WO2010009524A1 - Use of the angiotensin-(1-7) peptide, analogues, agonists and derivatives thereof for the treatment of painful conditions - Google Patents

Use of the angiotensin-(1-7) peptide, analogues, agonists and derivatives thereof for the treatment of painful conditions Download PDF

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WO2010009524A1
WO2010009524A1 PCT/BR2009/000217 BR2009000217W WO2010009524A1 WO 2010009524 A1 WO2010009524 A1 WO 2010009524A1 BR 2009000217 W BR2009000217 W BR 2009000217W WO 2010009524 A1 WO2010009524 A1 WO 2010009524A1
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ang
induced
angiotensin
effect
antinociception
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Kátia CONCEIÇÃO
Igor Dimitri Gama Duarte
Robson Augusto Souza Dos Santos
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Universidade Federal De Minas Gerais-Ufmg
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A61K38/085—Angiotensins

Definitions

  • the present invention relates to the use of angiotensin- (1-7) peptide or analogs thereof for the treatment of painful conditions in mammals.
  • the invention further discloses the mechanism of action involved in peripheral antinoceptive action in mammals, which mechanism is based on the involvement of the L-arginine / NO / cGMP / K + AT p pathway.
  • angiotensinogen The classic view of circulating SARS presupposes the formation of angiotensinogen by the liver and renin production by the juxtaglomerular apparatus. These two substances are released into the bloodstream, where angiotensinogen will be hydrolyzed by renin, forming the decapeptide angiotensin I (Ang I), which, mainly in the pulmonary circulation, gives rise to angiotensin II (Ang II) by the action of the enzyme converting enzyme. angiotensin. It will then perform its actions on target organs distant from the site of release (SANTOS, RA, CAMPAGNOLE-SANTOS, MJ, ANDRADE, SP, (2000). Angiotensin- (1-7): an update. Regul Pept.
  • Ang II was considered to be the only biologically active product of SARS (DIZAU, D.V. (1988) Circulating versus local renin-angiotensin system in cardiovascular homeostasis. Circulation 77: 4-13).
  • angiotensinergic peptides including angiotensins-III and IV (Ang III and Ang IV) and angiotensin- (1-7) [Ang- ( 1-7)].
  • Ang- (1-7) exerts its effects through interaction with the Mas receptor (SANTOS, RAS; SIMOES AND SILVA, AC, WALTHER, T. (2003) Angiotensin- (1-7) is in the endogenous ligand for the G protein-coupled receptor Mas. Proc. Natl Acad. Sci USA 100: 8258-8263).
  • AVE 0991 the first physiologically tolerable synthetic compound that mimics Ang- (1-7) actions on vessels, kidneys and heart (SANTOS, RAS & FERREIRA, AJ (2006) Pharmacological Effects of AVE 0991, Nonpeptide Angiotensin- (1-7) Cardiovascular Agonist Receptor Drug Reviews Vol. 24, No. 3-4, pp. 239-246).
  • Pain can be defined as an unpleasant, subjective sensory and emotional experience that assumes different expressions as a function of multiple factors related to both the characteristics of the harmful event and the circumstances in which it occurs (LIMA, D .; ALMEIDA, A. (2002 ) The medullary dorsal reticular nucleus as a pronociceptive center of the pain control system. Neurobiol. 66 (2): 81-108).
  • PRADO et al. demonstrated antinociception induced by the injection of SARS peptides, including Ang I, Ang II and Ang III, in specific areas of periaqueductal gray matter (PAG), with the antinociceptive effect reversed by saralasin (PRADO, WA; PELEGRINI-DA -SILVA A., MARTINS AR (2003) Microinjection of renin-angiotensin system peptides in discrete sites within the periaqueductal gray matter elicits antinociception rat (Brain Res. 972: 207-215).
  • DUARTE & FERREIRA (1992) showed that intracerebroventricular injection-induced analgesia of carbachol was inhibited by the administration of a NOS inhibitor (L-NIO) and methylene blue (DUARTE, ID; FERREIRA, SH (1992). central analgesia induced by morphine or carbachol and the L-arginine-nitric oxide-cGMP pathway (Eur J Pharmacol 221 (1): 171-4).
  • Glucose, sulfonyureas, and neurotransmitter release role of ATP-sensitive K + chanels. Science 247: 852-854).
  • NO acts as a second messenger with the ability to diffuse across cell membranes, being an activator of the soluble guanylcyclase enzyme, leading to increased intracellular cGMP levels. Elevating cGMP levels may activate different types of K + channels in different tissue types (KUBO, M .; NKAYA, Y. MATSUOKA, S .; SAIKO, K .; KURODA, Y. (1994).
  • Atrial natriuretic factor and isosorbide dinitrate modulate the gating of ATP sensitive K channels in cultured vascular smooth cells Cir. Res. 74: 470-476).
  • DbGMP sodium nitroprusside-induced peripheral antinociception occurs by K + ATP activation has established the link between the L-arginine / NO / GMPc / K + AT p pathway (SOARES, AC; DUARTE, IDG (2001)
  • Dibutyryl-cyclic GMP induces peripheral antinociception via activation of ATP-sensitive K + channels in the PGE2-induced hyperalgesic paw (British Journal Pharmacology. 134: 127-131).
  • Ang- (1-7) an active SARS fragment, is capable of dose-dependent peripheral antinociception on PGE 2- induced hyperalgesia.
  • a single injection of PGE 2 is capable of sensitizing nociceptors to mechanical and chemical stimuli.
  • the use of PGE 2 as an inducer of hyperalgesia presents, over other models of hyperalgesia, such as carrageenan, the advantage of eliminating the possibility that the peripheral effects of the tested analgesic result from a blockage of the release or action of mediators produced during the inflammatory process (FERREIRA, SH (1972) Prostaglandins, aspirin-like drugs and analgesia. Nature New Biol. 204: 200-203). It was also found that the peak of antinociceptive action of Ang- (1-7) occurs ten minutes after its injection and that the effect lasts approximately 20 minutes.
  • A-779 has been described as a specific antagonist for Ang- (1-7) actions by SANTOS et al. (1994). In this study, A-779 blocked the antidiuretic effect of Ang- (1-7), as well as changes in blood pressure caused by heptapeptide injection into the dorsomedial and ventrolateral bulb; A 2 and AT 2 receptor antagonists have not been able to block such Ang- (1-7) actions. On the other hand, A-779 was not able to inhibit the pressor, dipsinogenic and myotrophic effect of Ang II. In addition, A-779 did not alter the antidiuretic effect of vasopressin or the contractile events caused by the administration of Ang III, bradykinin or substance P in isolated ileum rat.
  • A-779 is a potent and selective antagonist (SANTOS, RAS; CANAPGNOLE-SANTOS, MJ. (1994). Central and peripheral actions of angiotensin- (1- 7) .Braz. J. Med. Biol. Res. 27: 1033-47).
  • A-779 is the MasR specific blocker (SANTOS, RAS; SIMOES AND SILVA, AC WALTHER, T. (2003). )
  • Angiotensin- (1-7) is an endogenous ligand for the G protein-coupled receptor Mas. Proc. Natl Acad. Sci USA 100: 8258-8263).
  • the results presented in the present invention point to the presence of this receptor at the peripheral terminals of sensory neurons, since A-779 totally reversed the antinociceptive effect of Ang- (1-7).
  • NO acts as a second messenger with the ability to diffuse across cell membranes.
  • a soluble guanylcyclase enzyme activator leading to increased intracellular cGMP levels.
  • Nitric oxide by increasing cGMP levels, can activate different types of K + channels in different tissue types (KUBO, M .; NKAYA, Y. MATSUOKA, S .; SAITO, K .; KURODA, Y. (1994).
  • Ang- (1-7) actions have been closely related to NO release.
  • NOs inhibitors L-NOarg and L-NAME
  • BROSNIHAN the NO participation in Ang (1-7) endothelium-dependent vasodilatory action
  • BROSNIHAN KB
  • Effect of the angiotensin- (1-7) peptide on nitric oxide release Am. J. Cardiol. 82 (10A): 17S-19S.
  • NOs synthase blockers such as L-NAME, L-NOarg and L-NMMA
  • L-NAME L-NAME
  • L-NOarg L-NOarg
  • L-NMMA L-NMMA
  • DUARTE & FERREIRA demonstrated that the L-NAME has serious limitations as a specific inhibitor of the L-arginine / NO / cGMP pathway as it has an antinociceptive effect when administered alone as a stimulant of the same pathway (DUARTE, ID; FERREIRA, SH (1992). of central analgesia induced by morphine or carbachol and the L-arginine-nitric oxide-cGMP pathway Eur J Pharmacol 221 (1): 171-4).
  • L-NAME can be considered an inappropriate drug for the study of analgesia and may cause errors in the interpretation of results.
  • ODQ is a specific soluble guanylcyclase inhibitor synthesized by no activity on membrane guanylcyclase or adenylyl cyclase. Has no effect on NOs isoforms or NO (GARTHWAITE, J .; SOUTHAM,
  • Methylene blue is now considered a weak inhibitor of soluble guanylcyclase, more efficient in releasing superoxide anions and inhibiting NOs (MAYER, B.; BRUNNER, F .; SCHIMIDT, K. (1993) Inhibition of nitric oxide synthesis by methylene blue Biochem Parmacol 45: 2547-2549). Therefore, ODQ is considered, among these substances, the best pharmacological tool for the study of the actions of soluble guanylcyclase. From this perspective, some studies have used ODQ to investigate NOs actions on analgesia (BRITO, G.A .; SACHS, D .; CUNHA,
  • WO03039434A2 (WO03039434A3), Millán, Dos Santos et. al. (2003) developed an invention in which he describes the characterization of the process of preparing Angiotensin- (1-7) peptide formulations and their analogs, agonists and antagonists using cyclodextrins, their derivatives, liposomes and biodegradable polymers and / or mixtures thereof. systems and / or derived products, which can be used to treat various conditions such as high blood pressure, other cardiovascular diseases and their complications, wounds, burns, erythema, tumors, diabetes mellitus and others.
  • WO06128266A2 (WO06128266A3), Dos Reis, Millán et. al. (2006) discloses an invention characterized by the pharmaceutical composition of the Angiotensin- (1-7) peptide and its analogs, agonists and antagonists for use in controlling reproductive system functions.
  • WO03072059A2 (WO03072059A3), Tallant, Gallagher, Ferrario (2003) describes a technology that makes use of the Angiotensin- (1-7) peptide and its agonists as an anti-cancer therapy.
  • the main point of the present invention is the demonstration for the first time that Ang- (1-7) has antinociceptive effect and the main pathway involved in this action is L-arginine / NO / cGMP.
  • This invention contributes to the broadening of the concept of SARS, indicating that Ang- (1-7) as well as SARS may be involved in controlling pain sensitivity.
  • the discovery opens perspectives for creation of a new class of angiotensinergic analgesic drugs, without the side effects of opioid analgesics or non-steroidal anti-inflammatory drugs.
  • PGE 2 Prostaglandin E 2 (PGE 2 , Calbiochem, USA). Kept in the freezer in a stock solution dissolved in ethanol at a concentration of 1 mg / ml. Immediately prior to injections, the PGE 2 solution was diluted in sterile physiological saline and kept at low temperature in an ice-cold Styrofoam box.
  • Angiotensin- (1-7) (Ang- (1-7), Bachem, Germany). Dissolved in sterile saline.
  • Angiotensin II (Ang-11, Bachen, Germany). Dissolved in sterile saline.
  • N G -Nitro-L-arginine (L-Noarg, RBI, USA). Dissolved in sterile saline.
  • ODQ (Tocris, USA). Kept in freezer in stock solution dissolved in DMSO at a concentration of 10 mg / ml. Immediately prior to injections, the ODQ solution was diluted in physiological saline and kept at a low temperature in an ice-cold Styrofoam box.
  • EXAMPLE 03 Nociceptive Test - Rat Paw Hyperalgesia
  • Hyperalgesia was measured using the compressed rat paw withdrawal method originally described by RANDALL & SELITTO (1957). These authors developed a technique for measuring antinociceptive activity based on the principle that inflammation increases sensitivity to painful stimuli and that increased sensitivity is susceptible to drug modification. To perform the hyperalgesia measurements, the Ugo Basile (Italy) algesimetric device was used.
  • the compressing part of the apparatus consists of two surfaces, one flat, on which the animal's foot rests, and the other tapered, with an area of 1.75 mm 2 at the end, whereby pressure is applied to the surface. mouse plantar.
  • the intensity of the applied pressure increases at a constant rate of 32 g / s by the pedal being operated by the experimenter.
  • the experimenter disengages the pedal, thus interrupting the increase of the pressure imposed on the paw.
  • the last value, which corresponds to the nociceptive threshold is indicated on the scale of the apparatus and expressed in grams.
  • the nociceptive threshold is defined as the pressure applied to the animal's paw, which presents the nociceptive response described above.
  • the nociceptive threshold of the animals was monitored as a function of the action of different drugs against prostaglandin E 2 induced hyperalgesic challenge.
  • hyperalgesia as the nociceptive threshold decrease, its intensity was evaluated in some experiments by the difference ( ⁇ ) of the nociceptive threshold measured in the third hour after PGE 2 injection in relation to that value obtained at the beginning of the experiment, before any injection. (zero hour).
  • Figure 1 shows an example of the calculation of nociceptive threshold ⁇ .
  • EXAMPLE 05 This example describes the effect of Angiotensin (1-7) on intraplantar hyperalgesia induced by different doses of Prostaglandins E 2 (PGE 2 ).
  • Figure 2 shows that intra-plant administration of PGE 2 (0.25; 0.5; 1 and 2 ⁇ g) induced a dose-dependent decrease in nociceptive threshold in relation to the control group (2% ethanol in saline). This hyperalgesic effect was detected only after the second hour, with maximum intensity in the third hour after PGE 2 administration. The 2 ⁇ g dose was able to produce higher intensity hyperalgesia and was chosen for subsequent experiments. Intraplantar subcutaneous administration of Ang- (1-7) produced dose-dependent antinociception ( Figure 3), ie increasing doses of Ang- (1-7) were able to proportionally reduce hyperalgesia induced by PGE 2 (2 ⁇ g).
  • EXAMPLE 06 Effect Induced by Intraplantar Administration of Mas A-779 Receptor Antagonist on Ang-Induced Peripheral Antinociception (1-7)
  • Figure 4 shows that increasing doses of antagonist A-779 (2, 4 and 8 ⁇ g / paw) reversed Ang- (1-7) -induced antinociception at the dose of 4 ⁇ g / paw.
  • A-779 is fully reversed the antinociceptive action of Ang- (1-7), indicating the exclusive participation of the angiotensinergic receptor Mas in this response.
  • EXAMPLE 07 Evaluation of L-Arginine / NO / GMPc / K + ATP Pathway Participation in Ang- (1-7) -induced Peripheral Antinociception
  • experiments were performed using NOs (L-NOarg) and guanylylcyclase (ODQ) inhibitors to evaluate the participation of this pathway in Ang- (1-7) -induced antinociception.
  • EXAMPLE 08 Effect induced by L-NOarg administration on Ang-induced peripheral antinociceptive effect (1-7)
  • Figure 5 shows the dose-dependent inhibition of antinociception induced by 4 ⁇ g Ang- (1-7) by administration of L-NOarg (12, 18 and 24 ⁇ g). This result indicates that NO is an important mediator in antinociceptive activity of Ang- (1-7). There was no hyperalgesic or nociceptive action of the inhibitor.
  • EXAMPLE 09 Effect induced by ODQ administration on Ang-induced peripheral antinociception (1-7)
  • Soluble guanyl projectlase inhibitor ODQ dose-dependent inhibited the antinociceptive effect induced by Ang- (1-7) 4 ⁇ g, pointing to the action of the L-arginine / NO / GMPc pathway ( Figure 6).
  • the ODQ did not present hyperalgesic or nociceptive activity.
  • EXAMPLE 10 Effect Induced by Glibenclamide Administration on Ang ⁇ Peripheral Antinociceptive Activity (1-7)
  • Figure 7 shows that glibenclamide, an ATP-sensitive K + channel blocker, was also reversed by the Ang- (1-7) -induced peripheral antinociceptive effect at a dose of 4 ⁇ g. The highest dose of the inhibitor produced complete block and showed no hyperalgesic or antinociceptive effect.
  • EXAMPLE 10 Effect induced by administration of the naloxone opioid receptor antagonist on Ang- (1-7) -induced antinociception
  • Intraplantar administration of naloxone did not induce any change in Ang- (1-7) -induced antinociception (Figure 8).
  • This result indicates that opioid receptors are not involved in Ang- (1-7) -induced antinociception, as the 50 ⁇ g / paw dose of naloxone is capable of reversing morphine-induced antinociception at the 200 ⁇ g / paw dose. .
  • nociceptive threshold D The two left columns refer to the measurement of the nociception threshold expressed in grams (g) before intraplantar (0 hour) administration of carrageenan (Cg, 250 mg) or saline (Sal) and the two central columns three hours after this administration (time 3). The two right columns refer to the differences between these measurements (nociceptive threshold D).
  • FIGURE 02. Temporal development of the hyperalgesia effect induced by intra-plant injection of different PGE 2 doses. Each symbol represents the mean + SEM of the measurement of nociceptive threshold expressed in grams (g) for 5 animals. * indicates statistical significance (P ⁇ 0.05) compared to the control group (2% ethanol in saline).
  • Each bar represents the mean + SEM of the nociceptive threshold ⁇ measurement expressed in grams (g) of the right paw for five animals.
  • * and # indicate statistical significance (P ⁇ 0.05) in relation to the groups (PGE 2 + Sal + Salt) and [PGE 2 + Sal + Ang- (1-7)], respectively.
  • Ang Ang- (1-7).
  • FIGURE 05 Effect induced by intra-plantar administration of different doses of L-NOarg on 4 ⁇ g / Ang- paw-induced peripheral antinociception (1-7).
  • Each bar represents the mean + SEM of the nociceptive threshold ⁇ measurement expressed in grams (g) of the right paw for five animals.
  • * and # indicate statistical significance (P ⁇ 0.05) in relation to the groups (PGE 2 + Sal + Salt) and [PGE 2 + Sal + Ang- (1-7)], respectively.
  • Ang Ang- (1-7).
  • FIGURE 06. Effect induced by intraplantar administration of different doses of ODQ on peripheral antinociception induced by 4 ⁇ g / paw of Ang- (1-7).
  • Each bar represents the mean + SEM of the nociceptive threshold ⁇ measurement expressed in grams (g) of the right paw for five animals.
  • * and # indicate statistical significance (P ⁇ 0.05) in relation to the groups (PGE 2 + 20% DMSO + Salt) and [PGE 2 + 20% DMSO + Ang- (1-7)], respectively.
  • Ang Ang- (1-7).
  • Each bar represents the mean + SEM of the nociceptive threshold ⁇ measurement expressed in grams (g) of the right paw for five animals.
  • * and # indicate statistical significance (P ⁇ 0.05) in relation to the groups (PGE 2 + Salt + Salt) and [PGE 2 + Salt + Ang- (1-7)], respectively.
  • Ang Ang- (1-7).
  • FIGURE 08. Effect induced by intraplantar administration of Nx on peripheral antinociception induced by 4 ⁇ g / Ang- paw (1-7). Each bar represents the mean + SEM of the nociceptive threshold ⁇ measurement expressed in grams (g) of the right paw for five animals. * indicates statistical significance (P ⁇ 0.05) in relation to the control group (PGE 2 + Salt + Salt). Ang Ang- (1-7).

Abstract

The present invention relates to the use of the angiotensin-(1-7) peptide or analogues thereof for the treatment of painful conditions in mammals. The present invention also discloses the action mechanism involved in the peripheral antinociception action in mammals, a mechanism involving the L-arginin/NO/GMP/K+ ATP channel.

Description

"USO DO PEPTÍDEO ANGIOTENSINA-(1-7), SEUS ANÁLOGOS, AGONISTAS OU DERIVADOS PARA O TRATAMENTO DE CONDIÇÕES DOLOROSAS"  "USE OF ANGIOTENSIN- (1-7) PEPTIDE, ANALOGS, AGONISTS OR DERIVATIVES FOR THE TREATMENT OF PAINFUL CONDITIONS"
A presente invenção diz respeito ao uso do peptídeo angiotensina-(1-7) ou seus análogos para o tratamento de condições dolorosas em mamíferos. A invenção revela ainda o mecanismo de ação envolvido na ação antinoceptiva periférica em mamíferos, mecanismo este baseado no envolvimento da via L- arginina/NO/GMPc/K+ ATp. The present invention relates to the use of angiotensin- (1-7) peptide or analogs thereof for the treatment of painful conditions in mammals. The invention further discloses the mechanism of action involved in peripheral antinoceptive action in mammals, which mechanism is based on the involvement of the L-arginine / NO / cGMP / K + AT p pathway.
A visão clássica do SRA circulante pressupõe a formação de angiotensinogênio pelo fígado e a produção de renina pelo aparelho justaglomerular. Essas duas substâncias são liberadas na corrente sanguínea, onde o angiotensinogênio será hidrolisado pela renina, formando o decapeptídeo angiotensina I (Ang I), o qual, principalmente na circulação pulmonar, dá origem a angiotensina II (Ang II) pela ação da enzima conversora de angiotensina. Ela exercerá, então, suas ações em órgãos alvos distantes do sítio de liberação (SANTOS, R.A, CAMPAGNOLE-SANTOS, M.J., ANDRADE, S.P, (2000) Angiotensin-(1-7): an update. Regul Pept. 28;91 (1-3):45-62). Até o início da década de 80, a Ang II era considerada o único produto biologicamente ativo do SRA (DIZAU, D.V. (1988) Circulating versus local renin-angiotensin system in cardiovascular homeostasis. Circulation 77: 4-13). Entretanto, observações subsequentes indicam que importantes ações periféricas e centrais do SRA podem ser mediadas por sequências menores de peptídeos angiotensinérgicos, incluindo as angiotensinas-lll e IV (Ang III e Ang IV) e a angiotensina-(1-7) [Ang-(1-7)]. A Ang-(1-7) exerce seus efeitos por meio da interação com o receptor Mas (SANTOS, R.A.S.; SIMOES E SILVA, AC, WALTHER, T. (2003) Angiotensin-(1-7) is na endogenous ligand for the G protein-coupled receptor Mas. Proc. Natl Acad. Sei USA 100: 8258- 8263).  The classic view of circulating SARS presupposes the formation of angiotensinogen by the liver and renin production by the juxtaglomerular apparatus. These two substances are released into the bloodstream, where angiotensinogen will be hydrolyzed by renin, forming the decapeptide angiotensin I (Ang I), which, mainly in the pulmonary circulation, gives rise to angiotensin II (Ang II) by the action of the enzyme converting enzyme. angiotensin. It will then perform its actions on target organs distant from the site of release (SANTOS, RA, CAMPAGNOLE-SANTOS, MJ, ANDRADE, SP, (2000). Angiotensin- (1-7): an update. Regul Pept. 28; 91 (1-3): 45-62). Until the early 1980s, Ang II was considered to be the only biologically active product of SARS (DIZAU, D.V. (1988) Circulating versus local renin-angiotensin system in cardiovascular homeostasis. Circulation 77: 4-13). However, subsequent observations indicate that important peripheral and central actions of SARS may be mediated by smaller sequences of angiotensinergic peptides, including angiotensins-III and IV (Ang III and Ang IV) and angiotensin- (1-7) [Ang- ( 1-7)]. Ang- (1-7) exerts its effects through interaction with the Mas receptor (SANTOS, RAS; SIMOES AND SILVA, AC, WALTHER, T. (2003) Angiotensin- (1-7) is in the endogenous ligand for the G protein-coupled receptor Mas. Proc. Natl Acad. Sci USA 100: 8258-8263).
Dentre os agonistas do receptor Mas, destaca-se o AVE 0991 , o primeiro composto sintético fisiologicamente tolerável que mimetiza as ações da Ang-(1-7) sobre vasos, rins e coração (SANTOS, R.A.S & FERREIRA, A.J (2006) Pharmacological Effects of AVE 0991 , a Nonpeptide Angiotensin-(1-7) Receptor Agonist. Cardiovascular Drug Reviews Vol. 24, No. 3-4, pp. 239-246).  Among the receptor agonists, however, stands out AVE 0991, the first physiologically tolerable synthetic compound that mimics Ang- (1-7) actions on vessels, kidneys and heart (SANTOS, RAS & FERREIRA, AJ (2006) Pharmacological Effects of AVE 0991, Nonpeptide Angiotensin- (1-7) Cardiovascular Agonist Receptor Drug Reviews Vol. 24, No. 3-4, pp. 239-246).
A dor pode ser definida como uma experiência sensorial e emocional desagradável, subjetiva, que assume diferentes expressões como função de múltiplos fatores relacionados tanto às características do evento nocivo quanto às circunstâncias em que ela ocorre (LIMA, D.; ALMEIDA, A. (2002) The medullary dorsal reticular nucleus as a pronociceptive centre of the pain control system. Prog. Neurobiol. 66(2): 81-108). Apesar da sensação dolorosa ser indispensável à preservação tecidual e à sobrevivência do indivíduo, existem várias situações em que a intervenção farmacológica se torna necessária, por exemplo, no caso da dor inflamatória, presente em pacientes no quadro pós-cirúrgico ou pós-trauma, ou mesmo em indivíduos com doenças inflamatórias crónicas, como a artrite reumatóide, e ainda nos casos de dor neuropática, proveniente de lesões neuroniais, ou de dor espontânea, em que não existe um motivo aparente (CLIFFORD, J.; WOOLF, M.D. (2004) Moving from symptom control toward mechanism-specific pharmacologic management. Ann. Intern. Med. 140: 441-451). Pain can be defined as an unpleasant, subjective sensory and emotional experience that assumes different expressions as a function of multiple factors related to both the characteristics of the harmful event and the circumstances in which it occurs (LIMA, D .; ALMEIDA, A. (2002 ) The medullary dorsal reticular nucleus as a pronociceptive center of the pain control system. Neurobiol. 66 (2): 81-108). Although painful sensation is indispensable to the tissue preservation and survival of the individual, there are several situations in which pharmacological intervention is necessary, for example, in the case of inflammatory pain present in patients after surgery or after trauma, or even in individuals with chronic inflammatory diseases such as rheumatoid arthritis, and even in cases of neuropathic pain from neuronal injury or spontaneous pain where there is no apparent reason (CLIFFORD, J .; WOOLF, MD (2004)). Moving from symptom control toward mechanism-specific pharmacological management (Ann. Intern. Med. 140: 441-451).
Na década de 80 surgiram evidências mais diretas que a hipertensão crónica ou aguda era acompanhada por analgesia. No estudo de NARANJO & FUENTES (1985), ratos com hipertensão experimental renal ou no modelo DOCA-salt apresentaram hipoalgesia passível de reversão com naloxona. Além disso, esta hipoalgesia apresenta-se diretamente ligada à elevação da pressão, já que a normalização da mesma elimina o comportamento antinociceptivo (NARANJO, J. R.; FUENTES, J. A. (1985) Association between hypoalgesia and hypertension in rats after sort-term isolation. Neuropharmacology 24: 167-171). Entretanto, a principal evidência de que hipertensão e hipoalgesia estão associadas é o alto limiar à estimulação elétrica na polpa dentária, encontrado em pacientes não tratados com quadro de hipertensão espontânea (ZAMIR, N.; MAIXNER W. (1986) The relationship between cardiovascular and pain regulatory systems. Ann. N. Y. Acad. Sei. 467: 371- 384).  In the 1980s, more direct evidence emerged that chronic or acute hypertension was accompanied by analgesia. In the study by NARANJO & FUENTES (1985), rats with experimental renal hypertension or the DOCA-salt model presented reversible hypoalgesia with naloxone. In addition, this hypoalgesia is directly linked to the increase in pressure, since its normalization eliminates antinociceptive behavior (NARANJO, JR; FUENTES, JA (1985). Association between hypoalgesia and hypertension in rats after sort-term isolation. 24: 167-171). However, the main evidence that hypertension and hypoalgesia are associated is the high threshold for electrical pulp stimulation found in untreated patients with spontaneous hypertension (ZAMIR, N .; MAIXNER W. (1986). pain regulatory systems, Ann, NY Acad. Sci. 467: 371-384).
Sabendo-se da extrema importância do Sistema Renina Angiotensina (SRA) no controle da homeostase cardiovascular e que disfunções deste sistema podem levar à hipertensão, começou-se a acreditar que o mesmo poderia ter importante papel na analgesia, onde a elevação dos níveis de Ang-ll poderia promover alterações nos níveis de peptídeos envolvidos no processamento da dor (IRVINE J.; WHITE J. M.; HEAD RJ. (1995). The renin angiotensin system and nociception in spontaneously hypertensive rats. Life Sciences 56 (13): 1073-1078).  Knowing the extreme importance of the Renin Angiotensin System (SARS) in the control of cardiovascular homeostasis and that dysfunctions of this system can lead to hypertension, we began to believe that it could play an important role in analgesia, where the elevation of Ang levels -ll could promote changes in the levels of peptides involved in pain processing (IRVINE J .; WHITE JM; HEAD RJ. (1995). The renin angiotensin system and nociception in spontaneously hypertensive rats. Life Sciences 56 (13): 1073-1078 ).
IRVINE et al (1995) verificaram que a hipoalgesia de animais SHR IRVINE et al (1995) found that hypoalgesia in SHR animals
(Spontaneous Hypertensive Rats), detectada com as técnicas de retirada da cauda e placa quente, desaparece durante o tratamento com captopril e losartan, mas não com hidralasina, indicando que essa alteração na sensibilidade dolorosa não é proveniente de uma simples elevação na pressão arterial e envolve participação do SRA e receptores AT1. Posteriormente, o peptídeo Ang II, ao ser injetado por via intratecal em ratos, induziu antinocicepção no teste de retirada da cauda (IRVINE J.; WHITE J. M.; HEAD R.J. (1995). The renin angiotensin system and nociception in spontaneously hypertensive rats. Life Sciences 56 (13): 1073-1078). O efeito antinociceptivo era revertido nos animais pré-tratados com losartan, salarasina e naloxona, mas não nos tratados com hidralasina, destacando a participação de receptores angiotensinérgicos, AT-i principalmente, na liberação de peptídeos opióides (TOMA N.; SGAMBATO, V.; COUTURE, R. (1997) Effect of angiotensin II on a spinal nociceptive reflex in the rat: receptor and mechanism of action. Life Sei. 61 : 503-13). (Spontaneous Hypertensive Rats), detected with tail and hot plate removal techniques, disappears during treatment with captopril and losartan, but not hydralasine, indicating that this change in pain sensitivity is not due to a simple elevation in blood pressure and involves participation of SARS and AT1 receptors. Subsequently, the Ang II peptide, when injected intrathecally in rats induced antinociception in the tail removal test (IRVINE J.; WHITE JM; HEAD RJ (1995). The renin angiotensin system and nociception in spontaneously hypertensive rats. Life Sciences 56 (13): 1073-1078). The antinociceptive effect was reversed in losartan, salarasine and naloxone pretreated animals, but not in hydralasin-treated animals, highlighting the participation of angiotensinergic receptors, AT-i mainly, in the release of opioid peptides (TOMA N.; ; COUTURE, R. (1997) Effect of angiotensin II on a nociceptive spinal reflex in the rat: receptor and mechanism of action (Life Sci. 61: 503-13).
Em outro estudo, PRADO et al. (2003) demonstraram a antinocicepção induzida pela injeção de peptídeos do SRA, incluindo Ang I, Ang II e Ang III, em áreas específicas da substância cinzenta periaquedutal (PAG), sendo o efeito antinociceptivo revertido por saralasina (PRADO, W.A.; PELEGRINI-DA-SILVA A., MARTINS A.R. (2003) Microinjection of renin-angiotensin system peptides in discrete sites within the rat periaqueductal gray matter elicits antinociception. Brain Res. 972: 207-215). O mesmo grupo de pesquisadores verificou, posteriormente, que além do efeito antinociceptivo induzido pela injeção destes peptídeos na PAG, existe, na mesma área, a participação da Ang II endógena no controle tónico da nocicepção em ratos mediada por receptores AT-, e AT2 (PELEGRINI-DA-SILVA A.; MARTINS A.R.; PRADO, W.A. (2005) A new role for the renin-angiotensin system in the rat periaqueductal gray matter: angiotensin receptor-mediated modulation of nociception. Neuroscience 132: 453-463). In another study, PRADO et al. (2003) demonstrated antinociception induced by the injection of SARS peptides, including Ang I, Ang II and Ang III, in specific areas of periaqueductal gray matter (PAG), with the antinociceptive effect reversed by saralasin (PRADO, WA; PELEGRINI-DA -SILVA A., MARTINS AR (2003) Microinjection of renin-angiotensin system peptides in discrete sites within the periaqueductal gray matter elicits antinociception rat (Brain Res. 972: 207-215). The same group of researchers later found that in addition to the antinociceptive effect induced by the injection of these peptides into PAG, there is, in the same area, the participation of endogenous Ang II in tonic control of nociception in mice mediated by AT-, and AT 2 receptor receptors. (PELEGRINI-DA-SILVA A.; MARTINS AR; PRADO, WA (2005) A new role for the renin-angiotensin system in the rat periaqueductal gray matter: receptor mediated angiotensin modulation of nociception. Neuroscience 132: 453-463).
DUARTE & FERREIRA (1992) mostraram que a analgesia induzida pela injeção intracerebroventricular de carbacol foi inibida pela administração de um inibidor da NOS (L-NIO) e pelo azul de metileno (DUARTE, I. D.; FERREIRA, S. H. (1992) The molecular mechanism of central analgesia induced by morphine or carbachol and the L-arginine-nitric oxide-cGMP pathway. Eur J Pharmacol. 221 (1):171-4).  DUARTE & FERREIRA (1992) showed that intracerebroventricular injection-induced analgesia of carbachol was inhibited by the administration of a NOS inhibitor (L-NIO) and methylene blue (DUARTE, ID; FERREIRA, SH (1992). central analgesia induced by morphine or carbachol and the L-arginine-nitric oxide-cGMP pathway (Eur J Pharmacol 221 (1): 171-4).
ZHUO et al. (1993) demonstraram que o efeito analgésico da injeção intratecal de drogas muscarínicas é diretamente dependente da produção de NO e GMPC (ZHUO, H.; FUNG, S.J.; BARNES, CD. (1993) Opioid action on spinal cord reflexes due to dorsolateral pontine tegmentum stimulation. Neuropharmacol. 32(7): 621-31). ZHUO et al. (1993) demonstrated that the analgesic effect of intrathecal injection of muscarinic drugs is directly dependent on NO and GMP C production (ZHUO, H .; FUNG, SJ; BARNES, CD. (1993). Opioid action on spinal cord reflexes due to dorsolateral. pontine tegmentum stimulation Neuropharmacol 32 (7): 621-31).
Em experimentos realizados pelos pesquisadores da presente invenção, as sulfoniluréias tolbutamida e glibenclamida reverteram, de forma dose-dependente, o efeito antinociceptivo periférico induzido pelo NPS e DbGMPc (SOARES, A.C.; DUARTE, I.D.G. (2001) Dibutyryl-cyclic GMP induces peripheral antinociception via activation of ATP-sensitive K+ channels int the PGE2-induced hyperalgesic paw. British J. Pharmacol. 134: 127-31). Essas drogas bloqueiam especificamente canais para potássio sensíveis ao ATP (K+ ATp)> não apresentando efeito sobre canais para potássio ativados por cálcio ou dependentes de voltagem (AMOROSO, S.; SHMID- ANTOMARCHI, H.; FOSSET, M.; LAZDUNSKI, M. (1990) Glucose, sulfonyureas, and neurotransmitter release: role of ATP-sensitive K+ chanels. Science 247: 852-854). No sistema nervoso e em outros tecidos, o NO age como segundo mensageiro com capacidade de difundir-se através das membranas celulares, sendo um ativador da enzima guanililciclase solúvel, levando ao aumento dos níveis de GMPc intracelular. A elevação dos níveis de GMPc, pode ativar diferentes tipos de canais de K+ em diferentes tipos de tecidos (KUBO, M.; NKAYA, Y. MATSUOKA, S.; SAIKO, K.; KURODA, Y. (1994) Atrial natriuretic fator and isosorbide dinitrate modulate the gating of ATP sensitive K channels in cultured vascular smooth cells. Cir. Res. 74: 470-476). A demonstração de que a antinocicepção periférica induzida pelo nitroprussiato de sódio de DbGMP ocorre pela ativação de K+ ATP estabeleceu a ligação entre a via L- arginina/NO/GMPc/K+ ATp (SOARES, A.C; DUARTE, I.D.G. (2001) Dibutyryl-cyclic GMP induces peripherical antinociception via activation of ATP-sensitive K+ channels in the PGE2-índuced hyperalgesic paw. British Journal Pharmacology . 134:127-131). In experiments conducted by the researchers of the present invention, the sulfonylureas tolbutamide and glibenclamide dose-dependently reversed the peripheral antinociceptive effect induced by NPS and DbGMP c (SOARES, AC; DUARTE, IDG (2001). via activation of ATP-sensitive K + channels int the PGE2-induced hyperalgesic paw. British J. Pharmacol. 134: 127-31). These drugs specifically block ATP-sensitive potassium channels (K + AT p) > having no effect on calcium-activated or voltage-dependent potassium channels (AMOROSO, S .; SHMID-ANTOMARCHI, H .; FOSSET, M .; LAZDUNSKI , M. (1990) Glucose, sulfonyureas, and neurotransmitter release: role of ATP-sensitive K + chanels. Science 247: 852-854). In the nervous system and other tissues, NO acts as a second messenger with the ability to diffuse across cell membranes, being an activator of the soluble guanylcyclase enzyme, leading to increased intracellular cGMP levels. Elevating cGMP levels may activate different types of K + channels in different tissue types (KUBO, M .; NKAYA, Y. MATSUOKA, S .; SAIKO, K .; KURODA, Y. (1994). Atrial natriuretic factor and isosorbide dinitrate modulate the gating of ATP sensitive K channels in cultured vascular smooth cells Cir. Res. 74: 470-476). The demonstration that DbGMP sodium nitroprusside-induced peripheral antinociception occurs by K + ATP activation has established the link between the L-arginine / NO / GMPc / K + AT p pathway (SOARES, AC; DUARTE, IDG (2001) Dibutyryl-cyclic GMP induces peripheral antinociception via activation of ATP-sensitive K + channels in the PGE2-induced hyperalgesic paw (British Journal Pharmacology. 134: 127-131).
A ação antinociceptiva de várias substâncias também parece estar relacionada com a ativação de canais para potássio sensíveis ao ATP. A administração de sulfoniluréias antagonizou o efeito antinociceptivo central induzido pela morfina em ratos (WILD, K.D.; VANDERAH, T.; MOSBERG, H.I.; PORRECA, F. (1991) Opioid delta receptor subtypes are associated with different potassium channels. Eur. J. Pharmacol. 193(1): 135-6).  The antinociceptive action of various substances also appears to be related to activation of ATP-sensitive potassium channels. Administration of sulfonylureas antagonized the morphine-induced central antinociceptive effect in rats (WILD, KD; VANDERAH, T.; MOSBERG, HI; PORRECA, F. (1991) Opioid delta receptor subtypes are associated with different potassium channels. Eur. J. Pharmacol 193 (1): 135-6).
Os resultados da presente invenção mostraram que a Ang-(1-7), um fragmento ativo do SRA, é capaz de produzir antinocicepção periférica, de maneira dose- dependente, sobre a hiperalgesia produzida pela PGE2. The results of the present invention have shown that Ang- (1-7), an active SARS fragment, is capable of dose-dependent peripheral antinociception on PGE 2- induced hyperalgesia.
Segundo FERREIRA (1972), uma única injeção de PGE2 é capaz de sensibilizar nociceptores a estímulos mecânicos e químicos. A utilização da PGE2 como indutor de hiperalgesia apresenta, sobre outros modelos de hiperalgesia, como por exemplo, a carragenina, a vantagem de eliminar a possibilidade de que os efeitos periféricos do analgésico testado resultem de um bloqueio da liberação ou ação de mediadores produzidos durante o processo inflamatório (FERREIRA, S.H. (1972) Prostaglandins, aspirin-like drugs and analgesia. Nature New Biol. 204: 200-203). Verificou-se, também, que o pico da ação antinociceptiva da Ang-(1-7) ocorre dez minutos após a sua injeção e que o efeito tem duração de aproximadamente 20 minutos, o que era esperado visto que os peptídeos são altamente sensíveis a peptidases e, por isso, são rapidamente degradados nos tecidos (GANTE, J. (1994) Peptidomimetics-Tailored Enzyme Inhibitors Angew. Chem. Int. Ed. Engl. 33: 1699- 1720). According to FERREIRA (1972), a single injection of PGE 2 is capable of sensitizing nociceptors to mechanical and chemical stimuli. The use of PGE 2 as an inducer of hyperalgesia presents, over other models of hyperalgesia, such as carrageenan, the advantage of eliminating the possibility that the peripheral effects of the tested analgesic result from a blockage of the release or action of mediators produced during the inflammatory process (FERREIRA, SH (1972) Prostaglandins, aspirin-like drugs and analgesia. Nature New Biol. 204: 200-203). It was also found that the peak of antinociceptive action of Ang- (1-7) occurs ten minutes after its injection and that the effect lasts approximately 20 minutes. minutes, which was expected since peptides are highly sensitive to peptidases and therefore are rapidly degraded in tissues (GANTE, J. (1994). Peptidomimetics-Tailored Enzyme Inhibitors Angew. Chem. Int. Ed. Engl. 33: 1699-1720).
A possibilidade de um efeito local para a Ang-(1-7) foi confirmada pelos pesquisadores da presente invenção, uma vez que foi descoberta que a administração do peptídeo na pata esquerda não alterou a iperalgesia avaliada na pata contralateral. Em nossos experimentos, a PGE2 foi administrada nas duas patas traseiras e verificamos que a dose de 4 μg, responsável pela reversão total da hiperalgesia produzida pela PGE2, não causou efeito não local. The possibility of a local effect for Ang- (1-7) was confirmed by the researchers of the present invention as it was found that administration of the peptide to the left paw did not alter the evaluated iperalgesia in the contralateral paw. In our experiments, PGE 2 was administered on both hind legs and we found that the 4 μg dose responsible for the total reversal of PGE 2 produced hyperalgesia did not cause a non-local effect.
O A-779 foi descrito como antagonista específico para as ações da Ang-(1-7) por SANTOS et al. (1994). Neste trabalho, o A-779 bloqueou o efeito antidiurético da Ang-(1-7), bem como as alterações na pressão arterial, provocadas pela injeção do heptapeptídeo no bulbo dorsomedial e ventrolateral; antagonistas dos receptores A^ e AT2, não foram capazes de bloquear tais ações da Ang-(1-7). Por outro lado, o A- 779 não foi capaz de inibir o efeito pressor, dipsinogênico e miotrófico da Ang II. Além disso, o A-779 não alterou o efeito antidiurético da vasopressina ou os eventos contráteis causados pela administração de Ang III, bradicinina ou substância P no rato de íleo isolado. Estudos com a técnica de binding vieram a dar suporte a estes resultados, indicando que o A-779 é um antagonista potente e seletivo (SANTOS, R.A.S.; CANAPGNOLE-SANTOS, MJ. (1994) Central and peripheral actions of angiotensin-(1-7). Braz. J. Med. Biol. Res. 27: 1033-47). A-779 has been described as a specific antagonist for Ang- (1-7) actions by SANTOS et al. (1994). In this study, A-779 blocked the antidiuretic effect of Ang- (1-7), as well as changes in blood pressure caused by heptapeptide injection into the dorsomedial and ventrolateral bulb; A 2 and AT 2 receptor antagonists have not been able to block such Ang- (1-7) actions. On the other hand, A-779 was not able to inhibit the pressor, dipsinogenic and myotrophic effect of Ang II. In addition, A-779 did not alter the antidiuretic effect of vasopressin or the contractile events caused by the administration of Ang III, bradykinin or substance P in isolated ileum rat. Studies with the binding technique have supported these results, indicating that A-779 is a potent and selective antagonist (SANTOS, RAS; CANAPGNOLE-SANTOS, MJ. (1994). Central and peripheral actions of angiotensin- (1- 7) .Braz. J. Med. Biol. Res. 27: 1033-47).
Com a caracterização do MasR e definição da Ang(1-7) como o seu ligante endógeno, foi confirmado que o A-779 é o bloqueador específico para o MasR (SANTOS, R.A.S.; SIMOES E SILVA, A.C. WALTHER, T. (2003) Angiotensin-(1-7) is an endogenous ligand for the G protein-coupled receptor Mas. Proc. Natl Acad. Sei USA 100: 8258-8263). Os resultados apresentados na presente invenção apontam para a presença desse receptor nos terminais periféricos dos neurónios sensoriais, posto que o A-779 reverteu totalmente o efeito antinociceptivo da Ang-(1-7). Dessa forma, acredita-se que os outros receptores angiotensinérgicos não estejam envolvidos e o MasR seria o principal responsável na ação antinociceptiva da Ang-(1- 7). Ao ser administrado isoladamente, o A-779 (8 μg/pata) não apresenta efeito hiperalgésico ou antinociceptivo.  By characterizing MasR and defining Ang (1-7) as its endogenous ligand, it has been confirmed that A-779 is the MasR specific blocker (SANTOS, RAS; SIMOES AND SILVA, AC WALTHER, T. (2003). ) Angiotensin- (1-7) is an endogenous ligand for the G protein-coupled receptor Mas. Proc. Natl Acad. Sci USA 100: 8258-8263). The results presented in the present invention point to the presence of this receptor at the peripheral terminals of sensory neurons, since A-779 totally reversed the antinociceptive effect of Ang- (1-7). Thus, it is believed that the other angiotensinergic receptors are not involved and MasR would be the main responsible for the antinociceptive action of Ang- (1-7). When administered alone, A-779 (8 μg / paw) has no hyperalgesic or antinociceptive effect.
No sistema nervoso e em outros tecidos, o NO age como um segundo mensageiro com capacidade de difundir-se através das membranas celulares, sendo um ativador da enzima guanililciclase solúvel, levando ao aumento dos níveis de GMPc intracelular. O óxido nítrico, elevando os níveis de GMPc, pode ativar diferentes tipos de canais de K+ em diferentes tipos de tecidos (KUBO, M.; NKAYA, Y. MATSUOKA, S.; SAITO, K.; KURODA, Y. (1994) Atrial natriuretic fator and isosorbide dinitrate modulcate the gating of ATP sensitive K channels in cultured vascular smooth cells. Cir. Res. 74: 470-476). A demonstração de que a antinocicepção periférica induzida pelo nitroprussiato de sódio de DbGMP ocorre pela ativação de K+ ATP estabeleceu a ligação entre a via L-arginina/NO/GMPc/K+ ATp (SOARES, A.C.; DUARTE, I.D.G. (2001) Dibutyryl-cyclic GMP induces peripheral antinociception via activation of ATP-sensitive K+ channels in the PGE2-induced hyperalgesic paw. British J. Pharmacol. 134: 127-31 ). In the nervous system and other tissues, NO acts as a second messenger with the ability to diffuse across cell membranes. a soluble guanylcyclase enzyme activator, leading to increased intracellular cGMP levels. Nitric oxide, by increasing cGMP levels, can activate different types of K + channels in different tissue types (KUBO, M .; NKAYA, Y. MATSUOKA, S .; SAITO, K .; KURODA, Y. (1994). ) Atrial natriuretic factor and isosorbide dinitrate modulating the gating of sensitive ATP channels in cultured vascular smooth cells. Cir. Res. 74: 470-476). The demonstration that DbGMP sodium nitroprusside-induced peripheral antinociception occurs by activation of K + A TP has established the link between the L-arginine / NO / GMPc / K + AT p pathway (SOARES, AC; DUARTE, IDG (2001 ) Dibutyryl-cyclic GMP induces peripheral antinociception via activation of ATP-sensitive K + channels in the PGE2-induced hyperalgesic paw (British J. Pharmacol. 134: 127-31).
As ações da Ang-(1-7) têm se mostrado estreitamente relacionadas à liberação de NO. Vários trabalhos, por exemplo, verificaram, através da utilização de inibidores da NOs (L-NOarg e o L-NAME), a participação NO na ação vasodilatadora dependente do endotélio exercida pela Ang(1 -7) (BROSNIHAN, K.B. (1998) Effect of the angiotensin-(1-7) peptide on nitric oxide release. Am. J. Cardiol. 82(10A): 17S-19S).  Ang- (1-7) actions have been closely related to NO release. Several studies, for example, have verified, through the use of NOs inhibitors (L-NOarg and L-NAME), the NO participation in Ang (1-7) endothelium-dependent vasodilatory action (BROSNIHAN, KB (1998) Effect of the angiotensin- (1-7) peptide on nitric oxide release (Am. J. Cardiol. 82 (10A): 17S-19S).
O envolvimento da via L-arginina/NO/GMPc/ K+ ATP na antinocicepção vem sendo demonstrado em vários trabalhos. Estudos realizados no laboratório dos pesquisadores da presente invenção constataram o envolvimento dessa via na antinocicepção periférica induzida por algumas substâncias, como: o agonista delta opióide SNC80 (PACHECO, D. F.; DUARTE, I. D. (2005) Delta-opioid receptor agonist SNC80 induces peripheral antinociception via activation of ATP-sensitive K+ channels. Eur J Pharmacol. 512(1): 23-8); a bremazocina, um agonista kappa opióide (AMARANTE, L.H.; DUARTE, I.D. (2002) The kappa-opioid agonist (+/-)-bremazocine elicits peripheral antinociception by activation of the L-arginine/nitric oxide/cyclic GMP pathway. Eur. J. Pharmacol. 454(1 ): 19-23); do antiinflamatório não esferoidal diclofenaco (ALVES, D.P; DUARTE, I.D. (2002) Involvement of ATP-sensitive K(+) channels in the peripheral antinociceptive effect induced by dipyrone. Eur. J. Pharmacol. 444 (1-2): 47-52). The involvement of the L-arginine / NO / cGMP / K + ATP pathway in antinociception has been demonstrated in several studies. Studies conducted in the laboratory of the researchers of the present invention found the involvement of this pathway in peripheral antinociception induced by some substances, such as: the opioid delta agonist SNC80 (PACHECO, DF; DUARTE, ID (2005). activation of ATP-sensitive K + channels Eur J Pharmacol 512 (1): 23-8); bremazocine, an opioid kappa agonist (AMARANTE, LH; DUARTE, ID (2002) The kappa-opioid agonist (+/-) - bremazocine elicits peripheral antinociception by activation of the L-arginine / nitric oxide / cyclic GMP pathway. Eur. J. Pharmacol 454 (1): 19-23); diclofenac non-spheroidal anti-inflammatory drug (ALVES, DP; DUARTE, ID (2002) Involvement of ATP-sensitive K (+) channels in the peripheral antinociceptive effect induced by dipyrone. Eur. J. Pharmacol. 444 (1-2): 47- 52).
Na presente invenção revela-se a participação da referida via na antinocicepção mediada por Ang-(1-7), a partir da inibição da NOs (L-NOarg), da guanilato ciclase (ODQ) e dos K+ ATp (glibenclamida). The participation of said pathway in Ang- (1-7) -mediated antinociception is disclosed by inhibition of NOs (L-NOarg), guanylate cyclase (ODQ) and K + AT p (glibenclamide). .
Os bloqueadores da NOs sintase, como o L-NAME, o L-NOarg e o L-NMMA, são amplamente utilizados como ferramenta farmacológica para o estudo das ações desta enzima na analgesia. Entretanto, DUARTE & FERREIRA demonstraram que o L-NAME possui sérias limitações enquanto inibidor específico da via L- arginina/NO/GMPc, pois apresenta efeito antinociceptivo, ao ser administrado isoladamente, por ser um estimulador da mesma via (DUARTE, I. D.; FERREIRA, S. H. (1992)The molecular mechanism of central analgesia induced by morphine or carbachol and the L-arginine-nitric oxide-cGMP pathway. Eur J Pharmacol. 221 (1): 171- 4). NOs synthase blockers, such as L-NAME, L-NOarg and L-NMMA, are widely used as a pharmacological tool to study the actions of this enzyme in analgesia. However, DUARTE & FERREIRA demonstrated that the L-NAME has serious limitations as a specific inhibitor of the L-arginine / NO / cGMP pathway as it has an antinociceptive effect when administered alone as a stimulant of the same pathway (DUARTE, ID; FERREIRA, SH (1992). of central analgesia induced by morphine or carbachol and the L-arginine-nitric oxide-cGMP pathway Eur J Pharmacol 221 (1): 171-4).
Dessa forma, o L-NAME pode ser considerado um fármaco inadequado para o estudo da analgesia, podendo ocasionar erros na interpretação dos resultados. Nessa intenção, verificamos a participação da NOs na analgesia induzida por Ang-(1-7) utilizando o L-NOarg.  Thus, L-NAME can be considered an inappropriate drug for the study of analgesia and may cause errors in the interpretation of results. In this intention, we verified the participation of NOs in Ang- (1-7) induced analgesia using L-NOarg.
O ODQ é um inibidor específico da guanililciclase solúvel sintetizado por sem atividade sobre guanililciclase de membrana ou sobre adenililciclase. Não apresenta efeitos sobre a isoformas da NOs, nem sobre o NO (GARTHWAITE, J.; SOUTHAM, ODQ is a specific soluble guanylcyclase inhibitor synthesized by no activity on membrane guanylcyclase or adenylyl cyclase. Has no effect on NOs isoforms or NO (GARTHWAITE, J .; SOUTHAM,
E. ; BOULTON, C. L; NIELSEN, E. B.; SCHMIDT, K,; MAYER, B. (1995) Potent and selective inhibition of nitric oxide-sensitive guanylyl cyclase by 1 H-[1 ,2,4]oxadazolo[4,3- a]quinoxalin-1-one. Am. Soe. Pharmacol. Exp. Ther. 48: 184-188). Duas outras substâncias são frequentemente empregadas como ferramentas farmacológicas para a inibição da gualinilciclase solúvel: o LY- 83583 e o azul de metileno. Já o azul de metileno é, atualmente, considerado um fraco inibidor da guanililciclase solúvel, mais eficiente na liberação de ânions superóxido e inibição da NOs (MAYER, B.; BRUNNER, F.; SCHIMIDT, K. (1993) Inhibition of nitric oxide synthesis by methylene blue. Biochem. Parmacol. 45: 2547-2549). Portanto, o ODQ é considerado, dentre as referidas substâncias, a melhor ferramenta farmacológica para o estudo das ações da guanililciclase solúvel. Nessa perspectiva, alguns estudos têm utilizado o ODQ na investigação das ações da NOs sobre a analgesia (BRITO, G.A.; SACHS, D.; CUNHA,AND. ; BOULTON, C.L; Nielsen, E. B .; SCHMIDT, K; MAYER, B. (1995) Potent and selective inhibition of nitric oxide-sensitive guanylyl cyclase by 1 H- [1,2,4] oxadazolo [4,3-a] quinoxalin-1-one. Am. Sound. Pharmacol. Exp. Ther. 48: 184-188). Two other substances are often employed as pharmacological tools for inhibiting soluble gualinyl cyclase: LY-83583 and methylene blue. Methylene blue is now considered a weak inhibitor of soluble guanylcyclase, more efficient in releasing superoxide anions and inhibiting NOs (MAYER, B.; BRUNNER, F .; SCHIMIDT, K. (1993) Inhibition of nitric oxide synthesis by methylene blue Biochem Parmacol 45: 2547-2549). Therefore, ODQ is considered, among these substances, the best pharmacological tool for the study of the actions of soluble guanylcyclase. From this perspective, some studies have used ODQ to investigate NOs actions on analgesia (BRITO, G.A .; SACHS, D .; CUNHA,
F. Q.; VALE, M.L.; LOTUFO, C.M.; FERREIRA, S.H.; RIBEIRO, R.A.(2006) Peripheral antinociceptive effect of pertussis toxin: activation of the arginine/NO/cGMP/PKG/ATP- sensitive K channel pathway). F. Q .; VALE, M.L .; LOTUFO, C.M .; FERREIRA, S.H .; RIBEIRO, R.A. (2006) Perinatal antinociceptive effect of pertussis toxin: activation of the arginine / NO / cGMP / PKG / ATP-sensitive K channel pathway).
Os resultados da presente invenção demonstraram que a sulfoniluréia glibenclamida reverteu, de forma dose-dependente, o efeito antinociceptivo periférico induzido pela Ang-(1-7) ao ser injetada cinco minutos antes desses agentes. Além disso, a referida substância não causou nenhum efeito hiperalgésico ou antinociceptivo quando administrada isoladamente. A sensibilidade às sulfoniluréias é comumente utilizada para caracterizar os K+ ATp (BABENCO, A.P.; AGUILAR-BRYAN, L; BRYAN, J. (1998) A view of Sur/KIR 6.X ATP channels. Annu. Rev. Physiol. 60: 667-687). Essas drogas bloqueiam especificamente estes canais, sem apresentar efeito em canais para potássio ativados por cálcio ou dependentes de voltagem (EDWARDS, G.; WESTON, A. H. (1993) The pharmacology of ATP-sensitive potassium channels. Annu. Rev. Pharmacol. Toxicol. 33: 597-637). A reversão do efeito nociceptivo mediante a administração intraplantar de glibenclamida indica a participação de K+ ATp na ação antinociceptiva da Ang-(1 -7). The results of the present invention demonstrated that glibenclamide sulfonylurea dose-dependently reversed the Ang- (1-7) -induced peripheral antinociceptive effect by being injected five minutes prior to these agents. Furthermore, said substance did not cause any hyperalgesic or antinociceptive effect when administered alone. Sensitivity to sulfonylureas is commonly used to characterize K + AT p (BABENCO, AP; AGUILAR-BRYAN, L.; BRYAN, J. (1998). A view of Sur / KIR 6.X ATP channels. Annu. Rev. Physiol. 60: 667-687). These drugs specifically block these channels, with no effect on calcium-activated or voltage-dependent potassium channels (EDWARDS, G.; WESTON, AH (1993). The pharmacology of ATP-sensitive potassium channels. Annu. Rev. Pharmacol. Toxicol. 33: 597-637). Reversal of the nociceptive effect upon intraplantar administration of glibenclamide indicates the participation of K + AT p in the antinociceptive action of Ang- (1-7).
Os resultados mostrados na presente invenção estão de acordo com a literatura que evidencia a ação antinociceptiva de várias substâncias, relacionada com a ativação de K+ATP- administração de sulfoniluréias antagonizou o efeito antinociceptivo central (WILD, K.D.; VANDERAH, T.; MOSBERG, H.I.; PORRECA, F. (1991 ) Opioid delta receptor subtypes are associated with different potassiem channels. Eur. J. Pharmacol. 193(1 ): 135-6) e periférico, (RODRIGUES, A R. A.; DUARTE I.D.G. (2000) The peripheral antinociceptive effect induced by morphine is associated with ATP-sensitive K+ channels. Brít. J. of Pharmacol. 129: 1 10-14), induzido pela morfina; o efeito antinociceptivo periférico induzido pelo NPS e DbGMPG (SOARES, A.C.; DUARTE, I.D.G. (2001 ) Dibutyryl-cyclic GMP induces peripheral antinociception via activation of ATP-sensitive K+ channels int the PGE2-induced hyperalgesic paw. British J. Pharmacol. 134: 127-31 ); a ação antinociceptiva central do agonista R-PIA do receptor A-1 de adenosina (OCANA, M.; BAEYENS, J.M. (1994) Role of ATP-sensitive K+ channels in antinociception induced by R-PIA, an adenosine A1 receptor agonist. Naunyn Schmiedebergs Arch Pharmacolol. 350: 57-62); a ação antinociceptiva induzida pela prolactina (SHEWADE, D.G.; RAMASWAMY, S. (1995) Prolactin induced analgesia is dependent on ATP sensitive potassium channels. Clin. Exp. Pharmacol. Physiol. 22(9): 635-636); o efeito antinociceptivo central de vários agonistas do receptor 5-HT1A de serotonina (8-OH-DPAT, buspirona, lesopitron e tandospirona). The results shown in the present invention are consistent with the literature showing the antinociceptive action of various substances related to K + ATP activation. Sulfonylurea administration antagonized the central antinociceptive effect (WILD, KD; VANDERAH, T .; MOSBERG, HI; PORRECA, F. (1991) Opioid delta receptor subtypes are associated with different potassium channels Eur. J. Pharmacol. 193 (1): 135-6) and peripheral, (RODRIGUES, A RA; DUARTE IDG (2000) The peripheral antinociceptive effect induced by morphine is associated with ATP-sensitive K + channels (Br. J. of Pharmacol. 129: 110-14), morphine-induced; the peripheral antinociceptive effect induced by NPS and DbGMP G (SOARES, AC; DUARTE, IDG (2001) Dibutyryl-cyclic GMP induces peripheral antinociception via activation of ATP-sensitive K + channels in the PGE2-induced hyperalgesic paw. British J. Pharmacol. 134 : 127-31); the central antinociceptive action of adenosine receptor A-1 agonist R-PIA (OCANA, M .; BAEYENS, JM (1994) Role of ATP-sensitive K + channels in antinociception induced by R-PIA, an adenosine A1 receptor agonist. Naunyn Schmiedebergs Arch Pharmacolol 350: 57-62); prolactin-induced antinociceptive action (SHEWADE, DG; RAMASWAMY, S. (1995) Prolactin induced analgesia is dependent on ATP sensitive potassium channels. Clin. Exp. Pharmacol. Physiol. 22 (9): 635-636); the central antinociceptive effect of various serotonin 5-HT 1A receptor agonists (8-OH-DPAT, buspirone, lesopitron and tandospirone).
Vários trabalhos evidenciam a participação do sistema opióide na hipoalgesia mediada pelo SRA. Foi demonstrado que a injeção intracerebroventricular de Ang-ll, bem como a analgesia por estresse eram bloqueadas por salarasina, um antagonista de receptores para angiotensina, e naloxona, evidenciando a participação do sistema opioidérgico (HAULICA, I.; NEAMTU C; STRATONE, A.; PETRESCU, G.; BRANISTEANUY, D.; SALTINEANU S. (1986). Evidence for the involvement of cerebral renin-angiotensin system (RAS) in stress analgesia. Pain 27: 237-45).  Several studies show the participation of the opioid system in SARS-mediated hypoalgesia. Intracerebroventricular injection of Ang-11 as well as stress analgesia were shown to be blocked by salarasine, a receptor antagonist for angiotensin, and naloxone, evidencing the participation of the opioid system (HAULICA, I .; NEAMTU C; STRATONE, A PETRESCU, G; BRANISTEANUY, D; SALTINEANU S. (1986) Evidence for involvement of cerebral renin-angiotensin system (RAS) in stress analgesia. Pain 27: 237-45).
O estudo realizado por RAGHAVENDRA et aí. (1999) mostra que a injeção intratecal de Ang II produz analgesia passível de reversão com o uso e losartan ou naloxona (RAGHAVENDRA V.; CHOPRA K.; KULKARNI S. K. (1999) Brain renin angiotensin system (RSA) in stress-induced analgesia and impaired retention. Peptides 20: 335-342). Além disso, foi verificado por TOMA (1997) que a injeção intratecal de Ang-II inibe a transmissão do estímulo térmico através de um mecanismo que envolve receptores ΑΤΊ e peptídeos opióides, posto que o efeito analgésico foi revertido por naloxona e losartan (TOMA N.; SGAMBATO, V.; COUTURE, R. (1997) Effect of angiotensin II on a spinal nociceptive reflex in the rat: receptor and mechanism of action. Life Sei. 61 : 503-13). Entretanto, nos resultados da presente invenção não encontramos evidências a respeito da participação do sistema de opióides endógenos no efeito antinociceptivo periférico induzido por Ang-(1-7), uma vez que a administração de intraplantar de naloxona, um antagonista de receptores opióides, em uma dose utilizada normalmente em nosso laboratório para reverter a antinocepção periférica induzida pela morfina (RODRIGUES, A R. A.; DUARTE I.D.G. (2000) The peripheral antinociceptive effect induced by morphine is associated with ATP-sensitive K+ channels. Brit. J. of Pharmacol. 129: 110-14), não antagonizou o efeito antinociceptivo do heptapetídeo estudado. The study by RAGHAVENDRA et al. (1999) shows that intrathecal injection of Ang II produces reversible analgesia with the use of losartan or naloxone (RAGHAVENDRA V .; CHOPRA K .; KULKARNI SK (1999). Brain renin angiotensin system (RSA) in stress-induced analgesia and impaired retention. Peptides 20: 335-342). In addition, it was found by TOMA (1997) that intrathecal injection of Ang-II inhibits the transmission of thermal stimulus through a mechanism involving ΑΤΊ receptors and opioid peptides, as the analgesic effect was reversed by naloxone and losartan (TOMA N SGAMBATO, V; COUTURE, R. (1997) Effect of angiotensin II on a spinal nociceptive reflex in the rat: receptor and mechanism of action. Life Sci. 61: 503-13). However, in the results of the present invention we found no evidence regarding the participation of the endogenous opioid system in the Ang- (1-7) -induced peripheral antinociceptive effect, since the administration of intraplantar naloxone, an opioid receptor antagonist, in a dose commonly used in our laboratory to reverse morphine-induced peripheral antinoception (RODRIGUES, A RA; DUARTE IDG (2000) The peripheral antinociceptive effect induced by morphine is associated with ATP-sensitive K + channels. : 110-14) did not antagonize the antinociceptive effect of the heptapetide studied.
Na patente WO03039434A2 (WO03039434A3), Millán, Dos Santos et. al. (2003) desenvolveram uma invenção na qual relata a caracterização do processo de preparação de formulações do peptídeo Angiotensina-(1-7) e seus análogos, agonistas e antagonistas usando as ciclodextrínas, seus derivados, lipossomas e os polímeros biodegradáveis e/ou misturas desses sistemas e/ou dos produtos derivados, que podem ser utilizados no tratamento de diversas patologias como hipertensão arterial, outras doenças cardiovasculares e suas complicações, feridas, queimaduras, eritemas, tumores, diabetes melitus dentre outras.  In WO03039434A2 (WO03039434A3), Millán, Dos Santos et. al. (2003) developed an invention in which he describes the characterization of the process of preparing Angiotensin- (1-7) peptide formulations and their analogs, agonists and antagonists using cyclodextrins, their derivatives, liposomes and biodegradable polymers and / or mixtures thereof. systems and / or derived products, which can be used to treat various conditions such as high blood pressure, other cardiovascular diseases and their complications, wounds, burns, erythema, tumors, diabetes mellitus and others.
A patente de número WO06128266A2 (WO06128266A3), Dos Reis, Millán et. al. (2006) apresenta uma invenção que caracteriza-se pela composição farmacêutica do peptídeo Angiotensina-(1-7) e seus análogos, agonistas e antagonistas para uso no controle de funções do sistema reprodutivo.  WO06128266A2 (WO06128266A3), Dos Reis, Millán et. al. (2006) discloses an invention characterized by the pharmaceutical composition of the Angiotensin- (1-7) peptide and its analogs, agonists and antagonists for use in controlling reproductive system functions.
Já o documento de patente WO03072059A2 (WO03072059A3), Tallant, Gallagher, Ferrario (2003) descreve uma tecnologia que faz uso do peptídeo Angiotensina-(1-7) e seus agonistas como uma terapia anti-câncer.  WO03072059A2 (WO03072059A3), Tallant, Gallagher, Ferrario (2003) describes a technology that makes use of the Angiotensin- (1-7) peptide and its agonists as an anti-cancer therapy.
O ponto principal da presente invenção é a demonstração, pela primeira vez, que Ang-(1-7) apresenta efeito antinociceptivo e a principal via envolvida nesta ação é L-arginina/NO/GMPc. Esta invenção contribui com a ampliação do conceito sobre o SRA, indicando que a Ang-(1-7), bem como o SRA, podem estar envolvidos no controle da sensibilidade dolorosa. Além disso, a descoberta abre perspectivas para a criação de uma nova classe de drogas analgésicas angiotensinérgicas, sem os efeitos colaterais próprios dos analgésicos opióides ou dos antiinflamatórios não esteroidais. The main point of the present invention is the demonstration for the first time that Ang- (1-7) has antinociceptive effect and the main pathway involved in this action is L-arginine / NO / cGMP. This invention contributes to the broadening of the concept of SARS, indicating that Ang- (1-7) as well as SARS may be involved in controlling pain sensitivity. In addition, the discovery opens perspectives for creation of a new class of angiotensinergic analgesic drugs, without the side effects of opioid analgesics or non-steroidal anti-inflammatory drugs.
A presente invenção pode ser mais claramente explicada, de forma não-restrita ou limitante, através dos exemplos a seguir:  The present invention may be more clearly explained, without limitation or limitation, by the following examples:
EXEMPLO 01: Animais de experimentação, drogas e solventes  EXAMPLE 01: Experimental Animals, Drugs and Solvents
Foram utilizados ratos da linhagem Wistar, machos, pesando entre 180 e 250 gramas, provenientes do Centro de Bioterismo do Instituto de Ciências Biológicas da UFMG (Cebio-ICB/UFMG). Esses animais foram mantidos em caixas plásticas com forragem, tendo livre acesso à ração e à água. No dia anterior a realização dos experimentos, os animais eram levados a uma sala termicamente controlada (23 a 25 °C) e com ciclo claro-escuro de 12 horas, para ambientalização. Todos os experimentos foram realizados no período da manhã, na fase clara. As drogas e os solventes utilizados foram:  Male Wistar rats weighing between 180 and 250 grams, from the Bioterismo Center of the UFMG Institute of Biological Sciences (Cebio-ICB / UFMG), were used. These animals were kept in plastic boxes with forage, having free access to feed and water. On the day before the experiments, the animals were taken to a thermally controlled room (23 to 25 ° C) and with a 12-hour light-dark cycle for ambientalization. All experiments were performed in the morning, in the clear phase. The drugs and solvents used were:
A. Prostaglandina E2 (PGE2, Calbiochem, EUA). Mantida, "no freezer", em solução-estoque, dissolvida em etanol, na concentração de 1 mg/ml. Imediatamente antes das injeções, a solução de PGE2 era diluída em salina fisiológica estéril e mantida a baixa temperatura em uma caixa de isopor com gelo. A. Prostaglandin E 2 (PGE 2 , Calbiochem, USA). Kept in the freezer in a stock solution dissolved in ethanol at a concentration of 1 mg / ml. Immediately prior to injections, the PGE 2 solution was diluted in sterile physiological saline and kept at low temperature in an ice-cold Styrofoam box.
B. Angiotensina-(1-7) (Ang-(1-7), Bachem, Alemanha). Dissolvida em salina, estéril.  B. Angiotensin- (1-7) (Ang- (1-7), Bachem, Germany). Dissolved in sterile saline.
C. Angiotensina II (Ang-ll, Bachen, Alemanha). Dissolvida em salina, estéril. C. Angiotensin II (Ang-11, Bachen, Germany). Dissolved in sterile saline.
D. A-779 (Bachen, Alemanha). Dissolvido em salina, estéril.  D. A-779 (Bachen, Germany). Dissolved in sterile saline.
E. Naloxona (Nx, Sigma, EUA). Dissolvida em salina, estéril.  E. Naloxone (Nx, Sigma, USA). Dissolved in sterile saline.
F. NG-Nitro-L-arginine (L-Noarg, RBI, EUA). Dissolvido em salina, estéril. F. N G -Nitro-L-arginine (L-Noarg, RBI, USA). Dissolved in sterile saline.
G. ODQ (Tocris, EUA). Mantido, "no freezer", em solução estoque, dissolvido em DMSO, na concentração de 10 mg/ml. Imediatamente antes das injeções, a solução de ODQ era diluída em salina fisiológica e mantida em baixa temperatura em uma caixa de isopor com gelo.  G. ODQ (Tocris, USA). Kept in freezer in stock solution dissolved in DMSO at a concentration of 10 mg / ml. Immediately prior to injections, the ODQ solution was diluted in physiological saline and kept at a low temperature in an ice-cold Styrofoam box.
H. Glibenclamida (Gli, Sigma, EUA). Dissolvida em solução de tween 85 a 2%  H. Glibenclamide (Gly, Sigma, USA). Dissolved in 2% tween 85 solution
EXEMPLO 02: Administração de drogas  EXAMPLE 02: Drug Administration
Todas as drogas foram administradas pela via subcutânea na superfície plantar da pata posterior do rato (via intraplantar). O volume de injeção foi de 0,05 mi para todas as drogas, exceto os agentes hiperalgésico e antinociceptivo que foram injetados num volume de 0,1 ml. Em todos os experimentos, foi utilizada a pata posterior direita dos animais, com exceção do protocolo utilizado para excluir a possibilidade de que um efeito sistémico do agente testado fosse responsável pelo efeito observado, para o qual ambas as patas traseiras foram utilizadas. All drugs were administered subcutaneously on the plantar surface of the rat hind paw (intraplantar). The injection volume was 0.05 ml for all drugs except hyperalgesic and antinociceptive agents which were injected in a volume of 0.1 ml. In all experiments, the right hind paw of the animals was used, except for the protocol used to exclude possibility that a systemic effect of the agent tested was responsible for the observed effect, for which both hind legs were used.
EXEMPLO 03: Teste Nociceptivo - Hiperalgesia da pata do rato  EXAMPLE 03: Nociceptive Test - Rat Paw Hyperalgesia
A medida da hiperalgesia foi realizada com a utilização do método de retirada da pata do rato submetida à compressão, descrito originalmente por RANDALL & SELITTO (1957). Esses autores desenvolveram uma técnica para medir a atividade antinociceptiva, baseada no princípio de que a inflamação aumenta a sensibilidade ao estímulo doloroso e que essa sensibilidade aumentada é susceptível à modificação por drogas. Para a realização das medidas de hiperalgesia, foi utilizado o aparelho algesimétrico da Ugo Basile (Itália).  Hyperalgesia was measured using the compressed rat paw withdrawal method originally described by RANDALL & SELITTO (1957). These authors developed a technique for measuring antinociceptive activity based on the principle that inflammation increases sensitivity to painful stimuli and that increased sensitivity is susceptible to drug modification. To perform the hyperalgesia measurements, the Ugo Basile (Italy) algesimetric device was used.
Durante o teste, o animal foi cuidadosamente mantido em posição horizontal sobre a bancada, por uma das mãos do experimentador, enquanto a pata sob teste é apresentada, por sua superfície plantar, à parte compressora do aparelho. A parte compressora do aparelho consiste em duas superfícies, sendo uma plana, sobre a qual se apóia a pata do animal, e outra cónica, com uma área de 1 ,75 mm2 na extremidade, por meio da qual é aplicada uma pressão na superfície plantar do rato. A intensidade da pressão aplicada aumenta a uma taxa constante de 32 g/s, mediante o acionamento de um pedal pelo experimentador. Ao observar a resposta nociceptiva do animal, o experimentador desaciona o pedal, interrompendo, assim, o aumento da pressão imposta à pata, sendo que o último valor, que corresponde ao limiar nociceptivo, fica indicado na escala do aparelho e expresso em gramas. During the test, the animal was carefully kept horizontally on the bench by one of the experimenter's hands, while the test leg is presented, by its plantar surface, to the compressing part of the apparatus. The compressing part of the apparatus consists of two surfaces, one flat, on which the animal's foot rests, and the other tapered, with an area of 1.75 mm 2 at the end, whereby pressure is applied to the surface. mouse plantar. The intensity of the applied pressure increases at a constant rate of 32 g / s by the pedal being operated by the experimenter. By observing the nociceptive response of the animal, the experimenter disengages the pedal, thus interrupting the increase of the pressure imposed on the paw. The last value, which corresponds to the nociceptive threshold, is indicated on the scale of the apparatus and expressed in grams.
O aprendizado para a medida do limiar nociceptivo na pata do rato, consiste no treinamento do experimentador para detectar o momento em que o animal percebe o estímulo doloroso e desenvolve uma reação. É considerada como resposta o instante inicial dessa reação. Nesse momento, observa-se um reflexo de retirada da pata e, eventualmente, o animal pode desenvolver uma fasciculação (sucessivas ondas de contração muscular, sensíveis ao tato através da pele do dorso). É importante ressaltar que o animal é ambientalizado ao aparelho no dia que antecede o teste. Na ambientalização submeter o animal é submetido a mesma situação que será vivenciada no dia do experimento. A pata do animal é apresentada ao aparelho várias vezes, até que o mesmo não manifeste mais uma reação de fuga. Esse procedimento é muito importante, pois permite uma melhor observação da resposta nociceptiva do animal, que durante o teste deve permanecer quieto, evitando que desenvolva uma reação aversiva simplesmente devido a situação estranha imposta a ele. EXEMPLO 04: Medida do limiar nociceptivo, hiperalgesia e antinocicepçãoLearning to measure the nociceptive threshold in the rat paw consists of training the experimenter to detect the moment when the animal perceives the painful stimulus and develops a reaction. The initial moment of this reaction is considered as a response. At this moment, a paw withdrawal reflex is observed and, eventually, the animal may develop a fasciculation (successive waves of muscle contraction, sensitive to the touch through the skin of the back). Importantly, the animal is acclimatized to the device on the day before the test. In environmentalization, the animal is subjected to the same situation that will be experienced on the day of the experiment. The animal's paw is presented to the device several times until it no longer manifests a flight reaction. This procedure is very important because it allows a better observation of the nociceptive response of the animal, which during the test should remain quiet, avoiding developing an aversive reaction simply due to the strange situation imposed on him. EXAMPLE 04: Measurement of nociceptive threshold, hyperalgesia and antinociception
Como já exposto, o limiar nociceptivo é definido como a pressão, aplicada à pata do animal, em que o mesmo apresenta a resposta nociceptiva descrita anteriormente. Na maioria dos experimentos foi realizado o acompanhamento temporal do limiar nociceptivo dos animais em função da ação de diferentes drogas frente ao desafio hiperalgésico induzido por prostaglandina E2. Considerando hiperalgesia como a diminuição do limiar nociceptivo, sua intensidade foi avaliada, em alguns experimentos, pela diferença (Δ) do limiar nociceptivo medido na terceira hora após a injeção de PGE2 em relação àquele valor obtido no início do experimento, antes de qualquer injeção (zero hora). Na Figura 1 observa-se um exemplo do cálculo do Δ do limiar nociceptivo. As already stated, the nociceptive threshold is defined as the pressure applied to the animal's paw, which presents the nociceptive response described above. In most experiments, the nociceptive threshold of the animals was monitored as a function of the action of different drugs against prostaglandin E 2 induced hyperalgesic challenge. Considering hyperalgesia as the nociceptive threshold decrease, its intensity was evaluated in some experiments by the difference (Δ) of the nociceptive threshold measured in the third hour after PGE 2 injection in relation to that value obtained at the beginning of the experiment, before any injection. (zero hour). Figure 1 shows an example of the calculation of nociceptive threshold Δ.
EXEMPLO 05: Esse exemplo descreve o efeito da Angiotensina (1-7) na hiperalgesia induzida pela administração intraplantar de diferentes doses de Prostaglandinas E2 (PGE2) EXAMPLE 05: This example describes the effect of Angiotensin (1-7) on intraplantar hyperalgesia induced by different doses of Prostaglandins E 2 (PGE 2 ).
Na Figura 2, observa-se que a administração intraplantar de PGE2 (0,25; 0,5; 1 e 2 μg) induziu diminuição dose-dependente do limiar nociceptivo em relação ao grupo controle (etanol 2% em solução salina). Esse efeito hiperalgésico foi detectado apenas a partir da segunda hora, com intensidade máxima na terceira hora após a administração de PGE2. A dose de 2 μg foi capaz de produzir hiperalgesia de maior intensidade, tendo sido escolhida para os experimentos subsequentes. A administração de Ang-(1-7) por via subcutânea intraplantar produziu antinocicepção de maneira dose-dependente (Figura 3), ou seja, doses crescentes de Ang-(1-7) foram capazes de reduzir de forma proporcional a hiperalgesia induzida por PGE2 (2 μg). Figure 2 shows that intra-plant administration of PGE 2 (0.25; 0.5; 1 and 2 μg) induced a dose-dependent decrease in nociceptive threshold in relation to the control group (2% ethanol in saline). This hyperalgesic effect was detected only after the second hour, with maximum intensity in the third hour after PGE 2 administration. The 2 μg dose was able to produce higher intensity hyperalgesia and was chosen for subsequent experiments. Intraplantar subcutaneous administration of Ang- (1-7) produced dose-dependent antinociception (Figure 3), ie increasing doses of Ang- (1-7) were able to proportionally reduce hyperalgesia induced by PGE 2 (2 μg).
EXEMPLO 06: Efeito induzido pela administração intraplantar do antagonista do receptor Mas A-779 sobre a antinocicepção periférica induzida pela Ang-(1-7)  EXAMPLE 06: Effect Induced by Intraplantar Administration of Mas A-779 Receptor Antagonist on Ang-Induced Peripheral Antinociception (1-7)
A Figura 4 mostra que doses crescentes do antagonista A-779 (2, 4 e 8 μg/pata) reverteram a antinocicepção induzida pela Ang-(1-7), na dose de 4 μg/pata. Além disso, observou-se que a maior dose, o A-779 é reverteu totalmente a ação antinociceptiva da Ang-(1-7), indicando a participação exclusiva do receptor angiotensinérgico Mas nesta resposta. O A-779 (8 μg) isoladamente, como apresentado nos controles, não apresenta efeito antinociceptivo ou hiperalgésico.  Figure 4 shows that increasing doses of antagonist A-779 (2, 4 and 8 μg / paw) reversed Ang- (1-7) -induced antinociception at the dose of 4 μg / paw. In addition, it was observed that the highest dose, A-779 is fully reversed the antinociceptive action of Ang- (1-7), indicating the exclusive participation of the angiotensinergic receptor Mas in this response. A-779 (8 μg) alone, as presented in the controls, has no antinociceptive or hyperalgesic effect.
EXEMPLO 07: Avaliação da participação da via L-arginina/NO/GMPc/K+ ATP Na antinocicepção periférica induzida por Ang-(1-7) Nessa parte do estudo, foram realizados experimentos utilizando inibidores da NOs (L-NOarg) e da guanililciclase (ODQ), com o objetivo de avaliar a participação dessa via na antinocicepção induzida pela Ang-(1-7). EXAMPLE 07: Evaluation of L-Arginine / NO / GMPc / K + ATP Pathway Participation in Ang- (1-7) -induced Peripheral Antinociception In this part of the study, experiments were performed using NOs (L-NOarg) and guanylylcyclase (ODQ) inhibitors to evaluate the participation of this pathway in Ang- (1-7) -induced antinociception.
EXEMPLO 08: Efeito induzido pela administração de L-NOarg sobre o efeito antinociceptivo periférico induzido por Ang-(1-7)  EXAMPLE 08: Effect induced by L-NOarg administration on Ang-induced peripheral antinociceptive effect (1-7)
Verifica-se, na Figura 5, a inibição dose-dependente da antinocicepção induzida por 4 μg de Ang-(1-7) pela da administração do L-NOarg (12, 18 e 24 μg). Este resultado indica que o NO é um importante mediador na atividade antinociceptiva da Ang-(1-7). Não foi verificada ação hiperalgésica ou nociceptiva do inibidor.  Figure 5 shows the dose-dependent inhibition of antinociception induced by 4 μg Ang- (1-7) by administration of L-NOarg (12, 18 and 24 μg). This result indicates that NO is an important mediator in antinociceptive activity of Ang- (1-7). There was no hyperalgesic or nociceptive action of the inhibitor.
EXEMPLO 09: Efeito induzido pela administração de ODQ sobre a antinocicepção periférica induzida por Ang-(1-7)  EXAMPLE 09: Effect induced by ODQ administration on Ang-induced peripheral antinociception (1-7)
O inibidor da guanil cliclase solúvel ODQ inibiu de forma dose dependente (25, 50 e 100 μg) o efeito antinociceptivo induzido pela Ang-(1-7) 4 μg, apontando para a atuação da via L-arginina/NO/GMPc (Figura 6). O ODQ não apresentou atividade hiperalgésica ou nociceptiva.  Soluble guanyl cliclase inhibitor ODQ dose-dependent (25, 50 and 100 μg) inhibited the antinociceptive effect induced by Ang- (1-7) 4 μg, pointing to the action of the L-arginine / NO / GMPc pathway (Figure 6). The ODQ did not present hyperalgesic or nociceptive activity.
EXEMPLO 10: Efeito induzido pela administração da glibenclamida sobre a atividade antinociceptiva periférica da Ang~(1-7)  EXAMPLE 10: Effect Induced by Glibenclamide Administration on Ang ~ Peripheral Antinociceptive Activity (1-7)
Na Figura 7, verifica-se que a glibenclamida, um bloqueador de canais para K+ sensíveis ao ATP, foi também reverteu o efeito antinociceptivo periférico induzido pela Ang-(1-7) na dose de 4 μg. A maior dose do inibidor produziu bloqueio completo e não apresentou efeito hiperalgésico ou antinociceptivo. Figure 7 shows that glibenclamide, an ATP-sensitive K + channel blocker, was also reversed by the Ang- (1-7) -induced peripheral antinociceptive effect at a dose of 4 μg. The highest dose of the inhibitor produced complete block and showed no hyperalgesic or antinociceptive effect.
EXEMPLO 10: Efeito induzido pela administração do antagonista de receptores opióides naloxona sobre a antinocicepção induzida pela Ang-(1-7) EXAMPLE 10: Effect induced by administration of the naloxone opioid receptor antagonist on Ang- (1-7) -induced antinociception
A administração intraplantar de naloxona, mesmo na dose de 100 μg, não induziu qualquer alteração na antinocicepção induzida pela Ang-(1-7) (Figura 8). Este resultado indica que os receptores opióides não estão envolvidos na antinocicepção induzida por Ang-(1-7), visto que a dose de 50 μg/pata de naloxona é capaz de reverter a antinocicepção induzida por morfina na dose de 200 μg/pata.. Intraplantar administration of naloxone, even at a dose of 100 μg, did not induce any change in Ang- (1-7) -induced antinociception (Figure 8). This result indicates that opioid receptors are not involved in Ang- (1-7) -induced antinociception, as the 50 μg / paw dose of naloxone is capable of reversing morphine-induced antinociception at the 200 μg / paw dose. .
BREVE DESCRIÇÃO DAS FIGURAS FIGURA 01. Parâmetros utilizados para o cálculo do D do limiar nociceptivo: As duas colunas da esquerda se referem à medida do limiar de nocicepção expresso em gramas (g) antes da administração intraplantar (0 hora) de carragenina (Cg, 250 mg) ou salina (Sal) e as duas colunas centrais três horas após essa administração (3a hora). As duas colunas da direita se referem às diferenças entre essas medidas (D do limiar nociceptivo). BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 01. Parameters used for the calculation of nociceptive threshold D: The two left columns refer to the measurement of the nociception threshold expressed in grams (g) before intraplantar (0 hour) administration of carrageenan (Cg, 250 mg) or saline (Sal) and the two central columns three hours after this administration (time 3). The two right columns refer to the differences between these measurements (nociceptive threshold D).
FIGURA 02. Desenvolvimento temporal do efeito hiperalgesia induzida pela injeção intraplantar de diferentes doses PGE2. Cada símbolo representa a média + E.P.M. da medida do limiar nociceptivo expresso em gramas (g), referente a 5 animais. * indica significância estatística (P<0,05) em relação ao grupo controle (etanol 2% em solução salina). FIGURE 02. Temporal development of the hyperalgesia effect induced by intra-plant injection of different PGE 2 doses. Each symbol represents the mean + SEM of the measurement of nociceptive threshold expressed in grams (g) for 5 animals. * indicates statistical significance (P <0.05) compared to the control group (2% ethanol in saline).
FIGURA 03. Desenvolvimento temporal induzido pela administração intraplantar de diferentes doses de Ang-(1-7) sobre a hiperalgesia induzida por PGE2. Cada símbolo representa a média + E.P.M. da medida do limiar nociceptivo expresso em gramas (g), referente a cinco animais. * indica significância estatística (P<0,05) em relação ao grupo controle (PGE + Sal). Ang = Ang-(1-7). FIGURE 03. Temporal development induced by intra-plantar administration of different doses of Ang- (1-7) on PGE 2 -induced hyperalgesia. Each symbol represents the mean + SEM of the measurement of nociceptive threshold expressed in grams (g) for five animals. * indicates statistical significance (P <0.05) in relation to the control group (PGE + Sal). Ang = Ang- (1-7).
FIGURA 04. Efeito induzido pela administração intraplantar de diferentes doses de A-779 sobre a antinocicepção periférica induzida por 4 μg/pata de Ang-(1-7). Cada barra representa a média + E.P.M. da medida do Δ do limiar nociceptivo expresso em gramas (g) da pata direita, referente a cinco animais. * e # indicam significância estatística (P<0,05) em relação aos grupos (PGE2+Sal+Sal) e [PGE2+Sal+Ang-(1-7)], respectivamente. Ang = Ang-(1-7). FIGURE 04. Effect induced by intraplantar administration of different doses of A-779 on peripheral antinociception induced by 4 μg / Ang- paw (1-7). Each bar represents the mean + SEM of the nociceptive threshold Δ measurement expressed in grams (g) of the right paw for five animals. * and # indicate statistical significance (P <0.05) in relation to the groups (PGE 2 + Sal + Salt) and [PGE 2 + Sal + Ang- (1-7)], respectively. Ang = Ang- (1-7).
FIGURA 05. Efeito induzido pela administração intraplantar de diferentes doses de L-NOarg sobre a antinocicepção periférica induzida por 4 μg/pata de Ang-(1-7). Cada barra representa a média + E.P.M. da medida do Δ do limiar nociceptivo expresso em gramas (g) da pata direita, referente a cinco animais. * e # indicam significância estatística (P<0,05) em relação aos grupos (PGE2+ Sal + Sal) e [PGE2+ Sal + Ang-(1-7)], respectivamente. Ang = Ang-(1-7). FIGURE 05. Effect induced by intra-plantar administration of different doses of L-NOarg on 4 μg / Ang- paw-induced peripheral antinociception (1-7). Each bar represents the mean + SEM of the nociceptive threshold Δ measurement expressed in grams (g) of the right paw for five animals. * and # indicate statistical significance (P <0.05) in relation to the groups (PGE 2 + Sal + Salt) and [PGE 2 + Sal + Ang- (1-7)], respectively. Ang = Ang- (1-7).
FIGURA 06. Efeito induzido pela administração intraplantar de diferentes doses de ODQ sobre a antinocicepção periférica induzida por 4 μg/pata de Ang-(1-7). Cada barra representa a média + E.P.M. da medida do Δ do limiar nociceptivo expresso em gramas (g) da pata direita, referente a cinco animais. * e # indicam significância estatística (P<0,05) em relação aos grupos (PGE2+ DMSO 20% + Sal) e [PGE2+ DMSO 20% + Ang-(1-7)], respectivamente. Ang = Ang-(1-7). FIGURE 06. Effect induced by intraplantar administration of different doses of ODQ on peripheral antinociception induced by 4 μg / paw of Ang- (1-7). Each bar represents the mean + SEM of the nociceptive threshold Δ measurement expressed in grams (g) of the right paw for five animals. * and # indicate statistical significance (P <0.05) in relation to the groups (PGE 2 + 20% DMSO + Salt) and [PGE 2 + 20% DMSO + Ang- (1-7)], respectively. Ang = Ang- (1-7).
FIGURA 07. Efeito induzido pela administração intraplantar de diferentes doses de glibenclamida (Gli) sobre a antinocicepção periférica induzida por 4 μg pata Ang-(1- 7). Cada barra representa a média + E.P.M. da medida do Δ do limiar nociceptivo expresso em gramas (g) da pata direita, referente a cinco animais. * e # indicam significância estatística (P<0,05) em relação aos grupos (PGE2 + Sal + Sal) e [PGE2 + Sal + Ang-(1-7)], respectivamente. Ang = Ang-(1-7). FIGURE 07. Effect induced by intraplantar administration of different doses of glibenclamide (Gli) on peripheral antinociception induced by 4 μg paw Ang- (1-7). Each bar represents the mean + SEM of the nociceptive threshold Δ measurement expressed in grams (g) of the right paw for five animals. * and # indicate statistical significance (P <0.05) in relation to the groups (PGE 2 + Salt + Salt) and [PGE 2 + Salt + Ang- (1-7)], respectively. Ang = Ang- (1-7).
FIGURA 08. Efeito induzido pela administração intraplantar de Nx sobre a antinocicepção periférica induzida por 4 μg/pata de Ang-(1-7). Cada barra representa a média + E.P.M. da medida do Δ do limiar nociceptivo expresso em gramas (g) da pata direita, referente a cinco animais. * indica significância estatística (P<0,05) em relação ao grupo controle (PGE2+ Sal + Sal). Ang = Ang-(1-7). FIGURE 08. Effect induced by intraplantar administration of Nx on peripheral antinociception induced by 4 μg / Ang- paw (1-7). Each bar represents the mean + SEM of the nociceptive threshold Δ measurement expressed in grams (g) of the right paw for five animals. * indicates statistical significance (P <0.05) in relation to the control group (PGE 2 + Salt + Salt). Ang = Ang- (1-7).

Claims

REIVINDICAÇÕES
1. Uso de formulações do peptídeo angiotensina-(1-7) ou seus análogos, agonistas e derivados caracterizado por ser na preparação de um medicamento para tratar condições dolorosas em mamíferos.  Use of formulations of the angiotensin- (1-7) peptide or its analogs, agonists and derivatives characterized in that it is in the preparation of a medicament for treating painful conditions in mammals.
2. Uso de formulações do peptídeo angiotensina-(1-7) ou seus análogos, agonistas e derivados, de acordo com a reivindicação 01 , caracterizado pela condição dolorosa ser de origem nociceptiva periférica.  Use of formulations of angiotensin- (1-7) peptide or analogs, agonists and derivatives thereof according to claim 01, characterized in that the painful condition is of peripheral nociceptive origin.
3. Uso de formulações de peptídeo angiotensina-(1-7) ou seus análogos, agonistas e derivados, de acordo com as reivindicações 01 e 02, caracterizadas por serem administradas pelas vias oral, intramuscular, intravenosa, transdérmica ou como dispositivos que possam ser implantados ou injetados.  Use of angiotensin- (1-7) peptide formulations or analogs, agonists and derivatives thereof according to claims 01 and 02, characterized in that they are administered by oral, intramuscular, intravenous, transdermal or as devices which may be implanted or injected.
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EP3110435A4 (en) * 2014-02-25 2017-12-06 Tarix Pharmaceuticals Ltd. Methods and compositions for the delayed treatment of stroke
JP2020514253A (en) * 2017-01-09 2020-05-21 アリゾナ ボード オブ リージェンツ オン ビハーフ オブ ザ ユニヴァーシティー オブ アリゾナ ANG (1-7) derivative oligopeptide for pain treatment
US11104706B2 (en) 2014-07-21 2021-08-31 Arizona Board Of Regents On Behalf Of The University Of Arizona Ang (1-7) derivative oligopeptides and methods for using and producing the same

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3110435A4 (en) * 2014-02-25 2017-12-06 Tarix Pharmaceuticals Ltd. Methods and compositions for the delayed treatment of stroke
US11104706B2 (en) 2014-07-21 2021-08-31 Arizona Board Of Regents On Behalf Of The University Of Arizona Ang (1-7) derivative oligopeptides and methods for using and producing the same
JP2020514253A (en) * 2017-01-09 2020-05-21 アリゾナ ボード オブ リージェンツ オン ビハーフ オブ ザ ユニヴァーシティー オブ アリゾナ ANG (1-7) derivative oligopeptide for pain treatment
EP3565575A4 (en) * 2017-01-09 2020-11-18 The Arizona Board of Regents On Behalf of the University of Arizona Ang (1-7) derviative oligopeptides for the treatment of pain
JP7235658B2 (en) 2017-01-09 2023-03-08 アリゾナ ボード オブ リージェンツ オン ビハーフ オブ ザ ユニヴァーシティー オブ アリゾナ ANG(1-7) derivative oligopeptides for pain therapy

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