**4. Perspective in developing new antiemetic candidates**

#### **4.1. Antiemetic efficacy of LTCC blockers in the least shrew model of emesis**

Nifedipine along with amlodipine, are among the most studied of Ca2+ channels blockers, and both belong to the dihydropyridine subgroup of LTCC antagonists. Relative to nifedipine, a fast and short-acting LTCC antagonist with a plasma half-life of 1.2 h, amlodipine is slow and longer acting, more extensively bound to plasma protein, with a larger volume of distribution, more gradual elimination, with a half-life of over 30 h [130–134]. We have evaluated the antiemetic efficacy of both nifedipine and amlodipine (**Figure 2**) by assessing mean emesis frequency and the percentage of shrews vomiting, and demonstrated that both LTCC blockers [45, 46] behave as broad-spectrum antiemetics when delivered systemically against diverse specific emetogens, including FPL 64176 (10 mg/kg, i.p.), the peripherally-acting and non-selective 5-HT<sup>3</sup> R agonist 5-HT (5 mg/kg, i.p.), the peripherally/centrally-acting and more selective 5-HT<sup>3</sup> R agonist 2-Methyl-5-HT (5 mg/kg, i.p.), the dopamine D<sup>2</sup> R-preferring agonist quinpirole (2 mg/kg, i.p.), the non-selective dopamine D<sup>2</sup> R agonist apomorphine (2 mg/kg, i.p.), the nonselective cholinergic agonist pilocarpine (2 mg/kg, i.p.), the M<sup>1</sup> -preferring cholinergic agonist McN-A343 (2 mg/kg, i.p.), and the selective neurokinin NK<sup>1</sup> R agonist GR73632 (5 mg/kg, i.p.). The vomiting behavior was recorded for 30 min. Our results suggest that both amlodipine and nifedipine act by suppressing the influx of extracellular Ca2+, thereby delay the onset as well as protecting least shrews from vomiting, further supporting our proposed Ca2+ hypothesis of emesis. Nifedipine appears to be more potent than amlodipine against vomiting caused by FPL64176, 5-HT, 2-Methyl-5-HT, GR73632, quinpirole and McN-A343. These potency disparities could be explained in terms of their pharmacokinetic and pharmacodynamic differences [130–139].

and vomiting. Indeed, dexamethasone (**Figure 2**), one of the clinically used glucocorticoids, is effective in reducing both acute and delayed chemotherapy-induced nausea and vomiting, and

high emetogenic chemotherapy [6]. Glucocorticoids' antiemetic effect has been related to its inhibitory effects in the following facets: (i) glucocorticoids control the inflammatory response involved in mediating chemotherapy-induced nausea and vomiting by reducing the production of inflammatory mediators such as cytokines, chemokines, inducible nitric oxide synthase, and increasing the gene transcription of anti-inflammatory proteins [6]; (ii) glucocorticoids can inhibit 5-HT and substance P release, both of which can evoke emesis [6, 121], (iii) glucocorticoids can cross the blood-brain barrier and can exert direct central inhibitory effects on the nucleus tractus solitarius [6], which may be due to a decrease in abnormal elevation of cytosolic Ca2+ concentration as well as downstream Ca2+ signals and the maintenance of Ca2+ homeostasis within the cell [122], (iv) inhibitory actions of glucocorticoid could also be due to increased release of endocannabinoids, anandamide and 2-arachidonoylglycerol, evoked by glucocor-

glucocorticoid facilitation of synaptic γ-aminobutyric acid (GABA) release and suppression of glutamate release [123, 124]. The endocannabinoid system is composed of CBRs, endocannabinoids and the enzymes involved in their synthesis. Anandamide and 2-arachidonoylglycerol are among the well-studied endocannabinoids and endogenous activators of CBRs [125]. The

dexamethasone may decrease motion sickness through modulation of the endocannabinoid/

inhibition of transmitter release from interneurons of the nucleus tractus solitarius [99, 126]. Selective elevation of 2-arachidonoylglycerol by inhibition of its major metabolic enzyme monoacylglycerol lipase, have been shown to suppress lithium chloride evoked vomiting in the house musk shrew (*Suncus murinus*) [127]. However, intraperitoneal administration of the endocannabinoid 2-arachidonoylglycerol can evoke vomiting in the least shrew in a dose-dependent manner probably via its rapid metabolism to arachidonic acid which is also a potent emetogen in this species [128]. Moreover, the cancer chemotherapeutic agent cisplatin can increase 2-arachidonoylglycerol but not anandamide levels in the least shrew brain [129].

receptor system on the terminals of the nucleus tractus solitarius neurons that project to the

R agonists as antiemetics was discussed in Section 2.2. It has been suggested that

antagonists, it is utilized in patients receiving

R activation as well as

receptor system-mediated

or neurokinin NK1

ticoid administration which will then be followed by subsequent CB<sup>1</sup>

output neurons of the DMNV as well as by endocannabinoid/CB<sup>1</sup>

**4. Perspective in developing new antiemetic candidates**

**4.1. Antiemetic efficacy of LTCC blockers in the least shrew model of emesis**

Nifedipine along with amlodipine, are among the most studied of Ca2+ channels blockers, and both belong to the dihydropyridine subgroup of LTCC antagonists. Relative to nifedipine, a fast and short-acting LTCC antagonist with a plasma half-life of 1.2 h, amlodipine is slow and longer acting, more extensively bound to plasma protein, with a larger volume of distribution, more gradual elimination, with a half-life of over 30 h [130–134]. We have evaluated the antiemetic efficacy of both nifedipine and amlodipine (**Figure 2**) by assessing mean emesis frequency and the percentage of shrews vomiting, and demonstrated that both LTCC blockers [45, 46] behave as broad-spectrum antiemetics when delivered systemically against diverse specific emetogens,

when combined with 5-HT<sup>3</sup>

118 Calcium and Signal Transduction

role of CB1

CB1

Unlike the above tested emetogens which can evoke vomiting within minutes of administration, cisplatin (10 mg, i.p.) requires more exposure time (30–45 min) to begin to induce emesis since only its metabolites are emetogenic. The relative efficacy of amlodipine (5 mg/kg., i.p.) in reducing the frequency of cisplatin-evoked early vomiting by 80% compared with the observed lack of antiemetic action of nifedipine up to 20 mg/kg [45, 46], could be explained in terms of positively charged amlodipine associating more slowly with LTCCs, requiring more exposure time not only to reach its sites of action, but also to compensate for its slower receptor binding kinetics, which can lead to a more gradual onset of antagonism [140]. In addition, intracerebroventricular microinjection of another LTCC antagonist, nitrendipine, has been shown to attenuate nicotine-induced vomiting in the cat [141], which further supports the discussed broad-spectrum antiemetic efficacy of nifedipine and amlodipine as observed in the least shrew model. Cisplatin-based chemotherapeutics induce both immediate and delayed vomiting in humans and in vomit-competent animals [16, 142, 143]. In the least shrew, cisplatin (10 mg/kg, i.p.) causes emesis over 40 h with respective peak early- and delayed-phases occurring at 1–2 and 32–34 h post-injection [144]. Amlodipine, due to its unique pharmacokinetics, may offer practical advantages over other calcium antagonists in cisplatin-evoked delayed emesis.

#### **4.2. Potentiation of antiemetic efficacy of 5-HT<sup>3</sup> R antagonists when combined with LTCC blockers**

In 1996 Hargreaves and co-workers [20] demonstrated that members of all three major classes of LTCC antagonists can prevent the ability of the 5-HT<sup>3</sup> receptor-selective agonist 1-(m-chlorophenyl)-biguanide to increase intracellular Ca2+ concentration in cell lines that possess either one or both of these two different Ca2+-ion channels. The latter interaction is not competitive since the binding site for the different classes of LTCC antagonists appear not to be the same as the serotonin 5-HT<sup>3</sup> R binding site itself (i.e., the orthosteric site) but instead, is an allosteric site in the 5-HT<sup>3</sup> receptor channel complex. Furthermore, 5-HT release from enterochromaffin cells can be prevented by antagonists of both 5-HT<sup>3</sup> Rs and LTCCs [145, 146]. These findings provide possible mechanisms via which antagonists of both LTCCs and 5-HT<sup>3</sup> Rs can mutually prevent the biochemical and behavioral effects of their corresponding selective agonists, including the vomiting behavior induced by their corresponding selective agonists FPL64176 and 2-Methyl-5-HT as we reported previously [45]. We have further demonstrated that when non-effective antiemetic doses of their selective antagonists (nifedipine and palonosetron, respectively) are combined [45], the combination significantly and in an additive manner attenuate both the frequency and the percentage of shrews vomiting in response to either FPL 64176 or 2-Methyl-5-HT. Furthermore, although nifedipine alone up to 20 mg/kg dose failed to protect shrews from acute cisplatin-induced vomiting, its 0.5 mg/kg dose, significantly potentiated the antiemetic efficacy of a non-effective (0.025 mg/kg) as well as a semi-effective (0.5 mg/kg) dose of palonosetron. In another study we also utilized a combination of non-effective doses of amlodipine (0.5 mg/kg or 1 mg/kg) with a non- or semieffective dose of the 5-HT<sup>3</sup> R antagonist palonosetron (0.05 or 0.5 mg/kg) [46]. The combined antiemetic doses produced a similar additive efficacy against vomiting induced by either FPL 64176 or cisplatin. In fact relative to each antagonist alone, the combination was at least 4 times more potent in reducing the vomit frequency and provided more protection against FPL 64176-induced vomiting. The observed additive antiemetic efficacy of a combination of 5-HT<sup>3</sup> - (and/or possibly NK<sup>1</sup> -) with LTCC-antagonists in the least shrew suggests that such a combination should provide greater emesis protection in cancer patients receiving chemotherapy in a manner similar to that reported between 5-HT<sup>3</sup> - and NK1 -receptor antagonists both in the laboratory [144, 147] and in the clinic [148]. Although in our investigation, the mechanism underlying the additive antiemetic efficacy of combined low doses of LTCC antagonists with 5-HT<sup>3</sup> R antagonists was not directly studied, the published literature points to their interaction at the signal transduction level involving Ca2+ [20, 149, 150].

during neuronal depolarization to generate a transient increase in cytosolic Ca2+ [160–162].

coupling has also been proposed as one of the mechanisms connecting depletion of internal Ca2+ stores with stimulation of extracellular Ca2+ influx [163]. For example, Ca2+ release from

mediated vomiting triggered by 5 mg/kg 2-Methyl-5-HT is insensitive to the intracellular Ca2+

the RyR antagonist, dantrolene [25]. Furthermore, a combination of the semi-effective doses of amlodipine and dantrolene was more potent than each antagonist being tested alone [25]. Significant reductions (70–85%) in the frequency of Ca2+ mobilizer thapsigargin-evoked vomiting (see Section 1.2) were observed when shrews were pretreated with antagonists of either

Rs (2-APB at 1 and 2.5 mg/kg, i.p.)- or RyRs (dantrolene at 2.5 and 5 mg/kg, i.p.)-ER luminal Ca2+ release channels. Moreover, while a mixture of 2-APB (1 mg/kg) and dantrolene (2.5 mg/kg) did not offer additional protection than what was afforded when each drug administered alone, a combination of the latter doses of 2-APB plus dantrolene with a partially effective dose of nifedipine (2.5 mg/kg), led to a complete elimination of thapsigargin-evoked vomiting [70].

emetogens and their antagonists provide further efficacy when combined with LTCC antagonists (**Figure 2**). Suppression of Ca2+ release from the sarco/endoplasmic reticulum stores

In mammals, cyclic AMP (cAMP) is synthesized by 10 adenylate cyclase isoforms [168]. One of the best-studied second messenger molecules downstream of selected G-protein coupled receptors is cAMP. It is an example of a transient and diffusible second messenger involved in signal propagation by integrating multiple intracellular signaling pathways [169]. cAMP activates protein kinase A (PKA) which results in phosphorylation of downstream intracellular signals. The adenylyl cyclase/cAMP/PKA signaling pathway can phosphorylate Ca2+ ion-

respectively increase extracellular Ca2+-influx and intracellular Ca2+-release [110]. The emetic role of cAMP has been well established (**Figure 5**), since microinjection of cAMP analogs (e.g., 8-bromocAMP) or forskolin (to enhance endogenous levels of cAMP) in the brainstem dorsal vagal complex emetic locus area postrema, not only can increase electrical activity of local neurons, but also induces vomiting in dogs [170]. Moreover, administration of 8-chlorocAMP as a potential chemotherapeutic in cancer patients can evoke nausea and vomiting [171]. Furthermore, phosphodiesterase inhibitors (PDEI) such as rolipram prevent cAMP metabolism and consequently increase cAMP tissue levels, which leads to excessive nausea and vomiting in humans [172]. In fact, one major side-effect of older PDEIs is excessive nausea and vomiting which often precludes their use in the clinical setting [173]. In addition, we have

Rs and RyRs may be additional targets for the prevention of nausea and vomiting.

In another set of experiments [167], we found that pretreatment with the IP<sup>3</sup>

Rs was shown to couple with extracellular Ca2+ influx through LTCCs in non-excitable cells such as Jurkat human T lymphocytes [164] and drosophila S2 cells [165], as well as in excitable cells such as submucosal neurons in the rat distal colon [166]. We have found that 5-HT<sup>3</sup>

Rs to plasma membrane Ca2+ influx channels through conformational

R antagonist 2-APB, but in contrast, was dose-dependently suppressed by

R agonist GR73632-induced emesis, however the RyR

Rs can be differentially modulated by various

R-

121

Role of Calcium in Vomiting

http://dx.doi.org/10.5772/intechopen.78370

R inhibitor 2-APB

Rs [110]. These Ca2+ channels

Physical attachment of IP<sup>3</sup>

release channel IP<sup>3</sup>

causes a significant reduction in NK<sup>1</sup>

*4.5.1. The role of cAMP-PKA in vomiting*

inhibitor dantrolene did not. Thus, RyRs and IP<sup>3</sup>

**4.5. Ca2+-related signaling pathways in emesis**

channels found on the plasma membrane and intracellular IP<sup>3</sup>

IP3

IP3

through IP<sup>3</sup>

#### **4.3. Clinical use of LTCC blockers as anti-nausea/antiemetic medication**

There are several published clinical case reports that demonstrate Ca2+ channel blockers may provide protection against several causes of nausea and vomiting. The LTCC antagonist flunarizine (**Figure 2**) was shown to reduce cyclic vomiting on acute basis in one patient [151] and prophylactically in 8 other patients [152]. Gabapentin is a gamma-aminobutyric acid (GABA) analog and is predominantly used in the clinic for the management of pain [3]. Gabapentin binds to the alpha-2/delta auxiliary subunits of voltage-gated Ca2+ channels (VGCCs) (i.e., LTCCs), and exerts inhibitory actions on trafficking and activation kinetics of VGCCs [153] (**Figure 2**). Moreover, several other reports indicate that gabapentin can also be used as a well-tolerated, less-expensive and promising anti-nausea and antiemetic agent in diverse conditions including: postoperative nausea and vomiting [154, 155], moderately or highly emetogenic chemotherapy-induced nausea and vomiting, particularly effective against delayed chemotherapy-induced nausea and vomiting [156], and both acute and delayed nausea induced by chemotherapy [157], as well as hyperemesis gravidarum [158]. When combined with dexamethasone, gabapentin can also significantly reduce the 24-h incidence of postoperative nausea and vomiting [159]. Alpha-2/delta subunits of VGCCs control transmitter release and further facilitate excitatory transmission [153]. Gabapentin's interaction with neuronal alpha-2/delta subunits of VGCCs and subsequent downregulation of neuronal Ca2+ signaling in emesis relevant sites, such as the dorsal vagal complex, is postulated to play a critical role in its anti-nausea and anti-vomiting effects [3].

#### **4.4. Intracellular Ca2+ release channels: possible targets for suppression of emesis**

A functional and physical linkage between LTCCs and RyRs appears to exist and plays an important role in intracellular Ca2+ release following voltage-dependent Ca2+ entry through LTCCs during neuronal depolarization to generate a transient increase in cytosolic Ca2+ [160–162]. Physical attachment of IP<sup>3</sup> Rs to plasma membrane Ca2+ influx channels through conformational coupling has also been proposed as one of the mechanisms connecting depletion of internal Ca2+ stores with stimulation of extracellular Ca2+ influx [163]. For example, Ca2+ release from IP3 Rs was shown to couple with extracellular Ca2+ influx through LTCCs in non-excitable cells such as Jurkat human T lymphocytes [164] and drosophila S2 cells [165], as well as in excitable cells such as submucosal neurons in the rat distal colon [166]. We have found that 5-HT<sup>3</sup> Rmediated vomiting triggered by 5 mg/kg 2-Methyl-5-HT is insensitive to the intracellular Ca2+ release channel IP<sup>3</sup> R antagonist 2-APB, but in contrast, was dose-dependently suppressed by the RyR antagonist, dantrolene [25]. Furthermore, a combination of the semi-effective doses of amlodipine and dantrolene was more potent than each antagonist being tested alone [25]. Significant reductions (70–85%) in the frequency of Ca2+ mobilizer thapsigargin-evoked vomiting (see Section 1.2) were observed when shrews were pretreated with antagonists of either IP3 Rs (2-APB at 1 and 2.5 mg/kg, i.p.)- or RyRs (dantrolene at 2.5 and 5 mg/kg, i.p.)-ER luminal Ca2+ release channels. Moreover, while a mixture of 2-APB (1 mg/kg) and dantrolene (2.5 mg/kg) did not offer additional protection than what was afforded when each drug administered alone, a combination of the latter doses of 2-APB plus dantrolene with a partially effective dose of nifedipine (2.5 mg/kg), led to a complete elimination of thapsigargin-evoked vomiting [70]. In another set of experiments [167], we found that pretreatment with the IP<sup>3</sup> R inhibitor 2-APB causes a significant reduction in NK<sup>1</sup> R agonist GR73632-induced emesis, however the RyR inhibitor dantrolene did not. Thus, RyRs and IP<sup>3</sup> Rs can be differentially modulated by various emetogens and their antagonists provide further efficacy when combined with LTCC antagonists (**Figure 2**). Suppression of Ca2+ release from the sarco/endoplasmic reticulum stores through IP<sup>3</sup> Rs and RyRs may be additional targets for the prevention of nausea and vomiting.
