Loss of adaptation following reversion suggests trade-offs in host use by a seed beetle.

Experimental evolution has provided little support for the hypothesis that the narrow diets of herbivorous insects reflect trade-offs in performance across hosts; selection lines can sometimes adapt to an inferior novel host without a decline in performance on the ancestral host. An alternative approach for detecting trade-offs would be to measure adaptation decay after selection is relaxed, that is, when populations newly adapted to a novel host are reverted to the ancestral one. Lines of the seed beetle Callosobruchus maculatus rapidly adapted to a poor host (lentil); survival in lentil seeds increased from 2% to > 90% in < 30 generations. After the lines had reached a plateau with respect to survival in lentil, sublines were reverted to the ancestral host, mung bean. Twelve generations of reversion had little effect on performance in lentil, but after 25-35 generations, the reverted lines exhibited lower survival, slower development and smaller size. The most divergent pair of lines was then assayed on both lentil and mung bean. Performance on lentil was again much poorer in the reverted line than in the nonreverted one, but the lines performed equally well on mung bean. Moreover, the performance of the nonreverted line on mung bean remained comparable to that of the original mung-bean population. Our results thus present a paradox: loss of adaptation to lentil following reversion implies a trade-off, but the continued strong performance of lentil-adapted lines on mung bean does not. Genomic comparisons of the reverted, nonreverted and ancestral lines may resolve this paradox and determine the importance of selection vs. drift in causing a loss of adaptation following reversion.

Cartilage proteoglycan degradation by a mouse transformed macrophage cell line is mediated by macrophage metalloelastase.

OBJECTIVE AND DESIGN: Identify and characterize the matrix metalloproteinase responsible for cartilage proteoglycan degradation mediated by a macrophage cell line in a cell culture model that resembles some aspects of rheumatoid pannus. MATERIALS OR SUBJECTS: Supernatants from the transformed mouse macrophage cell line J774A.1 were used to purify the proteoglycan degrading activity. METHODS: J774A.1 macrophage culture supernatants were purified by sequential column chromatography and proteins were identified by zymography, western blotting and amino acid sequence analysis. Cartilage degradation was measured using 35S labeled bovine nasal cartilage. RESULTS: The cartilage degrading proteolytic activity in the mouse macrophage supernatants proved to be due to two major proteins with approximate molecular masses of 48 kDa and 22 kDa that were identified as macrophage metalloelastase (MME). Incubation of purified MME at 37 degrees C for up to 16 h resulted in the processing of the 48 kDa protein to several novel bands including a previously undescribed protein of approximately 25 kDa without accumulation of fully processed 22 kDa protein. A number of proteinases increased the rate of this processing. J774A.1 macrophage metalloelastase degraded cartilage proteoglycan with an efficiency approximately equal to human macrophage metalloelastase (MMP-12) and matrilysin (MMP-7) and twice that of stromelysin-1 (MMP-3). CONCLUSIONS: These data identify the cartilage proteoglycan degrading metalloproteinase secreted by J774A.1 macrophages in this cell culture model as MME, and describes mechanisms of activation and processing of this enzyme that may play an important role in cartilage degradation.

Inhibition of human neutrophil elastase. 4. Design, synthesis, X-ray crystallographic analysis, and structure-activity relationships for a series of P2-modified, orally active peptidyl pentafluoroethyl ketones.

A series of P2-modified, orally active peptidic inhibitors of human neutrophil elastase (HNE) are reported. These pentafluoroethyl ketone-based inhibitors were designed using pentafluoroethyl ketone 1 as a model. Rational structural modifications were made at the P3, P2, and activating group (AG) portions of 1 based on structure-activity relationships (SAR) developed from in vitro (measured Ki) data and information provided by modeling studies that docked inhibitor 1 into the active site of HNE. The modeling-based design was corroborated with X-ray crystallographic analysis of the complex between 1 and porcine pancreatic elastase (PPE) and subsequently the complex between 1 and HNE.

Inhibition of cartilage degradation in rat collagen-induced arthritis but not adjuvant arthritis by the neutrophil elastase inhibitor MDL 101,146.

OBJECTIVE AND DESIGN: The neutrophil elastase inhibitor MDL 101,146 was examined for its anti arthritic effect and to determine the role of neutrophil elastase in collagen-induced arthritis and adjuvant arthritis. MATERIAL: The collagen-induced arthritis model was performed in female DA rats immunized on day 0 an 1.7 with chick type II collagen in Freund's incomplete adjuvant. Adjuvant arthritis was induced in female Lewis rats by an intradermal injection of heat-killed Mycobacterium tuberculosis H37RA. METHODS: The clinical signs of arthritis were assessed, joint swelling was measured using calipers and bone degradation, new bone proliferation, pannus formation and cartilage degradation were evaluated histologically. RESULTS: MDL 101,146 administered orally inhibited the severity of collagen-induced arthritis as measured by a reduction in clinical score and joint swelling. Histological evaluation demonstrated a bone and cartilage sparing effect of MDL 101,146 in the tibio-tarsal joint of animals with collagen-induced arthritis. CONCLUSIONS: These results suggest that destruction of the joint in collagen-induced arthritis is at least partially due to neutrophil elastase.

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)

Inhibition of human neutrophil elastase. 3. An orally active enol acetate prodrug.

Several analogs of N-[4-(4-morpholinylcarbonyl)benzoyl]-L-valyl-N-[3,3,4,4,4-penta fluoro-1- (1-methylethyl)-2-oxobutyl]-L-prolinamide (1), in which the chiral center of the P1 residue has been eliminated, were synthesized and tested as inhibitors of human neutrophil elastase (HNE). Observations made during the course of this work led to the development of a single-step, stereoselective synthesis of E-enol acetate derivatives from HNE inhibitors containing a mixture of epimers at P1. In vitro studies, in the presence of added esterase, and 19F NMR studies, in biological media, indicated that the E-enol acetate derivatives should act as prodrugs in vivo. The ED50 value for (E)-N-[4-(4-morpholinylcarbonyl)benzoyl]-L-valyl-N-[2- (acetyloxy)-3,3,4,4,4-pentafluoro-1-(1-methylethyl)-1-buteny l]-L-prolinamide (20), when administered orally in the hamster lung hemorrhage model, was 9 mg/kg.

Inhibition of human neutrophil elastase with peptidyl electrophilic ketones. 2. Orally active PG-Val-Pro-Val pentafluoroethyl ketones.

Valylprolyvalyl pentafluoroethyl ketones with different N-protecting groups were evaluated in vitro and in vivo as inhibitors of human neutrophil elastase (HNE). Several of these compounds were found to be orally active in HNE-induced rat and hamster lung hemorrhage models. The compound with 4-(4-morpholinylcarbonyl)benzoyl as the protecting group, 71 (MDL 101,146), was studied in greater detail. Hydration and epimerization studies were performed on 71 and related compounds in various media, including human blood serum. High-performance liquid chromatography studies on a reversed-phase system as a measure of the lipophilicity of 71 and related compounds revealed a small range of relative retention times wherein the orally active compounds fell. The Ki value determined for 71 vs HNE was 25 nM.

Pharmacology of N-[4-(4-morpholinylcarbonyl)benzoyl]-L-valyl-N- [3,3,4,4,4-pentafluoro-1-(1-methylethyl)-2-oxobutyl]-L-prolinamide (MDL 101,146): a potent orally active inhibitor 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 a number of chronic inflammatory diseases. N-[4-(4-morpholinylcarbonyl)benzoyl]-L-valyl-N- [3,3,4,4,4-pentafluoro-1-(1-methylethyl)-2-oxobutyl]-L-prolinamide (MDL 101,146), a potent reversible inhibitor of HNE, was evaluated for its ability to inhibit connective tissue degradation in vitro and in vivo. HNE-mediated degradation of proteoglycan and elastin in vitro was inhibited by MDL 101,146 in a dose-related manner. Intratracheal instillation of HNE into rodents induces acute pulmonary hemorrhage that can be measured by hemoglobin content in the bronchoalveolar fluid. Oral administration of MDL 101,146 to hamsters at 10, 25 and 50 mg/kg before an intratracheal instillation of HNE inhibited pulmonary hemorrhage with an ED50 of 15 mg/kg. The duration of action of MDL 101,146 (50 mg/kg p.o.) for the inhibition of HNE-induced hemorrhage was between 2 and 4 hr. HNE-induced pulmonary hemorrhage was inhibited by a single bolus i.v. injection of MDL 101,146 (ED50 of 0.5 mg/kg); the duration of action of the compound (10 mg/kg i.v.) was between 60 and 120 min. Intratracheal administration of MDL 101,146 inhibited HNE-induced pulmonary hemorrhage with an ED50 of 0.5 microgram/hamster (5 microgram/kg) and a duration of action of between 6 and 18 hr. MDL 101,146 inhibited HNE-induced pulmonary hemorrhage by 75% when administered as a single i.v. bolus after lung hemorrhage had occurred. MDL 101,146 had no effect on thermolysin-induced pulmonary hemorrhage, which demonstrated the specificity of MDL 101,146 for HNE in vivo. MDL 101,146 is a potent, versatile compound with potential value in a number of clinical situations in which there is an imbalance between HNE and endogenous inhibitors.

Pharmacokinetics and metabolism of vigabatrin following a single oral dose of [14C]vigabatrin in healthy male volunteers.

14C-labeled vigabatrin (50 microCi), an antiepileptic drug, was administered to six healthy male volunteers as a single oral dose containing 1500 mg of vigabatrin to determine the disposition profile of 14C and parent drug, and to investigate the metabolism of vigabatrin in humans. Vigabatrin was well tolerated by all subjects. There were no clinically important changes in any clinical laboratory parameter. Plasma concentration profiles of both 14C and vigabatrin exhibited biexponential decay. AUC, Cmax, Tmax, and terminal phase t1/2, for 14C were consistently greater than vigabatrin (248.2 vs. 176.0 micrograms-hr/ml; 48.8 vs. 42.8 micrograms/ml; 0.7 vs. 0.6 hr; and 9.5 vs. 7.7 hr, respectively). Mean renal clearance, oral clearance, and volume of distribution for 14C was consistently lower than vigabatrin (1.20 vs. 1.45 ml/min/kg; 1.26 vs. 1.77 ml/min/kg; and 1.01 vs. 1.18 liters/kg, respectively). The mean percentage of recovery of 14C in urine was higher than vigabatrin (95.4 vs. 82.0%). The mean percentage of recovery of 14C in feces was 1.0%. Concentration ratios of 14C showed that vigabatrin distributes into red blood cells at a concentration of 30-80% of that in plasma. The concentration of vigabatrin in saliva was approximately 10% of that in the plasma. The main radioactive component eliminated in the urine was vigabatrin. Two minor urinary metabolites (< 5% of total dose) of vigabatrin were detected using liquid scintillation counting of HPLC eluant fractions. One urinary metabolite was identified by thermospray-LC/MS as the lactam metabolite of vigabatrin.