Inhibition of stromelysin-1 (MMP-3) by P1'-biphenylylethyl carboxyalkyl dipeptides.

Carboxyalkyl peptides containing a biphenylylethyl group at the P1' position were found to be potent inhibitors of stromelysin-1 (MMP-3) and gelatinase A (MMP-2), in the range of 10-50 nM, but poor inhibitors of collagenase (MMP-1). Combination of a biphenylylethyl moiety at P1', a tert-butyl group at P2', and a methyl group at P3' produced orally bioavailable inhibitors as measured by an in vivo model of MMP-3 degradation of radiolabeled transferrin in the mouse pleural cavity. The X-ray structure of a complex of a P1-biphenyl inhibitor and the catalytic domain of MMP-3 is described. Inhibitors that contained halogenated biphenylylethyl residues at P1' proved to be superior in terms of enzyme potency and oral activity with 2(R)-[2-(4'-fluoro-4-biphenylyl)ethyl]-4(S)-n-butyl-1,5-pentane dioic acid 1-(alpha(S)-tert-butylglycine methylamide) amide (L-758,354, 26) having a Ki of 10 nM against MMP-3 and an ED50 of 11 mg/kg po in the mouse pleural cavity assay. This compound was evaluated in acute (MMP-3 and IL-1 beta injection in the rabbit) and chronic (rat adjuvant-induced arthritis and mouse collagen-induced arthritis) models of cartilage destruction but showed activity only in the MMP-3 injection model (ED50 = 6 mg/kg iv).

Structure-activity relationships of C1 and C6 side chains of zaragozic acid A derivatives.

Systematic modification of the C6 acyl side chain of zaragozic acid A, a potent squalene synthase inhibitor, was undertaken to improve its biological activity. Simplification of the C6 side chain to the octanoyl ester has deleterious effects; increasing the linear chain length improves the in vitro activity up to the tetradecanoyl ester. An omega-phenoxy group is a better activity enhancer than an omega-phenyl group. A number of C6 carbamates, ethers, and carbonates were prepared and found to have similar activity profiles as the C6 esters. In the preparation of C6 ethers, C4 and C4,6 bisethers were also isolated; their relative activity is: C6 > C4 > C4,6. These C6 long-chain derivatives are subnanomolar squalene synthase inhibitors; they are, however, only weakly active in inhibiting hepatic cholesterol synthesis in mice. The C6 short-chain derivatives are much less active in vitro, but they all have improved oral activity in mice. Modification of the C1 alkyl side chain of the n-butanoyl analogue (ED50 4.5 mg/kg) did not improve the po activity further. A number of these C6 long-chain derivatives are also potent antifungal agents in vitro.

Development, synthesis, and biological evaluation of (-)-trans-(2S,5S)-2-[3-[(2-oxopropyl)sulfonyl]-4-n-propoxy-5-(3- hydroxypropoxy)-phenyl]-5-(3,4,5-trimethoxyphenyl)tetrahydrofuran, a potent orally active platelet-activating factor (PAF) antagonist and its water-soluble prodrug phosphate ester.

(-)-trans-(2S,5S)-2-[3-[(2-Oxopropyl)sulfonyl]-4-n-propoxy-5-(3- hydroxypropoxy)phenyl]-5-(3,4,5-trimethoxyphenyl)tetrahydrofuran (10) is one of the most potent platelet-activating factor (PAF) antagonists in vitro and in vivo developed to date. This diaryltetrahydrofuran derivative evolved from modifications of MK 0287 which has been evaluated in clinical studies for asthma. Two structural modifications of MK 0287 were made: (1) elaboration of the 3'-[(hydroxyethyl)sulfonyl] group to a beta-keto propylsulfonyl, and (2) replacement of the 5'-methyl ether by a 3-hydroxypropyl ether. Compound 10 potently and specifically inhibits the binding of [3H]-C18-PAF to human platelet membranes (Ki 1.85 nM) and PMN membranes (Ki 2.89 nM). In vivo, 10 inhibits PAF-induced plasma extravasation and elevated N-acetyl-beta-D-glucosaminidase (NAGA) levels in male rats with ED50 values of 60 micrograms/kg, po and 4 micrograms/kg, iv respectively, and inhibits PAF-induced bronchoconstriction in guinea pigs with an ED50 value of 15 micrograms/kg after intraduodenal administration. Compound 15, a water-soluble phosphate ester prodrug derivative of 10 is at least equipotent to 10 in the in vivo models. Compound 19S, the primary and major metabolite of 10 and 15, is equipotent in in vitro and in vivo models.

Metabolism of the platelet-activating factor antagonist (+/-)-trans-2-(3'-methoxy-5'-methylsulphonyl-4'-propoxyphenyl)-5-(3",4" ,5"- trimethoxyphenyl)tetrahydrofuran (L-659,989) in rhesus monkeys.

1. The plasma concentration, main route of metabolism and excretion of 3H-L-659,989 were studied in male and female rhesus monkeys by dosing either i.v. or orally at 10 mg/kg. 2. The percentage of the AUC for the plasma radioactivity concentration-time curve of oral vs i.v. dosed monkeys was 78% for males and 90% for females, indicating that the dose was well absorbed. 3. The bioavailability of the drug was low (less than or equal to 10%) for all monkeys, probably due to rapid first pass metabolism. The drug was metabolized-predominantly at the C-4'-propoxy side-chain. The two major plasma metabolites were identified as the 4'-2-(hydroxy)propoxy metabolite (3H-trans-4'-HP) and the 4'-hydroxy metabolite (3H-4'-hydroxy) which was isolated as a 2:1 mixture of (+/-)trans: (+/-)cis. 4. Approx. 80% of the radiolabelled dose was excreted equally in the urine and faeces in 96 h, with the largest percentage of the tritiated dose (31 +/- 4%) in the 0-24 h urine. 5. The major metabolites in the excreta were the (+/-)trans/(+/-)cis mixture of 3H-4'-hydroxy and the glucuronide conjugate of 3H-trans-4'-hydroxy. The glucuronide conjugate of 3H-trans-4'-hydroxy was excreted in the urine of i.v. and orally dosed monkeys and represented an average of 21% and 5.1% of the dose, respectively. 3H-4'-Hydroxy was excreted in both the urine and faeces, accounting for less than or equal to 0.1% and 7.4% of the dose in i.v. and orally dosed monkeys, respectively.

Purification and characterization of recombinant human farnesyl diphosphate synthase expressed in Escherichia coli.

We previously reported the isolation of a partial-length human fetal-liver cDNA encoding farnesyl diphosphate (FPP) synthase (EC 2.5.1.10) and the expression of an active FPP synthase fusion protein in Escherichia coli. The expressed human FPP synthase fusion protein has now been purified to apparent homogeneity by using two chromatographic steps. The purification scheme allowed the preparation of 1.8 mg of homogeneous protein from 149 mg of crude extract in a 64% yield with a 52-fold enrichment. A single band with a subunit molecular mass of 39 kDa was observed by Coomassie Blue staining after SDS/PAGE. A molecular mass of 78-80 kDa was calculated for the native form of the fusion protein by h.p.l.c. on a SEC-250 column, suggesting that the active fusion protein is a dimer. The purified fusion protein has FPP synthase condensation activities in the presence of both substrates, isopentenyl diphosphate and geranyl diphosphate. Enzyme activity was inhibited by a bisubstrate analogue of isopentenyl diphosphate and dimethylallyl diphosphate, and a small amount of higher prenyltransferase was observed. Michaelis constants for isopentenyl diphosphate and geranyl diphosphate were 0.55 and 0.43 microM respectively, and Vmax for synthesis of farnesyl diphosphate from these substrates was 1.08 mumol/min per mg. These results suggest that the structure and catalytic properties of the expressed FPP synthase fusion protein are virtually identical with those of the native human liver enzyme.

Glycolipids as host resistance stimulators.

6-(5-Cholesten-3 beta-yloxy)hexyl 1-thio-beta-D-mannopyranoside (L-644,257) enhances natural host resistance in cyclophosphamide-treated mice against Pseudomonas aeruginosa in a dose-dependent manner. It is active sc, im, and ip but not orally. L-644,257 is substantially more protective against P. aeruginosa than its alpha anomer. The beta-L-fucose glycolipid is more effective when given im and ip than sc. The lactose and beta-D-glucose glycolipids were only marginally effective to nonprotective. The 17 beta-steroidal side chain of L-644,257 can be modified without substantial loss of protective activity.

Steroidal glycolipid, L-644,257, is a potent enhancer of nonspecific host resistance.

A steroidal glycolipid that enhances the nonspecific cellular response to opportunistic infection in an immunocompromised host has been discovered. A dose dependent response with 6-(5-cholesten-3 beta-yloxy)hexyl 1-thio-beta-D-mannopyranoside, L-644,257, was observed against several infective agents including bacterial, fungal, and viral pathogens in cyclophosphamide-treated mice. A mechanism for this protective action is proposed.

Biochemical and pharmacological characterization of L-659,989: an extremely potent, selective and competitive receptor antagonist of platelet-activating factor.

L-659,989 [trans-2-(3-methoxy-5-methylsulfonyl-4-propoxy-phenyl)-5-(3,4,5- trimethoxyphenyl)tetrahydrofuran] is a p.o. active and extremely potent, selective and competitive platelet-activating factor (PAF) receptor antagonist. It inhibited [3H]PAF binding to either rabbit platelet or rabbit polymorphonuclear leukocyte membranes with an equilibrium inhibition constant (Kl) of 1.1 nM; whereas in human platelet, human polymorphonuclear leukocyte or human lung membranes, L-659,989 was about 10 times less potent with a Kl of 14.3 nM. The structural specificity of L-659,989 was demonstrated by the low activity of its cisisomer which was about 100 to 200 times less potent, also the (-)-L-659,989 was found to be 20- to 30-fold more potent than (+)-L-659,989. In both [3H]PAF binding and PAF-induced platelet aggregation inhibition, L-659,989 was found to be a competitive inhibitor with an equilibrium dissociation constant (KB) of 1.5 and 1.7 nM, respectively, in rabbit platelets. Even up to 6 microM concentration, L-659,989 showed no inhibition on the aggregation of rabbit platelets in plasma induced by ADP, arachidonic acid, collagen or thrombin. In an in vivo model, it inhibited guinea pig bronchoconstriction induced by i.v. infusion of 75 ng/kg of PAF with ED50 values of 13 micrograms/kg i.v. and 0.5 mg/kg p.o.

(+/-)-trans-2-(3-Methoxy-5-methylsulfonyl-4-propoxyphenyl)-5-(3,4,5- trimethoxyphenyl)tetrahydrofuran (L-659,989), a novel, potent PAF receptor antagonist.

The title compound, L-659,989, is a highly potent, competitive, and selective antagonist of the binding of [3H]PAF to its receptors in platelet membranes from rabbits and humans. It exhibits equilibrium inhibition constants for PAF binding of 1.1 nM (rabbit) to 9.0 nM (human), values that are at least 1-2 orders of magnitude lower than those of other PAF antagonists tested. L-659,989 potently inhibits PAF-induced aggregation of rabbit platelets and degranulation of rat (ED50 4.5 nM) and human (ED50 10 nM) neutrophils. L-659,989 inhibits PAF-induced extravasation and lysosomal enzyme release in rats, and is active orally in female rats (ED50 0.2 mg/kg) with an extraordinary oral duration of action of 12 to 16 hours at 1.0 mg/kg p.o.

Inhibition of delayed hypersensitivity reactions by cinnamyl 1-thioglycosides.

Cinnamyl 1-thio-alpha-D-manno(and L-rhamno)pyranosides have good inhibitory effects in an antigen-specific T cell proliferation assay. The beta anomers are slightly less effective than the alpha anomers. The 6-substituted analogues of cinnamyl 1-thio-alpha-D-mannopyranoside such as 6-deoxy and 6-O-methyl derivatives also block macrophages in presenting the antigen to T cells. D-Mannose and L-rhamnose, when tested by themselves with no modifications, did not block at concentrations up to 1 mM. These cinnamyl 1-thioglycosides when given ip or po at 3-30 mg/kg to mice significantly inhibited the delayed type hypersensitivity reaction as measured by footpad swelling.

Structure-activity relationships of kadsurenone analogues.

Kadsurenone, a specific receptor antagonist of platelet-activating factor (PAF), and its analogues were prepared from derivatives of cinnamyl alcohol and (allyloxy)phenol. Racemic kadsurenone, resolvable by a Chiralpak column at low temperatures, has an IC50 value of 2 X 10(-7) M, which is about 50% of the activity of the natural product (IC50 = 1 X 10(-7) M). The structural specificity of kadsurenone was further demonstrated by the low PAF-receptor-blocking activities of denudatin B, mirandin A, desallylkadsurenone, and the 2-epimer of kadsurenone.

Targeting of synthetically glycosylated human placental glucocerebrosidase.

Human placental beta-glucocerebrosidase modified by covalent attachment of N2-(N2, N6-bis [3-(alpha-D-mannopyranosylthio)propionyl]-L- lysyl)-N6-[3-(alpha-D-mannopyranosylthio)propionyl]-L-lysine was administered to rats by intravenous injection. Comparison of enzyme distribution in isolated liver cell populations indicates an increase in enzyme-specific activity of 18-fold in nonparenchymal cells and only 1.5-fold to hepatocytes compared to uninjected control animals. This macrophage-specific delivery of an active lysosomal enzyme has potential for application in enzyme replacement trials.

Methyl beta-glycosides of N-acetyl-6-O-(omega-aminoacyl)muramyl-L-alanyl-D-isoglutamines, and their conjugates with meningococcal group C polysaccharide.

Spacer arms 2.1-3.7 nm (21-37 A) long were prepared, and coupled with the methyl beta-glycoside of N-acetylmuramyl-L-alanyl-D-isoglutamine benzyl ester, to give blocked 6-acylates. Deprotection was effected with palladium chloride and triethyl-silane. Chemical conjugates of MDP-meningococcal group C polysaccharide were then synthesized, in attempts to enhance the immunogenicity of the polysaccharide antigen.

omega-Aminoalkyl beta-glycosides of N-acetylmuramyl-L-alanyl-D-isoglutamine, and their conjugates with meningococcal group C polysaccharide.

The 6-aminohexyl beta-glycoside of N-acetylmuramyl-L-alanyl-D-isoglutamine and its spacer-arm-linked analog (3.8 nm) were synthesized from 2-methyl-(3,4,6-tri-O-acetyl-1,2-dideoxy-alpha-D-glucopyrano)-[2,1-d]-2- oxazoline, and coupled with meningococcal group C polysaccharide in attempts to enhance the immunogenicity of the polysaccharide antigen.

Stability of carbohydrate-modified vesicles in vivo: comparative effects of ceramide and cholesterol glycoconjugates.

The stability and tissue distribution of lipid vesicles modified at the surface by the incorporation of either a galactosyl ceramide (GalCer) or a galactosyl cholesterol (GalChol) glycoconjugate have been studied in mice by measuring the release of vesicle-entrapped 111In. Although the tissue distributions of both vesicle types were similar, the GalCer-containing vesicles were markedly less stable than those prepared with GalChol, whether administered orally or by intraperitoneal injection. Physical characterization of the vesicles in vitro suggests that the increased disruption rate for GalCer vesicles in vivo is related to structural instabilities induced by the cerebroside, which can then result in either an increased rate of vesicle uptake by tissues or a greater susceptibility to lysis. These studies demonstrate the importance of the nonpolar anchoring groups in determining the fate of surface-modified vesicles in vivo.

Synthetic glycopeptide substrates for receptor-mediated endocytosis by macrophages.

Mammalian macrophages contain a transport system that binds and internalizes glycoproteins with exposed mannose residues. This system and analogous systems on other types of cells require substrates to bear multiple nonreducing terminal residues of the appropriate sugar for effective uptake. Small multivalent synthetic glycopeptides with mannose residues covalently linked through a spacer arm to the alpha- and epsilon-amino groups of lysine, dilysine, and trilysine are competitive inhibitors of rat alveolar macrophage uptake of the neoglycoprotein mannosyl-bovine serum albumin with inhibition constants in the microM range. Various compounds could be covalently attached to the alpha-carboxyl group of these glycopeptides with substantial retention of inhibitory potency. This uptake system does not recognize galactose residues, and the galactosyl analog of an inhibitory mannosylpeptide did not inhibit uptake of mannosyl-bovine serum albumin. The trimannosyldilysine ligand is not only an inhibitor but also a substrate for specific uptake by macrophage, as shown with an 125I-labeled derivative. Macrophages bound 6.4 x 10(5) molecules per cell at 0 degrees C with a dissociation constant of 2 microM. At 21 degrees C the cells could internalize the labeled conjugate with an apparent Michaelis constant of 6 microM and a maximal velocity of 1.7 x 10(5) molecules per min per cel. The dissociation constant and Michaelis constant are similar to the inhibition constant of 9 microM determined at 21 degrees C for inhibition by this conjugate at mannosyl-bovine serum albumin uptake. These synthetic substrates may be useful in targeting pharmacologic agents to macrophages, and analogous compounds may target such agents to other types of cell.

Cell-specific ligands for selective drug delivery to tissues and organs.

Various numbers of D-mannose residues have been attached via spacer arms to lysine, dilysine, and oligolysine backbones. These D-mannosyl peptide analogues were found to be potent competitive inhibitors of the uptake of 125I-labeled D-mannose-bovine serum albumin conjugate by rat alveolar macrophages. The inhibitory potency of these synthetic ligands increased with increasing number of carbohydrate moieties. The chirality of the peptide backbone did not appear to play a major role in binding, whereas variations of the length and linkage of the spacer arm notably affected the inhibitory activities. The saccharide specificity of the macrophage receptor was demonstrated by the inactivity of the corresponding D-galactosyl peptide analogues. The L-fucosyl peptide derivative was only weakly active. The trimannosyldilysine ligand (KI = 3.9 microM) and its analogues are potentially useful in selective delivery of therapeutic agents to macrophages.

Effect of surface modification on aggregation of phospholipid vesicles.

Phospholipid vesicles have been extensively investigated because of their usefulness as models for biological membranes and their potential application as carriers for drug delivery. However, preparations of small sonicated vesicles tend to aggregate and fuse (on storage at room temperature and at 4 degrees C), resulting in significant changes in turbidity, rate of uptake by macrophage, and proton NMR linewidths. By modification of the surface of phospholipid vesicles with charged groups such as beta-aminogalactose that extend significantly from the vesicle surface, it is possible to obtain preparations that are stable for greater than 7 days.