Pharmacological evaluation of selected, orally active, peptidyl inhibitors of human neutrophil elastase.

Human neutrophil elastase (HNE) is a serine proteinase capable of degrading a number of connective tissue macromolecules and has been implicated in the destructive processes associated with several chronic inflammatory diseases. A large series of peptidyl electrophilic ketones have been shown to be potent inhibitors of HNE in vitro and in vivo. We report the pharmacology and pharmacokinetics of selected inhibitors from this series. MDL 101, 146, MDL 102, 111, MDL 102,823 and MDL 100,948A are -Val-Pro-Val-pentafluoroethylketones with various amino-terminal protecting groups. Although their Ki values varied considerably, (25-170 nM), these compounds demonstrated similar ED50 values after oral administration in the HNE-induced hemorrhage model in hamsters and rats. The duration of action of MDL 102,111 was shorter than that of the other analogs in the HNE-induced pulmonary hemorrhage model in both species. The duration of action of all of the compounds was longer in the rat than in the hamster. Isolated sections of rat jejunum were used to determine the in situ absorption of these compounds. MDL 102,111 showed the greatest extent of absorption, with MDL 102,823, MDL 100,948A and MDL 101,146 following in descending rank order. The comparative metabolic stability of these analogs was measured over a 2-hr incubation period using rat liver homogenates. MDL 101,146 was the most stable, followed by MDL 102,823, MDL 102,111 and MDL 100,948A. MDL 101,146 was more stable in a liver homogenate from rats compared with a liver homogenate from hamsters.(ABSTRACT TRUNCATED AT 250 WORDS)

Disposition kinetics of ML-1035 sulfoxide enantiomers and the prochiral sulfide in rats.

ML-1035, 4-amino-5-chloro-2-[2-(methylsulfinyl)ethoxy]-N-[2- (diethyl-amino)ethyl]benzamide, is a sulfoxide compound and a racemic gastroprokinetic agent with a chiral center at the sulfur atom. We have investigated the disposition kinetics of (R)-ML-1035 sulfoxide (R) and (S)-ML-1035 sulfoxide (S) after the single enantiomers and the racemic mixture were administered to rats in separate experiments. There was no noticeable chiral inversion after either enantiomer dose. Both enantiomers were rapidly absorbed. After dosing with enantiomers or with the racemate, the resulting plasma concentration-time curve of R was closely parallel to that of S in both intravenous and oral experiments, suggesting that the two enantiomers have approximately the same disposition kinetics. After intravenous enantiomer doses, only S underwent conversion to sulfide, suggesting that sulfidation in the liver is enantioselective. However, the enantioselective sulfidation after intravenous dosing did not introduce a difference in the global plasma disposition profiles between R and S, since the reduction reaction is a minor metabolic process. Other metabolic reactions such as sulfonation and mono-N-desethylations were not enantioselective. After oral administration, conversion to sulfide was observed for both enationers, implicating the existence of a nonhepatic pathway in sulfidation. Administration of a prochiral sulfide dose was associated with an enantioselective sulfoxidation, in which the R/S concentration ratios increased as a function of time. In addition, enatiomeric interaction causing changes in pharmacokinetic parameters was observed after the oral racemate dose, while the interaction is negligible after an intravenous racemate dose, indicating a route dependency in enantiomeric interaction.

Determination of ML-1035 enantiomers in plasma by chiral high-performance liquid chromatography.

ML-1035, is a gastroprokinetic agent structurally related to metoclopramide. Because ML-1035 contains an asymmetric chiral sulphoxide moiety, a chiral HPLC method has been developed to separate and quantitate its R- and S-enantiomers in plasma. The ML-1035 enantiomers present in plasma are extracted with dichloroethane under alkaline conditions, the extract evaporated to dryness and reconstituted in the mobile phase. Samples are chromatographed on a Chiralcel OD HPLC column with hexane-absolute ethanol (1% TEA) (1:1, v/v) as the mobile phase. The enantiomers of the unchanged drug are determined by fluorescence measurement (ex: 310 nm, em: 350 nm). The method provides a linear response for both enantiomers over a concentration range of 25 (limit of determination) to 2500 ng ml-1 with correlation coefficients of 0.9987 or greater. The inter-assay precision is 9.5% or less and the accuracy ranges from 93.9 to 103.4% of the theoretical value. The method is used to determine the plasma concentrations of the R- and S-enantiomers following oral and intravenous administration of R- or S-enantiomers to dogs. The method is also adapted to measure enantiomer levels from in vitro reaction mixtures so that the possibility of metabolic inversion may be assessed. The data suggest that no significant level of inversion between the enantiomers occurred either in vivo or in vitro.

Contribution of wood smoke to in vivo formation of N-nitrosothiazolidine-4-carboxylic acid: initial studies.

The effect of administering liquid smoke or smoked food products to rats on in vivo formation of N-nitrosamino acids was investigated. Rats treated by gavage with either cysteine, formaldehyde or nitrite excreted urine containing no detectable levels of N-nitrosothiazolidine-4-carboxylic acid (NTCA). All three precursor compounds were required for NTCA formation. Two liquid smokes, when administered to rats in combination with cysteine and nitrite also produced measurable quantities of NTCA. Ascorbate inhibited in vivo formation of NTCA by approximately 90% when given to rats simultaneously with cysteine, formaldehyde, and nitrite. alpha-Tocopherol was much less effective than ascorbate in blocking NTCA formation. When smoked bacon, smoked Swiss cheese, and chicken barbecued with a sauce containing smoke flavouring were fed to rats along with nitrate, NTCA was again detected in the urine.

Use of Antioxidant-Coated Salts as N-Nitrosamine Inhibitors in Dry- and Brine-Cured Bacon 1.

Several experiments were completed to further evaluate use of α-tocopherol-coated salts as inhibitors of N-nitrosamine formation in fried bacon. Studies with dry-cured bacon prepared with various levels of α-tocopherol indicated that the chemical did not contribute to formation of N-nitrosodimethylamine (NDMA). N-Nitrosopyrrolidine levels for the α-tocopherol-treated bacon samples were generally below 5 µg/kg, which represents an average reduction of approximately 70%. Experiments were also done to evaluate the role of lecithin as a possible precursor of NDMA in brine-cured bacon. At concentrations used to disperse α-tocopherol in the curing brine, lecithin did not contribute to NDMA formation in bacon prepared with α-tocopherol-coated salts.

Further factors influencing N-nitrosamine formation in bacon.

The possible relationship of unsaturated fatty acids in adipose tissue to the formation of N-nitrosamines in bacon was evaluated by trials in which pigs were fed regular (control), tallow-, coconut fat- and corn oil-supplemented diets. Bacon prepared from pigs fed corn oil-supplemented diets contained significantly higher levels of N-nitrosopyrrolidine and N-nitrosodimethylamine than did control bacon samples; however, bacon produced from pigs fed a coconut fat-supplemented diet contained significantly lower levels of N-nitrosopyrrolidine. Fatty acid analyses of the adipose tissue of the bacon samples indicated that N-nitrosopyrrolidine levels in bacon correlated well with the degree of unsaturation of the adipose tissue. N-nitrosothiazolidine was detected in both brine-cured and dry-cured bacon at levels generally below 4 micrograms/kg. However, its formation was greatly reduced by the inclusion of alpha-tocopherol in the cure. The role of woodsmoke in N-nitrosothiazolidine formation in bacon is discussed.