Smooth muscle and parasympathetic nerve terminals in the rat urinary bladder have different subtypes of alpha(1) adrenoceptors.

Neurally evoked contractions and release of (3)H- acetylcholine (ACh) during electrical field stimulation were measured in rat urinary bladder strips. The alpha(1) agonist phenylephrine (PE, 2-8 microM) increased the amplitude of neurally evoked contractions, facilitated the release of ACh and increased the baseline tone of the bladder strips. The PE-induced facilitation of the contractions did not significantly change during a prolonged exposure to PE (120 min), whereas the PE-induced rise in baseline tone gradually decreased to 65% of the initial value. Low concentrations of specific alpha(1A) antagonists, 5-methyl urapidil (5-MU), REC15/2739 and WB-4101 competitively inhibited the facilitation of the neurally-evoked contractions (pA(2:) 8.77; 9.59 and 9.62, respectively), whereas higher concentrations of 5-MU (IC(50): 48 nM) were required to suppress the PE-rise in baseline. WB-4101 (100 microM) inhibited the PE-induced facilitation of ACh release. The irreversible alpha(1B) antagonist chloroethyl-clonidine (CEC, 10-50 microM) inhibited the PE-evoked rise in base line tone, but did not affect the PE-induced facilitation of the neurally evoked contractions nor the facilitation of ACh release. However, CEC increased the area and amplitude of the neurally-evoked contractions by 261+/-33 and 47.2+/-8.4%, respectively. Atropine significantly inhibited the CEC evoked increase in area and amplitude of the electrically evoked contractions (76.5+/-4.8 and 40.8+/-3%, respectively) indicating that CEC facilitated the cholinergic responses of the electrically stimulated bladder strips. It is concluded that alpha(1A) and CEC sensitive alpha(1B) and/or alpha(1D) adrenoceptors are expressed in the rat bladder in different locations. On the cholinergic nerve terminals alpha(1A) adrenoceptors mediate prejunctional facilitation, whereas postjunctional alpha(1B)/alpha(1D) adrenoceptors mediate smooth muscle contraction.

Active site-directed thrombin inhibitors: alpha-hydroxyacyl-prolyl-arginals, new orally active stable analogues of D-Phe-Pro-Arg-H.

D-alpha-Hydroxyacyl-prolyl-arginals, a new type of analogues of D-Phe-Pro-Arg-H (R1), have been prepared and evaluated. Unlike R1, whose terminal group is NH2, the new analogues with a terminal OH group are stable, as are the N-substituted derivatives of R1, that is, D-MePhe-Pro-Arg-H (R2), the highly potent and selective thrombin inhibitor, and Boc-D-Phe-Pro-Arg-H (R3), the much less favorable analogue. The most notable of the new analogues corresponds to the general formula D-Xaa-Pro-Arg-H, wherein Xaa means the acyl residue of mandelic acid (Man, 1), diphenyllactic acid (Dpl, 2), hexahydrophenyllactic acid (Hpl, 3), or hexahydromandelic acid (Hma, 4). In plasma clotting assays, 1 to 4 appeared to inhibit thrombin as well as some other clotting enzymes involved in thrombin generation, whereas R1 and R2 seemed to produce anticoagulation through inhibition of thrombin only. In the fibrin plate assay, 1 to 4 possessed even more moderate antifibrinolytic activities than R2. In in vivo evaluation in rats and rabbits, 2 to 4 proved to be potent anticoagulants/antithrombotics even on oral administration in a dose of 5 mg/kg. In view of these findings with the alpha-hydroxyacyl-prolyl-arginals, it is very likely that the less favorable biologic properties of Boc-D-Phe-Pro-Arg-H are due to the hydrophobicity and bulkiness of the terminal Boc-NH rather than its neutrality.

Effect of peptide aldehydes with IL-1 beta converting enzyme inhibitory properties on IL-1 alpha and IL-1 beta production in vitro.

Tripeptide and pentapeptide aldehydes as substrate-base inhibitors of cysteine proteases were designed in our laboratory for the inhibition of interleukin-1 beta converting enzyme (ICE), a recently described cysteine protease responsible for the processing of IL-1 beta. The biological effectivity of the peptide aldehydes was studied in THP-1 cells and human whole blood. The released and cell-associated IL-1 alpha and IL-1 beta levels were determined by ELISA from the supernatants and cell lysates, respectively. The total IL-1 like bioactivity was assayed by the D10 G4.1 cell proliferation method. The tripeptide aldehyde (Z-Val-His-Asp-H) and pentapeptide aldehyde (Eoc-Ala-Tyr-Val-Ala-Asp-H) significantly reduced IL-1 beta levels in the supernatants in relatively high concentrations (10-100 microM), but the IL-1 alpha release was unaffected by these peptides. However, a considerable decrease in the cell-associated IL-1 beta and IL-1 alpha levels was observed. N-terminal extension of the tripeptide aldehyde yielded even more potent inhibitors. Amino acid substitution at the P2 position did not cause considerable changes in the inhibitory activity. The peptide aldehydes suppressed the IL-1 beta production in a reversible manner, whereas dexamethasone, a glucocorticoid, had a prolonged inhibitory effect. The inhibitory effect of these peptides and that of dexamethasone appeared to be additive. These findings indicate that these peptide aldehydes might be used as IL-beta inhibitory agents in experimental models in which IL-1 beta is a key mediator or ICE is implicated.

Active site-directed thrombin inhibitors: alpha-hydroxyacyl-prolyl-arginals. New orally active stable analogs of D-Phe-Pro-Arg-H.

D-alpha-Hydroxyacyl-prolyl-arginals have been designed and synthesized as orally active stable analogs of D-Phe-Pro-Arg-H, the active site-directed peptidyl thrombin inhibitor prototype. Many of the new analogs possess high in vitro anticoagulant activity while having little effect on fibrinolysis. Compounds GYKI-66104 (2), -66131 (3) and -66132 (5) effectively delay the clotting time in rabbits ex vivo and prevent thrombus formation in various thrombosis models in rabbits and rats when applied in a single oral dose of 5 mg kg-1.

Screening for fibrinolysis inhibitory effect of synthetic thrombin inhibitors.

Fibrin plate assay (FPA) and thrombelastography (TEG) were used to assess the antifibrinolytic effects of D-Phe-Pro-Arg-H (1), the prototype of peptide aldehyde inhibitors of thrombin, and two of its more stable derivatives, D-MePhe-Pro-Arg-H (2) and Boc-D-Phe-Pro-Arg-H (3). Inhibition of plasmin generation by tissue plasminogen activator, urokinase and streptokinase were studied by both FPA and TEG while that of plasmin could only be examined by FPA. TEG was more sensitive than FPA in general and for the detection of streptokinase inhibition in particular. Derivative (3) was 2-50 times more inhibitory than (1) or (2) depending on the enzyme studied and the assay system used. The thrombin selectivities of (1)-(3) were defined as the thrombin to fibrinolytic enzyme potency ratios. Data obtained by the FPA and thrombin time assay indicated (1) and (2) to be 2-80 times more selective for thrombin than (3). On the other hand, the values determined by TEG and recalcification assay showed the thrombin selectivity of (2) to be two to three times higher than that of (1), and (3) to have no such selectivity. According to TEG studies, (1) and (2) assisted rather than inhibited fibrinolysis by reducing the elasticity of human plasma clots.

Comparative studies in vitro and ex vivo on the anticoagulant effect of a reversible and an irreversible tripeptide inhibitor of thrombin.

Comparative studies on the anticoagulant effect of D-Phe-Pro-Arg-H (ALD) and D-Phe-Pro-Arg-CH2Cl (CMK) were carried out in order to estimate whether the reversible or the irreversible tripeptide inhibitor of thrombin would be more suitable to develop as a novel anticoagulant. Conventional screening assay methods in vitro were focused on the functional stability of the compounds in whole blood and blood components while ex vivo the changes in whole blood clotting time under parenteral application of the inhibitors were investigated. The efficacy of ALD relative to that of CMK was found to depend on the complexity of the test systems. Thus CMK was the more inhibitory in citrated plasma, but ALD showed the higher potency in whole blood. When incubated in various systems such as human whole blood, serum, solutions of isolated plasma proteins, digestive juices and tissue homogenates, respectively, the inhibitory activity of ALD showed only slight decreases for several hours while marked or substantial loss of activity was observed with CMK under identical conditions. ALD administered parenterally to rabbits proved to be powerful anticoagulant; CMK exhibited only a weak and transient anticoagulant effect presumably due to its ability to bind irreversibly to various plasma and tissue proteins. Accordingly, the reversible inhibitor ALD should be more suitable to develop as an anti-coagulant than CMK, its irreversibly acting analogue.

In vitro inhibition of blood coagulation by tripeptide aldehydes--a retrospective screening study focused on the stable D-MePhe-Pro-Arg-H.H2SO4.

A series of peptide aldehydes synthetized in our institute during the last 15 years were screened to detect their inhibitory effect on blood coagulation. Simple conventional clotting assays, platelet function tests and fibrinolytic methods were used to evaluate the inhibitory potency of the compounds in complex clotting systems as well as their supposed antifibrinolytic effect in vitro. Special attention was paid to the possible interactions with blood cells and plasma proteins, and to the functional stability of the inhibitors in several tissue homogenates. D-Phe-Pro-Arg-H (GYKI-14166, RGH-2958), Boc-D-Phe-Pro-Arg-H (GYKI-14451) and D-MePhe-Pro-Arg-H (GYKI-14766) were found to be the most potent inhibitors. The peptide aldehydes via formation of reversible complexes with thrombin impede the enzyme to react with the coagulation factors, platelet membrane and vessel wall. The compounds inhibit platelet aggregation induced by thrombin specifically without changing the sensitivity of platelets to other inducers. D-Phe-Pro-Arg-H and D-MePhe-Pro-Arg-H showed no antifibrinolytic effect. D-MePhe-Pro-Arg-H and Boc-D-Phe-Pro-Arg-H proved to be stable in dry state for years and in solution at room temperature for several days. The anticoagulant activity of the compounds was declared in NIH antithrombin units.

In vivo anticoagulant and antiplatelet effect of D-Phe-Pro-Arg-H and D-MePhe-Pro-Arg-H.

D-Phe-Pro-Arg-H and D-MePhe-Pro-Arg-H synthetized in our institute were administered to mice, rats, rabbits and beagle dogs. The kinetics of the anticoagulant and antiplatelet effect was recorded by measuring various clotting parameters, platelet count and aggregation, and evaluated as proposed by Verstraete and Verwilghen. The minimum effective doses were found to be 0.25-0.5 mg kg-1h-1 by intravenous continuous infusions and 0.5-1.0 mg/kg by single injections. The dose-dependent prolongation of clotting times appeared after application within minutes and returned to baseline values as a function of dose. Blood level of the inhibitors was determined by a bioassay. Unlike heparin, no higher starting dose was required to reach the anticoagulant threshold level, i.e. 0.03-0.1 microgram/ml whole blood. The peptides did not cause significant changes in platelet count and function or in hemodynamic parameters (blood pressure, heart rate and ECG) and in respiration. They blocked platelet aggregation induced by thrombin ex vivo specifically. No rebound effect or bleeding could be demonstrated even after subtoxic doses of the compounds. The onset of the anticoagulant and antithrombotic effect appeared within 60 min after single oral doses and lasted for 3-6 h. In close correlation with the anticoagulant effect a complete or significant inhibition of platelet aggregation induced by thrombin ex vivo could also be recorded by using 5-10 mg/kg doses.

Oligopeptides interfering with calcium channels inhibit prolactin and growth hormone release by cultured anterior pituitary cells of the rat.

A number of oligopeptides, protected at their N termini and possessing an aldehyde residue at their C terminal amino acids, are able to inhibit 45Ca2+ influx into anterior pituitary cells grown in monolayer culture and depolarized with high extracellular potassium concentration. In addition, the same oligopeptides interfere with hormone release, especially with that produced by lactotrophs. Our findings imply that oligopeptides may represent a new class of calcium channel ligands, and the pituitary cells are sensitive targets for them.

Highly active and selective anticoagulants: D-Phe-Pro-Arg-H, a free tripeptide aldehyde prone to spontaneous inactivation, and its stable N-methyl derivative, D-MePhe-Pro-Arg-H.

D-Phe-Pro-Arg-H sulfate (GYKI-14166) is a highly active and selective inhibitor of thrombin both in vitro and in vivo. Recent studies on the stability of D-Phe-Pro-Arg-H in neutral aqueous solution at higher temperature have revealed that it is transformed into inactive 5,6,8,9,10,10a-hexahydro-2-(3'- guanidinopropyl)-5-benzyl-6-oxo- imidazo[1,2-a]pyrrolo[2,1-c]pyrazine. No such inactivation could be observed with Boc-D-Phe-Pro-Arg-H (GYKI-14451), but this compound was far less specific than the free peptide as it inhibited thrombin and, for instance, plasmin equally well. Assuming that the transformation of free tripeptide aldehyde, mentioned above, can only be initiated by a primary amino terminus, the N-alkyl derivatives of D-Phe-Pro-Arg-H were prepared. Of the new analogues, D-MePhe-Pro-Arg-H (GYKI-14766) proved to be as highly active and selective anticoagulant as its parent compound and was not inactivated by transformation into a heterocyclic compound.

Experimental oral anticoagulation by a directly acting thrombin inhibitor (RGH-2958).

D-Phenylalanyl-L-prolyl-L-arginine aldehyde sulfate (RGH-2958), a directly acting synthetic thrombin inhibitor proved to be effective by experimental oral application. The rapid onset of its action has a special importance both from theoretical and practical point of view. Its antiplatelet effect relates to thrombin induced PA and runs parallel with the anticoagulant effect. RGH-2958 significantly reduced the thrombus weight in an experimental thrombosis model in rabbits. In the light of the therapeutic indices of the compound there is a real possibility to open a third way in the prevention and therapy of thrombosis.

Specific inhibition of human granulocyte elastase with peptide aldehydes.

The kinetic features of human granulocyte elastase, chymotrypsin, porcine pancreatic elastase and elastomucoproteinase were compared. Amino acyl ester substrates were assayed and Km and kcat values were defined. Aldehyde analogues of the p-nitroanilide substrates designed for granulocyte elastase as optimal for Km appeared to be potent inhibitors. Suc-D-Phe-Pro-valinal (Ki = 40 microM) was found to inhibit granulocyte elastase competitively and specifically when measured with synthetic substrates, and the Ki was 3 microM with the natural protein substrate, elastin.

Design and synthesis of peptide inhibitors of blood coagulations.

Inhibition of blood coagulation by peptide aldehydes has been studied. Amino acid sequences were assembled from the P1-P2 portion of the cleavage sites(s) of clotting factors and residues selected experimentally. The thrombin-fibrinogen reaction could effectively be inhibited by D-Phe-Pro-Arg-H (GYKI-14,166) and Boc-D-Phe-Pro-Arg-H (GYKI-14,451). Plasmin digestion of fibrin could be retarded by Boc-Gln-Phe-Lys-H (GYKI-14,605) derived from a susceptible fragment, i.e. Asn-Phe-Lys decreases to Ser. However, such peptides could not retard the zymogen activations proceeding in Ca++ complexes (which seemed to be uneffected by heparin-antithrombin III, too). Inhibition of enzymes by peptide aldehydes showed marked substrate dependence.

Inhibition of thrombin and trypsin by tripeptide aldehydes.

Inhibitory effects of certain tripeptide aldehydes on both thrombin and trypsin have been found to be strongly substrate-dependent. These compounds should therefore be considered as inhibitors of the particular proteolytic reaction for which they had been designed rather than real enzyme inhibitors, i.e. protein or polypeptide proteinase inhibitors of natural origin.