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Antiplatelet activity of histamine receptor antagonists
2-PYRIDYLI~*DRAZONES DERIVATIVES AS PLATELET AGGREGATION INHIBITORS. A.L.P. Miranda; A.1L Todeschini; E J . Barreiro. LASSBIo -- Laboratbrio de Avalia~o e Sintese de Snbst~ncias Bioaflvas, Faculty of Pharmacy, University Federal of Rio de Janetro, RJ., Brazil.
EFFECT OF NEW REGULATORS IN ~ M O S T A S I S . V.A.Makarov, G,N, Petrukhina, G.G.Belozerskay~,N.N,Drozd, G.V.Bashkov, N.T.Miftanova, S.A.Kalugin. National Research Centre for Hematology, Moscow, Russia. Specific pharmecological effects of new hemostatic regulators have been studied. In particular, we investigated a pharmacological action of chitorin - a new anticoagulant whichpresents a combination of heparin and chltosan a polysulfate Of a certain molecular mass and degree of sulfation. Chitorin produces an anticoagulant effect similar to heparin, but itlis characterized by a lower hemorrhagic actlvity and a weak effect on platelets. As newir~ibitors of the platelet aggregation we studied stable derivatives of prostaglandines and polyunsaturated fat acids. They don't only affect the pla telets, b u t a l s o activate the fibrlnolytic activity of t h e v e s s e l wall.Plasminogen tissue activator manifested itself mere strongly against the background of adrenalin and immobilized stress.The possibility to strengthen the antlthrombic activity of the vessel wall was shown due t o t h e combination of drugs activating the ATP synthesis and adenylate cyclase reaction.To activate the lysls of thrombus we studied new substituted derivatives of the streptokinasa.plasminogen c o m p l e x .
A series of 2-pyridylihydrazones derivatives was sinthesized as hioisoslers of dual lnhihitors of CO/5-LO and thromboxane synthelase. The general structure of derivatives w~s designed based ml the bloiso~ertsm concept between the pyridyl and the pyrazol ring's ~vhose platelet anti-aggregating activities were described for a series of 5pyrazolylhydrazones and 4-acylpyrazolylhydrazones (Siivelra e t a l , J.Phm~a. Pharmacol.,45, 646,1993 and Miranda el al, XII IUPHAR Congress,1994). The objective of this .study was to evaluate the in vitro anti-aggregating activity in citrated platelet-rich rabbit plasma (PRP) induced by ADP (SgM), collagen (Sgghnl) and arachidonic acid (AA 200gM) of 15 compounds of this class, which were tested at 100gM concentration. None of the compomlds showed inhibitory activity on ADP indueed rabbit platelet aggregation. Compounds AS, A7, A l l , A14 were the most active inhibitors with 100% of inhibition on AA induced platelet aggregation. Some of these compounds showed analgesic but none anttinflanmtory activity, suggesting an interference on the AA metabolism, probably on the COX-1. Also, the presence of a furyi-ring linked to the hydrazone s e e m s t o be outstanding for the activity, the latter was previously obselwed for the two differents series above cited.
A7='~OH A14 S u p p o r t e d by: C N P q , C E P G (Brazil).
TWO DIFFERENT RESPONSES IN HUMAN PLATELETS STIMULATED BY CALCIUM IONOPHORES. A. Montero-Lastres,N. Fraiz, R. Laguna,R. Villar, E. cano. Laboratorio de Farmacoloxfa, Facultade de Farmacia, Universidade de Santiago de Cornpostela,15706Santiagode Compostela,Espa~a(Spain).
ANTIPLATELET A C T I V I T Y OF HISTAMINE R E C E P T O R ANTAGONISTS R.NOS~I, V.Jan~inov~, M.Petrikov~ Institute of Experimental P h a r m a c o l o g y SAS, Dubravska 2, 842 16 Bratislava, Slovakia
Changes in the cytoplasmic free Ca.2 concentration have a fundamental role in the regulation of platelet functions. Activation of human p[atelets using several agonists is followed by an intracellu[ar calcium increase that might be caused by mobilization from internal Ca + 2 storage and/or by influx of external Ca+ 2 . Thus, the addition of calcium ionophores to induce intracellular calcium increase is frecuently used to study calcium-depending ce lular responses. By means of calcium ionophore ~1~7 (calCiomycin) and IonomyCin we have studied piateiet responses induced by the increase of intracellular calcium due to influx through the plasma membrane. Two different patterns of platelet response have been noted. Low ionophore concentration (A231~775-100nM;Ionomycin 25-37nM) caused aggregation (evaluated by turbidimetry and optical observation) and phosphorylation 'of 20KDa and 47KDa proteins (myosin light chain and pleckstrin respectively). This response has been inhibited by inhibitors of calmodulin (W-7), PKC (Ro-318220), by a fibrinogen receptor antagonist (RGDS) and by a stimulating agent of adenylatecyclase (forskolin); aspirin produced a weak inhibition. Using a higher concentration of both ionophores (A23187llxM; Ionomicyn 0.5~M) a decrease in light transmition was produced but aggregates were not observed. However, generation of platelet microvesic[es and calciumactivated neutral protease (calpain) activation have been observed. Protein phosphorylations (20 KDa and 47 KDa) were immediately caused, but very fast dephosphorylation have been observed due to protein-phosphatase activity. These facts are probably more associated with cytotoxic damage by massive influx of external Ca÷2 than with a physiological=response. Since calcium ionophores are frecuently used in the higher concentration that we used, special care should be taken when selecting calcium ionophore concentration in order to avoid cytotoxic problems and to facilitate the study of physiological activation process in platelets and, probably, other cells.
Cationic amphiphilic drugs(CAD) interact w i t h cells and tissues via receptor m e d i a t e d and r e c e p t o r - i n d e p e n d e n t pathways. The antiplatelet effect of b e t a a d r e n o c e p t o r blocking drugs calcium antagonists and chloroquine was d e m o n strated.As most of HI-antagonists are CAD it was of interest to investigate the effect of bromadryl(BR0),dithiaden(DIT) and two derivatives of p h e n i r a m i n e ( P A ; b r o m - B P A , c h l o r - C P A ) o n aggregation, m a l o n d i a l d e h y d e formation (MDA) and thromboxane g e n e r a t i o n ( T X B 2) of b l o o d pla telets in vitro.All tested Hl-antagonists inhibited ADP- and thrombin-stlmulated aggregation,MDA formation and TXB 2 generation in a d o s e - d e p e n d e n t way.DIT was 5 to i0 times m o r e active than BRO.Halogen substitution on the aromaticc ring of PA increased the p a r t i t i o n coefficients and inhibition of the aggregat i o n , M D A formation and TXB 2 generation in the rank order of p o t e n c y : P A < C P A < BPA.PA halogenization increased the availability of the drug for lipophilic as well as electrostatic interactions w i t h p l a t e l e t membranes.It seems likely that the non-receptor membrane interactions play an important role in the inhibitory effect of Hl-antagonoists on platelet f u n c t i o n s . F r o m the interaction of these drugs with stimulated arachidonic acid pathway it is suggested that investigated drugs may inhibit p l a t e l e t phospholipase A 2 activation.
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EFFECT OF NEW REGULATORS IN ~ M O S T A S I S . V.A.Makarov, G,N, Petrukhina, G.G.Belozerskay~,N.N,Drozd, G.V.Bashkov, N.T.Miftanova, S.A.Kalugin. National Research Centre for Hematology, Moscow, Russia. Specific pharmecological effects of new hemostatic regulators have been studied. In particular, we investigated a pharmacological action of chitorin - a new anticoagulant whichpresents a combination of heparin and chltosan a polysulfate Of a certain molecular mass and degree of sulfation. Chitorin produces an anticoagulant effect similar to heparin, but itlis characterized by a lower hemorrhagic actlvity and a weak effect on platelets. As newir~ibitors of the platelet aggregation we studied stable derivatives of prostaglandines and polyunsaturated fat acids. They don't only affect the pla telets, b u t a l s o activate the fibrlnolytic activity of t h e v e s s e l wall.Plasminogen tissue activator manifested itself mere strongly against the background of adrenalin and immobilized stress.The possibility to strengthen the antlthrombic activity of the vessel wall was shown due t o t h e combination of drugs activating the ATP synthesis and adenylate cyclase reaction.To activate the lysls of thrombus we studied new substituted derivatives of the streptokinasa.plasminogen c o m p l e x .
A series of 2-pyridylihydrazones derivatives was sinthesized as hioisoslers of dual lnhihitors of CO/5-LO and thromboxane synthelase. The general structure of derivatives w~s designed based ml the bloiso~ertsm concept between the pyridyl and the pyrazol ring's ~vhose platelet anti-aggregating activities were described for a series of 5pyrazolylhydrazones and 4-acylpyrazolylhydrazones (Siivelra e t a l , J.Phm~a. Pharmacol.,45, 646,1993 and Miranda el al, XII IUPHAR Congress,1994). The objective of this .study was to evaluate the in vitro anti-aggregating activity in citrated platelet-rich rabbit plasma (PRP) induced by ADP (SgM), collagen (Sgghnl) and arachidonic acid (AA 200gM) of 15 compounds of this class, which were tested at 100gM concentration. None of the compomlds showed inhibitory activity on ADP indueed rabbit platelet aggregation. Compounds AS, A7, A l l , A14 were the most active inhibitors with 100% of inhibition on AA induced platelet aggregation. Some of these compounds showed analgesic but none anttinflanmtory activity, suggesting an interference on the AA metabolism, probably on the COX-1. Also, the presence of a furyi-ring linked to the hydrazone s e e m s t o be outstanding for the activity, the latter was previously obselwed for the two differents series above cited.
A7='~OH A14 S u p p o r t e d by: C N P q , C E P G (Brazil).
TWO DIFFERENT RESPONSES IN HUMAN PLATELETS STIMULATED BY CALCIUM IONOPHORES. A. Montero-Lastres,N. Fraiz, R. Laguna,R. Villar, E. cano. Laboratorio de Farmacoloxfa, Facultade de Farmacia, Universidade de Santiago de Cornpostela,15706Santiagode Compostela,Espa~a(Spain).
ANTIPLATELET A C T I V I T Y OF HISTAMINE R E C E P T O R ANTAGONISTS R.NOS~I, V.Jan~inov~, M.Petrikov~ Institute of Experimental P h a r m a c o l o g y SAS, Dubravska 2, 842 16 Bratislava, Slovakia
Changes in the cytoplasmic free Ca.2 concentration have a fundamental role in the regulation of platelet functions. Activation of human p[atelets using several agonists is followed by an intracellu[ar calcium increase that might be caused by mobilization from internal Ca + 2 storage and/or by influx of external Ca+ 2 . Thus, the addition of calcium ionophores to induce intracellular calcium increase is frecuently used to study calcium-depending ce lular responses. By means of calcium ionophore ~1~7 (calCiomycin) and IonomyCin we have studied piateiet responses induced by the increase of intracellular calcium due to influx through the plasma membrane. Two different patterns of platelet response have been noted. Low ionophore concentration (A231~775-100nM;Ionomycin 25-37nM) caused aggregation (evaluated by turbidimetry and optical observation) and phosphorylation 'of 20KDa and 47KDa proteins (myosin light chain and pleckstrin respectively). This response has been inhibited by inhibitors of calmodulin (W-7), PKC (Ro-318220), by a fibrinogen receptor antagonist (RGDS) and by a stimulating agent of adenylatecyclase (forskolin); aspirin produced a weak inhibition. Using a higher concentration of both ionophores (A23187llxM; Ionomicyn 0.5~M) a decrease in light transmition was produced but aggregates were not observed. However, generation of platelet microvesic[es and calciumactivated neutral protease (calpain) activation have been observed. Protein phosphorylations (20 KDa and 47 KDa) were immediately caused, but very fast dephosphorylation have been observed due to protein-phosphatase activity. These facts are probably more associated with cytotoxic damage by massive influx of external Ca÷2 than with a physiological=response. Since calcium ionophores are frecuently used in the higher concentration that we used, special care should be taken when selecting calcium ionophore concentration in order to avoid cytotoxic problems and to facilitate the study of physiological activation process in platelets and, probably, other cells.
Cationic amphiphilic drugs(CAD) interact w i t h cells and tissues via receptor m e d i a t e d and r e c e p t o r - i n d e p e n d e n t pathways. The antiplatelet effect of b e t a a d r e n o c e p t o r blocking drugs calcium antagonists and chloroquine was d e m o n strated.As most of HI-antagonists are CAD it was of interest to investigate the effect of bromadryl(BR0),dithiaden(DIT) and two derivatives of p h e n i r a m i n e ( P A ; b r o m - B P A , c h l o r - C P A ) o n aggregation, m a l o n d i a l d e h y d e formation (MDA) and thromboxane g e n e r a t i o n ( T X B 2) of b l o o d pla telets in vitro.All tested Hl-antagonists inhibited ADP- and thrombin-stlmulated aggregation,MDA formation and TXB 2 generation in a d o s e - d e p e n d e n t way.DIT was 5 to i0 times m o r e active than BRO.Halogen substitution on the aromaticc ring of PA increased the p a r t i t i o n coefficients and inhibition of the aggregat i o n , M D A formation and TXB 2 generation in the rank order of p o t e n c y : P A < C P A < BPA.PA halogenization increased the availability of the drug for lipophilic as well as electrostatic interactions w i t h p l a t e l e t membranes.It seems likely that the non-receptor membrane interactions play an important role in the inhibitory effect of Hl-antagonoists on platelet f u n c t i o n s . F r o m the interaction of these drugs with stimulated arachidonic acid pathway it is suggested that investigated drugs may inhibit p l a t e l e t phospholipase A 2 activation.
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