European Journal of Pharmacology 684 (2012) 1–7

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European Journal of Pharmacology

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Review

Pharmacological mechanisms of 5-HT3 and tachykinin NK1 receptor antagonism toprevent chemotherapy-induced nausea and vomiting

Camilo Rojas a, Barbara S. Slusher a,b,c,⁎a Brain Science Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USAb Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USAc Department of Psychiatry, Johns Hopkins University School of Medicine, Baltimore, MD, USA

⁎ Corresponding author at: Brain Science InstituteJohns Hopkins University School of Medicine, 6611 Tr21224-6515, USA. Tel.: +1 1 1 410 614 0662; fax: +1

E-mail address: bslusher@jhmi.edu (B.S. Slusher).

0014-2999/$ – see front matter © 2012 Elsevier B.V. Alldoi:10.1016/j.ejphar.2012.01.046

a b s t r a c t

a r t i c l e i n f o

Article history:Received 8 December 2011Received in revised form 23 January 2012Accepted 28 January 2012Available online 9 March 2012

Keywords:PalonosetronOndansetronGranisetronNetupitant5-HT3 receptorNK1 receptor

Nausea and vomiting are among the most common and distressing consequences of cytotoxic chemotherapies.Nausea and vomiting can be acute (0–24 h) or delayed (24–72 h) after chemotherapy administration. The intro-duction of 5-HT3 receptor antagonists in the 90s was a major advance in the prevention of acute emesis. Thesereceptor antagonists exhibited similar control on acute emesis but had no effect on delayed emesis. These find-ings led to the hypothesis that serotonin plays a central role in themechanism of acute emesis but a lesser role inthe pathogenesis of delayed emesis. In contrast, delayed emesis has been largely associatedwith the activation ofneurokinin 1 (NK1) receptors by substance P. However, in 2003, a new 5-HT3 receptor antagonist was introducedinto the market; unlike first generation 5-HT3 receptor antagonists, palonosetron was found to be effective inpreventing both acute and delayed chemotherapy induced nausea and vomiting. Recent mechanistic studieshave shown that palonosetron, in contrast to first generation receptor antagonists, exhibits allosteric bindingto the 5-HT3 receptor, positive cooperativity, persistent inhibition of receptor function after the drug is removedand triggers 5-HT3 receptor internalization. Further, in vitro and in vivo experiments have shown that palonose-tron can inhibit substance P-mediated responses, presumably through its unique interactions with the 5-HT3 re-ceptor. It appears that the crossroads of acute and delayed emeses include interactions among the 5-HT3 andNK1

receptor neurotransmitter pathways and that inhibitions of these interactions lend the possibility of improvedtreatment that encompasses both acute and delayed emeses.

© 2012 Elsevier B.V. All rights reserved.

Contents

1. Chemotherapy-induced nausea and vomiting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12. 5-HT3 receptor antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23. Tachykinin NK1 receptor antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24. Molecular pharmacology of 5-HT3 receptor antagonists: a direct comparison among ondansetron, granisetron and palonosetron . . . . . . . . 3

4.1. Allosteric binding and 5-HT3 receptor function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34.2. Receptor internalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34.3. Inhibition of 5-HT3/NK1 receptor crosstalk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

5. Current studies and future possibilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Neurotranslational Program,ibutary Street, Baltimore, MD1 1 410 614 0659.

rights reserved.

1. Chemotherapy-induced nausea and vomiting

Chemotherapy-induced nausea and vomiting (CINV) is a commonand distressing consequence of cytotoxic chemotherapies and a majorreason for non-compliance with cancer treatment (Feyer and Jordan,2011). CINV can be acute if it occurs within the first 24 h after chemo-therapy administration or delayed if the symptoms persist beyond24 h; the terms acute and delayed are approximations and do not

Fig. 1. Activation of emetic pathways — Cytotoxic chemotherapies can damage the gas-trointestinal tract and activate abdominal vagal afferents. Serotonin, SP and dopamineacting on 5-HT3, NK1 and D2 dopamine receptors respectively are thought to play piv-otal roles in the neurotransmission that culminates in emesis. The dorsal vagal complexencompasses the emetic center, the area postrema and the vagal afferent terminals.Sensory inputs are integrated at the dorsal vagal complex resulting in activation ofabdominal muscles, diaphragm, stomach and esophagus triggering the emetic response.

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provide a clear distinction of where acute emesis ends and delayedemesis begins. However, the terminology points to the realizationthat different pathological pathways are involved (Darmani andRay, 2009; Hesketh et al., 2003; Rudd and Andrews, 2005). Activationof emetic pathways by chemotherapy occurs through a complex net-work of neuroanatomical centers and neurotransmitters. Neuroana-tomical centers that have been identified include first, the emeticcenter found in the brainstem, second, the area postrema located atthe floor of the fourth ventricle and third, the vagal nerve afferentsthat project from the gastrointestinal tract to the emetic center directlyor indirectly through the area postrema. The emetic center is thought ofas a network of loosely organized neurons throughout the medullaoblongata rather than a discrete anatomical entity. During chemothera-py, cytotoxic agents damage the intestinal tract and activate abdominalvagal afferents; sensory inputs in the vagal afferents and area postremaare then consolidated at the dorsal vagal complex resulting in activationof abdominal muscles, diaphragm, stomach and esophagus culminatingin the emetic response (Fig. 1) (Darmani and Ray, 2009; Feyer andJordan, 2011; Hornby, 2001; Rubenstein et al., 2006).

Even though multiple neurotransmitters are involved in triggeringemesis, dopamine, serotonin (5-HT) and substance P (SP) are thoughtto play the largest roles. Receptors for these transmitters are found inthe vagal afferents by the gastrointestinal tract (Fig. 1). Drugs thatblock these neurotransmitter systems have been shown to be effectivetherapeutics for CINV (Jordan et al., 2007; Rubenstein et al., 2006).

When cisplatin and other highly emetogenic cytotoxins were in-troduced in the late 1970s, nausea and vomiting rapidly became amajor problem for patients receiving chemotherapy. At that timethe best available treatment for CINV included the use of corticosteroids,antihistamines and dopamine receptor antagonists. The efficacy of treat-ment was limited to less than half of CINV patients (Herrstedt, 2005;Rubenstein et al., 2006). Antidopaminergic agents were shown to workthrough neuronal blockade of the D2 subtype of dopamine receptorsin both the area postrema and the emetic center. Metoclopramide, themost efficacious non selective antidopaminergic agent, was alsoshown to be a weak antagonist at the 5-HT3 receptor which suggestedthe possibility of using 5-HT3 receptor antagonists for the treatment ofCINV (Bianchi et al., 1970; Fontaine and Reuse, 1973; Miner andSanger, 1986).

2. 5-HT3 receptor antagonists

Miner and Sanger in the mid 1980s were the first to report that aselective 5-HT3 receptor antagonist could attenuate cisplatin-inducedemesis in ferrets (Miner and Sanger, 1986). Cytotoxic chemotherapiesare toxic to enterochromaffin cells lining the upper small intestinecausing free radical generation and serotonin release. Serotoninbinds to 5-HT3 receptors on vagal afferents thus contributing sensoryinputs that cause emesis (Fig. 1). The antiemetic effect of 5-HT3 recep-tor antagonists is thought to be largely mediated through antagonismof 5-HT3 receptors located in the gut; however, blockade of centrallylocated 5-HT3 receptors could also be playing a role (Higgins et al.,1989). The introduction of ondansetron in 1991 was a pivotal advancein the prevention of acute emesis. Other 5-HT3 receptor antagonistslike granisetron and dolasetron soon followed; even though theyexhibited differences in 5-HT3 receptor binding affinity, serum half-life, and metabolism, they exhibited similar control on acute emesiscompared to ondansetron and had no major effect on delayed emesis(del Giglio et al., 2000). These clinical results led to the hypothesis thatserotonin plays a central role in the mechanism of acute emesis but alesser role in the pathogenesis of delayed emesis.

3. Tachykinin NK1 receptor antagonists

In an effort to further optimize antiemetic therapy, aprepitant, adrug belonging to a new class of antiemetic was introduced in 2003.

Aprepitant counteracts the activity of SP, the preferred ligand at NK1

receptors. These receptors are located in the gut, the area postremaand the nucleus tractus solitarius; all areas involved in the emeticreflex. Like serotonin, SP is released by emetogenic chemotherapiesbut it appears to act largely on receptors that are centrally located.Consequently, NK1 receptor antagonists require entry into the centralnervous system to have an antiemetic effect (Tattersall et al., 1996).Aprepitant remains the only available agent in this class. However,other NK1 receptor antagonists like netupitant and rolapitant are inclinical trials in the emesis field (Feyer and Jordan, 2011) and it isexpected that new agents belonging to this class will soon becomeavailable. The use of 5-HT3 receptors and aprepitant in clinical trialsfurther confirmed the hypothesis of acute and delayed emeses havingseparate pathophysiologies. A retrospective analysis of two phase IIclinical trials using ondansetron or granisetron and aprepitant providedsubstantial evidence that serotonin mediates acute emesis occurring8–12 h after chemotherapy and that SP-mediated emesis is the domi-nant factor at later times (Hesketh et al., 2003).

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4. Molecular pharmacology of 5-HT3 receptor antagonists: a directcomparison among ondansetron, granisetron and palonosetron

Palonosetron, a 5-HT3 receptor antagonist came to the market in2003, the same year aprepitant was introduced; unlike first generation5-HT3 receptor antagonists, palonosetron was found to be effective inpreventing both acute and delayed CINV (Aapro et al., 2006; Eisenberget al., 2003; Gralla et al., 2003; Saito et al., 2009). The effect of palonose-tron on delayed emesis was initially received with skepticism by theclinical community. There was no apparent reason why one 5-HT3 re-ceptor antagonist should be more efficacious against delayed emesisthan another. Palonosetron does not bind to the NK1 receptor (Wonget al., 1995) so its effect on delayed emesis was reasoned to be throughanother mechanism. Even though palonosetron has a higher bindingaffinity (pKi=10.45) (Wong et al., 1995) and a longer plasma half lifethan other 5-HT3 receptor antagonists (Constenla, 2004; Stoltz et al.,2004), these attributes are not sufficient to explain its distinct clinicalefficacy. Enhanced binding affinity could be countered by administeringless potent drugs at higher doses provided the receptor is not saturated.Longer half-life could be addressed by administering drugs with ashorter half-life more often. However, ondansetron was found not tomimic palonosetron's protective action in delayed emesis even whenit was administered at higher doses and beyond 24 h after chemother-apy (Geling and Eichler, 2005). Further, the longer duration of actionof palonosetron did not account for its greater efficacy in protectingpatients from emesis within 24 h after moderately emetogenic chemo-therapy compared to ondansetron or dolasetron (Eisenberg et al., 2003;Gralla et al., 2003).

In an effort to determine if there was a difference in the molecularpharmacology of palonosetron vs. other 5-HT3 receptor antagoniststhat could help explain palonosetron's clinical results, a series of parallelexperimentswas carried out using palonosetron and the other twomostwidely used 5-HT3 receptor antagonists, ondansetron and granisetron.

4.1. Allosteric binding and 5-HT3 receptor function

Analyses of binding isotherms using Scatchard and Hill plots sug-gested positive cooperativity for palonosetron and simple bimolecularbinding for both granisetron and ondansetron (Rojas et al., 2008). Inseparate experiments, equilibriumdiagnostic tests discriminated differ-ential effects of palonosetron on [3H]-ligand binding clearly indicatingthat palonosetron was an allosteric receptor antagonist whereas grani-setron and ondansetron were competitive receptor antagonists. Inanother set of experiments, using dissociating rate strategies (Limbird,2005), palonosetron was shown to be an allosteric modifier that

Fig. 2. Dissociation kinetics test for allosteric interaction (Limbird, 2005) — (A) Control expeing 5-HT3 receptors and allowed to reach equilibrium (not shown). Dissociation of [3H]-ondbound radioactivity was measured at various times after initiation of dissociation. Half-lifeassociation phase was the same as control; [3H]-ondansetron dissociation was initiated by t1.4 min. (C) Effect of palonosetron on [3H]-ondansetron dissociation: association phase wasexcess of both ondansetron and palonosetron. Half-life of dissociation was 0.5 min. Resultsmodifier that accelerates the rate of dissociation of [3H]-ondansetron. Palonosetron also aaffected the rate of dissociation of [3H]-palonosetron (Rojas et al., 2008).

accelerated the rate of dissociation from the receptor of both [3H]-ondansetron (Fig. 2) and [3H]-granisetron (Rojas et al., 2008). Thedifference in binding to the receptor could be the result of palonose-tron's different structure: most 5-HT3 receptor antagonists incorporatea 3-substituted indole resembling serotonin whereas palonosetron ex-hibits a fused tricyclic ring attached to a quinuclidine moiety (Fig. 3).Differential binding pointed to palonosetron interacting with 5-HT3receptors at different or additional sites than those binding granisetronor ondansetron.

Differences in binding in turn suggested the potential for differenteffects on receptor function. 5-HT3 receptor antagonists block serotonin-induced calcium-ion influx into cells via 5-HT3 receptor channels.When HEK293 cells expressing the 5-HT3 receptor were incubatedwith ondansetron, granisetron or palonosetron and calcium-ion influxmeasurementsweremadewhile the receptor antagonistswere present,all three compounds inhibited calcium-ion influx. The resultswere con-sistent with these agents being 5-HT3 receptor antagonists. However, ifcells were first preincubated with each receptor antagonist followed byrinsing of cells to remove the compounds from the media, cells thatwere preincubated with palonosetron surprisingly still exhibited sub-stantial inhibition of calcium-ion influx. In contrast, cells that hadbeen preincubated with either granisetron or ondansetron followedby infinite dilutions and dissociation, recovered their ability to respondto serotonin-induced calcium-ion influx. (Rojas et al., 2010b).

4.2. Receptor internalization

One possible explanation for these findings was that palonosetroncould trigger 5-HT3 receptor internalization. Internalization of recep-tors would cause a reduction in receptor population at the cell surfaceand result in persistent inhibition of receptor function. The effectwould last longer than simple binding equilibria between ligandand receptor; this equilibrium is altered when receptor antagonistsare removed by infinite dilution and/or rinsing in cell culture or bynormal circulationwithin an organism. In order to determine if receptorinternalization could be occurring with any of these three receptorantagonists, a series of four independent experiments was carried out.First, when incubating [3H]-palonosetron with cells expressing the5-HT3 receptor for as little as 15 min followed by dissociation condi-tions, [3H]-palonosetron remained associated with whole cells but notto cell-free membranes. Second, [3H]-palonosetron's binding to cellswas resistant to both protease and acid treatments designed to dena-ture cell surface proteins suggesting the ligand-receptor complex wasinside the cells rather than at the surface (Fig. 4 and Table 1). Third,cells pretreated with unlabeled palonosetron subsequently exhibited

riment: [3H]-ondansetron was added to N1E-115 cell membrane preparations contain-ansetron from receptor was initiated by the addition of excess ondansetron. Receptor

of dissociation was 1.2 min. (B) Effect of granisetron on [3H]-ondansetron dissociation:he addition of excess of both ondansetron and granisetron. Half-life of dissociation wasthe same as control; dissociation of [3H]-ondansetron was initiated by the addition ofare representative of three independent determinations. Palonosetron is an allostericccelerated the dissociation of [3H]-granisetron. Neither ondansetron nor granisetron

Fig. 3. Chemical structures of serotonin and 5-HT3 receptor antagonists — Most 5-HT3 receptor antagonists are based on a 3-substituted indole structure (in red) that resemblesserotonin. Palonosetron's structure has a tricyclic ring system attached to a quinuclidine moiety.

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reduced cell surface 5-HT3 receptor binding. Finally, palonosetron-triggered receptor internalization was visualized by confocal fluores-cence microscopy using cells transfected with 5-HT3 receptor fused toenhanced cyan fluorescent protein. In contrast, parallel studies in eachset of experiments with granisetron and ondansetron showed minimalandno effect on receptor internalization respectively (Rojas et al., 2010b).

Even though receptor antagonist-driven internalization is less com-mon than agonist-driven internalization and possibly requires a morenuanced interaction between ligand and receptor than simple receptorblockade, there is no a priori reason why it could not occur. In fact, re-ceptor antagonist-driven internalization has been reported for severalreceptor systems (Lin et al., 2000; Pheng et al., 2003; Roettger et al.,1997). 5-HT3 receptors are known to be internalized by their naturalligand serotonin; after serotonin-induced internalization free receptorreappears at the surface within 1 h (Freeman et al., 2006). The bindingaffinity of palonosetron is approximately 2500-fold higher than that ofserotonin (Wong et al., 1995; Yan et al., 1999); upon palonosetron-triggered receptor internalization, this compound would be expected

Fig. 4. Rationale of protease and/or acid treatments to detect receptor internalization—Whecells to protease (Simantov and Sachs, 1973) or acid (Haigler et al., 1980) denatures the recewhen there is binding and internalization, protease or acid treatments will still release som[3H]-receptor antagonist-receptor complex will remain inside the cells.

to remain bound to the receptor much longer than serotonin, preventrecycling and cause a reduction in receptor density at the cell surface.Pertinent to this point was the finding that after palonosetron-inducedreceptor internalization, asmeasured by reduced binding of [3H]-palono-setron, 5-HT3 receptor remained internalized for at least 2.5 h after incu-bation with palonosetron (Rojas et al., 2010b).

4.3. Inhibition of 5-HT3/NK1 receptor crosstalk

Palonosetron-triggered receptor internalization would be expectedto influence cell processes including receptor signaling and crosstalk.Crosstalk between NK1 and 5-HT3 receptor signaling pathways hasbeen reported by different laboratories. For example, SP, an agonistacting largely at the NK1 receptor, was shown to potentiate 5-HT3 re-ceptor mediated inward current in rat trigeminal ganglion neurons(Hu et al., 2004). In independent studies, 5-HT3 receptor antagonistswere shown to block SP-mediated vagal afferent activation. In addition,NK1 antagonism blocked serotonin-induced vagal afferent activation

n [3H]-receptor antagonist binds to receptor at the cell surface only, limited exposure ofptor at the surface and releases all [3H]-receptor antagonist into the media. In contrast,e [3H]-receptor antagonist bound to receptors at the cell surface; however, internalized

Table 1Protease and acid treatments to demonstrate receptor internalization.

% [3H]-receptor antagonist remaining with cells after each treatment

Protease treatment Acid treatment

[3H]-ondansetron (30 nM) 3±1 2±0.1[3H]-granisetron (5 nM) 11±5 3±0.6[3H]-palonosetron (1 nM) 62±8 53±2

HEK293 cells expressing 5-HT3 receptors were incubated with excess [3H]-receptor an-tagonist (5-fold Kd) for 24 h. Media were removed and cells were exposed to protease(trypsin) for 5 min at 37 °C or acetic acid (pH 2.5) for 6 min on ice. Cells were subse-quently washed and radioactivity present in each wash and in the cells was measuredand percent radioactivity in the cell fraction was calculated. Values shown are percent ofreceptor antagonist remaining with cells. Data are the average of four independent deter-minations±S.E.M. After protease and acid treatments about 50–60% of [3H]-palonosetronremained with the cells consistent with receptor internalization. In contrast, most[3H]-ondansetron and [3H]-granisetron were found in the washes and only trace orminimal amounts remained with cells. Similar results were obtained when chymotrypsinwas used instead of trypsin (Rojas et al., 2010b).

Fig. 5. Effect of 5-HT3 receptor antagonists on cisplatin-induced neuronal response toSP — (A) Cisplatin (5 mg/kg) was administered to rats and 10 h later single nodoseneuronal activity in response to SP (10 μg/kg) was measured. Receptor antagonistswere administered 10–30 min before SP administration. Receptor antagonist dosestook into account differences in clinical dose and were higher than those used in ani-mal models where efficacy with these receptor antagonists was observed. Basal mea-surements were approximately 8 impulses/10 s; administration of receptorantagonists alone did not have an effect on base line. Data are the average of at least12 independent neuronal measurements from at least seven rats (***, pb0.001 com-pared to SP); error bars correspond to ±S.E.M. Adapted from (Rojas et al., 2010a).Only palonosetron inhibited the cisplatin-induced neuronal response to SP. (B) Illustra-tion of 5HT3/NK1 receptor crosstalk — Palonosetron does not bind to the NK1 receptordirectly (Wong et al., 1995) but it could still inhibit the SP response through inhibitionof receptor signaling crosstalk. Even though the mechanism is not understood, cross-talk between the 5-HT3 and NK1 receptors has been reported by several laboratories(Darmani et al., 2011; Hu et al., 2004; Minami et al., 2001).

Table 2Summary of comparison among palonosetron, ondansetron and granisetron.

Palonosetron Ondansetron Granisetron

Half life (h)* >40 5–6 12Binding affinity (PKi) 10.45 8.19 8.91Positive cooperativity YES NO NOInhibition of receptor function Long lasting Short lasting Short lastingReceptor internalization YES NO NOInhibition of 5-HT3/NK1 receptorcrosstalk

YES NO NO

*Literature sources for the data in the table are as follows: half life — Constenla (2004)and Stoltz et al. (2004); binding affinity —Wong et al. (1995); positive cooperativity —

Rojas et al. (2008); inhibition of receptor function and receptor internalization — Rojaset al. (2010b); inhibition of 5-HT3/NK1 receptor crosstalk — Rojas et al. (2010a).

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(Minami et al., 2001). Evidence of receptor signaling crosstalk raised theinteresting possibility that palonosetron's unique efficacy in delayedemesis could be due to differential inhibition of the 5-HT3/NK1 receptorcrosstalk. Consequently, studies were carried out to study the effect ofpalonosetron, granisetron and ondansetron on SP-induced responsesin vitro and in-vivo. For the in vitro experiments, NG108-15 cells wereused because they express both 5-HT3 and NK1 receptors (Emerit etal., 1993; Reiser and Hamprecht, 1989). First, serotonin was shown toenhance SP-induced calcium-ionmobilization to demonstrate crosstalkbetween the two receptor systems in these cells. Next, NG108-15 cellswere preincubated with palonosetron, granisetron or ondansetronand then rinsed to remove receptor antagonists from the media; finallythe effect on serotonin enhancement of SP-induced calcium releasewas measured. Following preincubation with palonosetron, but notondansetron or granisetron, the serotonin enhancement of the SP re-sponse was inhibited. Importantly, in parallel studies in vivo, ratswere treated with cisplatin and palonosetron or granisetron or ondan-setron at different times after cisplatin administration. Pretreatment ofthe animals with cisplatin is known to induce a 3 to 6-fold increase ofthe neuronal response in nodose ganglia to SP (Wu et al., 2009); 10 hafter cisplatin administration single neuronal recordings from nodoseganglia were collected following stimulation with SP. Palonosetron, butnot ondansetron or granisetron, inhibited the cisplatin-enhanced SP re-sponse (Fig. 5). This inhibition was dose dependent and was observedeven when palonosetron was administered before cisplatin (Rojas et al.,2010a). The results indicated that palonosetron uniquely could inhibit5-HT3/NK1 receptor crosstalk both in vitro and in vivo. Palonosetrondoes not bind to the NK1 receptor directly (Wong et al., 1995) but it in-hibits the SP response through inhibition of receptor signaling crosstalk(Fig. 5). Even though 5HT3/NK1 receptor crosstalk has been reported byseveral laboratories (Darmani et al., 2011; Hu et al., 2004; Minami et al.,2001) the mechanistic details have not been elucidated.

Table 2 and Fig. 6 summarize the differences among palonosetron,ondansetron and granisetron detailed above. Taken together, thesedata suggest that palonosetron's allosteric interactions and positivecooperativity trigger receptor internalization resulting in persistentinhibition of 5-HT3 receptor function as well as inhibition of 5-HT3/NK1

receptor signaling crosstalk. These molecular interactions lead ultimatelyto a distinct inhibition on the SP response associatedwith delayed emesisthat is not observed with ondansetron or granisetron. These molecularpharmacology studies provide a rationale to explain palonosetron'sunique efficacy against delayed emesis observed in the clinic.

5. Current studies and future possibilities

Current guidelines for patients receiving highly emetogenic che-motherapy recommend the use of a 5-HT3 receptor antagonist,

dexamethasone and a NK1 receptor antagonist (Basch et al., 2011;Ettinger et al., 2011; Roila et al., 2010). Palonosetron is recommendedby the National Comprehensive Cancer Network (NCCN) as the

Fig. 6. Summary of pharmacological differentiation of palonosetron vs. other 5-HT3 receptor antagonists. All 5-HT3 receptor antagonists compete with serotonin and exhibit com-petitive binding (Grunberg and Hesketh, 1993). Palonosetron in addition to competing with serotonin exhibits allosteric binding and positive cooperativity (Rojas et al., 2008).Allosteric binding induces a conformational change that brings about an increased binding affinity between palonosetron and the 5-HT3 receptor. Increased binding affinity is possibly theresult of at least one additional palonosetron molecule binding to the same receptor. Palonosetron also triggers 5-HT3 receptor internalization (Rojas et al., 2010b) and inhibits 5-HT3/NK1

receptor crosstalk (Rojas et al., 2010a). These pharmacological differences help explain the ability of palonosetron, unique among 5-HT3 receptor antagonists, to inhibit delayed emesis.

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preferred 5-HT3 receptor antagonist for both high andmoderate emeticrisks to patients following intravenous chemotherapies to prevent eme-sis (Ettinger et al., 2011). The American Society of Clinical Oncology(ASCO) clinical guideline update recommends preferential use of palo-nosetron for moderate emetic risk regimens in combination with dexa-methasone. In addition, fosaprepitant, a prodrug of aprepitant, given ina single-day in intravenous formulation has been shown to be equiva-lent to aprepitant. Consequently, either aprepitant or fosaprepitant asthe NK1 receptor antagonist is considered an appropriate therapy(Basch et al., 2011).

Given the recommended therapy to treat CINV that includes theuse of an NK1 receptor antagonist, one question is whether palonose-tron's effect on prevention of delayed emesis would be masked by thepresence of an NK1 receptor antagonist or whether palonosetron'samelioration of delayed emesis is distinct and additive to that broughtabout by NK1 receptor antagonists. In short, does it make a differenceto use palonosetron vs. other 5-HT3 receptor antagonists when an NK1

receptor antagonist is also part of the therapy to treat CINV?Recent in vitro studies using NG108-15 cells which express NK1and

5-HT3 receptors have attempted to address this question. After preincu-bation with palonosetron, the SP response in these cells was inhibitedeven in the absence of added serotonin. This result indicated that palo-nosetron binding and subsequent 5-HT3 receptor internalization couldoccur without serotonin and that subsequent alteration of receptorcrosstalk and corresponding inhibition of the SP response could stillbe observed. In parallel experiments ondansetron and granisetron didnot inhibit the SP response (Rojas et al., 2011) (manuscript submitted).The finding with palonosetron allowed the experimental possibility ofdistinguishing the effect on the SP response from the serotonin re-sponse and a determination of whether palonosetron's inhibition of

the SP response could enhance inhibition of the NK1 receptor response.Accordingly, cellswere preincubatedwith palonosetron and netupitant,subsequently rinsed and the SP response was measured. Netupitantplus palonosetron exhibited a synergistic effect on inhibition of the SPresponse. This effect occurredwhen using concentrations of each recep-tor antagonist below their threshold for inhibition of the SP response oralternatively, when each receptor antagonist was used at concentra-tions where maximal inhibition of the SP response was observed(Rojas et al., 2011) (manuscript submitted). A recent in vivo studyusing the least shrew also reports synergistic antiemetic interactionsof the 5-HT3 receptor antagonist tropisetron and the NK1 receptorantagonist CP99, 994 (Darmani et al., 2011). However, synergism inthis case was observed only at a small tropisetron concentrationrange. The studies were limited due to tropisetron's ability to act as par-tial agonist at the 5-HT3 receptor when used at higher concentrations.The idea that palonosetron can enhance NK1 receptor antagonist effectsin contrast to ondansetron and granisetron has also been supported bythe results from a recent retrospective clinical study. The study showedthat the use of palonosetron with an NK1 receptor antagonist (aprepi-tant) during administration of highly emetogenic chemotherapy had alower risk for uncontrolled CINV events when compared to other5-HT3 receptor antagonists (ondansetron, granisetron and dolasetron)plus aprepitant (Schwartzberg et al., 2011).

In summary, significant progress has been made since the 1980swhen the best available treatment for CINV was a combination of anantidopaminergic agent and an anti-inflammatory steroid and lessthan half of the patients obtained relief. Current antiemetic treatmentinvolving 5-HT3 and NK1 receptor antagonists has largely alleviatedacute emesis and some delayed emesis. Delayed emesis however, re-mains an issue (Feyer and Jordan, 2011). Mechanistic studies using

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palonosetron suggest that the crossroads of acute and delayed emesisinclude interactions between the 5-HT3 and NK1 receptor neurotrans-mitter pathways and that inhibitions of these interactions present thepossibility of improved CINV treatment that encompasses both acuteand delayed emesis.

Acknowledgments

We thank Silvia Sebastiani and Silvia Olivari Tilola from HelsinnHealthcare for their help with the making of Table 2 and Fig. 6.

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