Title of Invention	THE USE OF PHENOLICACETATE DRUGS TO ENHANOE THE INTRACELLULAR NITRICOXIDE LEVEL IN HUMAN PLATELETS
Abstract	A phenolic acetate drug composition to enhance the intracellular nitric oxide level in human platelets comprising 100 µm phenolic acetate selected from 7,8-diacetoxy 4-methylcoumarin (DAMC), 7-acetoxy-4 methylcoumarin 7(AMC), 6-acetoxy benzenoid-3-one and carrier dimethyl sulphoxide (DMSO) 10 µl and 200 µm Adenine Dinucleotide Phosphate (ADP).

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This invention relates to a phenolic acetate drug composition to enhance the intracellular nitric oxide level in human platelets. BACKGROUND OF THE INVENTION There are numerous NOS inhibitors described in the literature and in use as pharmaceutical tools. Of these the most widely used have been L-NMMA (NG-nitro-L-arginine) and its methyl ester prodrug (NG-nitro-L-arginine methyl ester, 'L-NAME') and aminoguanidine Inhibitors of nitric oxide synthase have been described which interact with the nitric oxide synthase enzymes in a variety of ways: different sites, as well as differing time-and substrate-dependence, and mechanism of inhibition. Most inhibitors identified so far are competitive with the substrate L-arginine and have therefore been inferred to be binding at the arginine-binding site. In the instances in which data on nitric oxide synthase haem-domain crystal structures have been published with such inhibitors (aminoguanidine, S-ethylisothiourea, thiocitrulline), they do indeed bind in the active site, interacting with the conserved glutamate (Glu363 of bovine Enos, Glu371 of murine Inos) which also interacts with the guanidine group of L-arginine. However, with many arginine-site nitric oxide synthase inhibitors there are mechanisms involved in their effects on nitric oxide synthase beyond simple binding in competition with L-arginine. A substantial body of work has been published on the mechanisms of induced nitric oxide synthase inhibition by aminoguanidine and by the acetamidine inhibitors, N5-iminoethyl-L-omithine (L-NIO) and N6-iminoethyl-L-lysine (L-NIL) (85,86,87,88), GW273629 (S-[2-[(l-iminoethyl)-amino]ethyl]-4,4-dioxo-L-cysteine) and GW274150 (S-[2-[(l-iminoethyl)amino]ethyl]-L-homocysteine). These are all mechanism-based inhibrtors of induced nitric oxide synthase, requiring active enzyme and reduced nicotinamide adenine dinucleotide phosphate substrate to permit inhibition to proceed from the initial relatively weak binding to the enzyme: BhU-binding site is located adjacent to the arginine-binding region and the haem cofactor. A range of compounds have been identified which appear to interact with the pterin site as assessed by a range of criteria, e.g. mutual antagonism with BHU on activity, competition for binding of radiolabeHed BH» to full-length or haem domains of nitric oxide synthase. Some are obvious pterin analogues, e.g. 4-amino-BH4,BH2 and a range of synthetic analogues thereof, whereas others are not, e.g. 7-NI, 214-diamino-5-(3',4'dichlorophenyl)pyrimidine (11U50). The interactions of some of these pterin-site inhibitors with nitric oxide synthase reveals unexpected complexity. As another example, 7-NI competes with the binding of both [3H]nitro-L-arginine and B3H4 to nNOS haem domains, whereas the arginine-site inhibitor S-ethylisothiourea inhibits the binding of [3HJnitro-L-arginine but promotes the binding of B3H4, making it clear that there are allosteric interactions between the two binding sites. A further group of compounds interact directly with the haem. In monomeric murine-induced nitric oxide synthase haem domain crystallized in the presence of the inhibitor imidazole, two imidazole molecules have been shown to be bound to each monomer, one to the haem iron and one to the arginine-binding region (Glu371). Various anti-fungal imidazoles have been shown to inhibit nitric oxide synthase activity, not only by interacting with the haem at the active site but also by acting in competition with Calmodulin; these compounds have also been shown to affect the assembly of induced nitric oxide synthase monomers into active dimmer, either promoting or inhibiting dimerization. Most recently, a class of substituted pyrknidine imidazoles have been identified which do not directly inhibit nitric oxide synthase activity, but very potently inhibit dimerization of induced nitric oxide synthase during its synthesis and assembly. An example was shown from the crystal structure of its complex with monomeric induced nitric oxide synthase haern domain to be ligated to the haem iron of induced nitric oxide synthase. A range of inhibitors which have effects on a variety of either flavoproteins (e.g. diphenylenelodnium) or Calmodulin (e.g. trifluoperazine) have been shown to inhibit nitric oxide synthase. At present these do not appear to be a promising avenue for the discovery of selective inhibitors of nitric oxide synthases. OBJECTS OF THE INVENTION An object of this invention is to propose the use of phenolicacetate drugs to enhance intracellular nitricoxide level in human platelets. Another object of this invention is to propose transacetylase catalyzed modification of nitric oxide synthase activity in human platelets. Further object of this invention is to propose an unique mechanism to enhance the level of No in the human platelets in presence of the novel phenolicacetate drugs. Still further object of this invention is to propose the use of phenolicacetate drugs to inhibit platelets aggregation. DETAILED DESCRIPTION OF THE INVENTION According to this invention there is provided a phenolic acetate drug composition to enhance the intracellular nitric oxide level in human platelets comprising 100 \im phenolic acetate selected from 7,8-diacetoxy 4-methylcoumarin (DAMC), 7-acetoxy-4 methylcoumarin 7(AMC), 6-acetoxy benzenoid-3-one and carrier dimethyl sulphoxide (DMSO) 10 yl and 200 jam Adenine Dinucleotide Phosphate (ADP). The intracellular NO control many physiological process listed below: 1. NO relaxes smooth muscles in the walls of arterioles at each systole, the endothelial cells that line the blood vessels release puff of NO which diffuses into the underline muscle ceils causing them to relax. 2. NO produced by endothelium creates an active vasodilatory state responsible for haemostatic control of blood pressure and blood flow. There is abundant evidence that deficient production of NO contributes to the pathogenicsis of hypertension in animals and human beings with essential hypertension. 3. Over production of NO by iNOS in cytokine and Hpopolysaccharides exposed macrophages and smooth muscle ceils to contribute vasoditatation and hypotension of sepsis. 4. NO also inhibits the aggregation of platelets and thus keeps the inappropriate clotting from interfering with blood flow. 5. The release of NO around the glomeruH of kidney increase the blood flow through them thus increasing the rate of nitration and urine formation. 6. The erection of penis during sexual excitation is mediated by NO released from nerve ending close to blood vessels of penis. The popular prescription drug Sildenafil Citrate (VIAGRA) inhibits the breakdown of NO and thus enhancing its effect 7. Peristalsis-relaxing effect of NO on smooth muscle in GIT walls. 8. NO also inhibits the contractility of smooth muscle wall of uterus. 9. NO relaxes gastrointestinal smooth muscle and causes decrease motilrty, relaxation of sphincter of odd! and lower esophageal sphincter. 10. Deficiency of NO producing neurons hi GIT is beNeved to be responsible for Hirschsprung disease, achalasia, and chronic intestinal pseudoobst ruction. 11. Relaxation of bronchial smooth muscle can be provoked by inhalation of NO and endogenously produced NO may contribute to maintenance of basal bronchial and basal pulmonary arterial tone. 12.NO also effects the secretion from several endocrine glands like hypothalamus, exocrine portion of pancreas and adrenal medulla. 13. NO appears to inhibit atherogenesis by inhibiting leukocyte and platelet activation and by inhibiting smooth muscle cell proliferation. 14.NO Is the likely transmitter of non-adrenergic, non-cholinergic neurons and thereby participate in the regulation of myocardial contractility, heart rate, bronchial tone and penile erection. 15. NO is released by neurons in the hippocampus and stimulates the NMD A (N-methyl D-aspartate) receptors there that are responsible for long-term potentiation (LTP), a type of memory and learning. 16.Recent data demonstrates that NO increases oxygen affinity of sickle erythrocytes-suggesting potential role for inhaled NO in treatment of sickle cell disease. The platelets were incubated with phenolicacetate drugs 7,8-diacetoxy 4-methylcoumarin (DAMC) and it was observed that the reductase domain gets acetylated and in the case of DAMC it gets converted into DHMC. Thus, the phenolicacetate drugs activates reduced nicotinamide adenine dinucleotide phosphate (NADPH) cytochrome c reductase of platelets and as a result of which the intracellular level of No in the blood platelets enhances. This unique mechanism is shown in figure 1. No plays an important role in the regulation of platelet function and hence platelet function and hence platelet nitrlcoxide synthase assumes much importance. Platelets incubated with 7,8-diacetoxy 4-methyicoumarin and L-arginine, were found to produce No significantly higher than the ceNs treated with L-arginine alone. Further, the enhanced production of NO due to incubated with 7,8-diacetoxy 4-methylcoumarin, as described above. Hence it is evident that incubation of platelets with 7,8-diacetoxy 4-methylcoumarin, resulted in TAase catalyzed activation of platelet nitricoxide synthase. It is pertinent to point out that the activation of reductase domain of nitricoxide synthase could be responsible for the stimulation of nitricoxide synthase activity in platelets due to 7,8-diacetoxy 4-methylcoumarin. The activity of TAase was assayed in the platelet tysate by incubating it with 7,8-diacetoxy 4-methylcoumarin and liver cytosol (source of glutathione S-transferase ) followed by the addition of substrates of glutathlone S-transferase, 1-chloro-2,4-dinitrobenzene and reduced glutathione, in order to assay progress of glutathione S-transferase catalyzed reaction. The extent of inhibition of glutathione S-transferase was considered proportional to TAase activity. Earlier studies in our laboratory demonstrated purified buffalo liver TAase catalyzed transfer of acetyl groups from 7,8-diacetoxy 4-methylcoumarin to glutathione S-transferase and the acetyiation of glutathione S-transferase was established by MALOi-TOF/and LC/MS/MS. The specific activity of TAase in platelets was found to be 133 units/mg protein/10 minutes of preincubation as compared to Hver microsomes, which was shown to have TAase activity of 400 units/mg protein/10 minutes preincubation. The present investigation has revealed that platelets contain considerable activity of TAase. Efforts were made to characterize platelet TAase with respect to substrate concentration and specificity to various polyphenol acetates. Platelet TAase exhibited MichaeUls-Menten hyperbolic kinetics. Vmax & Km of platelet TAase with varying 7, 8-diacetoxy 4-methylcoumarin concentration (concentration of cytosolic glutathione S-transferase kept constant) as derived from double reciprocal plot were found to be 400 units and 1050 micro molar (µM) respectively under the conditions of the assay. The specificity of various aetoxy poryphenols to platelet TAase was studied and the results demonstrated that apart from 7,8-diacetoxy 4-methylcoumarin, TC, 7-acetoxy-4-methylcoumarin, 7-N-acetyl-4-methylcoumarin, methylene bis(7,8-diacetoxy-4-hydroxycoumarin), methyiene bis (4,7-diacetoxycoumarin), S-acetoxy xanthone and 7-acetoxy chromone were found to be acted upon by platelet TAase. Among the above mentioned acetoxy potyphenols, 7,8-diacetoxy 4-methylcoumarin yielded the maximum activity for TAase. 7-acetoxy-4-methylcoumarin yielded TAase activity approximately 50% of that 7,8-diacetoxy 4-methylcoumarin. Similarly 7-N-acetyl-4-methylcoumarin yielded activity similar to 7-acetoxy-4-methylcoumarin. These result reveal that more the number of acetoxy group on the benzenoid ring of coumarin, more was the activity of TAase. interestingly, TC (7-acetoxy-4-methyl thiocoumarln) gave platelet TAase activity comparable to 7,8-diacetory 4-methylcoumarin and much higher compared to 7-acetoxy-4-methylcoumar!n. It can be concluded that replacement of pyran carbonyl by C=S could lead to enhanced specificity of 7-acetoxy thiocoumarin to platelet TAase. The specificity of methylene bis (7,8-diacetoxy-4-hydroxycoumarin) and methylene bis (4,7-diacetoxycoumarin) (Table 1) to platelet TAase revealed that they yielded significantly less platelet TAase activity compared to 7,8-diacetoxy 4-methylcoumarin and 7-acetoxy-4-methylcoumarin respectively. It is possible that free rotation of the coumarin nucleus around the methylene group may contribute to the diminished activity of platelet TAase obtained with acetoxy biscoumarins compared to the corresponding acetoxy coumarins. Further platelets were incubated with 7,8-diacetoxy 4-methylcoumarin, followed by the addition of the substrates of cytocrhome c redutase, i.e. reduced nicotinamide adenine dinucleotide phosphate and cytochrome c in order to assay the reductase activity. The result shown in Table 2 indicate the time dependent activation of reduced nicotinamide adenine dinucleotide phosphate cytochrome c reductase by 7,8-diacetoxy 4-methylcoumarin. Likewise other acetoxy polypheonis such TC, 7-acetoxy-4-methylcoumarin, methylene bis (7,8-diacetoxy-4-hydroxy coumarin), methylene bis (4,7-diacetoxycoumarin), 5-acetoxy xanthone and 7-acetoxy chromone were effective to various extents in causing the activation of reduced nicotinamide adenine dinucleotide phosphate cytochrome c reductase. The ability of polyphenol acetates to activate reduced nicotinamide adenine dinucleotide phosphate cytochrome c reductase was found to be in tune with their specificities to TAase. TAase catalyzed activation of reduced nicotinamide adenine dinucleotide phosphate cytochrome c reductase of platelet was found to be linear wtth time of preincubation and also with the concentration of platelet protein (Table 2). Platelets preincubated wtth 7,8-diacetoxy 4-methylcoumarin and L-arginine exhibited enhanced levels of NO signifying the activation of plate nitric oxide synthase activity. It is evident from Figure 1 that 7,8-diacetoxy 4-methylcoumarin, when Incubated alone wtth platelets, exhibited control level of NO, as evident from dichloro fluoroscein diacetate fluorescence. The incubation of platelets wtth L-arginine and 7,8-diacetoxy 4-methylcoumarin resulted in four times the levels of NO production compared to incubation of platelet wtth L-arginine alone. Platelet incubated wtth L-arginine and 7-acetoxy-4-methylcoumarin resulted in marginal increase in, NO level. Preincubation of platelet wtth N-nitro-L-arginine methylester, along wtth L-arginine and 7,8-diacetoxy 4-methylcoumarin resulted in drastic reduction of nitric oxide synthase activity (Figure 3). The data described in Figure 4 demonstrates the platelet aggregation caused by the addition of ADP (control). PRP incubated wtth 7,8-dihydroxy 4-methylcoumarin (100 micro molar) had no effect on AOP induced aggregation. The incubation of PRP wtth 7,8-diacetoxy 4-methylcoumarin (100 micro molar), on the other hand produced profound inhibition of AOP induced aggregation, amounting to the abolishing the effect of AOP on aggregation. PRP was separately incubated wtth different concentration of 7,8-dihydroxy 4-methylcoumarin (25- 100 micro molar) in order to examine the effect on ADP induced platelet aggregation (Figure 5). 7,8-dihydroxy 4-methylcoumarin was found to cause dose-dependent inhibition of platelet aggregation due to ADP. The effect of incubation of TC with platelets on ADP induced aggregation was examined (Figure 6). TC was found to be highly effective in the inhibition of platelet aggregation due to ADP, although to a lesser extent compared to 7,8-dihydroxy 4-methylcoumarin. EXAMPLE 1: Demonstration of NOS activation by DAMC invtvo: Male rats (150-200g) were injected up with DAMC (300mg/Kg bodyweight) after 24 hours, the platelets were isolated and assayed for NOS activity. The result demonstrated that administration of DAMC to rats enhanced NOS activity. We have also demonstrated that NOS of tracheal smooth muscle cells are activated by DAMC as in the case of platelets. EXAMPLE 2 : The enhanced levels of NO in platelets caused by DAMC prompted us to examine whether DAMC could inhibit the platelet aggregation. For this purpose 0.2ml of PRP was mixed with ADP (200µM) and the extent of platelet aggregation was noted. When 0.2 ml of PRP was mixed with DAMC (100µM) in dimethyl sulphoxide (DMSO) and ADP (200uM), a complete inhibition of platelet aggregation due to ADP was observed DMSO, the vehicle used for DAMC in this study had no effect on platelet aggregation due to ADP. TABLE 1: Specificities of various acetoxy poiyphenols to platelet TAase. (Table Removed)'Platelet TAase activity was expressed in terms of percent inhibition of GST under assay conditions. The concentrations of acetoxy poiyphenols and platelet protein used in the assay were 100uM and 150ug respectively. The values are mean of 4 determinations with error less that 2%. Acetoxy polyphenols (Table Removed)TABLE 2: TAase catalyzed activation of platelet NADPH cytochrome c reductase by polyphenolic acetates. (Table Removed) *Polyphenol acetates were preincubated with platelet lysate followed by the addition of NADPH and cytochrome c in order to assay the reductase activity. The controls received DMSO in place of polyphenol acetates. The increment in the activity of NADPH cytochrome c reductase due to preincubation with polyphenolic acetates over the control is expressed as percent stimulation. Concentration of substrates and platelet protein was 25uM and 50ug respectively. The details of the assay are mentioned under "Materials and Methods". The values are mean of 4 determinations with error less than 2%. (Table Removed) We Claim: 1. A phenolic acetate drug composition to enhance the intracellular nitric oxide level in human platelets comprising 100 µm phenolic acetate selected from 7,8-diacetoxy 4-methylcoumarin (DAMC), 7- acetoxy-4 methylcoumarin 7(AMC), 6-acetoxy benzenoid-3-one and carrier dimethyl sulphoxide (DMSO) 10 µl and 200 µm Adenine Dinucleotide Phosphate (ADP). 2. A phenolic acetate drugs composition as claimed in claim 1 wherein the said phenolic acetate is preferably 7,8-diacetoxy 4-methylcoumarin (DAMC).

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