WO2005081613A2 - Pharmaceutical compositions of peptides secreted by the venom glands of snakes - Google Patents

Abstract

The present invention refers to the employment of pharmaceutical compositions of snake venom glands secreted peptides, particularly from Bothrops jararaca, EVASINS, and analogous and derivatives compounds, and associated products, as modulating agents of the acetylcholine receptors. The pharmaceutical compositions of EVASINS are characterized by the ability to prevent the binding of the cocaine to the acetylcholine receptors.

Description

"PHARMACEUTICAL COMPOSITIONS OF PEPTIDES, SECRETED BY the VENOM GLANDS OF SNAKES, ESPECIALLY BOTHROPS JARARACA, EVASINS, ANALOGUES, DERIVATIVES AND ASSOCIATED PRODUCTS FOR EMPLOYMENT AS MODULATORS OF ACETYLCHOLINE RECEPTOR FUNCTION" . The present invention is characterized by the use of pharmaceutical compositions of peptides secreted in the venom of snakes, such as Bothrops jararaca . Secreted peptides include EVASINS, its analogues, derivatives and associated products acting as modulators of acetylcholine receptors. The pharmaceutics compositions of the presented invention can act as agonists, antagonists, partial agonists or antagonists, or allosteric modulators of the acetylcholine receptor . The pharmaceutical compositions and/or associated products of peptide inhibitors of vasopeptidases, EVASINS, their analogues and derivatives, which are encapsulated in cyclodextrins or their derivatives, or associated or immobilized in pharmaceutically acceptable carriers and/or capsules, and are characterized for their employment as modulators of the acetylcholine receptor function. Another characteristic of the present invention is the micro-encapsulation of the EVASINS, their analogues and derivatives, when they are or not surrounded by cyclodextrins, in a controlled drug liberation system, such as liposomes or biodegradable polymers (and/or mixtures of both) and their employment as modulators of acetylcholine receptor function. The present invention includes yet the identification of other biochemical mechanisms of EVASIN action, such as the interaction with nicotinic or muscarinic acetylcholine receptors, for instance, in order to reverse the vasoconstriction caused in peripheral and blood brain vessels. Another characteristic of this invention is the employment of pharmaceutical compositions of EVASINS as modulators of cholinergic receptor function, such as the liberation of catecholamines, in addition to the direct or allosteric interaction with muscarinic acetylcholine receptors . The employment of the pharmaceutical compositions of EVASINS include also their use as protectors of the acetylcholine receptor function against inhibition by toxins such as phylantotoxins and cembranoides isolated from corals or tobacco, or spider toxins. Pharmaceutical compositions of EVASINS and their structural analogues and/or conformations characterized by the utilization of EVASINS and their respective analogues and derivatives including their employment as molecular models for the development of pharmacological active substances and/or pharmaceutical compositions based on peptidic and non-peptidic compositions for employment in diagnosis, prevention, study and treatment of diseases which are associated with the dysfunction of cholinergic receptor function, as it has been described before. Pharmaceutical compositions of EVASINS and their structural and/or conformational analogues, characterized by the utilization of EVASINS, and their respective analogues and derivatives, are characterized by their employment as molecular models for the development of pharmacological active substances and/or pharmaceutical compositions based on peptidic and non-peptidic compositions for diagnostic employment, for the prevention, study and treatment of hypertension and associated diseases, resulting from cholinergic receptor dysfunction. The pharmaceutical compositions of the referred EVASINS, their analogues and derivatives immobilized in cyclodextrins and derivatives, of this patent present an increase in the bioavailability, duration and/or efficiency of these compositions in modulating the acetylcholine receptors when administered by oral, intravenous, intramuscular, subcutaneous, transdermic and other application forms. In accordance with the present invention, the biological activities of bradykinin-potentiaiting peptides (EVASINS) inducing vasodilatation and reduction of blood pressure are directly related to the effects of the peptides on cholinergic receptor function. These compositions which act as positive modulators on the muscular subtype of the nicotinic acetylcholine receptor may also affect other types of cholinergic receptors such as neuronal nicotinic and muscarinic acetylcholine receptors and may act as agonists, partial agonists, antagonists or allosteric modulators. Therapeutic indications for the pharmaceutics compositions of EVASINS which act on cholinergic receptors include blood pressure regulation, rapid neuronal and neuromuscular signal transmission, sedation, anesthesia, reversion of effects induced by abused drugs such as cocaine and phencyclidine, reversion of intoxification by cocaine and animal toxins, reversion of the dysfunction of receptors during disease states such as convulsions and neurodegeneration, and their effects on Alzheimer' s disease and angiogenic properties of the nicotinic AChR. The present invention indicates that EVASINS modulate AchR function, protecting the receptor against inhibitors, and positively modulate nAChR function during hypotension, augmenting receptor activity. In addition EVASINS may be natural protectors against AchR inhibition caused by the amyloid protein in Alzheimer's disease and in other disease states involving memory and dementia. Angiogenesis such as the formation of endothelial system is promoted by the nicotinic acetylcholine receptor. EVASINS may be important in the regulation of angiogenesis, as they act positively or negatively acting on angiogenesis, showing the potency as therapeutic agents in angiogenic therapy. EVASINS which positively modulate nicotinic acetylcholine receptor function may have importance in processes of wound healing, promoting the formation of an endothelial net. In the process of alimentation of poisonous snakes, snakes subdue their preys by injecting toxins from venom glands into bloodstream of their victims, paralyzing and killing them. Two biomedical contributions of extraordinary importance came from the study of the molecular mechanism of intoxification by the Brazilian snake Bothrops j araraca ( Bj ) : The discovery of the hypotensive peptide bradykinin (Bk) [Rocha e Silva, M., Beraldo, W.T., and Rosenfeld, G. (1949] Bradykinin hypotensive and smooth muscle stimulating factor released from plasma globulins by snake venoms and by trypsine. Am. J. Physiol. 156, 261 - 273] and the discovery of the bradykinin-potentiating peptides (BPPs) which are produced in the venom glands of the snake [Ferreira, S.H., Greene, L.H., Alabaster, V.A., Bakhle, Y.S., and Vane, J.R. (1970) Activity of various fractions of bradykinin potentiating factor against angiotensin I converting enzyme. Nature 225 (230) , 379 - 380. ] . The synergistic actions of these compositions cause a vascular shock state in preys which normally include small mammalians. Typically, the BPPs isolated from snakes consist of oligopeptides of 5 - 13 aminoacids, which are rich in prolines and carry a pyroglutamic acid residue at their N-terminal end. [Ondetti, M.A. and Cushman, D.W. (1981) Inhibitors of angiotensin-converting enzyme, in Biochemical Regulation of Blood Pressure, (Softer, R.L., ed.), pp. 165-204. Wiley, New York] . The anti-hypertensive properties of BPPs of snake venom was demonstrated in model animals as well as human beings [Collier, J.G., Robinson, B.F. and Vane, J.R. (1973) Reduction of the pressure effects of angiotensin in man by a synthetic nonapeptide (BPP9a or SQ20,881) which inhibits converting enzyme. Lancet I, 72-74; Johnson, J.G., Black, W.D., Vukovich, R.A., Hatch, F.E.Jr., Friedman, B. I., Blackwell, C.F., Shenouda, A.N., Share, L., Shade, R.E., Acchiardo, S.R. and Muirhead, E.E. (1975) Treatment of patients with severe hypertension by inhibition of converting enzyme. Clin. Sci. Mol. Med. 48, 53s-56s; Gavras, H., Brunner, H.R., Laragh, J.H., Sealey, J.E., Gavras, I., and Vukovich, R.A. (1974) An angiotensin converting-enzyme inhibitor to identify and treat vasoconstrictor and volume factors in hypertensive patients. N. Engl. J. Med. 291 (16), 817 - 821]. The pharmaceutical and molecular properties of the BPPs allowed the discovery of the angiotensin-converting enzyme

(ACE) . ACE is a metallopeptidase with a key function for the treatment of human hypertension. The pharmaceutical and molecular properties of BPPs were also essential for the development of the first site-specific inhibitor of ACE, denominated captopril. Captopril is being used to treat hypertension in humans [Ondetti, M.A. and Cushman, D.W. (1981) Inhibitors of angiotensin-converting enzyme, in Biochemical Regulation of Blood Pressure, (Softer, R.L., ed.), pp. 165- 204. Wiley, New York; Vane, J.R. (1999) The history of inhibitors of angiotensin converting enzyme. J. Physiol. Pharmacol. 50, 489 - 498]. The angiotensin-converting enzyme (ACE, EC 3.4.15.1) is a zinc metallopeptidase which cleaves the C-terminal dipeptide of angiotensin I, producing an octapeptide, denominated angiotensin II with potent hypertensive activity, and also inactivates bradykinin which possesses hypotensive activity. The somatic form of ACE, which consists of two catalytic domains, is expressed in epithelial, neuroepithelial and endothelial cells. Recently, it has been demonstrated that some BPPs, nowadays called EVASINS (Endogenous Vasopeptidases Inhibitors) , are able to seletively block one of the two catalytic sites of the somatic ACE. Moreover, one of the members of this peptide family is the first described selective natural inhibitor for the C-site of somatic ACE [Cotton, J. , Hayashi, M.A.F., Cuniasse, P., Vazeux, G., Ianzer, D. , Camargo, A. CM. and Dive, V. (2002) Selective inhibition of the C-domain of angiotensin-I converting enzyme by bradykinin potentiating peptides. Biochemistry 41, 6065- 6071]. This property should be physiologically revelant since distinct peptide hormones have been described as being specific substrates for each of the active sites of somatic ACE. Although both catalytic domains convert angiotensin I (A- I) to angiotensin II (A-II), in addition to inactivating bradykinin [Jaspard, E., Wei, L. and Alhenc-Gelas, F. (1993) Differences in the properties and enzymatic specificities of the two active sites of angiotensin I-converting enzyme (kininase II). Studies with bradykinin and other natural peptides. J. Biol. Chem. 268, 9496-9503], the N-site is much more efficient in hydrolyzing the peptides angiotensin 1-7

[Deddish, P. A., Marcic, B., Jackman, H. L., Wang, H. Z.,

Skidgel, R. A. and Erdos, E. (1998) N-domain-specific substrate and C-domain inhibitors of angiotensin-converting enzyme: angiotensin- ( 1-7 ) and keto-ACE. Hypertension 31, 912- 917], and in hydrolyzing AcSDKP, which negatively regulates differentiation and proliferation of hematopoietic stem cells [Rousseau, A., Michaud, A., Chauvet, M. T., Lenfant, M. and Corvol, P. (1995) The haemoregulatory peptide N-acetyl-Ser- Asp-Lys-Pro is a natural and specific substrate of the N- terminal active site of human angiotesin-converting enzyme. J. Biol. Chem. 270, 3656-3661]. In 1997, the cDNA coding for the BPPs precursor was cloned for the first time, showing that six distinct BPPs are generated (in a total of seven peptides) from an unique precursor protein. The precursor protein presents BPP sequences in a tandem repeat (which means that BPPs, one after the other are intercalated by repetitive and highly conserved sequences) and a C-type natriuretic peptide (CNP) at its C- terminus . Northern blot analyses demonstrated the expression of messenger RNAs, which are homologous to that from the venom gland, in snake tissues that are not involved in the venom production, such as the CNS and the spleen [Murayama, N., Hayashi, M.A.F., Ohi, H., Ferreira, L.A.F., Hermann, V. V., Saito, H., Fujita, Y., Higuchi, S., Fernandes, B.L., Yamane, T., and Camargo, A. CM. (1997) Cloning and sequence analysis of a Bothrops jararaca cDNA encoding a precursor of seven brady.kinin-potentiating peptides and a C-type natriuretic peptide. Proc. Natl. Acad. Sci. USA 94, 1189-1193]. More recently, the cDNA coding for the BPP precursor has been cloned from the brain of B . jararaca (GenBank Ace. No AF171670), giving evidence that this neuronal precursor also contains seven sequences of BPPs in the N-domain with a single CNP at the C- terminus. The cDNA of the brain, consisting of 1724 bp, presents high similarity to its equivalent, which has been isolated from the venom gland (91.2% identity). Three of the five EVASINS present in the BPP precursor of the B . jarara ca brain are identical to those previously detected in gland venom precursor [Murayama, N., Hayashi, M.A.F., Ohi, H., Ferreira, L.A.F., Hermann, V. V., Saito, H., Fujita, Y., Higuchi, S., Fernandes, B.L., Yamane, T., and Camargo, A. CM. (1997) Cloning and sequence analysis of a Bothrops jararaca cDNA encoding a precursor of seven bradykinin-potentiating peptides and a C-type natriuretic peptide. Proc. Natl. Acad. Sci. USA 94, 1189-1193], whereas two of the EVASINS were different, being one of them characterized by its very high specificity for the N-site of somatic ACE [Hayashi, M.A., Murbach, A.F., Ianzer, D., Portaro, F.C, Prezoto, B.C., Fernandes, B.L., Silveira, P.F., Silva, C.A., Pires, R.S., Britto, L.R., Dive, V., and Camargo, A.C. (2003) The C-type natriuretic peptide precursor of snake brain contains highly specific inhibitors of the angiotensin- converting enzyme. J. Neurochem. 85, 969-977]. The EVASINS, identified in the cerebral precursor (called iBPP, in order to distinguish them from those secreted by the venom gland) are efficient inhibitors of the somatic ACE, being highly selective for one of the two sites of the somatic ACE (C or N- site) . They are also capable to potentiate the effect of bradykinin in ex vivo and in vivo experiments. The nicotinic acetylcholine rceptor (AChR) is a protype member of membrane proteins and structurally related to ligand-gated ion channels [Kandel, E.R., Schwatz, J.H. & Jessel, T.M. (1995) Essential of Neural Science and Behaviour

(Appleton & Lange, Stanford, CT) p, 5] . These proteins regulate intercellular communication between cells of the mammalian nervous system, a process considered to be essencial for brain function [Crick, F.H.C (1994) The Astonishing Hypothesis. The Scientific Search for the Soul (Macmillan, New York] . Many therapeutic agents and also abused drugs affect AChR function. For instance, AChR is inhibited by the anti- convulsant MK-801 [ (+ ) -dizocilpine] and the abused drug cocaine [Amador, M.& Dani, J.A. (1991) Synpase 7, 207-215, Karpen, J. W., Aoshima, H., Abood, L.G. & Hess, G.P (1982) Proc. Natl. Acad. Sci. USA 79, 2509-2513]. The family of muscarinic acetylcholine receptors consists of at least five different G-protein coupled seven- transmembrane proteins. The muscarinic receptors, denominated Ml - M5, are characterized by their selective pharmacology [Caulfield MP, Birdsall NJ. , International Union of Pharmacology XVII. Classification of muscarinic acetylcholine receptors, Pharmacol. Rev. 1998 Jun; 50 (2 ): 279-90] . The modulation of noradrenalin liberation in sympathic terminals by pre-synaptic muscarinic receptors is already known [Starke K. , Regulation of noradrenaline release by presynaptic receptor systems. Rev Physiol Biochem Pharmacol. 1977; 77:1- 124]. Apparently, muscarinic receptors can facilitate or as well inhibit the liberation of catecholamines [Boehm S, Kubista H., Fine tuning of sympathetic transmitter release via ionotropic and metabotropic presynaptic receptors, Pharmacol. Rev. 2002 Mar; 54 ( 1 ): 3-99] . The facilitating effect has been principally observed in Ml receptors, whereas opposite effects have been related to M2 receptors [Fuder H, Muscholl E., Heteroreceptor-mediated modulation of noradrenaline and acetylcholine release from peripheral nerves, Rev Physiol Biochem Pharmacol. 1995;126:265-412]. On the other hand, activation of M3 receptors of the vascular endothelium has a vasodilative effect [Casado MA, Sevilla MA, Alonso MJ, Marin J, Salaices M. , Muscarinic receptors involved in modulation of norepinephrine release and vasodilatation in guinea pig carotid arteries. J. Pharmacol. Exp Ther. 1994 Dec; 271 (3) : 1638-46] . The M3 receptor has also been implicated in the inhibition of noradrenalin in the atrium of guinea pigs [Nakatsuka H, Nagano 0, Foldes FF, Nagashima H, Vizi ES . , Effects of adenosine on norepinephrine and acetylcholine release from guinea pig right atrium: role of Al-receptors, Neurochem Int. 1995 Oct-Nov; 27 (4-5) : 345-53] . During the past decades, research on biological basis for the chemical addiction has contributed to establish the involvement of certain brain regions and neurotransmitters . It seems that addiction to alcohol, opioids, and cocaine are based on a common biochemical mechanism. A neuronal circuit in the brain, involving the limbic system and two regions, called nucleus accumbens and globus pallidus are important for symptoms of druged persons. Chronical use of cocaine, morphine, and alcohol has been shown to result in biochemical adaptations in the dopaminergic system [Blum Kenneth, US Patent 6, 132,724, 2000] . Signaling pathways in the brain, involved in multiple drug dependence, are directed to dopamine receptors (Dl, D2 , D3, D4, D5), with the D2 receptor apparently being the most important one. Despite the fact that each substance seems to act on different parts of the circuit, the final result with the liberation of dopamine in the nucleus accumbens and in the hippocampus is always the same. Dopamine acts here as main neurotrasnmitter [Koob and Bloom, "Cellular and molecular mechanisms of drug dependence," Science, 242:715-723, 1988]. Abnormalities in the dopamine metabolism are implicated in many disturbed behavorial states, such as sexual dysfunction, mania, schizophrenic behavior, altered or aggressive behavior among others. The liberation of dopamine is modulated by cholinergic receptor function [Boehm S, Kubista H., Fine tuning of sympathetic transmitter release via ionotropic and metabotropic presynaptic receptors, Pharmacol. Rev. 2002 Mar; 54 (1) :43-99] . In addition to genetic problems contributing to neurological dysfunction, neurotransmitters and pharmacological active substances have been studied in order to establish or abolish certain psychologic problems. In human beings, it has been suggested that the meso-frontal dopaminergic activity is involved in the human cognitive process. In patients suffering on Parkinson's disease and in patients with schizophrenic disease, both prefrontal activation during cognitive tasks and clinical symptoms of dysfunction of the dopaminergic system have been observed. Many neurotransmittes, especially dopamine, serotonin, norepinephrine, GABA, glutamine and opioid peptides, which fundamentally participate in cerebral functions and in regulation of mood, are dependent on the availability of their amino acid precursors. [Wurtman, Hefti, and Melamed, "Precursor control of neurotransmitter synthesis", Pharmacological Review, 32:315-335, 1981]. Experimental data suggest that these amino acid precursors and also inhibitors of enkephalinase affect the recuperation following consumption of alcohol and cocaine [Blum et al., "Methionine enkephalinase as a possible neuromodulator of regional cerebral blood flow, " Experimentia, 41:932-933, 1985; Blum et al . , "The D.sub.2 dopamine receptor gene as a determinant of reward deficiency syndrome," J. Royal Soc. of Med., 89:396-400, 1996b.; Blum, "A commentary on neurotransmitter restoration as a common mode of treatment for alcohol, cocaine and opiate abuse," Integrative Psychiatry, 6:199-204, 1989a.; Blum, Allison, Trachtenberg, Williams, Loeblich, "Reduction of both drug hunger and withdrawal against advice rate of cocaine abusers in a 30-day inpatient treatment program by the neuronutrient Tropamine, " Current Therapeutic Research, 43:1204-1214, 1988; Blum, Briggs, Trachtenberg, "Ethanol ingestive behavior as a function of central neurotransmission (Review)," Experientia, 45:444-452, 1989b; Blum, Cull, Braverman, Comings, "Reward deficiency syndrome," Am. Scientist, 114:132-145, 1996a. Blum, Cull, Chen, et al., "Clinical evidence for effectiveness of Phencal.TM. in maintaining weight loss in an open label controlled 2-year study," Current Therap. Res., 58:745-763, 1997b; Blum, Hamilton, Wallace, Alcohol and opiates: A review of common neurochemical and behavioral mechanisms, Editor: K. Blum, (pp. 203), Academic Press, New York, 1977; Blum, Noble, Sheridan, Finley, Montgomery, Ritchie, Ozkavagoz, Fitch, Sadlack, F. , Sheffield, Dahlmann, Halbardier, Nogami, "Association of the Al allele of the D.sub.2 dopamine receptor gene with severe alcoholism," Alcohol, 8 407-416, 1991b; Blum, Noble, Sheridan, Montgomery, Ritchie, Jagadeeswaren, Nogami, Briggs, Cohns, "Allelic association of human dopamine D.sub.2 receptor gene in alcoholism, " Journal of the American Medical Association, 263, 2055-2060, 1990b; Blum, Noble, Sheridan, Montgomery, Ritchie, Ozkaragoz, Fitch, Wood, Finley, Sadlack, "Genetic Predisposition in alcoholism: association of the D.sub.2 dopamine receptor Taql B.sub.l RFLP with severe alcoholism," Alcohol, 10:59-67, 1993; Blum, Trachtenberg, Cook, "Neuronutrient effect on weight loss in carbohydrate bingers: an open clinical trial," Current Therap. Res., 48:217-223, 1990c; Blum, Trachtenberg, Elliott, Dingier, Sexton, Samuels, Cataldie, "Enkephalinase inhibition and precursor amino acid loading improves inpatient treatment of alcohol and polydrug abusers: Double-blind placebo-controlled study of the nutritional adjunct," SAAVE . Alcohol, 5:481-493, 1989c; Blum, Wallace, Geller, "Synergy of ethanol and putative neurotransmitters : Glycine and serine, " Science, 176:292-294, 1972; Braun, Little, Reuter, Muller-Mysok, Koster, "Improved analysis of microsatellites using mass spectrometry, " Genomics, 46:18-23, 1997a; Braun, Little, Koster, "Detecting CFTR gene mutations by using primer oligo base extension and mass spectrometry," Clin.Chem., 43:1151-1158, 1997b; Braverman et al., "A commentary on brain mapping in 60 substance abusers: can the potential for drug abuse be predicted and prevented by treatment?" Cur . Ther . Res . , 48:549-585, 1990b; Braverman, Smith, Smayda, Blum, "Modification of P300 amplitude and other electrophysiological parameters of drug abuse by cranial electrical stimulation, " Current Therapueutic Research, 48:586-596, 1990c; Braverman and Blum, "Substance use disorder exacerbates brain electrophysiological abnormalitites in psychiatrically-ill population" Clin. EEC, 27 (4 supplement) : 1028, 1996a]. An important function of catecholaminergic innervation may be the control of attention. Both, cholinergic and dopaminergic systems have been implicated to fundamental functions of maintaing precise cognitive performance. Deficient precision induced by muscarinic or nicotinic receptor inhibition by scopolamine can be reverted in the presence of an inhibitor of the dopamine receptor. Cocaine addiction and toxicity affects more than three million people annually in the United States of America and the estimated costs for the society are much higher than 100 billion dollars [George P. Hess, et al. Proc . Na tl . Acad. Sci . USA, 91, 13895-13900] . Cocaine abuse is also frquent in Brazil, like in the U.S.A. or in Europe, and can result in cardiovascular diseases, myocardiac ischemia, epileptic attacks, hypothermy, heart attacks and neurological disorders, such as intracerebral hemorrhages, and vascular cerebral accidents [Lathers, CM., Tyan, L.S.Y., Spino, M.M., and Agarwal, I. (1988). Cocaine-induced seizures, arrhythmias and sudden death. J. Clin. Pharmacol. 28, 584-593; Das, C (1993). Cardiovascular effects of cocaine abuse. Int. J. Clin. Pharmacol. Ther. Tox. 31,521-528; Karch, B.S. (1993). The Pathology of Drug Abuse. CRC Press, Boca Raton, Ann Arbor, London, Tokyo.]. The fundamental mechanisms for the cardio- and neurotoxic effects of the cocaine in particular are yet to be discovered. Cocaine is characterized by two primary pharmacological properties which adversively affect heat and the vascular system. On one hand cocaine inhibits catecholamine reuptake (for instance the dopamine reuptake system) em neurons of the central and peripheral nervous system and, therefore, acts as a potent cardiac stimulant, lessening actions of the sympathic nervous system [Billman, G.E. (1995). Cocaine: a review of its toxic actions on cardiac function. Crit. Rev. Toxicol. 25, 113-132]. Cardiotoxic effects of cocaine result, for instance, in increased arrithmogenesis, cardiomyopathy, cardiac arrest, are among other reasons, and the consequence of inefficient signal transduction by muscarinic receptors which is caused by inhibition of these receptors by cocaine [Witkin, J.M., Goldenberg, S.R., Katz, J.L., and Kuhar, M.I. (1989). Modulation of the lethal effects of cocaine by cholimimetics . Life Sci. 45, 2295-2301; Sherief, H.T., and Carpenter, R.G. (1991). Electrophysiological mechanism of cocaine-induced cardiac arrest. A possible cause of sudden cardiac death. J. Electrocardiol . 24, 247-255.; Gantenberg, N.S., and Hageman, G.R. (1992). Cocaine-enhanced arrhythmogenesis : neural and nonneural mechanism. Can. J. Physiol. Pharmacol. 70, 240- 246.]. On the other hand, cocaine acts as a blocking agent of the fast neurotransmission by inhibiting the voltage-dependent ionic channels and the AchR functioning [Matthews, J.C, and Collins, A. (1983). Interaction of cocaine and cocaine congeners with sodium channels. Biochem. Pharmacol. 32, 450- 460.; Karpen, J.F., and Hess, G.P. (1986). Cocaine, phencyclidine, and procaine inhibition of the acetylcholine receptor: characterization of the binding site by stopped-flow measurements of receptor-controlled ion flux in membrane vesicles. Biochemistry 25, 1777-1785.; Niu, L., Abood, L.C, and Hess, G.P. (1995). Cocaine: Mechanism of inhibition of a muscle acetylcholine receptor studied by a laser-pulse photolysis technique. Proc. Natl. Acad. Sci. USA 92, 12008- 12012]. Nicotinic acetylcholine receptors have been also found on mammalian cerebral blood vessels, indicating their possible function in blood pressure regulation and stroke. In this regard, pharmaceutical compositions of EVASINS may be able to prevent cocaine binding to the AchR. In view of that, pharmaceutical compositions of EVASINS are important tools for the study and treatment of cardiovascular and cerebral adverse effects caused by cocaine and other agents which act in a similar manner. Further indications of pharmaceutical compositions of EVASINS include their employment as dopaminergic agents for the treatment of schizophrenia, Parkinson's disease, the Tourette's syndrome, drug dependence and hyperprolactinemia as well as for the treatment of central nervous system diseases related to dysfunction of the cholinergic system, such as Alzheimer's disease, extrapyramidal motor function disorders, such as Parkinson's disease, progressive supramuscular paralysis, Huntington' s disease, the Gilles de la Tourette's syndrome, and late dyskinesia, obesity, strong pain, desintoxification of abused drugs and tobacco, respiratory disease, mood disorders and emotional diosrders such as depression, anxiety e psychoses, diseases of motor functions and cognitive attention, disorders of cognitive function and concentration, memory loss, dementia (including dementia caused by AIDS) , neurodegenerative disorders, epilepsy, disorders involving convulsions, alimentation disorders inclding bulimi and anorexia, disorders of the autonomous nervous system, such as dysfunction of gastrointestinal motility, colon inflammation, irritated colon, diarrhea, constipation, ulcer, comedication during surgery and pheochromocytoma, hypertension and cardiovascular disease, nociception and pain control. In the state of the art, some technologies describing the employment of other types of pharmacological active substances and their employment as modulators of nicotinic acetylcholine receptor function are found. However, no records regarding the employment of EVASINS and their pharmaceutical compositions as modulators of nicotinic acetylcholine receptor function exist. The US patent 6,132,724, Blum, Kenneth (2000) describes the employment of an endorphinase inhibitor or encephalinase inhibitor and optionally a precursor of dopamine, serotonin, or GABA, or a liberator of endorphin, or certain herbal compositions including the extract of Rhodiola rosea and/or Huperzine. These compositions promote the restauration of normal neurotransmitter function and the combination of these compositions augment dopamine release in the nucleus accumbens . The US patent 6,362,009, Munoz, Benito and Chen, Chixu (2002) describe the synthesis of heterocyclic compositions by solid-phase and combinatorial synthesis, utilizing a strategy of activated resins with retention capability. Methods for production of dihydropyridones, tetrahydropyridones, pyridines, aminopyridines, N-acetyl tetrahydropiridines and combinatorial libraries containing these compounds are described. The patent encompasses methods for the screening and also pharmaceutical compositions containing these compounds. We claim the employment of these compounds for the treatment of central nervous system diseases, involving the acetylcholine receptor. The U.S. patent 6,638,621, Anderson, David (2003) describes the use of particles which are covered with lamellar materials, amorphous, and non-lamellar crystalline or semi- crystalline materials, including at least one nanostructural liquid phase, or a crystalline liquid nanostructutal phase or a combination of them, for being employed as release system for pharmacological active substances, nutrients and/or pesticides. These release systems may be used for diagnostics and diseases which involve the nicotinic acetylcholine receptor . The U.S. patent 6,670,356, Davies, Bonnie (2003) describes the use of galantamine and licoramine as modulators of nicotinic receptor function in humans and in animals. The inventors claim the employment of these compounds for the treatment of cognitive disorders and for receuperation from tobacco addiction as well as for Parkinson's disease. The American patent US 6,670,340, Zhang, Mingiang; Palin, Ronald, Bennett and David, Jonathan (2003) described the synthesis and the employment of a 6-mercapto-ciclodextrin derivative as agents for reversion of muscle blocks induced by drugs. Although the employment of cyclodextrins as carrier of active substances has not been described, the prevention and treatment of diseases related to the nicotinic acetylcholine receptor are the characteristics of this invention. The U.S. Patent 6,448,276, Yerxa, Benjamin R. (2002) claims the employment of a method for the treatment of vaginal lubrification with nicotinic receptor agonists. The claim states that the treatment augments vaginal lubrification and cervical tissues. The described method includes the employment of acetylcholine receptor agonists such as nicotine and its analogues, transmetanicotine and its anlogues, epibatidine and its analogues, lobeline and its analogues, pyridol and its analogues, para-alkylthio phenols and their derivatives, and imidaclopride and its analogues. Methods for drug application include topical administration via liquid or gel, cream, soap or tablet, systematic administration via drops or spray, inhalation or nebulation or via other disposal, in oral form liquid or pill, via injection, suppositorium or by transdermic applications . The US patent US 6,342,581, Rosen, Craig A.; Ruben, Steven M.; Olsen, Henrik S.; Ebner, Reinhard, (2002) claim the employment of a new protein HLHFP03, secreted by humans, and the isolation of nucleic acids containing the codifying regions of the gene which codes for the protein. Furthermore, there are claims for the use of vectors, host cells, antibodies and recombinant methods for the production of proteins secreted by humans. The invention also claims methods for diagnosis and therapy of disorders associated with the nicotinic acetylcholine receptor. The U.S. patent 6,288,055 Natarajan, Maya; Jenkins, Thomas E.; Griffin; John H., (2001) present the employment of a novel analgelsic agent, utilizing covalent-bound ligands to the nicotinic acetylcholine receptor, which are capable of modifying biological processes and functions. The U.S patent 6,100,269, Bencherif, Merouane; Caldwell, William Scott; Dull, Gary Maurice; Lippiello, Patrick Michael (2000) describes pharmaceutical compositions for the prevention and treatment of disorders of the central nervous system, based on the compounds [2.2.1] heptane e 2-azabicyclo [2.2.2] octane. The U.S. patent 6,077,846 Qian, Changgeng; Li, Tongchuan; Biftu, Tesfaye; Shen, Tsung-Ying (2000) describes the employment of epatidine and its derivatives as agonists and antagonists of cholinergic receptor function. The nicotinic agonists 7-azabicyclo [2.2.1] -heptane or heptene are claimed to be employed for the treatment of diseases related to excessive body weight, tabagism, Parkinson's disease, Alzheimer's disease, and Tourette's syndrome. The U.S. patent 5,922,723 Bencherif; Merouane; Caldwell; William Scott; Dull; Gary Maurice; Lippiello; Patrick Michael (1999) claims the use of pharmaceutical compositions to prevent and treat disorders of the CNS, such as senile dementia, dementias such as Alzheimer's disease, Parkinson's disease and other diseases of the central nervous system such as schizophrenia and Tourette's syndrome, employing the compounds 2-azabiciclo [2.2.1] hept-5-ene and 2- azabiciclo[2.2.2] oct-5-ene . The U.S. patent 5,583,140 Bencherif; Merouane; Caldwell; William S . ; Dull; Gary M.; Lippiello; Patrick M., (1996) describes pharmaceutical compositions for the treatment of disorders of the central nervous system, utilizing compounds based on 2-azabiciclo [2.2.1] hept-5-ene and 2- azabiciclo [2.2.2] oct-5-ene . In the patent US 6,178,349, Kieval, Roberts S. et. al.

(2001) developed a device for the liberation of pharmacological active substances for the treatment of cardiovascular diseases. This device consists of an electrode connected to the nerve, and a pulse generator which can be implanted and reservoir containing the pharmacological active substance. During the use of the device, the electrode and pharmacological active substance liberation stimulate the nerve, which then affects control over the cardiovascular system. In this regard, in the state of the art, the employment of peptides such as the EVASINS-type, encapsulated or not in cyclodextrins and microencapsulated in biodegradable polymers for the use in diagnosis, study, prevention and treatment of diseases related to cholinergic receptors are the characteristics of this invention. Both, muscarinic as well as nicotine receptors are present in vascular endothelial cells [Furchgott RF, Zawadzki JV, The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine, Nature. 1980 Nov 27;288 (5789) :373-6; Bruggmann D, Lips KS, Pfeil U, Haberberger RV, Kummer W., Multiple Nicotinic acetylcholine receptor alpha-subunits are expressed in the arterial system of the rat, Histochem Cell Biol. 2002 Dec; 118(6): 441-7]. In view of that, in the state of the art, the employment of peptides of the EVASIN type, encapsulated or not in cyclodextrins and microencapsulated in biodegradable polymers for the use in diagnosis, study, prevention and treatment of diseases related to cholinergic receptors are the characteristics of this invention. Pharmaceutical compositions and formulations of the present invention are characterized by the use of a mixture of pharmaceutically acceptable excipients combined with EVASINS, their analogues and derivatives. Exemples of excipients include water, saline solution, phosphate-buffered solutions, Ringer's solution, dextrose solution, Hank's solution, and biocompatible saline solutions containing or not polyethylene glycol. Other useful formulations include agents which increase viscosity such as sodium carboymethyl cellulose, sorbitol, or dextran. The excipients may also contain lower concentrations of additives, such as substances that augment isotonicity and chemical stability of substances and buffers. Exemples of buffers include phosphate buffer, bicarbonate and tris buffer, while exemples for preservatives include timerosal, meta- or ortho- cresol, formalin and benzylalcohol. The formulations may be liquid or solid. In this regard, the excipient in a pharmaceutical composition or non-liquid formulation may contain dextrose, human serum albumine, etc., to which sterile water or salt solution is being added prior to its administration. The present invention also encompassed the preparation of the controled release system containing the EVASINS, analogues and derivatives, for the use in the study, diagnosis, prevention and treatment of diseases associated with the cholinergic receptors, which are characteristic of the present invention. The system of controled release includes, but is not limited to the cyclodextrins, biocompatible polymers, biodegradable polymers, such as the PLA, PGLA, other polymeric matrix, capsules, microcapsules, microparticles, preparation in bol us, osmotic pumps, difusion devices, liposomes, lipospheres, and systems for transdermic administration. Other compositions of controled release of the present invention include liquids that after administration into animals form a solid or a gel in si tu . The cyclodextrins are cyclic oligosaccharides that include six, seven or eight unities of glucopyranose . Due to the steric interactions, the cyclodextrins (CDs) form a cyclic structure, which resembles a truncated cone with an apolar internal cavity. They are chemically stable compounds that might be modified in a region-selective manner. The cyclodextrins (host) form complexes with several hydrophobic molecules (guest), which are completely or partially included in the cavity. The CDs have been used for the solubilization and encapsulation of drugs, perfumes and flavorings such as described by Szejtli, J. , Chemical Reviews, (1998), 98, 1743- 1753. Szejtli, J. , J. Mater. Chem., (1997), 7, 575-587. As previously described [Rajewski, R.A., Stella, V., J. Pharmaceutical Sciences, (1996), 85, 1142-1169], a detailed toxicity, mutagenicity, teratogenicity and carcinogenicity study of the cyclodextrins showed a low toxicity, specially for the hydroxipropyl- (cyclodextrin) , as described in Szejtli, J. Ciclodextrins : Properties and aplications. Drug Investig. 2(suppl. 4): 11-21, 1990. Except for the high concentration of some derivatives, which determine erythrocyte damage, these products generally do not present heatlh risks. The use of cyclodextrins as nourish complement is already aproved in countries such as Japan and Hungary, and also for other more specific applications, in France and Denmark. All these features represent a good motivation to looking for new applications for the cyclodextrins. Besides the cyclodextrins, biodegradable polymers are also employed, which decrease the absorption rate of the drug in the body, through the use of the controled release system. In these systems the drugs are incorporated into a polymeric matrix based on the encapsulation of the drug into microspheres or nanospheres, which release the drug inside the body in small and daily controlled doses, during several days, months or even years. Several polymers were already assayed in the controled release system. Many of them, due to the physical properties such as the poly (uretanes) for the elasticity, poly (syloxanes) or silicone for the efficient isolation properties, poly (methyl-metacrylate) for the physical resistance, poly (vinyl alcohol) for the hydrophobicity and resistance, poly (ethylene) for the hardness and impermeability [Gilding, D. K. Biodegradable polymers. Biocompat. Clin. Implat. Mater. 2: 209-232, 1981] . 21

described by Szejtli, J. , Chemical Reviews, (1998), 98, 1743- 1753. Szejtli, J. , J. Mater. Chem., (1997), 7, 575-587. As previously described [Rajewski, R.A., Stella, V., J. Pharmaceutical Sciences, (1996), 85, 1142-1169], a detailed toxicity, mutagenicity, teratogenicity and carcinogenicity study of the cyclodextrins showed a low toxicity, specially for the hydroxipropyl- (cyclodextrin) , as described in Szejtli, J. Ciclodextrins : Properties and aplications. Drug Investig. 2(suppl. 4): 11-21, 1990. Except for the high concentration of some derivatives, which determine erythrocyte damage, these products generally do not present heatlh risks. The use of cyclodextrins as nourish complement is already aproved in countries such as Japan and Hungary, and also for other more specific applications, in France and Denmark. All these features represent a good motivation to looking for new applications for the cyclodextrins. Besides the cyclodextrins, biodegradable polymers are also employed, which decrease the absorption rate of the drug in the body, through the use of the controled release system. In these systems the drugs are incorporated into a polymeric matrix based on the encapsulation of the drug into microspheres or nanospheres, which release the drug inside the body in small and daily controlled doses, during several days, months or even years. Several polymers were already assayed in the controled release system. Many of them, due to the physical properties such as the poly (uretanes) for the elasticity, poly (syloxanes) or silicone for the efficient isolation properties, poly (methyl-metacrylate) for the physical resistance, poly (vinyl alcohol) for the hydrophobicity and resistance, poly (ethylene) for the hardness and impermeability [Gilding, D. K. Biodegradable polymers. Biocompat. Clin. Implat . Mater. 2: 209-232, 1981]. 22

However, for the human use, the material must be chemically inert and free of impurities. Some of the materials used in the controled release system are: poly (2-hydroxi- ethylmetacrylate) , polyacrylamide, polymers based on latic acid (PLA), based on glycolic acid (PGA) and respective so- polymers (PLGA) and the poly (anhydrides) such as the polymers based on sebastic acid (PSA) and the co-polymers with the hydrophobic polymers. Thus, we claim the formulations employing the cyclodextrins and the biodegradable polymers in the study, prevention and treatment of diseases associated with the nicotinic acethylcholine receptor, characterized in the present invention. Other characteristic of the present invention is the use of the liposomes as vector for the formulations of the EVASINS for the use in the study, prevention and treatment of diseases associated with cholinergic receptors. In the state of art, several patents describing the liposomes preparations are found [Pat US 4,552,803, Lenk; Pat US 4,310,506, Baldeschwieler ; Pat US 4,235,871, Papahadjopoulos; Pat US 4,224,179, Schneider; Pat US 4,078,052, Papahadjopoulos; Pat US 4,394,372, Alfaiate; Pat US 4,308,166, Marchetti; Pat US 4,485,054, Mezei; e Pat US 4,508,703, Redziniak; Woodle e Papahadjopoulos, Methods Enzymol. 171:193-215 (1989)]. The unilamelar liposomes have a single membrane that include the acqueous volume [Huang, Biochemistry 8:334-352 (1969)], while the multilamelar liposomes have inumerous concentric membranes [Bangham et al., J. Mol. Biol. 13:238-252 (1965)]. Veicles based on the use of liposomes were proposed for a wide variety of pharmacologically active compounds, including the antibiotics, hormones and anti-tumoral agents [Medical applications of liposomes (D.D. Lasic, D. Papahadjopoulos Ed.), Elsevier Science B.V., Holanda, 1998]. 23

Other liposomes preparation processes have been found in the state of art, [for a revision see, Cullis et al., in: Liposomes, From Biophysics to Therapeutics (M. Ostro, ed.), Marcel Dekker (New York), 1987, pp. 39-72; Woodle and Papahadjopoulos, Methods Enzymol. 171:193-215 (1989); Liposome technology (G. Gregoriadis ed.), CRC Press, Boca Raton, FL, 1993] . In the state of art is also found an example for the aplication of liposomes to last longer the effect of peptides, where the liposomes containing the Ang-(l-7) (LAng) are prepared and micro-injected into one side of the rostro ventrolateral bulb (BRVL) . The arterial pressure was recorded by telemetry during 10 seconds, at each 10 minutes, starting from 4 days before and until 12 days after, in not perturbed and free moving rats. The micro-injection of the LAng produced a significative pressor effect during the diurnal period maintened for 5 days. The highest PAM was obtained in the day 3 (114 ± 4 mmHg) , which was significantly different of the measurement of the day 0 (100 ± 3 mmHg) . As expected, the empty liposomes (Lvaz) did not determined a significative change in the PAM value (94 ± 5 mmHg in the day 3 vs 90 ± 5 mmHg in the day 0) . Moreover, the diurnal PAM value was significantly higher in the LAng group than in the Lvaz group in the days 1, 2, and 3. The nocturnal PAM value, in contrast to the diurnal PAM value, was not significantly affected by the micro-injection of the LAng. Previous works [Fontes MA, Pinge MC, Naves V, Campagnole-Santos MJ, Lopes OU, Khosla MC, Santos RA. Cardiovascular effects produced by microinjection of angiotensins and angiotensin antagonists into the ventrolateral medulla of freely moving rats. Brain Res. 1997 Mar 7;750 (1-2) : 305-10] have stablished that the micro- injection of the free Ang-(l-7) (not encapsulated form) into the BRVL, in a similar doses (25-50 ng) , produced an icrease of 155 mmHg for about 10 min. The short duration of this 24

effect was explained by the high metabolism of the peptide in vivo [Silva-Barcellos et al,, Hypertension, 38(6): 1266-71 (2001) ] . In spite of that, in the state of the art, the use the formulation of the EVASINS in liposomes and the combination of the cyclodextrins for the study, prevention and treatment of diseases associated with the cholinergic receptors has not been described. The pharmaceutical compostions of the EVASINS are characterized by the positive modulation of the muscle and neuronal-type of nicotinic acethylcholine receptors (AchR) functioning. As non-limiting examples of the present invention, it was verified that the EVASIN-5a modulates the muscle-type receptor expressed in the mammalian cells. Furthermore, it was also shown that the EVASIN-5a, in a final concentration of 200 μM in culture, reverses almost completely the inhibition determined by the anti-convulsive agent MK-801. Previous works have shown that the MK-801 and the addictive drugs, such as the cocaine and the phencyclidine, bind to the same site of the AchR [Hess, G.P., Ulrich, H., Breitinger, H.C, Niu, L., Gameiro, A.M., Grewer, C, Srivastava, S., Ippolito, J.E., Lee, S.M., Jayaraman, V., and Coombs, S.E. (2000) Mechanism-based discovery of ligands that counteract inhibition of the nicotinic acetylcholine receptor by cocaine and MK-801. Proc. Natl. Acad. Sci. USA 97, 13895 - 13900; Karpen, J.F., and Hess, G.P. (1986). Cocaine, phencyclidine, and procaine inhibition of the acetylcholine receptor: characterization of the binding site by stopped-flow measurements of receptor-controlled ion flux in membrane vesicles. Biochemistry 25, 1777-1785]. Thus, if the EVASINS are able to reverse the inhibition induced by the MK-801, this compound may also be effective to displace the cocaine and PCP from its site. 25

The cocaine dependence is widespread in Brazil, as also in USA and European countries, and can led to a cardiovascular diseases, myocardium ischemia, epileptic attack, hypothermia, heart stroke and neurological disfunctions such as intracerebral haemorhage and cerebrovascular accident [Lathers, CM., Tyan, L.S.Y., Spino, M.M., and Agarwal, I. (1988) Cocaine-induced seizures, arrhythmias and sudden death. J. Clin. Pharmacol. 28, 584-593; Das, G. (1993) Cardiovascular effects of cocaine abuse. Int. J. Clin. Pharmacol. Ther. Tox. 31, 521-528; Karch, B.S. (1993) The Pathology of Drug Abuse.

CRC Press, Boca Raton, Ann Arbor, London, Tokyo] . The mechanism responsible for these cardiotoxic and neurotoxic effects of the cocaine are still essentially to be discovered. Therefore, since the EVASINS prevent the binding of the cocaine to the AchR, and teoretically also to the muscarinic acethylcholine receptor, interfering in the dopamine reuptake process, they are strong candidates to become an essential tool for the treatment of cardivascular and cerebral effects of the cocaine and other agents that act in a similar way. The direct function of the nicotinic acethylcholine receptors in the arterial pressure control is supported by the work of Bruggmann and colaborators [Bruggmann, D., Lips, K.S., Pfeil, U., Haberberger, R.V., Kummer, W. (2002). Multiple nicotinic acetylcholine receptor alpha-subunits are expressed in the arterial system of the rat. Histochem. Cell Biol. 118, 441-447], where the expression of several alpha subunits were detected in the vascular system of the rat. Furthermore, the acethylcholine induces a vasodilation dependent of the concentration in skin slices affected by the nicotine, agonist/antagonist of this receptor [Black et al., 2001]. Addictive drugs such as the cocaine and the phencyclidine affect the AchR functioning [Matthews, J.C, and Collins, A. (1983). Interaction of cocaine and cocaine congeners with sodium channels. Biochem. Pharmacol. 32, 450- 26

460; Karpen, J.F., and Hess, G.P. (1986); Cocaine, phencyclidine, and procaine inhibition of the acetylcholine receptor: characterization of the binding site by stopped-flow measurements of receptor-controlled ion flux in membrane vesicles. Biochemistry 25, 1777-1785; Niu, L., Abood, L.G., and Hess, G.P. (1995) Cocaine: Mechanism of inhibition of a muscle acetylcholine receptor studied by a laser-pulse photolysis technique. Proc. Natl. Acad. Sci. USA 92, 12008 - 12012], blocking the ion influx into the cell and, therefore inhibiting the vasodilation action of the AchR. The cocaine affects the neuronal AchR of the cerebral vessels as much as the vessels out of the CNS [Kalaria, R.N., Homayoun, P., Whitehouse, P.J. (1994). Nicotinic cholinergic receptors associated with mammalian cerebral vessels. J. Auton. Nerv. Syst. 49, S3-7; Heeschen, C, Weis, M., Aicher, A., Dimmeler, S., Cooke, J.P. A novel angiogenic pathway mediated by non- neuronal nicotinic acetylcholine receptors. J. Clin. Invest. 110, 527-536] implying a function in the cerebral circulation and stroke. Recently, the mechanism of inhibition and protection of the AchR has been extensively studied, and the existence of compounds that reverse the inhibition of the receptor was presumed [Ulrich, H.; Ippolito, J. E.; Pagan, 0. R. ; Eterovic, V. E.; Hann, R. M.; Shi, H.; Lis, J. T . ; Eldefrawi, M. E.; Hess, G. P. (1998) In vitro selection of RNA molecules that displace cocaine from the nicotinic acetylcholine receptor. Proc. Natl. Acad. Sci. USA 95, 14051-14056; Hess, G.P., Ulrich, H., Breitinger, H.C, Niu, L., Gameiro, A.M., Grewer, C, Srivastava, S., Ippolito, J.E., Lee, S.M., Jayaraman, V., and Coombs, S.E. (2000) Mechanism-based discovery of ligands that counteract inhibition of the nicotinic acetylcholine receptor by cocaine and MK-801. Proc. Natl. Acad. Sci. USA 97, 13895 - 13900; Hess, G.P., Gameiro, A.M., Schoenfeld, R.C, Chen, Y., Ulrich, H., Nye, J.A., Caroll, F.I., and Ganem, B. (2003). Reversing the action of non-competitive inhibitors (MK-801 and cocaine) on a protein (nicotinic acetylcholine receptor) mediated reacted. Biochemistry 42, 6106-6114]. Using fast kinetics electrophysiological techniques named "cell flow" [Hess, G.P., Niu, L., and Wieboldt, R. Determination of the chemical mechanism of neurotransmitter receptor-mediated reactions by rapid chemical kinetic methods. Ann. N.Y. Acad. Sci. 757, 23-39], combmatory chemistry synthesis and drug design based on known structures led to the discovery of compounds that are able to displace the inhibitor of the regulatory site of the receptor, without the inhibition of the receptor. These works led to the development of a RNA aptamer and cocaine analogues (RTI-4229-70) , which protec the AchR [Hess, G.P., Ulrich, H., Breitmger, H.C, Niu, L., Gameiro, A.M., Grewer, C, Srivastava, S., Ippolito, J.E., Lee, S.M., Jayaraman, V., and Coombs, S.E. (2000) Mechanism- based discovery of ligands that counteract inhibition of the nicotinic acetylcholine receptor by cocaine and MK-801. Proc. Natl. Acad. Sci. USA 97, 13895 - 13900; Hess, G.P., Gameiro, A.M., Schoenfeld, R.C, Chen, Y., Ulrich, H., Nye, J.A., Caroll, F.I., and Gane , B. (2003). Reversing the action of non-competitive inhibitors (MK-801 and cocaine) on a protein (nicotinic acetylcholine receptor) mediated reacted. Biochemistry 42, 6106-6114]. Another characteristic of the presen invention is that the pharmaceutical compositions of EVASINS are able to protect the acetylclolme receptor (AchR) against inhibitors that bind to regulatory allosteric sites of the receptor. The present invention is characterized by the use of at least twenty-one bradykinin potentiating peptides identified in the venom and tissues of the snake Bothrops jararaca (geneπcally named as EVASINs or bradykinm-potentiatmg peptides), whose amino acid sequences were determined by mass spectrometry or were deduced from the cDNA coding for these 28

peptides precursor protein, expressed in tissues of this snake besides the venom gland (named as EVASINs or Endogenous VASopeptidase Inhibitors) , described in the patent file BR PI0205449-3 of Dec 09, 2002.

ID n° Nomenclature Sequence ID 1 EVASIN-5a <EKWAP ID 2 EVASIN-5b <EWPRP ID 3 EVASIN-5C <EKFAP ID 4 EVASIN-6a <ESWPGP ID 5 EVASIN-7a <EDGPIPP ID 6 EVASIN-9a <EWPRPQIPP ID 7 EVASIN-9b <ESWPGNIPP ID 8 EVASIN-lOa <ESWPGPNIPP ID 9 EVASIN-lOb <ENWPRPQIPP ID 10 EVASIN-lOc <ENWPHPQIPP ID 11 EVASIN-lOd <ESWPEPNIPP ID 12 EVASIN-lla <EWPRPTPQIPP ID 13 EVASIN-llb <EGRAPGPPIPP ID 14 EVASIN-llc <EGRAPHPPIPP ID 15 EVASIN-lld <EGRPPGPPIPP ID 16 EVASIN-lle <EARPPHPPIPP ID 17 EVASIN-12a <EGWAWPRPQIPP ID 18 EVASIN-12b <EWGRPPGPPIPP ID 19 EVASIN-13a <EGGWPRPGPEIPP ID 20 EVASIN-13b <EGGLPRPGPEIPP ID 21 EVASIN-13c <EGGWPRPGPQIPP

The majority of these peptides show a structural motif at the C-terminus PX¹X²PP, where X¹ might be any amino acid residue and X¹ normally is a isoleucine (I) amino acid residue, and the N-terminal amino acid is blocked, generally due to the presence of a pyroglutamic (<E) amino acid residue. The corresponding synthetic peptides were assayed as inhibitors of the C- and N-active sites of the recombinant 29

ACE , and also as potentiator of both contract ion ef fect of bradykinin on isolated guinea pig ileum and hypotensive activity of bradykinin in rats . The most selective and effective potentiating peptides of the contraction elicited by the bradykinin on the isolated guinea pig ileum and in the hipotens ive ef fect on the arterial blood pressure of the rat were those showing molecular weight between 500 and 1 , 700 daltons , comprising of 5 to 13 amino acid residues . These active molecules were chemically modified generating new peptides with qualitative similar features . The EVAS INS , oligopeptides of 5 to 13 amino acid residues , formulated in the present invention are described as follow :

Formules Sequences Nomenclature

I <E¹aa²aa³aa P⁵ Evasin- ^■ 5a, b, . • * r n

I I <E¹aa²aa³aa⁴aa⁵P⁶ Evasin- - 6a, b, . • • / n

I II <E¹aa²aa³aa⁴aa⁵P⁶P⁷ Evasin- ^■ 7a, b, . n

IV <E¹aa²aa³P⁴aa⁵aa⁶P⁷P⁸ Evasin- 8a, b, . _' • t n

V <E¹aa²aa³aa⁴aa⁵aa⁶aa⁷P⁸P⁹ Evasin- 9a, b, . n

VI <E¹aa²aa³aa aa⁵P⁶aa⁷aa⁸P⁹P¹⁰ Evasin- 10a, b, . n

VI I <E¹aa²aa³aa aa⁵aa⁶P⁷aa⁸aa⁹P¹⁰P¹¹ Evasin- 11a, b, . • • / n

VII I <E¹aa²aa³aa⁴aa⁵aa⁶aa⁷P⁸aa⁹aa¹⁰P¹¹P¹² Evasin- ^■12a, b, . • • / n

IX <E¹aa²aa³aa⁴aa⁵aa⁵aa⁷aa⁸P⁹aa¹⁰aa¹¹P¹²P¹³ EEvvaassiinn-- -1133aa,, bb,, . • • r n where : P is always proline. The remaining might be L- or D- amino acids and derivatives that are presented by one-letter code : aspartic acid (Asp, D) glutamic acid (Glu, E) alanine (Ala, A) arginine (Arg, R) asparagine (Asp, D) phenylalanine (Phe, F) glycine (Gly, G) glutamine (Gin, Q) histidine (His, H) isoleucine (lie, I) leucine (Leu, L) lysine (Lys, K) proline (Pro, P) serine (Ser, S) tyrosine (Tyr, Y) threonine (Thr, T) 30

tryptofane (Trp, W) valine (Val, V) aminobutiric acid (Abu) aminoisobutiric acid (Aib) diaminobutanoic acid (Dab) diaminopropionic acid (Dpr) hexanoic acid (ε-Ahx) isonipecotic acid (Isn) piroglutamic acid (Pyr, <E) tetrahydroisoquinoline-3-carboxilic acid (Tic) butyl-glycinacyclohexilalanine (Cha) citruline (Cit) statin and derivatives (Sta) phenylglycine (Phg) hydroxiproline (Hyp) homoserine (Hse) norleucine (Nle) norvaline (Nva) ornitine (Orn) penicylalanine (Pen) sarcosine (Sar) thietylalanine (Thi)

<E¹ piroglutamic acid is the N-terminal amino acid; aa² is an amino acid, usually W, S, or K for the formules I and II, generally D for the formule III and normally A, W, S, G or N for the formules IV to IX; aa³ is generally W, P, F or G for the formules I to III and normally A, P, G, W or R for the formules IV to IX; aa⁴ is an amino acid, usually P, A or R for the formules

I to III and generally P, L, Q, A, R or W for the formules IV to IX; aa⁵ is an amino acid, usually G, R or I for the formules

II and III and generally T, P, G, H, R, W or E for the formules IV to IX; aa⁶ is an amino acid, usually Q, N, P, T, H, R or G for the formules V, VII, VIII and IX; and normally I, A, T or Y for the formule IV; aa⁷ is an amino acid, usually P, N, Q, G or R for the formules VI, VIII and IX and generally I, A, T or Y for the formule V; aa⁸ is an amino acid, usually Q, P or G for the formules VII and IX and generally I, A, T or Y for the formule VI; 31 aa⁹ is an amino acid, usually P, Q, N or G for the formule VIII and normally I, A, T or Y for the formule VII; aa¹⁰ is an amino acid, usually Q and E for the formule IX and normally I, A, T or Y for the formule VIII; aa¹¹ for the formule IX is normally I, A, T or Y. Another characteristic of the present invention is the use of the conformationally modified EVASINS. Modifications of the lateral groups of the amino acids (χ-"constraints") claimed in the patent file BR PI0205449-3 in Dec 09^th, 2002, aiming improve the pharmacokinetics properties and the specificity in the action on different target molecules involved in cardiovascular pathologies, as either vasopeptidases inhibitor or acting in endothelial cells and smooth muscles. Besides the oral formulations, other routes for injection such as intravenous, intramuscular, topic, pulmonary inalation, intranasal, intrabuccal or as controlled release device using biodebradable polymers such as the PLA and PLGA or their mixture are non-limiting examples. The present invention is also characterized by the obtainment of systems for controlled release of the oligopeptides, EVASINS, analogues and derivatives, using the liposomes that increase the bioavailabilility of the peptide. Any application using the oligopeptides, EVASINS, analogues and derivatives, included in the cyclodextrins or derivatives, microencapsulated in biodegradable polymers such as the PLA or PLGA or mixture and the liposomes for use as modulators of the acetylcholine receptors was never described previously. The present invention is characterized by the use of different technologies, such as the molecular encapsulation of the oligopeptides, EVASINS and analogues, in cyclodextrins and the microencapsulation in biodegradable polymers or in liposomes and/or mixture of these materials, leading to an 32

increase of the bioavailability of the EVASINS in the compositions of oral and intravenous formulations, as non- limiting examples, when compared to the non-formulated compoud. Any pharmaceutical composition of the EVASINS and structural and/or conformational analogues characterized by the use of the EVASINS and respective analogues and derivatives, as molecular models for the development of the drugs and/or pharmaceutical compositions based on the peptidic and/on non-peptidic compounds for the use in the diagnosis, prevention, study and treatment of the diseases associated with the disfunctions of the colinergic receptors was never described previously. In the state of the art, it was not also found any pharmaceutical composition of the EVASINS and structrural and/or conformational analogues characterized by the use of the EVASINS and respective analogues and derivatives as molecular models for the development of drugs and/or pharmaceutical compositions based on peptidic and/or non- peptidic compounds for the use in the diagnosis, prevention, study and treatment of the hypertension and associated diseases, caused by cholinergic receptors disfunctions. Another feature of the present invention is the use of the pharmaceutical compositions of the EVASINS, analogues and derivatives, characterized by the inclusion and association compounds of the EVASINS, analogues and derivatives, included in the cyclodextrins and derivatives, microencapsulated or not in systems of controlled release, such as the liposomes and the biodegradable polymers, PLA, PLGA and/or mixtures, for the diagnosis, study and treatment of diseases or conditions associated with disfunctions of the cholinergic receptors, namely, blood pressure variations, sedation, anaesthesia, cocaine and phencyclidine addiction, cocaine and animal toxins intoxications, dopaminergic receptors disfunctions, in 33

diseases such as convulsions and neurodegenerations, e.g., Alzheimer' s disease and modulation of the angiogenic properties of the nicotinic acetylcholine receptor (AchR) . Another indications of the pharmaceutical compostions of the EVASINS, due to the effect on the nicotinic receptors, include the use as dopaminergic agents for the treatment of the schizophrenia, Parkinson's disease, Tourette's syndrome, drug dependence and hyperprolactinemia and for the treatment of diseases of the central nervous system correlated with the disfunctions of the nicotinic cholinergic systems, such as the Alzheimer's disease, extrapyramidal motor functions disorders, such as the Parkinson' s disease, progressive neuromuscular paralysis, Huntington' s disease, Gilles de la Tourette syndrome and late dyskinesia, obesity, strong pain, intoxication by drugs and tobacco, breathing disorders, humor and emotion disorders such as depression, anxiety and psycosis, motor and attention disfunctions, cognitive function disorders, loose of memory, madness (including insanity by AIDS), neurodegenerative disorders, epilepsy, convulsive disorders, nourishment disorders including the bulimia and anorexy, autonomic disorders such as disfunctions of the gastrointestinal motility, irritable colon, diarrhea, constipation, ulcers, comedication in surgical treatment and pheochromocytoma, hypertension and cardiovascular diseases, nociception and pain control. The pharmaceutical compositions of the EVASINS, analogues and derivatives, included into cyclodextrins and derivatives, or EVASINS, analogues and derivatives, associated or included into carriers and/or vehicles pharmaceutically acceptable, isolated or mixtured or associated with at least another pharmacologically active agent; or EVASINS, analogues and derivatives, included or not into the cyclodextrins, microencapsulated or not into systems of controlled release such as the liposomes and the biodegradable polymers PLA, PLGA 34

and/or mixtures characterized by the action as agonist, partial agonist, antagonist or allosteric modulators of the acetylcholine receptors. The present invention can be better understood by the following non-limiting examples:

Example 1 : Characterization of the protector effects of the BPPs on the nicotinic acetylcholine receptors . Muscle type BC3H1 cell culture BC3H1, is a clonal mammalian cell lineage, established from a tumor induced by injection of nitrosoethylurea in mouse, with features characteristic of the muscle, expressing the muscle-type fetal AchR [Schubert, D., Harris, A.J., Devine, C.E., and Heinemann, S. (1974). Characterization of a unique muscle cell line. J. Cell. Biol. 61, 398-413], and was cultivated as. described by Sine and Taylor [Sine, S.M. and Taylor, P. (1982). Local anesthetics and histrionicotoxin are allosteric inhibitors of the acetylcholine receptor. Studies of clonal muscle cells. J. Biol. Chem. 257, 8106-8114]. The products for the cell culture DMEM (Dulbecco^'s modified Eagle's medium), BFS (bovine fetal serum) and solution 10.000 U/mL of peniciline and 10 mg/mL of the streptomicine were all purchased from Invitrogen. During the proliferation period, the cells were cultivated in small cell culture bottles (Nunc, Denmark) , using DMEM culture media containing 10% BFS, lOOU/ml of penicilline and 0.1 mg/ml of streptomicine, which was replaced each 3 days. Confluent bottles were divided in 1:5, once a week, removing the media and washing the cell layer with PBSA, and adding 2 ml of trypsin solution 0.25%. In this step, the cells could be further proliferated or were plated on culture dishes of 35 mm diameter (Corning, RI) for the differentiation process. The cells were then incubated for 24 hours in culture media containing 10% BFS, to assure the adhesion of the cells on the plate, and then the media was 35

replaced by DMEM media containing 1% BFS, to allow the cells to differentiate. The media of the plated were changed every 5 days until the plate was used, between the 15^th to 30^th days of differentiation. The cells were maintened all the time at 37°C and 5% C0₂.

Transient transfection of the HEK cells with the neuronal-type acetylcholine receptors In this work, the mRNA of distinct nicotinic acetylcholine receptors such as the alpha7, alpha 2, alpha 3, beta 3, and beta 4 were transfected into HEK cells, as described in the example for the muscarinic receptors. The efficiency of the transient transfection is monitored by the co-transfection with a vector encoding the GFP (green fluorescent protein) [Grewer, C, Jager, J. , Carpenter, B.K., Hess, G.P. (2000) A new photolabile precursor of glycme with improved properties: A tool for chemical kinetic investigations of the glycine receptor. Biochemistry 39, 2063- 2070]. The functionality of the expressed receptors is verified by the whole-cell recording as explained below.

Rapid kinetic electrophysiological measurements (Whole-cell recording) The currents induced by the carbamoylcholine were recorded using the whole-cell configuration [Hamill, O.P., Marty, A., Neher, B., Sakmann, B., and Sigworth, F.J. (1981). Improved patch clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers

Arch. 391, 85-100] and the cell-flow technique, which allow a temporal resolution of 10 ms [Udgaonkar, J.B., and Hess, G.P.

(1987). Chemical kinetic measurements of a mammalian acetylcholine receptor using a fast reaction technique. Proc. Natl. Acad. Sci. USA 84, 8758-8762]. In the cell-flow technique, a U-tube, originally developed by Kπshtal and Pidoplichko [Krishtal, O.A., Pidoplichko, V.I. (1980). A 36

receptor for protons in the nerve cell membrane. Neuroscience 5, 2325-2327], was modified and used to apply a solution containing the ligant under a laminar flow controlled by a voltage converter (5 to 12 volts) specially made at Cornell Universtiy, Ithaca, New York. The cell-flow system is constituted by a tube (Kendall Healthcare, MA) , for the entrance of the ligant, connected to the U-tube at one end and to a tube for the exit of the ligant in the other end of the U-tube containing a solenoid valve (The Lee Company, CT) able to block the exit flow when commanded by the trigger box. The ligand solution moves through the circuit formed by the tubes, valves and U-tube by the action of a peristaltic pump (Rainin, CA) . A second similar system is used simutaneously to perform the pre-incubation of the ligands for 2 seconds in the cell- flow system. All the cell-flow system is tested before each experiment in order to certify a correct functioning and fidelity of the temporal resolution. This is done by obtaining measurements of the water being dispensed into a pipet without cell, in such a way that a regular junction potential variation is obtained. The currents were amplified using an amplifier model Axopatch 200B (Axon Instruments, Union City, CA) and filtered at 1kHz (using a low-pass Bessel filter of four poles incorporated to the amplifier) . The filtered signal was digitalized by using the DigiData 1322A digitalizer (Axon Instruments, Union City, CA) . The pipet contained intracellular buffer for BC3H1 (140mM KC1, lOmM NaCl, 2mM MgC12»6H20, ImM EGTA e 25mM HEPES), adjusted to pH 7.4; the buffer of the bain contained 145mM NaCl, 5.3mM KC1, 1.8mM CaC12*2H20, 1.7mM MgC12^«6H20 e 25mM HEPES, adjusted to pH 7.4. The pipets were prepared using a pipette-puller (Sutter

Instrument, CA) and borosilicate capillaries of 1.5 x 0.86 mm

(Harvard Apparatus, TN) and were polished by heating in a microforge (Narishige, Japao) in order to obtain a resistance typically around 2 to 4 MΩ. A compensation of the resistence 37

in series of 20-50% was used, via amplifier Axopatch 200B (Axon Instruments, Union City, CA) . Maximum currents for concentration of 100 μM of carbamoylcholine (control concentration) were typically around 1-5 nA. All experiments were performed at 22-24 °C, pH 7.4 and transmembranic voltage maintained at -60 mV.

Data analysis The obtained currents were recorded by the Clampex software (Axon Instruments) and saved as *.asci files. The Clamplex software (Axon Instruments) was used for the acquisition and initial analysis of the data. The MicroCal Origin software was used to estimate the kinetic of the current decrease in the presence of the agonist and antagonist. The equation 1 was adapted for the decreasing step of the current, and the maximum amplitude of the observed current was corrected by the dessensitivation of the receptor [Udgaonkar, J.B., and Hess, G.P. (1987). Chemical kinetic measurements of a mammalian acetylcholine receptor using a fast reaction technique. Proc. Natl. Acad. Sci. USA 84, 8758- 8762]. Equation 1: A(t) = Aιβ^{"t τl} + A₂e^{"t τ2} + A_e

A(t) - Amplitude of the current at the time t. Al, A2, Ae - Maximum amplitude in the first, second and equilibrium component . τl, x2 - Time constants for the first and second components.

Determination of the protector effect of the EVASINS on the nicotinic acetylcholine receptors The effect of the EVASINs was tesed by the cell-flow technique: at first, the amplitude of the current due to the response of a cell was observed with 100 μM of the agonist (carbamoylcholine) . In the next step, variations in this amplitude were observed after applying the same concentration of the agonist with different concentration of the EVASINS (10 - 500 μM) . The first possibility was that the amplitude of the 38

current changes, indicating that (1) the EVASINS binds to the right site and acts as inhibitor, or (n) the EVASINS binds to another site. In this case, the equation 2 is used to determine the dissociation constant of the EVASINS (Ki). Our interest was devoted to the EVASINS presenting another kind of activity when applied with the agonist, the normal response of the agonist is mamtened, that is, the binding of the EVASINS to the site does not cause the inhibition expected for a drug that binds to the same site. Thus, assays aiming to determine the ability of the EVASINS to protect the receptor against the inhibition by 500 μM MK-801 of the currents generated by 100 μM carbamoylcholine were performed. For this, the cells were pre-mcubated for 2 seconds at EVASINS concentrations ranging from 10 - 500 μM and after submitted to the same concentration of the EVASINS with the inhibitors (MK-801 or cocaine) and the agonist (carbamoylcholine), to generate the currents. In this case, the used equation (Equation 3), which considers the need of the presence of the inhibitor to verify the effect of the EVASINS, and the dissociation constant (K_BPP) is calculated [Ulrich, H., and Gameiro, A.M. (2001) Aptamers as tools to study dysfunction in the neuronal system. Curr. Med. Chem. - Central Nervous System Agents 1, 125-32].

Equation 2 A/A_R = 1 + [MK-801 J/K_M -₈₀i Equation 3 A/A_R , = 1 + [K_BPP/(K_BPP+[BPP])] [MK-80 l]/K_Mκ soi

Equations 1 and 2: determination of the mechanism of action of the BPPs isolated.

Inhibitors: MK-801 or cocaine. Where A is the amplitude of the control current, A_R is the amplitude of the current in the presence of the selected

EVASINS (EVASINS) and A_R, _τ is the amplitude of the current in the presence of the EVASINS and of the inhibitor (I) . K_R is the dissociation constant of the EVASINS and K_τ is the 39

dissociation constant of the inhibitor. It is assumed in the equation 3 that the receptor with a EVASINS bound to it forms an opened channel. Obtained results The funcioning of the muscle-type nicotinic acetylcholine receptors is blocked by the MK-801, an allosteric inhibitor that induces conformational modifications of the receptor, preventing the opening of the channel during the application of the ligand. The EVASIN (BPP5a) displaced the MK-801 from its allosteric site and reversed the inhibition of the receptor. The induced current in whole cell is inhibited by a factor of 6-7 times when 100 μM of Mk-801 is co-applied with 500 μM of carbamoylcholine. The EVASIN-5a allowed only a small inhibition of the AchR activity, even at concentrations such as 500 μM, when pre-incubated and co- applied with the carbamoylcholine. On the other hand, 200 μM of EVASIN reverted almost completely the inhibition of the AchR determined by 500 μM of the MK-801. These results are shown in the tables I and II.

Table II: Protective effect of the Evasin-5a on the muscle-type nicotinic acetylcholine receptor 40

Example 2. Example 10: Biodistribution of the EVASINS After the labeling reaction and purification by reverse- phase chromatography using a Sep-Pak C18 microcolumn, it was possible to determine the efficiency of the iodine incorporation by counting the cpm of the eluted product from the labeling reaction. In this way, the parameters for the labeling evaluation (Table 1) suggest that the used method was appropriate to provide a tracer with a good yield and specific activity of 100 μCi/μg, suitable to perform the pharmacokinetics assays.

PARAMETERS VALUES

TABLE 1. Parameters for evaluation of the labeling of EVASIN- 10c with ^l25l.

1.2. Biological activity of EVASIN-lOc labeled with iodine. In order to analyse the biological activity of the EVASIN-lOc after the incorporation of the ¹²⁵I, a labeling reaction using cold iodine was performed and the activity of the labeled peptide as bradykinin potentiator in guinea pig ileum assay and as inhibitor of the neprilisyn was evaluated. The results showed that both the labeled EVASIN-lOc and the non-labeled EVASIN-lOc present similar effect in the guinea pig ileum assay and the same inhibition constant (Ki) about 26 μM, confirming that the incorporation of iodine into the structure of this peptide did not modified its activity.

2.3. Biodistribution of EVASIN-lOc in mice. The distribution kinetics of the labeled EVASIN-lOc was analysed in different tissues at distinct times, 0, 5, 15, 30, 4 1

45, 60, 120 and 180 min after intraperitoneal injection. The obtained data, expressed in percentage of the administered dose per organ and per gram of organ are illustrated in the Tables 2 and 3, respectively. Between the analysed organs showed in the Table 2, in decreasing order, the kidney, small intestine, liver, large intestine and lung, showed the highest %DI, while the heart, spleen, brain and tesis, showed the lowest %DI between the distinct intervals of time. The data suggest that the maximum points for the labeled EVASIN-lOc capitation changes depending of the function of the analysed organ and of the time after the administration of the dose, such as in the liver and lung (16.145 e 6.788 %DI, respectively) at 1 min, heart and brain 1.909 e 0.758 %DI, respectively) at 5 min., spleen (0.932 %DI) at 15 min., brain and testis (0.758 %DI for both) at 5 and 30 min., respectively. At later times, the small intestine (14.4 %DI) at 45 min, large intestine (9.113 %DI) and stomach (5.957 %DI), both at 180 min after the administration of the dose. The tables 2 and 3 also show that the renal capitation of the EVASIN-lOc is significantly bigger when compared to other organs. The renal capitation is relatively fast with 12.492 %DI/organ and 32.815 %DI/g/organ at 5 min and keeps the average around 13.633 %DI/organ and 32.965 %DI/g/organ until 120 min (maximum peak of 15.752 %DI/organ and 38.929 %DI/g/organ at 30 min), with slight decrease up to 6.681 %DI/organ and 14.569 %DI/g/organ at 180 min. 42

TABLE 2 Kinetics of the distribution of the ^l25l labeled Evasm 10c at distinct time interval after i v administration in mice (n=10) Data are expressed in percentage of the dose administered per total organ evaluated (%DI/organ) average value and standard deviation (average ± SD) Other experiments involving the distribution kinetics of the labeled EVASIN-lOc were also performed. A solution mixture containing labeled and non-labeled ("cold") EVASIN-lOc was administered I.V. for a final dose of 0.8 μg/g and enalapril maleate for a final dose of 800 μg/g (1000 times bigger than the EVASIN-lOc dose) . The distribution of the labeled EVASIN- 0 lOc in this experiment was analysed at the same conditions, for time and collected organs, as those used for the animals treated only with the labeled EVASIN-lOc. The obtained data were expressed as the percentage of the administered dose per organ and per gram of the organ, and are showed in the Tables 5 4 and 5, respectively. 43

TABLE 3 Distribution kinetics of the ^l25l labeled Evasin-lOc, at distinct time interval, after i.v. administration in mice (n=10). Data expressed in percentage of the administered dose per gram of the evaluated organ (%Dl/g/organ), with the average and standard deviation (average ± SD). With the results obtained with the two experiments of distribution kinetics (%DI/organ or %DI/g/organ) in different tissues of the animals treated only with the EVASIN-lOc and with the EVASIN-lOc and enalapril maleate, it was possible to compare the percentage of the injected dose of the labeled EVASIN-lOc compared to the different organs. The data obtained showed that in both brain and skeletal muscle, enalapril determined a significant reduction of the radioactivity, which increased a little in the skeletal muscle at later times. Considering the high density of the nicotinic receptors in these organs, these data corroborate with the evidences obtained with the pathclamp, showing the binding of the EVASINS with the nicotinic receptors. 44

TABIE 4. Distribution kineticsof the ^l25l labeled Evasin-lOc. at distinct time intervals, after i.v. administration in mice treated with enalapril (n=10). Data expressed in percentage of the administered dose per gram of the evaluated organ (%DI/organ), with average and standard deviation (average ± SD)

45

TABLE 5. Distribution kinetics of the ^12SI labeled Evasin-lOc, at distinct time intervals, after i.v. administration in mice treated with enalapril (n=10). Data expressed in percentage of the administered dose per gram of the evaluated organ (%DI/g/organ), with average and standard deviation (average ± SD).

Example 3. Cardiovascular effects of the EVASIN-5a and EVASIN- 5a included into Hidroxipropil-βCD (HPβCD) . In order to evaluate the effect of the EVASIN-5a and the EVASIN-5a included into the HPβCD, transgenic TGR adult male rats (mREN2) 27 heterozigous, weighting between 300-400g, obtained from the Bioterio do Instituto de Ciencias Biolόgicas (CEBIO, UFMG) were used. The surgical procedure was performed one day before the experiment. After the anaesthesia with the ethylic ether, a polyethylene catheter (PE10 connected to a PE50) was inserted into the abdominal aorta, through the femoral artery, for the measurements of the arterial pressure. For the injection or the intravenous infusion, polyethylene canules were implanted into the femoral vessel. The canules, filled with 0.9 % saline solution and occluded with a metallic pin, were directed in the subcutaneous area until the inter- scapular region, exteriorized and fixed in the animal back. After the surgery, the rats were maintained in individual cages with free access to water and food. The average arterial pressure (PAM) and the cardiac frequency (FC) of the animals were monitorized by computer, through a data acquisition system (BIOPAC system) . The values were collected during all the experiment, performed 24 hours after the implantation of the canules. The PAM and FC of each rat were evaluated during 60 minutes before the injection of the EVASIN-5a, where the average arterial pressure and FC obtained in this period were considered as the basal values. The effect of the EVASIN-5a on the PAM and FC was observed after injection until the PAM value returned to the basal values. The used dose was of 0.71 nmol/Kg of rat (0.34mg /Kg of VA) . The magnitude of the effect was evaluated and the 46 duration of the EVASIN-5a included into the HPβCD was compared to the free EVASIN-5a. Effect of the injection of EVASIN-5a included or not into the HPβCD

The obtained data showed that the inclusion into the cyclodextrin significantly increased the duration of the EVASIN-5a effect, suggesting that this type of formulation can be used for the administration of these peptides with large advantage compared to the administration of the free form.

Claims

4 7CLAIMS :

1. Pharmaceutical compositions of peptides secreted by the snake venom gland, particularly from Bothrops jarara ca , EVASINS, analogues and derivatives and associated products, characterized by the employment of these peptides as acetylcholine receptors modulating agents.

2. Pharmaceutical compositions of peptides secreted by the snake venom gland, particularly from Bothrops jarara ca , EVASINS, analogues and derivatives and associated products, characterized by the fact that these peptides can be used as agonist, partial agonist, or as alosteric modulators of the acetylcholine receptors.

3. Pharmaceutical compositions of peptides secreted by the snake venom gland, particularly from Bothrops jarara ca , EVASINS, analogues and derivatives and associated products, characterized by the reversion, e.g., of the vasoconstriction determined by the cocaine in the peripheral blood vessels and brain.

4. Pharmaceutical compositions of peptides secreted by the snake venom gland, particularly from Bothrops jararaca , EVASINS, analogues and derivatives and associated products, according to claims 1, 2 and 3, characterized by the fact that the oligopeptide formulas are: Formulas Sequences Nomenclature II <E^xaa²aa³aa P⁵ Evasin-5a, b, ... n

II <E¹aa²aa³aaaa⁵P⁶ Evasin-6a, b, ... n

III <E¹aa²aa³aa⁴aa⁵P⁶P⁷ Evasin-7a, b, ... n

IV <E¹aa²aa³P⁴aa⁵aa⁶P⁷P⁸ Evasin-8a, b, ... n

V <E¹aa²aa³aa⁴aa⁵aa⁶aa⁷P⁸P⁹ Evasin-9a, b, ... n VVII <E¹aa²aa³aa⁴aa⁵P⁶aa⁷aa⁸P⁹P¹⁰ Evasin-lOa, b,... n

VII <E¹aa²aa³aa⁴aa⁵aa⁶P⁷aa⁸aa⁹P¹⁰Pⁿ Evasin-lla, b, ... n

VIII <E¹aa²aa³aa⁴aa⁵aa⁶aa⁷P⁸aa⁹aa¹⁰P¹¹P¹² Evasin-12a, b, ... n

IX <E¹aa²aa³aa⁴aaaa⁶aa⁷aa⁸P⁹aa¹⁰aa¹¹P¹²P¹³ Evasin-13a, b, ... n where : o

P is always proline. The remaining might be L- ou D-amino acids and derivatives that are presented by the one-letter code : aspartic acid (Asp, D) glutamic acid (Glu, E) alanine (Ala, A) arginine (Arg, R) asparagine (Asp, D) phenylalanine (Phe, F) glicine (Gly, G) glutamine (Gin, Q) histidine (His, H) isoleucine (lie, I) leucine (Leu, L) lysine (Lys, K) proline (Pro, P) serine (Ser, S) tyrosine (Tyr, Y) threonine (Thr, T) tryptophan (Trp, W) valine (Val, V) aminobutyric acid (Abu) aminoisobutyric acid (Aib) diaminobutanoic acid (Dab) diaminopropionic acid (Dpr) hexanoic acid (ε-Ahx) isonipecotic acid (Isn) piroglutamic acid (Pyr, <E) tetrahydroisoquinoline-3-carboxilic acid (Tic) butyl-glycinacyclohexilalanine (Cha) citrulin (Cit) statin and derivatives (Sta) phenylglycine (Phg) hydroxiproline (Hyp) homoserine (Hse) norleucine (Nle) norvalin (Nva) ornitin (Orn) phenycilalanine (Pen) sarcosine (Sar) thietylalanine (Thi) <E¹ piroglutamic acid is the N-terminal amino acid; aa² is an amino acid, usually , S, or K for the formules I and II , usually D for formule III and usually A, W, S, G or N for the formulas IV to IX; aa³ is usually , P, F or G for the formules I to III and usually A, P, G, W or R for the formules IV to IX; aa⁴ is an amino acid, usually P, A or R for the formules I to

III and usually P, L, Q, A, R or W for the formules IV to IX; 49

aa⁵ is an amino acid, usually G, R or I for the formules II and III and usually T, P, G, H, R, W or E for the formules IV to IX; aa⁶ is an amino acid, usually Q, N, P, T, H, R or G for the formules V, VII, VIII and IX; normally I, A, T or Y for the formule IV; aa⁷ is an amino acid, usually P, N, , G or R for the formules

VI, VIII and IX and is normally I, A, T or Y for the formule

V; aa⁸ is an amino acid, usually Q, P or G for the formules VII and IX and normally I, A, T or Y for the formule VI; aa⁹ is an amino acid, usually P, Q, N or G for the formule

VIII and normally I, A, T or Y for the formule VII; aa¹⁰ is an amino acid, usually Q and E for the formule IX and normally I, A, T or Y for the formule VIII; aa¹¹ for the formule IX is normally I, A, T or Y;

5. Pharmaceutical compositions and/or associated products of the peptidic inhibitors of the vasopeptidases, EVASINS and analogues, according to claims 1, 2, 3, and 4, characterized by the inclusion of these peptides into cyclodextrin and derivatives, or associated with or included into carriers and/or vehicles pharmaceutically acceptable to be used as modulators of the acetylcholine receptors .

6. Pharmaceutical compositions of peptides secreted by the snake venom gland, particularly from Bothrops jarara ca , EVASINS, analogues and derivatives and associated products, according to claims 1, 2, 3, 4 and 5, characterized by the microencapsulation of the EVASINS, analogues and derivatives, included or not into cyclodextrins, or into systems with controlled release such as the liposomes and/or biodegradable polymers and/or a mixture of these systems to be used as modulators of the acetylcholine receptors. 50

7. Pharmaceutical compositions of peptides secreted by the snake venom gland, particularly from Bothrops jararaca , EVASINS, analogues and derivatives and associated products, according to claims 1 to 5, characterized by the use of these compounds as modulators of the cholinergic and dopaminergic receptors, besides direct or allosteric interactions with muscarinic acetylcholine receptors.

8. Pharmaceutical compositions of peptides secreted by the snake venom gland, particularly from Bothrops jararaca , EVASINS, analogues and derivatives and associated products, according to claims 1 to 7, characterized by the use as protectors of the acetylcholine receptors against the action of toxins such as the phylantotoxins or cembranoids, isolated from the coral or tobacco, and also against spider toxins.

9. Pharmaceutical compositions of peptides secreted by the snake venom gland, particularly from Bothrops jararaca , EVASINS, analogues and derivatives and associated products, according to claims 1 to 7, the use of these compounds in the diagnosis, prevention, study and treatment of diseases associated with cholinergic receptors dysfunctions, characterized by the use of these pharmaceutical compositions of EVASINS, structural and/or conformational analogues and derivatives, for the development of the drugs and/or pharmaceutical compositions based on peptidic and/or non-peptidic compounds .

10. Pharmaceutical compositions of peptides secreted by the snake venom gland, particularly from Bothrops jararaca , EVASINS, analogues and derivatives and associated products, according to claims 1 to 7, for the use in the diagnosis, prevention, study and treatment of the hypertension and associated diseases, caused by dysfunctions of the cholinergic receptors, characterized 51 by the use of these pharmaceutical compositions as molecular models for the development of drugs and/or pharmaceutical compositions based on peptidic and/or non- peptidic compounds.

11. Pharmaceutical compositions of peptides secreted by the snake venom gland, particularly from Bothrops jararaca , EVASINS, analogues and derivatives and associated products, according to claims 1 to 7, when administered by distinct injection routes such as oral, nasal, intravenous, intramuscular, subcutaneous, transdermic, characterized by the increase in the bioavailability, duration and/or efficacy of the effect as modulators of the acetylcholine receptors.

12. Pharmaceutical compositions of the EVASINS, according to claims 1 to 11, for use in the diagnosis, study and treatment of the arterial hypertension and other cardiovascular diseases and related complications, associated to the dysfunctions of the cholinergic receptors, characterized by the use of compounds for inclusion or association of EVASINS, analogues and derivatives, microencapsulated or not into systems of controlled release, such as, for example, the liposomes and biodegradable polymers and/or mixtures.

13. Pharmaceutical compositions of the EVASINS, according to claims 1 to 11, for use in the diagnosis, study and treatment of diseases or conditions, such as the neural and neuromuscular synaptic fast transmission, sedation, anesthesia, reversion of the effect of drug dependence due to, e.g., cocaine or phencyclidine, reversion of the intoxication by the cocaine and toxins, reversion of the receptors dysfunctions during diseases such as convulsions and neurodegeneration such as Alzheimer' s disease and modulators of the angiogenic properties, characterized by the use of compounds for inclusion or association of 52

EVASINS, analogues and derivatives, microencapsulated or not into systems of controlled release, such as, for example, the liposomes and biodegradable polymers and/or mixtures .

14. Pharmaceutical compositions of the EVASINS, analogues and derivatives, according to claims 1 to 11, for use in the diagnosis, study and treatment of diseases or conditions, such as schizophrenia, Parkinson's disease, Tourette's Syndrome, drug dependence and hyperprolactinemy, and for the treatment of diseases of the central nervous system correlated with the dysfunctions of the cholinergic and/or nicotinic system, such as the Alzheimer's disease, extrapyramidal motor functions disorders, such as the Parkinson' s disease, characterized by the use of compounds for inclusion or association of EVASINS, analogues and derivatives, microencapsulated or not into systems of controlled release, such as, for example, the liposomes and biodegradable polymers and/or mixtures.

15. Pharmaceutical compositions of the EVASINS, analogues and derivatives, according to claims 1 to 11, for use in the diagnosis, study and treatment of diseases or conditions, such as supramuscular progressive paralysis, Huntington' s disease, Gilles de la Tourette's syndrome and late dyskinesia, characterized by the use of compounds for inclusion or association of EVASINS, analogues and derivatives, microencapsulated or not into systems of controlled release, such as, for example, the liposomes and biodegradable polymers and/or mixtures.

16. Pharmaceutical compositions of the EVASINS, analogues and derivatives, according to claims 1 to 11, for use in the diagnosis, study and treatment of diseases or conditions, such as obesity, strong pain, detoxification of drugs and tobacco, breathing dysfunctions, humor and emotional disorders such as depression, anxiety and psychosis, motor 53 and attention functions, cognitive function disorders, characterized by the use of compounds for inclusion or association of EVASINS, analogues and derivatives, microencapsulated or not into systems of controlled release, such as, for example, the liposomes and biodegradable polymers and/or mixtures.

17. Pharmaceutical compositions of the EVASINS, analogues and derivatives, according to claims 1 to 11, for use in the diagnosis, study and treatment of diseases or conditions, such as loose of memory, madness (including insanity by AIDS), characterized by the use of compounds for inclusion or association of EVASINS, analogues and derivatives, microencapsulated or not into systems of controlled release, such as, for example, the liposomes and biodegradable polymers and/or mixtures.

18. Pharmaceutical compositions of the EVASINS, analogues and derivatives, according to claims 1 to 11, for use in the diagnosis, study and treatment of diseases or conditions, such as neurodegenerative disorders, epilepsy, convulsive disorders, nourishment disorders including bulimia and anorexia, characterized by the use of compounds for inclusion or association of EVASINS, analogues and derivatives, microencapsulated or not into systems of controlled release, such as, for example, the liposomes and biodegradable polymers and/or mixtures.

19. Pharmaceutical compositions of the EVASINS, analogues and derivatives, according to claims 1 to 11, for use in the diagnosis, study and treatment of diseases or conditions, such as autonomic disorders such as the gastrointestinal motility disorders, irritable colon, diarrhea, constipation, ulcers, comedication in surgical treatment and pheochromocytoma, hypertension and cardiovascular diseases, nociception and pain control, characterized by the use of compounds for inclusion or association of 54

EVASINS, analogues and derivatives, microencapsulated or not into systems of controlled release, such as, for example, the liposomes and biodegradable polymers and/or mixtures .

20. Pharmaceutical compositions of the EVASINS, analogues and derivatives, according to claims 1 to 7, characterized by the increase in the bioavailabilility of the referred EVASINS once included into the cyclodextrins and derivatives, or when associated with or included into carriers and/or vehicles pharmaceutically acceptable, isolated or mixed.

21. Pharmaceutical compositions of the EVASINS, analogues and derivatives, according to claims 1 to 7, characterized by the increase in the duration and/or efficacy of the EVASINS effects, when included into cyclodextrins and derivatives, or associated or included into carriers and/or vehicles pharmaceutically acceptable, isolated or mixed.

22. Compositions and formulations, for intramuscular, intravenous, subcutaneous, topic, inhalation (pulmonary, intranasal, intra-buccal) or as a device that might be transplanted or injected, of EVASINS, analogues and derivatives, according to claims 1 to 7, characterized by the increase in the bioavailability of the referred EVASINS once included into the cyclodextrins and derivatives, or when associated with or included into carriers and/or vehicles pharmaceutically acceptable, isolated or mixed.

23. Compositions and formulations, for intramuscular, intravenous, subcutaneous, topic, inhalation (pulmonary, intranasal, intra-buccal) or as a device that might be transplanted or injected, of EVASINS, analogues and derivatives, according to claims 1 to 7, characterized by the increase in the bioavailabilility of the referred 55

EVASINS once included into the cyclodextrins and derivatives, or when associated with or included into carriers and/or vehicles pharmaceutically acceptable, isolated or mixed, or associated with at least another pharmacological agent active and/or microencapsulated or not into systems of controlled release such as the liposomes and biodegradable polymers and/or mixtures.

24. Compositions and formulations, for intramuscular, intravenous, subcutaneous, topic, inhalation (pulmonary, intranasal, intra-buccal) or as a device that might be transplanted or injected, of EVASINS, analogues and derivatives, according to claims 1 to 7, characterized by the increase in the duration and/or efficacy of the EVASINS effects, when included into cyclodextrins and derivatives, or associated or included into carriers and/or vehicles pharmaceutically acceptable, isolated or mixed.

25. Compositions and formulations, for intramuscular, intravenous, subcutaneous, topic, inhalation (pulmonary, intranasal, intra-buccal) or as a device that might be transplanted or injected, of EVASINS, analogues and derivatives, used for the study and treatment of the arterial hypertension and another cardiovascular diseases and complications, according to claims 1 to 7, characterized by the use of EVASINS, analogues and derivatives, once included into the cyclodextrins and derivatives, or when associated with or included into carriers and/or vehicles pharmaceutically acceptable, isolated or mixed, or associated with at least another pharmacological agent active and/or microencapsulated or not into systems of controlled release such as the liposomes and biodegradable polymers and/or mixtures.

26. Compositions and formulations, for intramuscular, intravenous, subcutaneous, topic, inhalation (pulmonary, 56 intranasal, intra-buccal) or as a device that might be transplanted or injected, of EVASINS, analogues and derivatives, used for the study and treatment of diseases or conditions, such as the neural and neuromuscular synaptic fast transmission, sedation, anesthesia, reversion of the effect of drug dependence due to, e.g., cocaine or phencyclidine, reversion of the intoxication by the cocaine and toxins, reversion of the receptors dysfunctions during diseases such as convulsions and neurodegeneration such as Alzheimer's disease and modulators of the angiogenic properties, according to the claims 1 to 7 and 13, characterized by the use of EVASINS, analogues and derivatives, once included into the cyclodextrins and derivatives, or when associated with or included into carriers and/or vehicles pharmaceutically acceptable, isolated or mixed, or associated with at least another pharmacological agent active and/or microencapsulated or not into systems of controlled release such as the liposomes and biodegradable polymers and/or mixtures.

27. Compositions and formulations, for intramuscular, intravenous, subcutaneous, topic, inhalation (pulmonary, intranasal, intra-buccal) or as a device that might be transplanted or injected, of EVASINS, analogues and derivatives, used for the study and treatment of diseases or conditions, such as schizophrenia, Parkinson's disease, Tourette's Syndrome, drug dependence and hyperprolactinemy, and for the treatment of diseases of the central nervous system correlated with the dysfunctions of the cholinergic and/or nicotinic system, such as the Alzheimer's disease, extrapyramidal motor functions disorders, such as the Parkinson's disease, according to claims 1 to 7 and 14, characterized by the use of EVASINS, analogues and derivatives, once included 57 into the cyclodextrins and derivatives, or when associated with or included into carriers and/or vehicles pharmaceutically acceptable, isolated or mixed, or associated with at least another pharmacological agent active and/or microencapsulated or not into systems of controlled release such as the liposomes and biodegradable polymers and/or mixtures.

28. Compositions and formulations, for intramuscular, intravenous, subcutaneous, topic, inhalation (pulmonary, intranasal, intra-buccal) or as a device that might be transplanted or injected, of EVASINS, analogues and derivatives, used for the study and treatment of diseases or conditions, such as supramuscular progressive paralysis, Huntington' s disease, Gilles de la Tourette's syndrome and late dyskinesia, according to claims 1 to 7 and 15, characterized by the use of EVASINS, analogues and derivatives, once included into the cyclodextrins and derivatives, or when associated with or included into carriers and/or vehicles pharmaceutically acceptable, isolated or mixed, or associated with at least another pharmacological agent active and/or microencapsulated or not into systems of controlled release such as the liposomes and biodegradable polymers and/or mixtures.

29. Compositions and formulations, for intramuscular, intravenous, subcutaneous, topic, inhalation (pulmonary, intranasal, intra-buccal) or as a device that might be transplanted or injected, of EVASINS, analogues and derivatives, used for the study and treatment of diseases or conditions, such as obesity, strong pain, detoxification of drugs and tobacco, breathing dysfunctions, humor and emotional disorders such as depression, anxiety and psychosis, motor and attention functions, cognitive function disorders, according to claims 1 to 7 and 16, characterized by the use of EVASINS, 58 analogues and derivatives, once included into the cyclodextrins and derivatives, or when associated with or included into carriers and/or vehicles pharmaceutically acceptable, isolated or mixed, or associated with at least another pharmacological agent active and/or microencapsulated or not into systems of controlled release such as the liposomes and biodegradable polymers and/or mixtures.

30. Compositions and formulations, for intramuscular, intravenous, subcutaneous, topic, inhalation (pulmonary, intranasal, intra-buccal) or as a device that might be transplanted or injected, of EVASINS, analogues and derivatives, used for the study and treatment of diseases or conditions, such as lack of memory, madness (including insanity by AIDS), according to claims 1 to 7 and 17, characterized by the use of EVASINS, analogues and derivatives, once included into the cyclodextrins and derivatives, or when associated with or included into carriers and/or vehicles pharmaceutically acceptable, isolated or mixed, or associated with at least another pharmacological agent active and/or microencapsulated or not into systems of controlled release such as the liposomes and biodegradable polymers and/or mixtures.

31. Compositions and formulations, for intramuscular, intravenous, subcutaneous, topic, inhalation (pulmonary, intranasal, intra-buccal) or as a device that might be transplanted or injected, of EVASINS, analogues and derivatives, used for the study and treatment of diseases or conditions, such as neurodegenerative disorders, epilepsy, convulsive disorders, nourishment disorders including bulimia and anorexia, according to claims 1 to 7 and 18, characterized by the use of EVASINS, analogues and derivatives, once included into the cyclodextrins and derivatives, or when associated with or included into 59 carriers and/or vehicles pharmaceutically acceptable, isolated or mixed, or associated with at least another pharmacological agent active and/or microencapsulated or not into systems of controlled release such as the liposomes and biodegradable polymers and/or mixtures.

32. Compositions and formulations, for intramuscular, intravenous, subcutaneous, topic, inhalation (pulmonary, intranasal, intra-buccal) or as a device that might be transplanted or injected, of EVASINS, analogues and derivatives, used for the study and treatment of diseases or conditions, such as autonomic disorders such as the gastrointestinal motility disorders, irritable colon, diarrhea, constipation, ulcers, surgical treatment and pheochromocytoma, hypertension and cardiovascular diseases, nociception and pain control, according to claims 1 to 7 and 19, characterized by the use of EVASINS, analogues and derivatives, once included into the cyclodextrins and derivatives, or when associated with or included into carriers and/or vehicles pharmaceutically acceptable, isolated or mixed, or associated with at least another pharmacological agent active and/or microencapsulated or not into systems of controlled release such as the liposomes and biodegradable polymers and/or mixtures.