Inhibitory effects of newly synthesized active center-directed trypsin-like serine protease inhibitors on the complement system.

OBJECTIVE: To obtain a synthetic anti-complement inhibitor which has stronger activity than FUT-175 (nafamostat mesilate), as a synthetic ester derivative containing amidino and guanidino groups. METHODS: We synthesized several modified compounds of FUT-175. The anti-complement activities were measured using synthetic substrates and complement-mediated hemolysis in vitro. The anti-complement activity in vivo was evaluated via Forssman systemic shock in guinea pigs. RESULTS: FUT-175 inhibited C1r and C1s with IC50s of 1.7x10(-6) and 3.2x10(-7) M, respectively. Inhibitory activities were decreased by substitution of the amidino group with a hydrogen atom (compound 2), but not the guanidino group with a hydrogen atom (compound 3). Compound 6, in which the benzene ring of compound 3 was substituted with a furan ring, inhibited C1r and the complement-mediated hemolysis in high-diluted serum with higher potency than FUT-175. The inhibitory activity of compound 6 in hemolysis was weakened in low diluted serum. Compound 7 had a guanidino group inserted into compound 6; however, Compound 7 strongly inhibited hemolysis even in low-diluted serum, and suppressed Forssman systemic shock more potently than both FUT-175 and compound 6. CONCLUSIONS: These data suggest that the 2-furylcarboxylic acid derivatives have a strong potential for inhibiting the activities of the complement, and the guanidino group was required to retain high inhibitory activities in vivo, and compound 7 is a hopeful anti-complement agent.

Benzenesulfonamide derivatives of 2-substituted 4H-3,1-benzoxazin-4-ones and benzthiazin-4-ones as inhibitors of complement C1r protease.

A series of 2-sulfonyl-4H-3,1-benzoxazinones was prepared that inhibit C1r protease in vitro. Several compounds were found to be selective for C1r verses the related serine protease trypsin. Selected compounds demonstrated functional activity in a hemolysis assay.

Regulation of the synthesis of C1 subcomponents and C1-inhibitor.

We have investigated the synthesis of C1q, C1r, C1s and C1-inhibitor in HepG2 cells, human umbilical vein endothelial cells (HUVEC), fibroblasts (skin and synovial membrane), chondrocytes and monocytes. C1q was only synthesised by monocytes, although the mRNAs for the C1qA and C1qC chains were expressed in HUVEC. C1r, C1s and C1-inhibitor were synthesised by all cell types. The secretion rates of C1r and C1s were approximately equimolar in fibroblasts and chondrocytes whereas the secretion rate for C1s exceeded that for C1r in the other cell types. Molar ratios of C1s to C1r were approximately 2:1 for HepG2 cells, 5:1 for monocytes and 10:1 for HUVEC. Stimulation with interferon-gamma resulted in increased expression of all four proteins. The C1s:C1r ratio did not alter in chondrocytes or fibroblasts, but approached unity in HepG2, monocytes and HUVEC, due to relatively greater stimulation of C1r gene expression.

[Preparation and clinical assays of Cl esterase inhibitors]. / Preparatyka i próby klinicznego zastosowania inhibitora Cl-esterazy.

Seven preparation of Cl-estrase inhibitor (Clinh) were prepared from the serum form which factor IX had been absorbed previously. Despite always the same technique and conditions of isolation the degree of Clinh recovery differed, being sometimes fairly low, and ranging from 24% to 56% in relation to the initial plasma. The preparations contained cartain amounts of other proteins from the groups of alpha- and beta-globulins, particularly coeruloplasmin and components of complement C3, C4 and C9. The concentration of these components was low in relation to the concentration of the inhibitor. Some preparations contained also low amount of albumins. The preparation (about 20 thousand Clinh units) was transfused to a patient with congenital deficiency of Cl-esterase inhibitor at the time free of symptoms. The investigation of the blood obtained 30 minutes after transfusion showed complete inhibition of C-L-esterase. Administration of the preparation to the patient at the time of acute oedema caused in 5 episodes rapid regression of oedema or inhibition of its increase, in two cases no evident positive effect was obtained.

Synthetic inhibitors of trypsin, plasmin, kallikrein, thrombin, C1r-, and C1 esterase.

p-Carbethoxyphenyl episol-guanidinocaproate and p-(p'-guanidinobenzoyloxy)-phenyl derivatives were prepared, and their inhibitory effects on trypsin, plasmin, plasma kallikrein, thrombin, C1r- and C1 esterase were examined. Among the various inhibitors tested, p-nitrophenyl p'-guanidinobenzoate, N,N-dimethylamino p-(p'-guanidinobenzoyloxy)-benzoyl glycolate and N,N-dimethylamino p-(p'-guanidinobenzoyloxy)-benzilcarbonyloxy glycolate were the most effective inhibitors of trypsin, plasmin, plasma kallikrien and thrombin, and they strongly inhibited the esterolytic activities of C1r- and C1 esterase.