**5. Potential and effective pharmaceutical applications**

### **5.1. Application of BPPs to treat CNS disorders and hypertension**

In the last few years, as presented here, it was possible to describe a number of new mechanisms of action for BRPs previously known only as potent ACE inhibitors. Taking this into account, many pharmaceutical applications could be possible suggested for these peptides solely based on the treatment of pathologies related with their targets, for instace the somatic ACE, AsS and so on.

Regarding AsS as a novel potential target for the development of new drugs based on BPPs structural model, it is worth mentioning that its action on L-arginine metabolims contributes to three main functions in the organism, depending on the cell or tissue type involved, including effects on detoxification of ammonia in the liver, production of L-arginine in the kidney to be distributed to the whole organism, and the synthesis of L-arginine for the production of NO in several other cells [94]. In addition to these three major functions, it has also been suggested that AsS plays an important role in neuromodulation by producing argininosuccinate [110].

that, contrary to what was thought for several decades, these peptides have different biological effects and therefore are an inexhaustible source of powerful biological tools not only for the study and discovery of new physiological pathways, but also as potentially useful compounds

Venom Bradykinin-Related Peptides (BRPs) and Its Multiple Biological Roles

http://dx.doi.org/10.5772/52872

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Therefore, a patent protecting the use of these oligopeptides capable of binding to diverse targets, determining the increase and the sustenance of nitric oxide (NO) production in mammalian cells by potentiating the endogenous argininosuccinate synthase activity present in animal cells and/or by increasing the intracellular bivalent free calcium ion in the cytosol of cells was filed (US20100035822). Pharmaceutical compositions containing one or more of these

NO is involved in vasodilatation and in many other physiological processes. Several lines of evidence have indicated that NO plays a complex and diverse role in the modulation of pain [122]. It has been shown that NO mediates the analgesic effect of opioids and other analgesic substances, opening opportunities of potential use of molecules able to regulate NO produc‐ tion in pain therapy. Modification of pre-existing analgesic and anti-inflammatory drugs by addition of NO-releasing moieties has been shown to improve the analgesic efficacy of these

NO donors have also been used with opioids to reduce pain in patients with cancer [124]. This strategy enhances the analgesic efficacy of morphine in patients with cancer pain, delaying the morphine tolerance and decreasing the incidence of the adverse effects of opioids [88, 125].

Nevertheless, the use of NO donors should be carefully evaluated, since the excessive levels of NO production can be deleterious to the organism [126]. Soon, keeping NO production at

Therefore, the BRPs that modulate AsS activity [20, 75] could be considered for pain treatment. The fact that this AsS activity interferes with L-arginine metabolism, a source of NO synthesis that has its own levels subjected to very precise mechanisms of physiological control, repre‐ sents the most likely target able to regulate NO production without generating undesired

Other family of BRPs, which should be considered in the treatment of pain and inflammation, are the antagonists of BK receptors (BIPs), namely helokinestatins. Although these BRPs were described as vasoactive peptides, due to their ability to antagonize the relaxation effect induced

Kinins formed following tissue trauma and in inflammatory processes, acting by means of the activation of B2 receptors, are among the most potent endogenous algogenic media‐ tors. Kinins through action on BK receptors can release a large number of inflammatory mediators, such as prostaglandins and neuropeptides such as neurokinins [128], that in turn amplify the nociceptive response. Therefore, these receptors play an important role in pain

by BK [35, 36], they could potencially be employed in the therapy of hyperalgesia.

peptides is also described and also disclosed in this same patent.

drugs, and also to reduce their side effects [123].

reactive by-products (review by [127]).

**5.2. Application of BIPs in the treatment of pain and inflammation**

a safe level, avoiding the deleterious threshold, is of particular interest.

for new drug development.

Due to its involvement in biochemical processes that generate physiological impacts on the organism, AsS is of great clinical value since its deficiency or excessive expression has been associated with some diseases, such as citrullinemia [111], hypertension [112, 113], and Alzheimer's disease [114, 115].

Since, AsS also participates in the L-arginine recycling, which contributes to the maintenance of NOS substrate,and AsS catalytic activity is considered the limiting step for NO production [116], the upregulation of this enzyme restores the balance of that system with consequent reduction in blood pressure [20].

Moreover, the identification of acetylcholine receptors as novel putative targets responsible for the vasodilatation promoted by the BRPs [19, 23, 75] also opens new avenues to the development of possible future therapeutic applications of BRPs related compounds for CNS diseases treatment.

Different experimental approaches demonstrate that acetylcholine muscarinic receptors are present in virtually all organs, tissues and cell types. The muscarinic receptors in the CNS are involved in the regulation of an extraordinary number of cognitive, behavioral, sensory, motor, and autonomous functions. Reduced or increased signalling of different subtypes of muscar‐ inic receptors are involved in the pathophysiology of several diseases of the CNS including Alzheimer's and Parkinson's diseases, depression, schizophrenia, and epilepsy [117].

The contribution of the muscarinic acetylcholine receptor, mAchR-M1, in the NO production stimulated by BPP-5a is therapeutically and scientifically interesting, since much effort has been undertaken in the search for mAchR-M1 agonists to treat cognitive disorders including the Alzheimer's disease [118, 119].

On other hand, the mAchR-M3 is mainly involved in the control of vascular tone. The main actions mediated by these peripheral muscarinic receptors include the reduction of HR, stimulation of glandular secretion, and smooth muscle relaxation [117]. Compounds that activate this receptor to promote vasorelaxation, such as pilocarpine, are used for the treatment of glaucoma, ocular hypertension [120, 121]. However, the BRPs as BPP-13a is not only able to activate mAchR-M3, but in the same time it acts modulating the AsS activity. Since both pathways contribute to the antihypertensive effects by controlling the NO production, BPP-13a represents a potent vasodilator compound with potential broad‐ er applications in the medicine [75].

Taking together, the comparison of the biological actions of BRPs found in the venom and brain of the pit viper *Bothrops jararaca* with those from different species, allowed us to describe that, contrary to what was thought for several decades, these peptides have different biological effects and therefore are an inexhaustible source of powerful biological tools not only for the study and discovery of new physiological pathways, but also as potentially useful compounds for new drug development.

Therefore, a patent protecting the use of these oligopeptides capable of binding to diverse targets, determining the increase and the sustenance of nitric oxide (NO) production in mammalian cells by potentiating the endogenous argininosuccinate synthase activity present in animal cells and/or by increasing the intracellular bivalent free calcium ion in the cytosol of cells was filed (US20100035822). Pharmaceutical compositions containing one or more of these peptides is also described and also disclosed in this same patent.

### **5.2. Application of BIPs in the treatment of pain and inflammation**

Regarding AsS as a novel potential target for the development of new drugs based on BPPs structural model, it is worth mentioning that its action on L-arginine metabolims contributes to three main functions in the organism, depending on the cell or tissue type involved, including effects on detoxification of ammonia in the liver, production of L-arginine in the kidney to be distributed to the whole organism, and the synthesis of L-arginine for the production of NO in several other cells [94]. In addition to these three major functions, it has also been suggested that AsS plays an important role in neuromodulation by producing

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

Due to its involvement in biochemical processes that generate physiological impacts on the organism, AsS is of great clinical value since its deficiency or excessive expression has been associated with some diseases, such as citrullinemia [111], hypertension [112, 113], and

Since, AsS also participates in the L-arginine recycling, which contributes to the maintenance of NOS substrate,and AsS catalytic activity is considered the limiting step for NO production [116], the upregulation of this enzyme restores the balance of that system with consequent

Moreover, the identification of acetylcholine receptors as novel putative targets responsible for the vasodilatation promoted by the BRPs [19, 23, 75] also opens new avenues to the development of possible future therapeutic applications of BRPs related compounds for CNS

Different experimental approaches demonstrate that acetylcholine muscarinic receptors are present in virtually all organs, tissues and cell types. The muscarinic receptors in the CNS are involved in the regulation of an extraordinary number of cognitive, behavioral, sensory, motor, and autonomous functions. Reduced or increased signalling of different subtypes of muscar‐ inic receptors are involved in the pathophysiology of several diseases of the CNS including

The contribution of the muscarinic acetylcholine receptor, mAchR-M1, in the NO production stimulated by BPP-5a is therapeutically and scientifically interesting, since much effort has been undertaken in the search for mAchR-M1 agonists to treat cognitive disorders including

On other hand, the mAchR-M3 is mainly involved in the control of vascular tone. The main actions mediated by these peripheral muscarinic receptors include the reduction of HR, stimulation of glandular secretion, and smooth muscle relaxation [117]. Compounds that activate this receptor to promote vasorelaxation, such as pilocarpine, are used for the treatment of glaucoma, ocular hypertension [120, 121]. However, the BRPs as BPP-13a is not only able to activate mAchR-M3, but in the same time it acts modulating the AsS activity. Since both pathways contribute to the antihypertensive effects by controlling the NO production, BPP-13a represents a potent vasodilator compound with potential broad‐

Taking together, the comparison of the biological actions of BRPs found in the venom and brain of the pit viper *Bothrops jararaca* with those from different species, allowed us to describe

Alzheimer's and Parkinson's diseases, depression, schizophrenia, and epilepsy [117].

argininosuccinate [110].

Applications

138

Alzheimer's disease [114, 115].

reduction in blood pressure [20].

the Alzheimer's disease [118, 119].

er applications in the medicine [75].

diseases treatment.

NO is involved in vasodilatation and in many other physiological processes. Several lines of evidence have indicated that NO plays a complex and diverse role in the modulation of pain [122]. It has been shown that NO mediates the analgesic effect of opioids and other analgesic substances, opening opportunities of potential use of molecules able to regulate NO produc‐ tion in pain therapy. Modification of pre-existing analgesic and anti-inflammatory drugs by addition of NO-releasing moieties has been shown to improve the analgesic efficacy of these drugs, and also to reduce their side effects [123].

NO donors have also been used with opioids to reduce pain in patients with cancer [124]. This strategy enhances the analgesic efficacy of morphine in patients with cancer pain, delaying the morphine tolerance and decreasing the incidence of the adverse effects of opioids [88, 125].

Nevertheless, the use of NO donors should be carefully evaluated, since the excessive levels of NO production can be deleterious to the organism [126]. Soon, keeping NO production at a safe level, avoiding the deleterious threshold, is of particular interest.

Therefore, the BRPs that modulate AsS activity [20, 75] could be considered for pain treatment. The fact that this AsS activity interferes with L-arginine metabolism, a source of NO synthesis that has its own levels subjected to very precise mechanisms of physiological control, repre‐ sents the most likely target able to regulate NO production without generating undesired reactive by-products (review by [127]).

Other family of BRPs, which should be considered in the treatment of pain and inflammation, are the antagonists of BK receptors (BIPs), namely helokinestatins. Although these BRPs were described as vasoactive peptides, due to their ability to antagonize the relaxation effect induced by BK [35, 36], they could potencially be employed in the therapy of hyperalgesia.

Kinins formed following tissue trauma and in inflammatory processes, acting by means of the activation of B2 receptors, are among the most potent endogenous algogenic media‐ tors. Kinins through action on BK receptors can release a large number of inflammatory mediators, such as prostaglandins and neuropeptides such as neurokinins [128], that in turn amplify the nociceptive response. Therefore, these receptors play an important role in pain transmission. BK produces a short-lived hyperalgesia, while des-Arg9-BK causes a longlasting hyperalgesia [1].

[5] Rocha ESM, Beraldo WT, Rosenfeld G. Bradykinin, a hypotensive and smooth mus‐ cle stimulating factor released from plasma globulin by snake venoms and by tryp‐

Venom Bradykinin-Related Peptides (BRPs) and Its Multiple Biological Roles

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However, most of the B2 receptor antagonists present partial agonist activity and fail to produce antinociception when given orally [1]. Non-peptide B2 receptor antagonists, although they are generally less potent when compared with Hoe 140, for instance, produce long-lasting oral antinociception with no evidence of partial agonistic activity [129-131]. In this way, further studies of BRPs applied in the pain treatment could provide valuable information for the development of novel peptidic or non-peptidic molecules to effectively relieve the pain of human patients.

The BRPs isolated from toad (*Bombina maxima*) defensive skin secretion, and their analogs thereof, prodrugs including the peptides, pharmaceutical compositions are protected by patent WO2004/068928. These BRPs and analogs thereof are antagonists of B2 receptor and they can be used to treat and/or prevent disorders associated with BK, including cardiovas‐ cular disorders, inflammation, asthma, allergic rhinitis, angiogenesis, pain and related pathologies.
