European Journal of Pharmacology 722 (2014) 147–155

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Pulmonary, gastrointestinal and urogenital pharmacology

Additive antiemetic efficacy of low-doses of the cannabinoid CB1/2 receptor agonist Δ9-THC with ultralow-doses of the vanilloid TRPV1 receptor agonist resiniferatoxin in the least shrew (Cryptotis parva) Nissar A. Darmani n, Seetha Chebolu, Weixia Zhong, Chung Trinh, Bryan McClanahan, Rajivinder S. Brar Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, 309 East Second Street, Pomona, CA 91766, USA

art ic l e i nf o

a b s t r a c t

Article history: Accepted 28 August 2013 Available online 22 October 2013

Previous studies have shown that cannabinoid CB1/2 and vanilloid TRPV1 agonists (delta-9-tetrahydrocannabinol (Δ9-THC) and resiniferatoxin (RTX), respectively) can attenuate the emetic effects of chemotherapeutic agents such as cisplatin. In this study we used the least shrew to demonstrate whether combinations of varying doses of Δ9-THC with resiniferatoxin can produce additive antiemetic efficacy against cisplatin-induced vomiting. RTX by itself caused vomiting in a bell-shaped dosedependent manner with maximal vomiting at 18 μg/kg when administered subcutaneously (s.c.) but not intraperitoneally (i.p.). Δ9-THC up to 10 mg/kg provides only 80% protection of least shrews from cisplatin-induced emesis with an ID50 of 0.3–1.8 mg/kg. Combinations of 1 or 5 μg/kg RTX with varying doses of Δ9-THC completely suppressed both the frequency and the percentage of shrews vomiting with ID50 dose values 5–50 times lower than Δ9-THC doses tested alone against cisplatin. A less potent TRPV1 agonist, capsaicin, by itself did not cause emesis (i.p. or s.c.), but it did significantly reduce vomiting induced by cisplatin given after 30 min but not at 2 h. The TRPV1-receptor antagonist, ruthenium red, attenuated cisplatin-induced emesis at 5 mg/kg; however, another TRPV1-receptor antagonist, capsazepine, did not. In summary, we present evidence that combination of CB1/2 and TRPV1 agonists have the capacity to completely abolish cisplatin-induced emesis at doses that are ineffective when used individually. & 2013 Elsevier B.V. All rights reserved.

Keywords: Least shrew Emesis Antiemetic Cisplatin Δ9-THC Resiniferatoxin Capsaicin Ruthenium red Capsazepine

1. Introduction The physiological processes involved in cisplatin-induced vomiting (CIV) are complex and involve release of multiple neurotransmitters (Darmani and Ray, 2009). Likewise, the emetic reflex arc is a highly complex system whose detailed circuitry is only partially characterized. Cisplatin-like drugs are thought to induce vomiting mainly via release of serotonin (5-HT) and substance P (SP) in the upper gastrointestinal tract (GIT) and brainstem which may directly activate their respective corresponding local 5-HT3- and tachykinin NK1-receptors (Darmani and Ray, 2009). Additionally they may indirectly activate brainstem emetic loci via stimulation of NK1- and 5-HT3-receptors present on vagal afferents in the GIT whose somata are in the nodose ganglion, and whose terminals are present in the area postrema (AP), the nucleus of the solitary tract (NTS), and the dorsal motor nucleus of the vagus nerve (DMNX) (Darmani and

n

Corresponding author. Tel.: þ 1 909 469 5654; fax: þ 1 909 469 5577. E-mail address: [email protected] (N.A. Darmani).

0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.08.051

Ray, 2009). The latter cluster of emetic nuclei is collectively described as the brainstem dorsal vagal complex (DVC). Cisplatin is a chemotherapeutic agent widely used for the treatment of several different types of cancers. However, it is highly emetogenic in both patients and animals (Warr, 2012; Darmani and Ray, 2009). This side-effect of cisplatin is biphasic in nature with the initial phase persisting up to 24 h post-administration in cancer patients, followed by a delayed phase occurring between days 3–7 after cisplatin treatment (Hesketh et al., 2003). Current prophylactic treatment for the prevention of cisplatin-induced vomiting includes the use of dexamethasone for both phases of CIV, plus a 5-HT3receptor antagonist for the suppression of the immediate emesis, as well as an NK1-receptor antagonist for the management of the delayed phase (Warr, 2012). The nausea and vomiting caused by cisplatin is distressing to patients and can reduce compliance and the quality of life of patients receiving such treatment (Lohr, 2008). Even with the use of triple antiemetic regimens, 20–30% of patients can still suffer from vomiting. The goal of modern antiemetic therapy is to abolish both CIV phases in order to allow all patients to continue effective antitumor therapy.

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Delta-9-tetrahydrocannabinol (Δ9-THC) is the main psychoactive component of marijuana and has been shown to possess significant antiemetic efficacy against both phases of CIV in animals and patients (Abrahamov et al., 1995; Darmani and Ray, 2009). Δ9-THC inhibits CIV via the stimulation of cannabinoid CB1 receptors in both the brainstem and the GIT (Darmani and Johnson, 2004; Ray et al., 2009; Van Sickle et al., 2003). Although the efficacy of Δ9-THC to suppress acute phase CIV is less than that of 5-HT3 receptor antagonists, the latter class of antiemetics is poorly effective against the delayed phase (Feyer and Jordan, 2011; Warr, 2012). Furthermore, a combination of Δ9-THC and 5-HT3 receptor antagonists leads to only minor or no additional antiemetic efficacy in both animals and patients (Meiri et al., 2007; Kwiatkowska et al., 2004; Wang et al., 2009). The transient receptor-potential vanilloid-1 receptor channel (TRPV1) is a target of the pungent component of chili peppers, capsaicin. Its ultrapotent analog resiniferatoxin (RTX) from the plant genus Euphorbia has been used in patients for pain relief and urinary incontinence (Kissin and Szallasi, 2011). RTX has also been shown to possess potent (100 μg/kg, s.c.) antiemetic properties against several centrally and peripherally acting emetogens, including cisplatin, in both ferrets and house musk shrews (Andrews and Bhandari, 1993; Andrews et al., 2000). However, RTX not only has undesirable actions on cardiovascular and pulmonary systems (Szallasi and Blumberg, 1999), but any dose larger than 10 μg/kg also causes significant vomiting by itself in the house musk shrew (Andrews et al., 2000). In an attempt to resolve the latter issues, investigators have used nonpungent TRPV1 agonists, such as olvanil, or synthetic hybrid agonists of CB1 and TRPV1 receptors, such as arvanil, for their antiemetic potential. However, these agents lack full antiemetic efficacy against CIV or copper sulphate-induced emesis (Chu et al., 2010; Sharkey et al., 2007). Since TRPV1- and CB1-receptors are known to co-localize in the brainstem emetic nuclei (Sharkey et al., 2007), it is possible that a combination of nonemetic ultralow dose(s) of RTX (e.g. 1–5 μg/kg) with low doses of Δ9-THC (e.g. 0.025–0.5 mg/kg) may provide additive antiemetic activity without producing significant side-effects. Thus, the present study was undertaken to investigate: (1) the emetic/ antiemetic potential of RTX alone in the least shrew, (2) whether a combination of ineffective low doses of RTX and Δ9-THC can prevent acute vomiting caused by a large dose of cisplatin, and (3) whether a less-potent analog of RTX capsaicin, or its nonselective antagonist ruthenium red, can affect cisplatin's emetic activity.

2.3. Experimental protocols

2. Materials and methods

2.4. Statistical analysis

2.1. Animals

The vomiting frequency data were analyzed using the Kruskal– Wallis non-parametric one-way analysis of variance (ANOVA) and post hoc analysis by Dunn's multiple comparisons test. The percentage of animals vomiting across groups at different doses was compared using the chi square test. Latency to the first vomit was analyzed by the Kruskal–Wallis ANOVA and post hoc analysis by Dunn's multiple comparison test. When an animal failed to vomit, the latency value equal to the corresponding total observation period (i.e. 30, 60 or 120 min) was noted. ID50 values were calculated using non-linear regression analysis (Graph Pad PRISM version 6, San Diego, CA). In all cases, a P-value o0.05 was necessary for statistical significance.

Adult least shrews were bred in the animal facility at the Western University of Health Sciences. Our initial studies have demonstrated no gender differences, so both males and females were used. Shrews were housed in groups of 5–10 on a 14:10 light: dark cycle, fed with food and water ad libitum as described previously (Darmani et al., 1999). All the shrews used were 45–60 days old and weighed between 4 and 6 g. All experiments were conducted between 9:00 and 16:00 h and in accordance with an approved Western University of Health Sciences IACUC protocol.

On the day of the experiment shrews were brought from the animal facility, separated into individual cages, and were allowed to adapt for at least two hours. Two hours before experimentation, daily food was withheld but shrews were given four mealworms each prior to injection to aid in identifying wet vomits as described previously (Darmani, 1998). The dose–response emetic potential of RTX was investigated following subcutaneous (s.c.) injection of either vehicle (i.e. 0 μg/kg, n¼7) or its varying doses (2, 5, 10, 18, 25 or 50 μg/kg, n¼6–15 shrews per dose). The shrews were observed for 30 min for vomiting behavior (number of animals vomiting within groups, frequency of vomits) and the latency to the first vomit was recorded. Least shrews significantly vomited in response to RTX at 10 μg/kg or larger doses. Thus, for the determination of antiemetic potential of RTX, only 5 μg/kg or lower doses were used. Each shrew was used once and then euthanized with an overdose of pentobarbital (100 mg/kg, i.p.) following the termination of each experiment. To investigate the direct antiemetic potential of RTX, varying doses were administered in different groups of shrews either subcutaneously or intraperitoneally (0, 1, and 5 μg/kg n¼ 7–10, s.c., or 0, 1, 2.5, and 5 μg/kg, n¼8–18, i.p.). Two hours later each shrew received a 20 mg/ kg i.p. dose of cisplatin and the frequency of vomits and the number of shrews vomiting was recorded for 1 h as described above. Subsequently, the antiemetic potential of RTX (1 or 5 μg/kg, s.c.) in combination with varying doses of Δ9-THC (0.1, 0.5, 2.5, 5, and 10 mg/kg, i.p.) was tested against acute cisplatin- (20 mg/kg, i.p.) induced vomiting. Cisplatin and Δ9-THC were administered simultaneously 2 h after the RTX injection, and the animals were monitored for 1 and 2 h postcisplatin injection. A 2 h maximum observation post-cisplatin injection was chosen since 95% of the induced vomiting occurred during this period following an acute administration of 20 mg/kg (i.p.) cisplatin in the least shrew (Darmani et al., 2009). The antiemetic potential of another TRPV1 agonist, E-capsaicin (0, 2.5, 5, and 10 mg/kg, n ¼6–10, i.p.) and the TRPV1 antagonist, ruthenium red (0, 1, 2.5, and 5 mg/kg, n ¼5–8, i.p.) were also tested against acute cisplatin- (20 mg/kg, i.p.) induced vomiting. The TRPV1 receptor agonist (E-capsaicin) was administered singly followed by cisplatin 30 min later, whereas the TRPV1 antagonist (ruthenium red) was injected along with cisplatin at the same time. The shrews were observed for vomiting behavior for 60 min post-cisplatin administration as described above.

2.2. Drugs 3. Results Cisplatin (cis-platinum (II) diamine dichloride (Pt(NH3)2)Cl2) and Δ9-THC were obtained from Sigma/RBI (St. Louis, MO). Resiniferatoxin, capsaicin, capsazepine and ruthenium red were purchased from Tocris (Minneapolis, MN). Cisplatin was dissolved in water and sonicated for 1 h. Ruthenium red was dissolved in saline. RTX, Δ9-THC, capsaicin and capsazepine were dissolved in alkamuls:ethanol:saline in a 1:1:18 ratio.

3.1. Proemetic effects of RTX Subcutaneous (s.c.) route of administration of RTX was used to assess the proemetic potential of RTX in the least shrew (Fig. 1). RTX increased the frequency of vomiting in shrews in a bell-shaped and dose-dependent manner (KW (6, 58) ¼ 32.04;

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Fig. 1. Dose–response proemetic effects of resiniferatoxin (RTX) in Cryptotis parva. Different groups of least shrews were injected with varying doses of RTX (s.c.) and were then observed for 30 min post-injection. The mean frequency (7 SEM) (graph A), the percentage (graph B) of shrews vomiting, and the mean latency ( 7 SEM) to the first vomit (graph C) were recorded. Significant differences relative to the 0 μg/kg control group are indicated as nPo 0.05, nnPo 0.01 and nnnP o0.001.

P o0.0001). The frequency of vomiting was maximally and significantly increased at 18 μg/kg (P o0.01) (Fig. 1A). In addition, the percentage of animals exhibiting emesis in response to RTX varied in a bell-shaped and dose-dependent manner (χ2 (6, 58) ¼ 26.78; P o0.001) with 100% vomiting occurring at 18 μg/kg (χ2 (1, 14) ¼ 16; P o0.001) (Fig. 1B). Significant increases (50 and 67%) in the number of animals vomiting also occurred at 10 (χ2 (1, 15) ¼ 4.985; P o0.05) and 25 (χ2 (1, 20) ¼8.556; P o0.01) μg/kg RTX doses. The latency to the first vomit altered in a U-shaped fashion with a significant reduction occurring at 18 μg/kg RTX (KW (6, 58) ¼ 32.06; Po0.01) (Fig. 1C). 3.2. Antiemetic effects of RTX against CIV The antiemetic dose–response potential of RTX administered via the s.c. (Fig. 2A–C) and the i.p. (Fig. 2D–F) routes 2 h prior to cisplatin (20 mg/kg) injection are depicted in Fig. 2. Relative to the vehicle-pretreated control group (0 mg/kg), the 5 μg/kg s.c. dose of RTX caused significant reductions in both the frequency (KW (2, 24)¼9.356; Po 0.01) (Fig. 2A) and the percentage of shrews vomiting (50% reduction) (χ2 (1, 15) ¼ 4.95; P o0.05) in response to cisplatin (Fig. 2B). Furthermore, relative to the 0 μg/kg control group, the 2.5 μg/kg i.p. dose of RTX completely suppressed both the frequency (KW (4, 41) ¼23.7; P o0.0001) (Fig. 2D) and percentage of shrews vomiting in response to cisplatin administration (Fig. 2E) (χ2 (1, 26) ¼17.95; Po 0.001). However, in approximately 20% of the 5 μg/kg i.p. RTX-pretreated shrews, cisplatin caused some vomiting (χ2 (1, 26) ¼10.81; P o0.01). Although RTX administration via the s.c. route did not cause a significant change in vomit latency (Fig. 2C), its i.p. injection significantly delayed (KW (3, 42) ¼17.82; P o0.01) time to the first vomit at 2.5 (P o0.01) and 5 (P o0.05) μg/kg doses (Fig. 2F). 3.3. Antiemetic effects of RTX in combination with Δ9-THC against CIV We have previously shown that Δ9-THC up to 10 mg/kg can suppress cisplatin (20 mg/kg)-induced emesis in 80% of least

shrews (Darmani, 2001). In the current experiment RTX (1 μg/kg, s.c.) by itself failed to significantly attenuate either the frequency or the percentage of shrews vomiting in response to cisplatin (i.e. V/V versus 0 mg/kg Δ9-THC group) (Fig. 3A and B). Shrews pretreated with RTX (1 or 5 μg/kg, s.c.) plus varying doses of Δ9-THC (i.p.) which were initially monitored for 1 h post-cisplatin treatment, exhibited dose-dependent decreases in the frequency and the percentage of shrews vomiting (Fig. 3A, B, D, and E). In fact, compared to the vehicle/vehicle (V/V) control group, which had received only the two vehicles, but no RTX or Δ9-THC, the 1 μg/kg RTX-pretreated shrews receiving varying doses of Δ9-THC exhibited significant reductions in vomit frequency [KW (6, 110) ¼ 60.98; P o0.0001] at its 0.5 mg/kg and larger doses with complete emesis protection at 10 mg/kg Δ9-THC (P o0.001 for all doses) with an ID50 of 0.016 (0.002–0.15) mg/kg (Fig. 3A). Thus, both RTX and Δ9-THC were required for optimal antiemetic effect. Furthermore, relative to the 1 μg/kg RTX-pretreated shrew control group that had received Δ9-THC's vehicle alone (i.e. 0 mg/kg), Δ9-THC caused significant reductions in vomit frequency from its 2.5 mg/ kg dose (P o0.01 for all) (Fig. 3A). In addition, compared to the V/V control group, varying doses of Δ9-THC in the 1 μg/kg RTXpretreated shrews caused significant reductions in the number of animals vomiting at its 0.1 (50%, χ2 (1, 21) ¼9.079; P o0.01), 0.5 (58%, χ2 (1, 39) ¼13.65; Po0.001), 2.5 (78%, χ2 (1, 36)¼ 22.3; Po 0.001) and 5 mg/kg doses (90%, χ2 (1, 33)¼27.79; Po 0.001), with 100% vomit protection at 10 mg/kg (χ2 (1, 23) ¼25; P o0.001) and ID50 of 0.34 (0.2–0.6) mg/kg (Fig. 3B). Moreover, relative to the 0 mg/kg Δ9-THC control group, significant reductions in percentage of shrews vomiting were seen at its 0.5 (58%), 2.5 (78%), 5 (90%) and 10 mg/kg (100%) doses (Fig. 3B). Relative to the V/V control group, a combination of 5 μg/kg RTX and 0.5 mg/kg Δ9-THC completely prevented both the frequency [KW (4, 67)¼40.19; Po 0.0001) and the percentage (χ2 (1, 27) ¼29; Po 0.001) of animals vomiting (Fig. 3D and E) with respective ID50 values of 0.0014 (0.0000003–0. 67) mg/kg and 0.015 (0.007–0.03) mg/kg. The 0.025 and 0.1 mg/kg doses of Δ9-THC (P o0.001 for both) also showed significant decreases in the frequency of emesis.

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Fig. 2. The antiemetic dose–response effects of resiniferatoxin (RTX) against cisplatin-induced vomiting (20 mg/kg, i.p.) in Cryptotis parva. Varying doses of RTX were administered s.c. (graphs A–C) or i.p. (graphs D–F) to different groups of least shrews 2 h prior to an injection of cisplatin (20 mg/kg, i.p.). The frequency (mean 7 SEM) (graphs A and D), the percentage of shrews vomiting (graphs B and E), and the mean latency ( 7SEM) to the first vomit (graphs C and F) were recorded for 1 h post-cisplatin injection. Significant differences relative to the corresponding 0 μg/kg control groups are indicated as nPo 0.05, nnP o0.01 and nnnP o 0.001.

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Fig. 3. Additive antiemetic dose–response effects of combinations of 1 (graphs A–C) and 5 μg/kg (graphs D–F) resiniferatoxin (RTX) with varying doses of Δ9-THC against cisplatin-induced vomiting in Cryptotis parva. Different groups of least shrews were injected with RTX (s.c.) and 2 h later were challenged with Δ9-THC (i.p.) and cisplatin (20 mg/kg, i.p.) simultaneously. The induced vomiting was then observed for 1 h. Both the frequency (mean 7 SEM) (graphs A and D) and the percentage of shrews vomiting (graphs B and E) were significantly reduced in a dose-dependent manner by the cited doses of Δ9-THC. The latency to the first vomit was significantly prolonged (graphs C and F). Significant differences relative to the resiniferatoxin vehicle/Δ9-THC vehicle control group (i.e. V/V) are indicated as þ P o 0.05, þ þ P o 0.01 and þ þ þ Po 0.001. Significant differences relative to the resiniferatoxin (1 or 5 μg/kg)/Δ9-THC vehicle control group (i.e. 0 mg/kg) are indicated as nPo 0.05, nnP o 0.01 and nnnPo 0.001.

Likewise, there were significant reductions in the percentage of shrews vomiting at its 0.025 (70%, χ2 (1, 23) ¼ 14.58; P o0.001) and at 0.1 (75%, χ2 (1, 25)¼ 16.88; P o0.001) mg/kg doses. However,

relative to the 0 mg/kg Δ9-THC control group, only the 0.5 mg/kg Δ9-THC dose significantly and completely protected shrews from vomiting (Fig. 3E). Relative to V/V control group, the combined

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Fig. 4. Additive antiemetic dose–response effects of combinations of 1 (graphs A-C) and 5 μg/kg (graphs D-F) resiniferatoxin (RTX) with varying doses of Δ9-THC against cisplatin-induced vomiting in Cryptotis parva. Different groups of least shrews were injected with RTX (s.c.) and 2 h later were challenged with Δ9-THC (i.p.) and cisplatin (20 mg/kg) simultaneously. The induced vomiting was then observed for 2 h. Both the frequency (mean 7 SEM) (graphs A and D) and the percentage of shrews vomiting (graphs B and E) were significantly reduced in a dose-dependent manner by the cited doses of Δ9-THC. The latency to the first vomit was significantly prolonged (graphs C and F). Significant differences relative to the resiniferatoxin vehicle/Δ9-THC vehicle control group (i.e. V/V) are indicated as þ P o 0.05, þ þ P o 0.01 and þ þ þ Po 0.001. Significant differences relative to the resiniferatoxin (1 or 5 μg/kg)/Δ9-THC vehicle control group (i.e. 0 mg/kg) are indicated as nPo 0.05, nnP o 0.01 and nnnPo 0.001.

1 μg/kg RTX plus all tested doses of Δ9-THC, except 0.1 mg/kg, exhibited significant delays in the onset of the first vomit. However, relative to the 0 mg/kg control group, Δ9-THC caused significant delays in vomit onset from 2.5 mg/kg (KW (6,110) ¼ 50; P o0.0001) (Fig. 3C). With the 5 μg/kg RTX plus all tested doses of Δ9-THC (i.e. 0–0.5 mg/kg), significant delays to the first vomit were seen only relative to the V/V control group (KW (4, 67)¼41.96; P o0.0001) and not with the 0 control group (Fig. 3F). To determine whether the additive antiemetic efficacy of RTX and Δ9-THC persists for longer periods, different groups of shrews were treated identically, but observed for 2 h post-cisplatin administration (Fig. 4). Although the antiemetic efficacy of combined doses of RTX and Δ9-THC tended to wane a little in the 2 h observation period, essentially similar dose-dependent additive antiemetic effects persisted (for 1 μg/kg RTX [KW (5, 59) ¼ 45.22; P o0.0001 versus V/V control, and KW (4, 45) ¼27.84; Po0.0001 versus 0 mg/kg Δ9-THC control]; for 5 μg/kg RTX [KW (4, 55) ¼ 32.83; P o0.0001 versus V/V control, and KW (3, 41) ¼ 10.45; P o0.05 versus 0 mg/kg Δ9-THC control]). For statistical details see Fig. 4A, B, D, and E. In addition, relative to the V/V group, significant delays to the first vomit in the 1 μg/kg RTX-pretreated shrews were seen at the 2.5, 5 and 10 mg/kg Δ9-THC doses (Fig. 4C) (KW (5, 59) ¼44.61; Po 0.0001). On the other hand, relative to the corresponding 0 control group, only the 10 mg/kg Δ9-THC dose caused a significant delay (P o0.0001) (Fig. 4C). In the 5 μg/kg RTX-pretreated shrews, the 0–0.5 mg/kg Δ9-THC doses showed significant increases in the onset of vomits relative to the V/V group only ( KW (4, 55) ¼ 36.97; Po 0.0001) (Fig. 4F). The

1 and 5 μg/kg doses of RTX did not cause vomiting by themselves in the above discussed combination experiments. 3.4. Effects of E-capsaicin on CIV Relative to the 0 mg/kg control group, i.p. administration of E-capsaicin caused significant decreases in the frequency of cisplatin-induced vomiting (KW (3, 32) ¼ 16.89; P o0.0001) at the 2.5 (P o0.05) and 10 mg/kg (P o0.001) (Fig. 5A). Statistically significant decreases in the percentage of shrews vomiting occurred with doses of 2.5 (50%, χ2 (1, 14) ¼ 4.36; P o0.05), 5 (50%, χ2 (1, 14) ¼4.36; P o0.05) and 10 mg/kg (90%, χ2 (1, 14) ¼ 12.34; P o0.001) (Fig. 5B). In addition, the latency to the first vomit showed significance from 2.5 mg/kg (KW (3, 32) ¼16.93; Po 0.001) but there was no differences among the E-capsaicin doses used (Fig. 5C). Furthermore, subcutaneous administration of E-capsaicin (2.5–10 mg/kg) failed to show a statistically significant decrease in these emetic parameters (data not shown). E-capsaicin by itself was not emetic via either route up to 10 mg/kg dose. 3.5. Effects of ruthenium red on CIV The nonselective TRPV1 antagonist ruthenium red was effective at reducing the frequency of vomiting (KW (3, 25) ¼12.19; Po 0.01) and the percentage of vomiting (62.5%, χ2 (1, 17) ¼5.078; Po 0.05) at its 5 mg/kg dose (Fig. 6A and B). Latency to the first vomit showed no significant change relative to the vehicle-treated control group (Fig. 6C).

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Fig. 5. The antiemetic dose–response effects of the TRPV1 agonist E-capsaicin (i.p.) against cisplatin-induced emesis in Cryptotis parva. Least shrews were challenged with cisplatin (20 mg/kg, i.p.) 30 min after administration of varying doses E-capsaicin (s.c.) and were observed for 1 h post-cisplatin injection. The mean frequency ( 7 SEM) of vomiting (graph A), the percentage of shrews vomiting (graph B), and the mean latency ( 7 SEM) to the first vomit (graph C) were recorded. Significant differences relative to the 0 mg/kg control group are indicated as nPo 0.05, nnPo 0.01 and nnnP o0.001.

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Ruthenium Red (mg/kg)

Fig. 6. The antiemetic dose–response effects of the nonselective TRPV1 antagonist ruthenium red (i.p.) against cisplatin-induced vomiting in Cryptotis parva. Least shrews were challenged with cisplatin (20 mg/kg, i.p.) and varying doses of ruthenium red (i.p.) simultaneously and were then observed for 1 h post-cisplatin injection. The mean frequency ( 7 SEM) (graph A), the percentage of shrews vomiting (graph B), and the mean latency ( 7 SEM) to the first vomit (graph C) were recorded. Significant differences relative to the 0 mg/kg control group are indicated as nPo 0.05, nnPo 0.01 and nnnPo 0.001.

4. Discussion Cisplatin is clinically effective against a variety of tumors (Reed and Chabner, 2010) but is also one of the most emetogenic antitumor therapies currently in use (Warr, 2012). As a result of this, and since no single antiemetic agent can prevent CIV,

combined prophylactic therapies are almost always a necessity. The different classes of antiemetics currently in use, including the serotonin 5-HT3-receptor antagonists, tachykinin NK1-receptor antagonists, anti-inflammatory steroids, and the cannabinoid CB1-receptor agonists, have been studied extensively and found to provide variable efficacy, either alone or in combination

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(Darmani and Ray, 2009; Feyer and Jordan, 2011; Hesketh et al., 2003; Warr, 2012). In addition, a significant body of the literature demonstrates that several distinct combination regimens can be superior than when each agent given alone in terms of antiemetic efficacy against CIV in both patients and animals: (i) a 5-HT3-receptor antagonist (e.g. ondansetron) and dexamethasone (du Sert et al., 2011; Warr, 2012) and (ii) a 5-HT3-receptor antagonist (e.g. tropisetron) and an NK1-receptor antagonist (e.g. aprepitant or CP99,994) (Darmani and Ray, 2009; Basch et al., 2011). On the other hand, combination of Δ9-THC with different 5HT3-receptor antagonists leads to only minor or no additional antiemetic efficacy in both patients and animals (Meiri et al., 2007; Kwiatkowska et al., 2004; Wang et al., 2009). The aim of the present study was to investigate the additive antiemetic potential of two plant-derived antiemetics, the TRPV1-receptor agonist RTX, and the cannabinoid CB1/2-receptor agonist, Δ9-THC. We focused on this antiemetic combination since recent evidence demonstrate that not only TRPV1- and CB1-receptors co-localize in the brainstem emetic nuclei (Sharkey et al., 2007), but also TRPV1mediated Ca2 þ ion entry is functionally coupled with CB1 receptors (Tsumara et al., 2012), making it more likely that these two diverse receptor systems functionally interact. Vanilloid TRPV1 receptors are nonselective cation channels involved in thermal, chemical and hyperalgesic responses. TRPV1 receptors are activated by vanilloids such as capsaicin and its ultrapotent analog, RTX. RTX-like agents are used in the clinic for the treatment of chronic pain (Lee et al., 2012; Kissin and Szallasi, 2011), urinary incontinence (Byeong et al., 2012; Kuo, 2008) and corneal injury (Bates et al., 2010). In addition, in the laboratory RTX has been shown to reduce vomiting by diverse emetogens including CIV in dogs, ferrets and house musk shrews (Andrews and Bhandari, 1993; Andrews et al., 2000; Chu et al., 2010; Yamakuni et al., 2002). However, RTX exhibits complex dosedependent pro/antiemetic effects in the house musk shrew. Thus, unlike ferrets and dogs, the house musk shrew vomits in response to administration of 10–1000 μg/kg (s.c.) doses of RTX, but at these doses RTX also potently suppresses emesis caused by diverse emetogens in this species (Andrews et al., 2000). In the present study, RTX caused vomiting in a dose- and route-dependent but bell-shaped manner in the least shrew, with a significant number of animals vomiting at 10 μg/kg (s.c.), and all tested shrews vomited with maximal frequency at 18 μg/kg. While the latter RTX dose had the shortest latency to the first vomit, the overall latency pattern was U-shaped in nature. Moreover, at 50 μg/kg, RTX induced vomiting in less than 10% of least shrews, whereas none of its tested i.p. doses (2, 5, 10, 18, 25 or 50 μg/kg) caused emesis (data not shown). Thus, the least shrew appears to be even more sensitive to the proemetic effects of subcutaneous RTX. The less-potent TRPV1 agonist E-capsaicin and its analog Z-capsaicin (data not shown), up to 10 mg/kg, did not cause vomiting via either the i.p. or s.c. routes in the least shrew. On the contrary, these congeners have been reported to be only seven times less potent than RTX in producing emesis when administered centrally in the house musk shrew (Rudd and Wai, 2001). Such species differences in the pharmacology of vanilloid receptors also occur in common laboratory animals (Szallasi, 1994). Likewise, the house musk shrew tachykinin NK1-receptor is different from the human NK1 receptor (Rudd and Wai, 2001), whereas the least shrew NK1-receptor exhibits over 90% sequence homology with its human counterpart (Darmani et al., 2013). Pharmacological, micro-injection and vagotomy studies in the house musk shrew suggest that the most likely explanation for RTX-induced vomiting is the stimulation of TRPV1 receptors present on vagal afferents in the GIT and the consequent release of substance P in the brainstem emetic nuclei (Andrews and Bhandari, 1993; Andrews et al., 2000). Thus, the inability of i.p. RTX to induce vomiting in the least shrew

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seems to be an enigma, unless it causes extremely rapid desensitization of vagal afferents via this route (see later) or it is very rapidly metabolized prior to reaching its main site of action in the brainstem via this route. However, RTX incubated in liver microsomes is not only quite stable for at least 60 min (Choi et al., 2009), but also possesses antiemetic activity via the i.p. route. In fact pretreatment with nonemetic ultralow doses of RTX (1, 2.5 or 5 μg/kg) via either the s.c. or the i.p. route, caused a similar and significant reductions, in both the frequency and the percentage of least shrews vomiting, in response to a very large dose of cisplatin (20 mg/kg, i.p.). The mechanism of the antiemetic action of RTX probably involves rapid desensitization of TRPV1 receptors present on vagal afferents, as well as reduction of further release of substance P and/or other emetic transmitters in the brainstem emetic nuclei, through inhibition of voltage-gated calcium channels (Andrews and Bhandari, 1993; Andrews et al., 2000; Shiroshita et al., 1997). Similarly, E-capsaicin (but not Z-capsaicin), albeit at relatively larger intraperitoneal (i.p.) doses (2.5–10 mg/kg), significantly reduced (490%) both the frequency and number of shrews vomiting caused by cisplatin when it was administered 30 min, but not 2 h (data not shown), prior to cisplatin injection. E-capsaicin (i.p.) also delayed the onset of the first vomit to near maximum at its lowest tested dose. However, when E-capsaicin was administered subcutaneously, no antiemetic effect was observed up to 10 mg/kg (data not shown). These findings suggest that at the doses tested, desensitization (antiemetic efficacy) caused by E-capsaicin is probably of shorter duration. In fact, desensitization caused by TRPV1 agonists is both time- and dose-dependent (Craft and Porreca, 1994). Ruthenium red, a nonselective TRPV1 antagonist, was also found to be capable of attenuating CIV in the least shrew, however, it did not delay cisplatin's onset to the first vomit. In fact, at a dose of 5 mg/kg (i. p.), ruthenium red significantly reduced both the percentage and the frequency of shrews vomiting by 63–88% reduction. Ruthenium red has also been shown to suppress vomiting caused by both resiniferatoxin and E-capsaicin in the house musk shrew (Cheng et al., 2005; Rudd and Wai, 2001). Thus, not only agonistinduced desensitization, but also blockade of TRPV1 receptors can lead to antiemetic activity. However, capsazepine, another wellinvestigated TRPV1 antagonist, failed to demonstrate antiemetic efficacy against cisplatin up to 20 mg/kg (s.c. or i.p.) in the least shrew (data not shown). The latter finding is not entirely surprising since capsazepine was also shown to be ineffective against resiniferatoxin-induced vomiting in the house musk shrew despite the fact it can compete for the binding site of [3H] resiniferatoxin (Jerman et al., 2000; Rudd and Wai, 2001). Since all of the published emesis-related RTX studies have been done via the s.c. route, we further investigated the additive antiemetic potential of combinations of nonemetic ultralow doses of RTX (e.g. 1 and 5 μg/kg, s.c.) with low doses of Δ9-THC (e.g. 0.025–0.5 mg/kg, i.p.). Previously we have shown that i.p. administration of Δ9-THC up to 10 mg/kg can only protect least shrews from cisplatin (20 mg/kg, i.p.)-induced emesis by 80% with an ID50 of 0.3–1.8 mg/kg (Darmani, 2001). A 2 h pretreatment with either 1 or 5 μg/kg RTX (s.c.) followed by administration of varying doses of Δ9-THC completely suppresses both the frequency and the percentage of shrews vomiting with ID50 dose values 5–50 times lower than Δ9-THC doses tested alone against cisplatin. In addition, the latency to the first vomit was further prolonged by Δ9-THC. Full antiemetic efficacy of this combined regimen tended to wane when the observation period was extended to 2 h postcisplatin injection. However, significant reductions in emesis parameters were still evident, but full protection against CIV at lower doses of Δ9-THC was lost. This is not too surprising since extremely low combination doses of both drugs were utilized in the latter experiments. Indeed, the antiemetic efficacy of a larger

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dose of resiniferatoxin (10 μg/kg) alone against CIV has been reported to disappear more slowly (up to 18–24 h post-cisplatin) in both dogs and ferrets receiving smaller doses of cisplatin (3.2 and 10 mg/kg, i.p., respectively) (Yamakuni et al., 2002). In fact the effect of RTX on TRPV1 receptors is characterized by fast desensitization and slow recovery from desensitization (Jiang et al., 2009). Thus, these findings suggest that utilization of a combination of low doses of Δ9-THC with ultralow doses of resiniferatoxin may reduce the reported side-effects (Sharma et al., 2012; Szallasi and Sheta, 2012) as well as providing full antiemetic protection in cancer patients receiving much lower and clinically-relevant doses of chemotherapeutic agents. The discussed findings indicate that desensitization of TRPV1 plays a significant role in potentiating the antiemetic efficacy of Δ9-THC. In fact as discussed earlier, intracellular calcium dynamics are affected by cannabinoid CB1- and TRPV1-receptor activation independently. In addition, both receptors co-localize in the brainstem emetic nuclei (Sharkey et al., 2007), and functionally interact to suppress Ca2 þ ion entry into cells (Tsumara et al., 2012). This provides a molecular basis for the observed additive antiemetic efficacy of these plant-derived agonist antiemetics. While 5-HT3 receptor antagonists generally affect both latency to the first vomit as well as the frequency and percentage of animals vomiting, some studies indicate that NK1 receptor antagonists do not alter the vomit latency until doses reached that almost completely prevent vomiting (Andrews and Rudd, 2004). As discussed earlier, the current study demonstrates differential dose-, route- and combination-dependent effects on vomit latency by drugs that target TRPV1 receptors. Overall, both RTX and Ecapsaicin tended to prolong the onset to the first vomit. In summary, our results extend previous observations that combinations of various antagonist antiemetics are more efficacious than each antagonist tested alone, by demonstrating that combinations of sub-therapeutic doses of agonist antiemetics such as RTX and Δ9-THC, have the potential to completely abolish CIV.

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