinin activity was not lost. Incubation of these peptide derivatives with either carboxypeptidase A or B did not result in any loss of the pharmacological effect. However, pancreatic protease extract produced a significant loss of the anti-oxytocic action on the isolated rat uterus preparation. H-D-Pro-Phe-Arg-NH-lauryl derivative also blocked the action of bradykinin and this effect sustained for a longer period of time comparative to the blockade with H-D-Pro-Phe-Arg-NH-heptyl derivative. In concentrations of 10(-7) M and 10(-8) M and 1 min incubation, which blocked the contractile action of bradykinin (1 nmole) on the isolated guinea pig ileum, these peptide derivatives did not block the action of acetylcholine, histamine, and serotonin. However, in concentrations of about 10(-6) M and higher with 5 min. incubation histamin is also blocked. On the isolated rat uterus preparation the contractile action of acetylcholine, angiotensin, oxytocin and vasopressin was blocked at concentrations of 10(-6) M. These findings warrant a differential pharmacological evaluation and in vivo testing of these peptide derivatives to investigate their therapeutic potential.

New chromogenic peptide substrates for factor X.

The factor Xa-sensitive substrate BZ-Ile-Glu-Gly-Arg-p-nitroanilide has been made more sensitive by making ester and amide derivatives of the gamma-carboxyl group of the glutamyl residue. The morpholinyl and piperidyl amides react 2.5 times more rapidly with factor Xa.