**3. Antinociceptive effects**

Of extreme importance for the organism, pain is an indicator of corporal integrity and has been considered since January 2000, by the Joint Commission on Accreditation on Health‐ care Organizations (JCAHO) as the fifth vital sign that should be assessed and recorded to‐ gether with other signals immediately after birth. According to the International Association for the Study of Pain (IASP), pain is defined as an unpleasant sensation and emotional expe‐ rience associated with actual or potential tissue damage. However, approximately one third of world population suffers from pathological persistent or recurrent pain, which is a com‐ mon complaint in patients with different diseases, and exerts great impact on their social life [13]. In these cases, treatment is a challenge for researchers and health professionals who constantly seek new therapeutic strategies, since most of these are inadequate or cause seri‐ ous side effects [14].

Analgesics and systemic conservative therapies are widely used for pain control. However, in many cases, especially in patients with neuropathic pain, more aggressive treatments are needed, which promote a significant clinical improvement but only in 30-50% of patients [15,16].

Although an injection of arthropod venoms is commonly reported to cause tonic pain and hyperalgesia, there is also evidence suggesting that these venoms might have antinocicep‐ tive effects on inflammation. Thus, nowadays, toxins isolated from arthropods are consid‐ ered powerful tools, since they have congruent targets of the impulse transmission of pain, and may provide an attractive alternative to opioid treatments.

### **3.1. Polypeptide toxins from Scorpion**

usage in some patients or may even function as a factor of impairment in people's quality of life. According to [6], none of antiepileptic drugs discovered in the last 20 years, was effi‐ cient to cure or even suppress seizures in epileptic patients. Therefore, there is a continued need for the discovery of novel drugs to treat most neurological and mental disorders [7].

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

This chapter will target the discussion of recent contributions of research on the compounds of arthropod venom, for the discovery of novel tools to study the functioning of the struc‐ tures of mammalian CNS, as well as the supply of novel alternatives to the treatment of neu‐ rological disorders. Among the major compounds, it will be highlighted those with the analgesic, anxiolytic, antiepileptic and neuroprotective effects, with emphasis on the most

**2. Main targets of the neuroactive compounds isolated from arthropod**

Venom isolated from bee, scorpion and spider have been used to the treatment of various diseases in Chinese and Korean traditional medicine, such as epilepsy, stroke, facial paraly‐ sis, arthritis, rheumatism, back pain, cancerous, tumors, and skin diseases [8-10]. Moreover, venoms of arthropod animals have been used to study various physiopathological process‐ es, and also offer opportunity to design and develop new therapeutic drugs [3,11,12] .

Arthropod venoms are rich in biologically active substances with different physiological ac‐ tions, specially the neurotoxins. So far, identified neurotoxins generally comprise the classes of peptides or acylpolyamines, acting with affinity and specificity over excitatory or inhibi‐ tory neurotransmissions (for revision see [12]. The actions of these compounds include the

ionotropic receptors for neurotransmitters as the excitatory neurotransmitter glutamate. At the presynaptic level, several studies have shown the interaction of arthropod neurotoxins with protein transporters of neurotransmitters, resulting in the facilitation or inhibition of

Of extreme importance for the organism, pain is an indicator of corporal integrity and has been considered since January 2000, by the Joint Commission on Accreditation on Health‐ care Organizations (JCAHO) as the fifth vital sign that should be assessed and recorded to‐ gether with other signals immediately after birth. According to the International Association for the Study of Pain (IASP), pain is defined as an unpleasant sensation and emotional expe‐ rience associated with actual or potential tissue damage. However, approximately one third of world population suffers from pathological persistent or recurrent pain, which is a com‐ mon complaint in patients with different diseases, and exerts great impact on their social life [13]. In these cases, treatment is a challenge for researchers and health professionals who

and Ca2+ ion channels, agonism or antagonism of metabotropic and

promising on preclinical or clinic evaluation.

, K+

**venoms**

Applications

92

interaction with Na+

**3. Antinociceptive effects**

their uptake.

The most studied Arthropod venom is extracted from the Asian scorpion *Mesobuthus mar‐ tensi* Karsch (BmK). It is composed of several toxins, and so far, ten have been described, which produce powerful antinociceptive effects. This is the case of the two β-excitatory antiinsect toxins BmK IT-AP (or Bm33-I) and BmK AngP1, two β- depressant anti-insect toxins BmK dITAP3 and BmK IT2, as well as six toxins yet without consensus classification, BmK AS, BmK AS1, BmK AGAP, BmK Ang M1, BmK AGP-SYPU1 and BmK AGP-SYPU2. These compounds probably belong to a family of peptides NaScTx that are composed of 60-76 ami‐ no acid residues with four disulfide bonds, the cysteine positions among these toxins are highly conserved [17,18]. Considering their structures, they might be able to bind to sodium channels impairing depolarization of the action potential in nerve and muscle, resulting in neurotoxicity [18], although it remains to be fully investigated.

The NaScTx family can be classified in at least two major families, α and β, according to the mode of action on Na+ channels [19]. The binding of α-toxins delays Nav channel inactiva‐ tion, while that of β-toxins shifts the membrane potential dependence of channel activation to more negative potentials. α and β-toxins also exhibit pharmacological preferences for mammals or insects sodium channels. Therefore, considering their pharmacological activi‐ ties, α and β NAScTx can be also divided into three groups:


Regarding the β-excitatory anti-insect toxins, BmK IT-AP (Insect Toxin-Analgesic Peptide), which was isolated in 1999, produces a potent antinociceptive effect in mouse-twisting mod‐ el, after i.v. injection [20]. The same toxin has also been sequenced by another group and named Bm K 33-I [21]. Later, Guan and colleagues [22] identified a novel toxin with analge‐ sic effects, BmK AngP1, which shows an evident analgesic effect with simultaneous excitato‐

ry insect toxicity, but is devoid of any toxicity on mice even at high dosages. The analgesic effect was assessed with a mouse-twisting model. The analgesic effect on mice of the AngP1 is at least 4-5 times weaker than that of IT-AP, but the toxicity to insects is twice as strong as that of IT-AP [20,22]

contribute to its analgesic activity, nine mutants of BmK AGAP were produced and tested. However, further studies are necessarily to elucidate the mechanism of action as well as to exploit its analgesic activity [36]. In relation to BmK Ang M1 [37], it also was reported to exhibit potential analgesic effect. Moreover, electrophysiological studies showed that BmK AngM1 at the concentration of 1 μM inhibited voltage-dependent Na+ current (INa) and voltage-de‐

It is important to note that the excitatory and depressant anti-insect toxins belong to differ‐ ent groups, which have distinct modes of interaction with receptors. Thus, one can infer that the analgesic effect of these peptides may have a molecular mode and mechanism different from that of insect toxicity. Still, the mechanisms by which these scorpion toxins can modu‐ late pain pathways remain to be clarified. According to [8], four different possibilities might

**iv.** pain alleviation is only apparent and results from misinterpretations that might

Another group of arthropods that have very promising antinociceptive compounds are spi‐ ders [41]. In 1996, Roerig & Howse reported the effect of ω-agatoxina IVA (Fig 1) isolated from funnel spider *Agelenopsis aperta* venom, against thermal stimulation in the tail flick test, when co-administrated with morphine intrathecal. Intrathecal injection of ω-agatoxin IVA (0.2 nmol/kg) also decreased the licking time in both the early and late response phases in a dose-dependent manner in the Formalin test [42]. The use of this peptide as an analgesic could be of particular benefit in patients tolerant or opioid-dependent, since this compound exhibits selectivity for the P/Q Ca2+ channels [43]. Other spider venom very promissory is the venom of the Brazilian armed spider *Phoneutria nigriventer*, the purified fraction 3 (PhTx3) contains 6 toxin isoforms (Tx3-1 to -6) [44,45] that target Ca2+ channels with different affinity patterns. Moreover, one toxin, Tx3-6 (Phα1β), demonstrated that it preferentially blocks the N-type calcium current [46] and produce a potent antinociceptive effect with higher therapeutic index [44]. Dalmolin and colleagues [45] showed that Tx3-3 (purified the same fraction) caused a short-lasting antinociceptive effect in the nociceptive pain test and a long-lasting antinociceptive effect in neuropathic pain models, without producing detecta‐ ble side effects. However, Tx3-3 did not change the inflammatory pain. Tx3-3 blockade of P/Q- and R-type Ca2+ channels and inhibit the glutamate release in rat brain cortical synapto‐ somes [47]. Other neurotoxin isolated from spider *Phoneutria nigriventer* is Phα1β, which is a potent toxin blocking neuronal voltage-sensitive Ca2+ channels. This peptide induced longer

current (IK), but had no effects on transient K+

New Perspectives in Drug Discovery Using Neuroactive Molecules From the Venom of Arthropods

channels involved in the pathway of pain,

current [37].

http://dx.doi.org/10.5772/ 52382

95

pendent delayed rectifier K+

**i.** peptides act directly Na+

**3.2. Polypeptide toxins from Spider**

**ii.** peptides modulate indirectly the pain sensation,

have occurred from animal models used.

**iii.** peptides also modulate other targets involved in pain pathway

antiallodynic effect than μ-conotoxin MVIIA and morphine in mice [48].

be described:

In relation of depressant toxins isolated from BmK venom, BmK IT2 has been more studied from the venom of BmK (Fig 1). Intraplantar injection of BmK IT2 inhibited thermal hyperal‐ gesia in carrageenan-treated rats and significantly prolonged paw withdrawal latency in normal rats [23]. This toxin also displays an inhibitory effect on the C component of the rat nociceptive flexion reflex by subcutaneous injection in vivo [24]. Peripheral or spinal deliv‐ ery of BmK IT2 suppressed formalin-induced nociceptive behaviors and c-Fos expression in spinal cord [25,26]. Both BmK IT2 and Bm K dIT-AP3 (depressant Insect Toxin-Analgesic Peptide 3) are toxic for insects, but not for mammals [27], and shows 86.7% of sequence simi‐ larity [23]. BmK dIT-AP3 also induces analgesia in the mouse-twisting model [18]. Using whole-cell patch clamp, it has been shown that BmK dIT-AP3 inhibits Nav currents of rat dorsal root ganglion (DRG) neurons, blocking more selectively the tetrodotoxin-resistant (TTX-R) component of the Na+ currents. These results suggest that the inhibition of the rat nociceptive flexion reflex by BmK dITAP3 may be attributed to modulation of the DRG's voltage-gated Na+ channels [24].

Wang and colleagues [28] isolated a new antinociceptive peptide, named BmK AGP-SYPU1. Recombinant BmK AGP-SYPU1 showed similar analgesic effects on mice compared to natu‐ ral when assayed using a mouse-twisting model [28]. More recently, BmK AGP-SYPU2 was purified and tested, also in mouse-twisting model. Sequence determination showed that the mature BmK AGP-SYPU2 peptide is composed of 66 amino acid residues, and BmK AGP-SYPU2 is identical to BmK alpha2 and BmK alphaTX11.

BmK AS had a strong analgesic effect on both visceral and somatic pain [29,30]. It relieves formalin-induced two-phase spontaneous flinching response and carrageenan-induced me‐ chanical hyperalgesia, probably by modulating the voltage-gated Na+ channels of sensory neurons [31,32]. Moreover, BmK AS showed activity nearly equivalent to that of morphine. Later, a new peptide that possesses 86.3% of similarity with BmK AS was identified. Both polypeptides have 66 amino acids cross-linked by four disulfide bridges [29]. In addition, these two peptides show a poor similarity with other known types of scorpion toxins. BmK AS and AS1 are not toxic against mammals and only have a weak toxicity to insects. BmK AS, then BmK AS1, have been found to significantly stimulate the binding of [3H]-ryano‐ dine to partially purified ryanodine receptors [33]. More recently, electrophysiological stud‐ ies have shown that they are able to inhibit Na+ currents in NG108-15 cells [34] and to depress TTX-sensitive and TTX-resistant Na+ currents in rat small DRG neurons. Interesting‐ ly, in rat models, BmK AS1 also displays antinociceptive effects according to [33]. These au‐ thors concluded that the effects could be mediated by the modulation of voltage-gated Na+ channels and they also suggested that BmK AS and BmK AS1 could form a new family of scorpion insect toxins.

BmK AGAP (antitumor-analgesic peptide), isolated in 2003, had strong inhibitory effect on both viscera and soma pain [35]. To evaluate the extent to which residues of the toxin core contribute to its analgesic activity, nine mutants of BmK AGAP were produced and tested. However, further studies are necessarily to elucidate the mechanism of action as well as to exploit its analgesic activity [36]. In relation to BmK Ang M1 [37], it also was reported to exhibit potential analgesic effect. Moreover, electrophysiological studies showed that BmK AngM1 at the concentration of 1 μM inhibited voltage-dependent Na+ current (INa) and voltage-de‐ pendent delayed rectifier K+ current (IK), but had no effects on transient K+ current [37].

It is important to note that the excitatory and depressant anti-insect toxins belong to differ‐ ent groups, which have distinct modes of interaction with receptors. Thus, one can infer that the analgesic effect of these peptides may have a molecular mode and mechanism different from that of insect toxicity. Still, the mechanisms by which these scorpion toxins can modu‐ late pain pathways remain to be clarified. According to [8], four different possibilities might be described:


### **3.2. Polypeptide toxins from Spider**

ry insect toxicity, but is devoid of any toxicity on mice even at high dosages. The analgesic effect was assessed with a mouse-twisting model. The analgesic effect on mice of the AngP1 is at least 4-5 times weaker than that of IT-AP, but the toxicity to insects is twice as strong as

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

In relation of depressant toxins isolated from BmK venom, BmK IT2 has been more studied from the venom of BmK (Fig 1). Intraplantar injection of BmK IT2 inhibited thermal hyperal‐ gesia in carrageenan-treated rats and significantly prolonged paw withdrawal latency in normal rats [23]. This toxin also displays an inhibitory effect on the C component of the rat nociceptive flexion reflex by subcutaneous injection in vivo [24]. Peripheral or spinal deliv‐ ery of BmK IT2 suppressed formalin-induced nociceptive behaviors and c-Fos expression in spinal cord [25,26]. Both BmK IT2 and Bm K dIT-AP3 (depressant Insect Toxin-Analgesic Peptide 3) are toxic for insects, but not for mammals [27], and shows 86.7% of sequence simi‐ larity [23]. BmK dIT-AP3 also induces analgesia in the mouse-twisting model [18]. Using whole-cell patch clamp, it has been shown that BmK dIT-AP3 inhibits Nav currents of rat dorsal root ganglion (DRG) neurons, blocking more selectively the tetrodotoxin-resistant

nociceptive flexion reflex by BmK dITAP3 may be attributed to modulation of the DRG's

Wang and colleagues [28] isolated a new antinociceptive peptide, named BmK AGP-SYPU1. Recombinant BmK AGP-SYPU1 showed similar analgesic effects on mice compared to natu‐ ral when assayed using a mouse-twisting model [28]. More recently, BmK AGP-SYPU2 was purified and tested, also in mouse-twisting model. Sequence determination showed that the mature BmK AGP-SYPU2 peptide is composed of 66 amino acid residues, and BmK AGP-

BmK AS had a strong analgesic effect on both visceral and somatic pain [29,30]. It relieves formalin-induced two-phase spontaneous flinching response and carrageenan-induced me‐ chanical hyperalgesia, probably by modulating the voltage-gated Na+ channels of sensory neurons [31,32]. Moreover, BmK AS showed activity nearly equivalent to that of morphine. Later, a new peptide that possesses 86.3% of similarity with BmK AS was identified. Both polypeptides have 66 amino acids cross-linked by four disulfide bridges [29]. In addition, these two peptides show a poor similarity with other known types of scorpion toxins. BmK AS and AS1 are not toxic against mammals and only have a weak toxicity to insects. BmK AS, then BmK AS1, have been found to significantly stimulate the binding of [3H]-ryano‐ dine to partially purified ryanodine receptors [33]. More recently, electrophysiological stud‐

depress TTX-sensitive and TTX-resistant Na+ currents in rat small DRG neurons. Interesting‐ ly, in rat models, BmK AS1 also displays antinociceptive effects according to [33]. These au‐ thors concluded that the effects could be mediated by the modulation of voltage-gated Na+ channels and they also suggested that BmK AS and BmK AS1 could form a new family of

BmK AGAP (antitumor-analgesic peptide), isolated in 2003, had strong inhibitory effect on both viscera and soma pain [35]. To evaluate the extent to which residues of the toxin core

currents. These results suggest that the inhibition of the rat

currents in NG108-15 cells [34] and to

that of IT-AP [20,22]

Applications

94

(TTX-R) component of the Na+

channels [24].

SYPU2 is identical to BmK alpha2 and BmK alphaTX11.

ies have shown that they are able to inhibit Na+

voltage-gated Na+

scorpion insect toxins.

Another group of arthropods that have very promising antinociceptive compounds are spi‐ ders [41]. In 1996, Roerig & Howse reported the effect of ω-agatoxina IVA (Fig 1) isolated from funnel spider *Agelenopsis aperta* venom, against thermal stimulation in the tail flick test, when co-administrated with morphine intrathecal. Intrathecal injection of ω-agatoxin IVA (0.2 nmol/kg) also decreased the licking time in both the early and late response phases in a dose-dependent manner in the Formalin test [42]. The use of this peptide as an analgesic could be of particular benefit in patients tolerant or opioid-dependent, since this compound exhibits selectivity for the P/Q Ca2+ channels [43]. Other spider venom very promissory is the venom of the Brazilian armed spider *Phoneutria nigriventer*, the purified fraction 3 (PhTx3) contains 6 toxin isoforms (Tx3-1 to -6) [44,45] that target Ca2+ channels with different affinity patterns. Moreover, one toxin, Tx3-6 (Phα1β), demonstrated that it preferentially blocks the N-type calcium current [46] and produce a potent antinociceptive effect with higher therapeutic index [44]. Dalmolin and colleagues [45] showed that Tx3-3 (purified the same fraction) caused a short-lasting antinociceptive effect in the nociceptive pain test and a long-lasting antinociceptive effect in neuropathic pain models, without producing detecta‐ ble side effects. However, Tx3-3 did not change the inflammatory pain. Tx3-3 blockade of P/Q- and R-type Ca2+ channels and inhibit the glutamate release in rat brain cortical synapto‐ somes [47]. Other neurotoxin isolated from spider *Phoneutria nigriventer* is Phα1β, which is a potent toxin blocking neuronal voltage-sensitive Ca2+ channels. This peptide induced longer antiallodynic effect than μ-conotoxin MVIIA and morphine in mice [48].

In addition to toxins calcium modulators, compounds isolated from spider that interact with other ionic channels have shown great potential. A new class of peptide toxins named is the Huwentoxin I (HWTX-I, Fig 1) that is the most abundant toxic component in the crude ven‐ om of the Chinese bird spider *Ornithoctonus huwena*. Whole-cell patch clamp records re‐ vealed that HWTX-I selectively inhibits N-type Ca2+ channels in NG108-15 cells, and it also can block transmitter release from nerve endings by preventing depolarization induced by calcium influx [38] Antinociception effect of the HWTX-I in formalin test was greater and lasted two-fold longer time compared to morphine [39]. Furthermore, Tao and collaborators [40] demonstrated that intrathecal administration of HWTX-I is effective in antinociception in the rat model of rheumatoid arthritis more effective than ibuprofen.

or formalin (only second phase) [56]. This molecule also inhibits P2X3 receptors in a power‐ ful and selective manner. These ATP-activated receptors are largely expressed in mammali‐ an sensory neurons play a key role in the pain perception. Thus, PT1 appears to be a promising

New Perspectives in Drug Discovery Using Neuroactive Molecules From the Venom of Arthropods

http://dx.doi.org/10.5772/ 52382

97

Bee venom has been traditionally used to relieve pain and treat chronic pain diseases (for revision see [57]). Moreover, acupoint stimulation into the subcutaneous region (acupunc‐ ture) rather than other injection sites may be important for the antinociceptive effects of this venom. There is increasing evidence suggesting that bee venom has antinociceptive effects on visceral nociceptive effects, mechanical and thermal hyperplasia, formalin-induced pain behavior and collagen-induced arthritic pain, as well as knee osteoarthritis (OA)-related pain [58-63]. BV contains at least 18 active components, including enzymes, peptides, and biogenic amines, which have a wide variety of pharmaceutical properties, and so multiple mechanisms associated to antinociceptive effects have been suggested, such as activation of the central and spinal opioid receptor and α2-adrenergic receptor, as well as activation of the

Melittin is a small protein containing 26 amino acid residues and is the major bioactive com‐ ponent in BV (Fig.1). This polypeptide readily integrates into and disrupts both natural and synthetic phospholipid bilayers [65,66]. Melittin also enhances the activity of PLA2 [67] and has a variety of effects on living cells possibly through the disruption of the membrane [68]. The decrease in cyclooxygenase (COX)-2 and phospholipase PLA2 expression and the de‐ crease in the levels of tumor necrosis factor alpha (TNF-α), interleukin (IL)-1, IL-6, nitric ox‐ ide (NO) and oxygen reactive species (ROS) are suggested to be associated with the antiarthritis effect of melittin [69]. This peptide has also been thought to play a role in production of anti-nociceptive and anti-inflammatory effects [64]. In addition, Merlo and colleagues [70] demonstrated the antinociceptive activity of the melittin in experimental models of nociceptive and inflammatory pain. Interestingly, melittin failed to increase the latency for the nociceptive response in the hot-plate model and in the first phase of the for‐ malin test, revealing that melittin presents an activity that resembles more that of anti-in‐ flammatory drugs and less that of centrally acting drugs [70]. Nevertheless, the molecular and cellular mechanisms underlying the anti-nociceptive effects of melittin are not entirely

clear and remain to be further clarified by further experimental studies [57].

tion of cyclooxygenase activity [71,72].

*dentalis*. The isolated peptide is a neurokinin named Thr6

Addition of melittin, adolapin has been isolated from BV and it demonstrated a potent anal‐ gesic effect in mouse-twisting model and the Randall-Sellito's test [71]. The anti-inflammato‐ ry activity of adolapin was evaluated and it had a pronounced activity in the following tests: carrageenan, PG, adjuvant rat hind paw edema and adjuvant polyarthritis. The effects of adolapin are presumably due to its ability to inhibit the prostaglandin synthesis via inhibi‐

Venoms of wasps also have analgesic peptides. Mortari and colleagues [73] isolated a com‐ pound with antinociceptive activity from the venom of the Brazilian social wasp *Polybia occi‐*


lead compound for the development of analgesics that target these receptors [56].

**3.3. Polypeptide toxins from Bees and Wasps**

descending serotonergic pathways (for revision see [64]).

Several studies have reported that intrathecal administration of non-selective blockers of Ca2+ channels shows antinociceptive effects in animals tested with thermal stimuli: hot plate and tail flick. According to [49], N and P/Q Ca2+ channels are probably involved in nocicep‐ tive behavior induced by formalin injection in rats, while the L-type channels has no effect. N- and P/Q-type Ca2+ channels are expressed specifically in the nervous system, and they have a major importance in controlling the excitation of spinal neurons from sensory affer‐ ents of inflamed tissues, relieving inflammatory pain.

A new class of peptide toxins named π-theraphotoxin-Pc1a (π-TRTX-Pc1a; also known as psalmotoxin-1 (PcTx1) was isolated from the venom of the spider neotropical *Psalmopoeus cambridgei* (Fig.1). π-TRTX-Pc1a is the most potent and selective blocker of ion channels sen‐ sitive to acid – ASICa [50]. These channels play important roles in pathological conditions such as cerebral ischemia or epilepsy, as well as being responsible for the sensation of pain that accompanies tissue acidosis and inflammation [51]. Since external acidification is a ma‐ jor factor in pain associated with inflammation (hematosis muscle and cardiac ischemia, or cancer), these neurotoxins can be used to control the pain sensation triggered by these chan‐ nels [52]. π-TRTX-Pc1a was shown to be an effective analgesic, comparable to morphine, in rat models of acute and neuropathic pain when injected directly in Central Nervous System [53] and intranasal administration of this peptide resulted in neuroprotection of neurons in a mouse model of ischemic stroke even when administered hours after injury [54].

Other important target in the search for new analgesics isolated from spider venoms are NaV channels, since modulatory compounds of these channels are the dominant pharmacological species in spider venoms, although still poorly characterized. In this context, Intrathecal ad‐ ministration of β-TRTX-Gr1b (formerly GsAFI), a peptide obtained from venom of *Grammos‐ tola spatulata*, the Chilean pink tarantula spider, induced analgesia in a variety of rat pain models such as the tail flick latency test, hot plate threshold test, von Frey threshold test, and formalin pain test, without any confounding side-effects. Moreover, the β-TRTX-Gr1b peptide did not exhibit cross tolerance with morphine [55].

Further on spider venoms, Purotoxin-1 (PT1) was recently isolated the, from the venom of the Central Asian spider *Geolycosa sp* [56]. PT1 is a 35-residue peptide with four disulfide bonds, and it exerts a potent analgesic effect in rat models of acute and chronic inflammatory pain by injection of either carrageenan or Freund's complete adjuvant, respectively. PT1 was also effective in reducing the number of nocifensive events triggered by the injection of capsaicin or formalin (only second phase) [56]. This molecule also inhibits P2X3 receptors in a power‐ ful and selective manner. These ATP-activated receptors are largely expressed in mammali‐ an sensory neurons play a key role in the pain perception. Thus, PT1 appears to be a promising lead compound for the development of analgesics that target these receptors [56].

### **3.3. Polypeptide toxins from Bees and Wasps**

In addition to toxins calcium modulators, compounds isolated from spider that interact with other ionic channels have shown great potential. A new class of peptide toxins named is the Huwentoxin I (HWTX-I, Fig 1) that is the most abundant toxic component in the crude ven‐ om of the Chinese bird spider *Ornithoctonus huwena*. Whole-cell patch clamp records re‐ vealed that HWTX-I selectively inhibits N-type Ca2+ channels in NG108-15 cells, and it also can block transmitter release from nerve endings by preventing depolarization induced by calcium influx [38] Antinociception effect of the HWTX-I in formalin test was greater and lasted two-fold longer time compared to morphine [39]. Furthermore, Tao and collaborators [40] demonstrated that intrathecal administration of HWTX-I is effective in antinociception

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

Several studies have reported that intrathecal administration of non-selective blockers of Ca2+ channels shows antinociceptive effects in animals tested with thermal stimuli: hot plate and tail flick. According to [49], N and P/Q Ca2+ channels are probably involved in nocicep‐ tive behavior induced by formalin injection in rats, while the L-type channels has no effect. N- and P/Q-type Ca2+ channels are expressed specifically in the nervous system, and they have a major importance in controlling the excitation of spinal neurons from sensory affer‐

A new class of peptide toxins named π-theraphotoxin-Pc1a (π-TRTX-Pc1a; also known as psalmotoxin-1 (PcTx1) was isolated from the venom of the spider neotropical *Psalmopoeus cambridgei* (Fig.1). π-TRTX-Pc1a is the most potent and selective blocker of ion channels sen‐ sitive to acid – ASICa [50]. These channels play important roles in pathological conditions such as cerebral ischemia or epilepsy, as well as being responsible for the sensation of pain that accompanies tissue acidosis and inflammation [51]. Since external acidification is a ma‐ jor factor in pain associated with inflammation (hematosis muscle and cardiac ischemia, or cancer), these neurotoxins can be used to control the pain sensation triggered by these chan‐ nels [52]. π-TRTX-Pc1a was shown to be an effective analgesic, comparable to morphine, in rat models of acute and neuropathic pain when injected directly in Central Nervous System [53] and intranasal administration of this peptide resulted in neuroprotection of neurons in a

mouse model of ischemic stroke even when administered hours after injury [54].

Other important target in the search for new analgesics isolated from spider venoms are NaV channels, since modulatory compounds of these channels are the dominant pharmacological species in spider venoms, although still poorly characterized. In this context, Intrathecal ad‐ ministration of β-TRTX-Gr1b (formerly GsAFI), a peptide obtained from venom of *Grammos‐ tola spatulata*, the Chilean pink tarantula spider, induced analgesia in a variety of rat pain models such as the tail flick latency test, hot plate threshold test, von Frey threshold test, and formalin pain test, without any confounding side-effects. Moreover, the β-TRTX-Gr1b

Further on spider venoms, Purotoxin-1 (PT1) was recently isolated the, from the venom of the Central Asian spider *Geolycosa sp* [56]. PT1 is a 35-residue peptide with four disulfide bonds, and it exerts a potent analgesic effect in rat models of acute and chronic inflammatory pain by injection of either carrageenan or Freund's complete adjuvant, respectively. PT1 was also effective in reducing the number of nocifensive events triggered by the injection of capsaicin

in the rat model of rheumatoid arthritis more effective than ibuprofen.

ents of inflamed tissues, relieving inflammatory pain.

Applications

96

peptide did not exhibit cross tolerance with morphine [55].

Bee venom has been traditionally used to relieve pain and treat chronic pain diseases (for revision see [57]). Moreover, acupoint stimulation into the subcutaneous region (acupunc‐ ture) rather than other injection sites may be important for the antinociceptive effects of this venom. There is increasing evidence suggesting that bee venom has antinociceptive effects on visceral nociceptive effects, mechanical and thermal hyperplasia, formalin-induced pain behavior and collagen-induced arthritic pain, as well as knee osteoarthritis (OA)-related pain [58-63]. BV contains at least 18 active components, including enzymes, peptides, and biogenic amines, which have a wide variety of pharmaceutical properties, and so multiple mechanisms associated to antinociceptive effects have been suggested, such as activation of the central and spinal opioid receptor and α2-adrenergic receptor, as well as activation of the descending serotonergic pathways (for revision see [64]).

Melittin is a small protein containing 26 amino acid residues and is the major bioactive com‐ ponent in BV (Fig.1). This polypeptide readily integrates into and disrupts both natural and synthetic phospholipid bilayers [65,66]. Melittin also enhances the activity of PLA2 [67] and has a variety of effects on living cells possibly through the disruption of the membrane [68]. The decrease in cyclooxygenase (COX)-2 and phospholipase PLA2 expression and the de‐ crease in the levels of tumor necrosis factor alpha (TNF-α), interleukin (IL)-1, IL-6, nitric ox‐ ide (NO) and oxygen reactive species (ROS) are suggested to be associated with the antiarthritis effect of melittin [69]. This peptide has also been thought to play a role in production of anti-nociceptive and anti-inflammatory effects [64]. In addition, Merlo and colleagues [70] demonstrated the antinociceptive activity of the melittin in experimental models of nociceptive and inflammatory pain. Interestingly, melittin failed to increase the latency for the nociceptive response in the hot-plate model and in the first phase of the for‐ malin test, revealing that melittin presents an activity that resembles more that of anti-in‐ flammatory drugs and less that of centrally acting drugs [70]. Nevertheless, the molecular and cellular mechanisms underlying the anti-nociceptive effects of melittin are not entirely clear and remain to be further clarified by further experimental studies [57].

Addition of melittin, adolapin has been isolated from BV and it demonstrated a potent anal‐ gesic effect in mouse-twisting model and the Randall-Sellito's test [71]. The anti-inflammato‐ ry activity of adolapin was evaluated and it had a pronounced activity in the following tests: carrageenan, PG, adjuvant rat hind paw edema and adjuvant polyarthritis. The effects of adolapin are presumably due to its ability to inhibit the prostaglandin synthesis via inhibi‐ tion of cyclooxygenase activity [71,72].

Venoms of wasps also have analgesic peptides. Mortari and colleagues [73] isolated a com‐ pound with antinociceptive activity from the venom of the Brazilian social wasp *Polybia occi‐ dentalis*. The isolated peptide is a neurokinin named Thr6 -Bradykinin. This neurokinin is a

small peptide consisting of nine amino acid residues, Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg-OH, which exhibits a high degree of homology with bradykinin (BK), except for the substitution of Thr for Ser in position 6 at BK. As a result, small changes in their secondary structures are observed [74]. This modification has been regarded as responsible for increas‐ ing B2 receptor affinity and potency of Thr6 -BK in relation to BK *in vitro* and *in vivo* [74,75]. Thr6 -BK antinociceptive effect was dose- and time- dependent, when injected directly into the CNS of rats in hot-plate and tail-flick tests, and it was three times more potent than mor‐ phine and 4 times more potent than BK in tail-flick test. Thr6 -BK induced antinociception by activating presynaptic B2 receptors, which activate descending adrenergic pathways. Studies investigating the role of kinins in the CNS provide new information on the supraspinal sys‐ tem of the pain control, whose modulation may represent a new strategy to control painrelated pathologies [76].

toxins may suggest a possible involvement of AMPA receptors in the spinal cord during the

New Perspectives in Drug Discovery Using Neuroactive Molecules From the Venom of Arthropods

http://dx.doi.org/10.5772/ 52382

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Neurodegenerative disorders comprise a wide range of conditions mostly characterized by a progressive loss of neuronal function and neuronal cell death. The incidence of these diseas‐ es in population differs greatly. In conditions such as Parkinson disease and Alzheimer, the number of cases significantly increases in elderly, whereas epileptic patients are mostly chil‐ dren and adolescents. Many processes may trigger neuronal cell death, such as trauma, stroke, tumors, infections, genetic factors and biochemical alterations. Among the latest, the alterations in Ca2+-mediated signaling is thought to play a key role in many neurodegenera‐ tive disorders and the increase in intracellular Ca2+ concentration might alter neuronal mem‐ brane potential [82]. Moreover, the hyperactivation of excitatory transmission mediated mostly by L-glutamate and its ionotropic receptors; kainate, AMPA and NMDA, is responsi‐ ble for the excessive cationic influx that depolarizes neuronal cells and lead to sustained hy‐ perexcitation observed in brain pathologies such as epilepsy [83]. This increase in glutamatergic activity often referred to as glutamate excitotoxicity [84], might also involve non-receptor neurochemical events such as failure in glutamate uptake system, which ends with an increase in the availability of this neurotransmitter in the synaptic cleft [85,86]. The importance of L-glutamate in neurological disorders relies on the fact that this neurotrans‐ mitter is release in the great majority of fast synapses in CNS [84,83]. In this context, many molecules mostly peptides and acylpolyamines, acting on ion channels, receptors and trans‐ porters were isolated from arthropod venoms, remarkably spiders, scorpions and wasps [3]. According to [82], polyamines are non-specific antagonist of ligand-gated ion channels, act‐ ing at glutamatergic and Ach receptors in an uncompetitive way, that is, the receptor must be activated in order to occur the blockade. This mode of action might diminish the side ef‐ fects of newly designed medicines, since it blocks only the activated receptors, but does not

The venom of the orb-web spider *Nephilia clavata* was one of the first venoms studied during the 80s, which resulted in the identification of small compounds named acylpolyamines, among whose we may find jorotoxin (JSTX), one of the first glutamate receptor uncompeti‐ tive antagonists [83,84]. Together with JSTX, another polyamines such as argiopin from the venom of the spider *Argiope lobata* [85] and philantotoxin (PhTx) from the venom of the soli‐ tary wasp *Philanthus triangulum* [86]. Following the structural characterization and studies in insect or crustaceans, the reports on the action of these polyamines in mammalian CNS started to take place, mostly during the 90s [87]. JSTX-1 and JSTX-3 are synthetic analogues of JSTX. The first inhibits kainate-induced seizures, whereas the latter block glutamate re‐ lease and hippocampal epileptic discharges [88,89]. Later, JSTX-3 was shown to inhibit the formation of superoxide dismutase-1 (SOD-1) aggregates that lead mutant motor neurons (mSOD-1) to death during the familiar form of the neurodegenerative disease, amyotrophic lateral sclerosis [90]. The authors concluded that increased Ca2+ influx mainly through AM‐

nociceptive excitatory stimulation [80,81].

prevent their opening.

**4. Antiepileptic and neuroprotective effects**

**Figure 1.** Tridimensional structure of antinociceptive peptides isolated from arthropod venoms. (A) BMK IT2; (B) HWTX 1; (C) ω-Agatoxin IVA; (D) π-Theraphotoxin-Pc1a; (E) Mellitin. Uniprot entry code: P68727, P56676, P30288, P60514 and P01501, respectively.

Besides peptides, some studies have evaluated the analgesic activity of acylpolyamines that can be used as new alternative drugs for the treatment of chronic pain, as well as tools for the study of the functional role of the AMPA/kainate receptors in the processing of nocicep‐ tive pain [77]. In this regard, intrathecal administration of different doses of these toxins blocked thermally induced allodynia [78] and hyperalgesia [79]. The effect of these neuro‐ toxins may suggest a possible involvement of AMPA receptors in the spinal cord during the nociceptive excitatory stimulation [80,81].
