Fused bicyclic Gly-Asp beta-turn mimics with potent affinity for GPIIb-IIIa. Exploration of the arginine isostere.

6-[4-Amidinobenzoyl]amino]-tetralone-2-acetic acid is a potent antagonist of GPIIb-IIIa. Substitution in the meta position of the benzamidine, or replacement with a heteroaryl amidine was tolerated in this series. Use of an acyl-linked 4-alkyl piperidine as an arginine isostere also provided active compounds. Compounds from this series provided substantial systemic exposure in the rat following oral administration.

Fused bicyclic Gly-Asp beta-turn mimics with specific affinity for GPIIb-IIIa.

Disubstituted isoquinolones 2 and 3 have affinity for GPIIb-IIIa and represent leads for further structural evaluation. Structure-activity studies centered on the bicyclic beta-turn mimic contained in these molecules indicated that this moiety could accommodate a variety of modifications. Specifically, monocyclic, 6, 5-bicyclic, and 6,7-bicyclic structures provide compounds with affinity for GPIIb-IIIa. Within the 6,6-series, isoquinoline, tetralin, tetralone, and benzopyran nuclei yield potent antagonists that are specific for GPIIb-IIIa. Attachment of the arginine isostere (benzamidine) to the supporting nucleus can be accomplished with an ether or amide linkage, although the latter enhances activity. Several compounds in this series provided measurable blood levels after oral dosing. Conversion of the acid moiety in these molecules to an ester generally provided compounds which gave greater systemic exposure after oral administration. Absolute bioavailabilities in the rat for the ethyl ester prodrug derivatives of the tetralin, tetralone, and benzopyran analogues of 3 were 28%, 23%, and 24%, respectively.

Use of conformationally restricted benzamidines as arginine surrogates in the design of platelet GPIIb-IIIa receptor antagonists.

The use of 5,6-bicyclic amidines as arginine surrogates in the design of a novel class of potent platelet glycoprotein IIb-IIIa receptor (GPIIb-IIIa) antagonists is described. The additional conformational restriction offered by the bicyclic nucleus results in 20-400-fold increases in potency compared to the freely flexible, acyclic benzamidine counterpart. The design, synthesis, structure-activity relationships (SAR), and in vitro activity of this novel class of GPIIb-IIIa antagonists are presented.

Non-peptide RGD surrogates which mimic a Gly-Asp beta-turn: potent antagonists of platelet glycoprotein IIb-IIIa.

Cyclic heptapeptide 1, which contains an Arg-Gly-Asp sequence, has good affinity for the platelet receptor GPIIb-IIIa and was chosen for study by 1H NMR techniques. The key RGD sequence of this molecule was found to reside in a conformationally defined type II' Gly-Asp beta-turn, and this information was used in the design of simple non-peptide RGD mimics. Disubstituted isoquinolones, bearing an acidic side chain at position 2 and a basic side chain at position 6, were prepared and were found to have modest affinity for GPIIb-IIIa. Systematic modification of the basic residue contained in these molecules yielded compounds with high affinity for GPIIb-IIIa.

Effects of a vitamin K-deficient diet and antibiotics in normal human volunteers.

Decreased concentrations of vitamin K-dependent plasma clotting factors are a well-documented response of vitamin K-deprived patients administered broad-spectrum antibiotics. It has recently been claimed that antibiotics containing a N-methylthiotetrazole (NMTT) side chain cause this response through a direct effect of NMTT on the vitamin K-dependent posttranslational carboxylation of these clotting factors. To further study these relationships, 11 groups of three volunteers were fed a synthetic vitamin K-free diet for 2 weeks. During the last 10 days of vitamin K restriction, seven of the volunteer groups received a therapeutic dose of antibiotics not containing NMTT: ampicillin, sulfamethoxazole-trimethoprim (Bactrim), cefoxitin, cefotaxime, ceftazidime, clindamycin, and piperacillin, and three groups received NMTT-containing antibiotics: moxalactam, cefamandole, and cefoperazone. Serum phylloquinone (vitamin K1) concentrations reflected dietary intake and fell from 1.4 +/- 0.9 ng/ml after 3 days of hospital diet to 0.4 +/- 0.3 ng/ml after 13 days of vitamin K-free diet. Median stool excretion of phylloquinone was 19 micrograms/day while subjects consumed the hospital diet, and fell to 3 micrograms/day by day 6 on vitamin K-free diet. Prothrombin times remained within the normal range throughout the study. Suppression of vitamin K-dependent clotting factor biosynthesis was evident by decreased factor VII levels in seven of the volunteers and by an increased concentration of des-gamma-carboxy (abnormal) prothrombin in 21 of the volunteers. The changes occurred in the control subjects and in subjects receiving all nine of the 10 antibiotics with no consistent pattern.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional properties of carbohydrate-depleted tissue plasminogen activator.

In order to evaluate the importance of the carbohydrate moiety of human tissue plasminogen activator (TPA), human melanoma (Bowes) cells were treated with a glycosylation inhibitor, tunicamycin (TM), and cellular fractions were assayed for fibrinolytic activity. Where glycosylation was inhibited by 90% and protein synthesis by 30%, TPA specific activity measured by fibrinolytic assays decreased 6-10-fold in the tissue culture medium and cell cytosol with a concomitant 2-fold increase in the 100000g microsomal pellet. In addition, TPA purified to apparent homogeneity was treated with endo-beta-N-acetylglucosaminidase H (Endo-H), producing a fraction that in contrast to native TPA did not adsorb to concanavalin A-Sepharose (Con A-Sepharose). This fraction represented TPA from which 85-90% of N-linked carbohydrate residues had been removed. Native TPA effectively activated plasminogen in the presence of fibrin (Km = 1 microM, kcat = 0.09 s-1) whereas saturation of the enzyme was not achieved at 100 microM plasminogen in the absence of fibrin. Glycosidase-treated and native TPA activated plasminogen at identical high rates in the presence and at identical negligible rates in the absence of fibrin. These studies indicate that the inhibition of glycosylation of TPA results in the inhibition of secretion of the molecule as has been observed for some other glycoproteins. The enzymatic removal of N-linked carbohydrate from purified TPA does not change its unique fibrin-directed properties.

A clinical study on the use of a fluorogenic substrate assay of plasminogen.

A fluorogenic substrate assay of plasminogen was compared with acceptable methodologies, i.e., caseinolytic and radial immunodiffusion assays. Reference intervals based on a population of 25 members of the laboratory staff were 2.5-3.8 CTA u/ml (fluorogenic), 2.2-4.0 CTA u/ml (caseinolytic) and 8.7-14.3 mg/dl (RID). Ninety eight determinations were performed on 65 patients. Regression analysis showed linear correlation between the fluorometric and caseinolytic assays (r = 0.8046, p less than 0.03) and between the fluorometric and immunologic assays (r = 0.8572, p less than 0.005). Pre-operative and post-operative determinations were performed on 33 patients undergoing coronary artery bypass surgery and there was a consistent and significant (10%-76%) decrease in the plasminogen levels post-operatively (p less than 0.01). Twenty-five patients with various malignancies were compared with the normal population; no significant difference in the plasminogen levels was observed between the two groups.

Fluorogenic substrate assay of antithrombin III: a clinical study.

The fluorogenic substrate assay of antithrombin III (AT III) was compared with a thrombin inhibition (two-stage) clotting test and a radial immunodiffusion assay. Normal ranges based on a population of 25 individuals were 88-121% (fluorogenic substrate), 72-111% (clotting) and 22.5-35.3 mg/dl (RID). A clinical comparison of the methods showed a linear relationship between the fluorogenic substrate and immunologic methods (r = 0.69, p less than 0.005), as well as between the fluorogenic substrate and clotting methods (r = 0.75, p less than 0.005). The fluorogenic substrate assay reflected the highest incidence of low AT III levels in all three clinical groups included (36 patients with arteriosclerosis, 18 with fractures and 37 with thrombosis/embolism).

Factor assay (VIII and IX) results in the College of American Pathologists Survey Program (1976-1979).

Data from the 1976-1979 CAP Surveys for Factor VIII assays are reviewed. Beginning in 1979, Factor IX assays were also performed by survey participants. Data was obtained from approximately 2500 laboratories and the different instrument-reagents are ranked according to precision and sensitivity. Also, the results of questionnaires on specific assay techniques (1977 through 1979) are analyzed. A trend was noted indicating an increasing number of laboratories are now performing factor assays. There is poor correlation between precision and sensitivity of most systems (i.e., instrument and reagent combination). For any given system, there was considerable variation in precision from a normal Factor VIII levels specimen to a low Factor VIII specimen. This was also the case with respect to Factor IX assays. With respect to the technical aspects of factor assays there was marked interlaboratory variation (i.e., dilution of normal plasma used to construct standard curve, etc.). The need for standardization of factor assays is evident from the survey data.

The effect of heparin on the activated partial thromboplastin time.

Data from the 1976 and 1977 CAP surveys were analyzed for response of the activated partial thromboplastin time (APTT) to heparin. Different sources and concentrations of heparin were used. The results indicate that the precision of the APTT is more dependent on instrumentation than on partial thromboplastin. This was true for all four of the heparinized specimens evaluated. A single exception was found with the "old" Dade reagent activated cephaloplastin. The mean difference in the activated partial thromboplastin times obtained with differing concentrations of heparin was entirely dependent on the partial thromboplastin reagent used. No significant difference in the results was found when equal concentrations of bovine lung and porcine intestinal mucosal heparin were compared.

Factor VIII (antihemophilic factor) assay results in the 1976 College of American Pathologists Survey Program.

Data from the 1976 CAP Survey for the performance of Factor VII (antihemophilic factor) assays were analyzed. The results indicated an undesirable variation on both normal and abnormal specimens. Reagent-instrumentation systems and procedures were examined. The need for standardization of factor VIII assays is evident from the results of this study.