EVALUATION OF SOME SELECTED MEDICINAL PLANTS AND THEIR COMBINATIONS IN CISPLATIN INDUCED VOMITING IN VOMIT MODEL(S); BEHAVIORAL NEUROCHEMICAL CORRELATES

Abstract

Cancer Chemotherapy Induced Vomiting (CIV) is one of the distressing untoward effects and a cause of non compliance and even refusal of treatment by the patients undergoing curative chemotherapy. Anti-emetics are therefore considered integral component of the anti-cancer therapeutic regimen. Numerous anti-emetics and their combinations are in clinical practice but none of them is capable of providing complete remission of CIV. The mechanistically multifactorial CIV, a challenge in clinics especially considering the delayed phase of vomiting necessitates the search for cost effective broad spectrum anti-emetic regimen for the management of CIV for prolong time periods (upto many days). In this study, extracts of some selected plants indigenous to Pakistan, were investigated for anti-emetic activity employing well known vomit models of pigeon and Suncus murinus (S. murinus). Vomiting was induced by highly emetogenic chemotherapeutic agent cisplatin in both models. Anti-emetic studies in pigeons were conducted at the bioassay laboratory of the Department of Pharmacy, University of Peshawar, Pakistan, while studies in S. murinus were carried out at the School of Biomedical Sciences, The Chinese University of Hong Kong, utilizing their state-of-the-art facilities. First of all, extracts of Cannabis sativa (CS; Hexane, n-butanol & methanol), Bacopa monniera (BM; methanol, & n-butanol) and Zingiber officinale (ZO; acetone) were prepared. Cannabis sativa and Zingiber officinale extracts were prepared by simple maceration method while Bacopa monniera was extracted by the method already developed by our laboratory. ivThe emetic, cisplatin was initially screened in a series of experiments to quantify its vomiting inducing potential; as a result the dose of 7 mg/kg was selected for pigeons, while 30 mg/kg dose was used in S. murinus. The behavior of the animals was observed using computer assisted video recording setup for each vomit model up to the desired period of time. The Retching plus Vomiting (R + V) episodes were then quantified from the video recordings for cisplatin control, standard & treatment groups. To find out the possible role of gastrointestinal (GIT) pro-kinetic properties on CIV in pigeon, we examined the impact of prokinetic/cholinergic agonist agents, utilizing charcoal propulsion method. High Performance Liquid Chromatography (HPLC) with UV detection was used for the quantification of bacosides whereas HPLC coupled with Electrochemical Detector (ECD) was employed for the measurement of neurotransmitters and their metabolites in specific brain areas and intestine involved in the act of vomiting of pigeons. Moreover, C-fos protein expression, a marker of neuronal excitation was also analyzed in hind brain areas; area postrema, nucleus tractus solitarius, dorsal motor nucleus of vagus nerve, and in the forebrain areas including dorsomedial and ventromedial nucleus of hypothalamus in the S. murinus model. CS hexane fraction (CS-HexFr) at the dose of 10 mg/kg was found to be effective in attenuating (P < 0.01) cisplatin induced R + V and has been proved to be superior to standard metoclopramide (30 mg/kg), while CS n-butanol and methanol fractions failed (P > 0.05) to do so in pigeons. BM fractions; methanol (BM-MetFr; 10, 20 & 40 mg) and n- butanol (BM-ButFr; 5, 10 & 20 mg), attenuated cisplatin induced R + V, dose dependently in both pigeons and S. murinus. The BM-ButFr was however, found to be more potent as vcompared to BM-MetFr in the vomit models of pigeon and S. murinus, as it reduced the number of R + V with high significance (P < 0.001) in pigeon (24 hr of observation period) and provided significant remission (P < 0.05) in the S. murinus for prolong time period (48 hr of observation period). In the pigeon model the anti-emetic effect of BM-MetFr and BM- ButFr was found to be pronounced as compared to standard metoclopramide (30 mg/kg), while in S. murinus the results proved to be analogous with the standard palonosetron (0.5 mg/kg). The strong suppression of cisplatin induced R + V by BM-ButFr may be attributed to the presence of high concentration of bacosides as HPLC - UV analysis revealed BM-ButFr to be rich in bacosides as compared to BM-MetFr, where the quantities of bacosides found were 115.74 μg/mg & 29.99 μg/mg of extract, respectively. The acetone fraction of Zingiber officinale (ZO-ActFr) also showed R + V reduction in pigeon model where, the dose of 50 mg/kg was found to be statistically significant (P < 0.05) while the R + V suppression achieved was found to be equivalent to standard metoclopramide (30 mg/kg). In combination studies; CS, BM & ZO showed variable protection against cisplatin induced R + V in pigeons. The combined treatment of CS-HexFr (10 mg) and BM-ButFr (5 mg) showed ~ 88.63 % protection (P < 0.001), where the protection provided by CS-HexFr (10 mg) and BM-ButFr (5 mg) alone were ~ 55.45 % (P < 0.01) & 68.08 % (P < 0.001), respectively. In S. murinus, Δ 9 -THC synthetic analogue, WIN 55, 212-2 (10 mg) in combination with BM-ButFr (5 mg) also enhanced protection against vomiting ~ 71.01 % (P > 0.05), where when tested alone the protection was found to be ~ 55.71 % & 57.97 % (P > 0.05) for WIN 55, 212-2 (10 mg) and BM-ButFr (5 mg), respectively. viCS-HexFr at its effective anti-emetic dose (10 mg) suppressed GIT motility ~ 26.62 % as compared to saline. The prokinetic agent metoclopramide (30 mg/kg) and cholinergic agonist carbachol (0.1 mg/kg) antagonized the suppression (P < 0.001) caused by CS-HexFr. Further, the combination of CS-HexFr (10 mg) with MCP (30 mg) or CS-HexFr (10 mg) with carbachol (0.1 mg) resulted in the enhancement of anti-emetic profile of CS-HexFr at delayed time point (12 hr +), however these combinations failed to show any synergism/potentiating at the acute time point (01 hr +). The neural data for acute vomiting response (03 hr) by cisplatin control group in this study revealed a significant upsurge of 5-hydroxytryptamine (5HT, serotonin) in the brain stem (BS; ~ 0.031 → 0.138 ng/mg tissue wet weight, P < 0.001) and intestine (~ 0.044 → 0.821 ng/mg tissue wet weight, P < 0.001) as compared to basal level, while for delayed response (18 hr) the significant increase in the concentration of dopamine (~ 0.535 → 13.43 ng/mg tissue wet weight, P < 0.001) and serotonin (~ 0.045 → 0.588 ng/mg tissue wet weight, P < 0.001) was observed in the area postrema (AP) and intestine, respectively in pigeon model. CS-HexFr (10 mg), BM-MetFr (10, 20 & 40 mg), BM-ButFr (5, 10 & 20 mg), ZO-ActFr (50 mg) & combination of CS-HexFr (10 mg) with BM-ButFr (5 mg) significantly decreased the concentration of 5HT (~ 0.438 → 0.006 ng/mg tissue wet weight, P < 0.001) and its metabolite; 5-hydroxy indole acetic acid (~ 5HIAA; 0.165 → 0.003 ng/mg tissue wet weight, P < 0.001) in the brain area of BS and intestine at acute time point (03 hr) as compared to cisplatin control. However, BM treatments failed to reduce 5HT concentration in AP any significantly (P > 0.05), and 5HIAA concentration in all the brain areas (AP & BS) and intestine. At the delayed time point (18 hr), BM-MetFr (10, 20 & 40 mg) and BM- ButFr (5, 10 & 20 mg) significantly decreased the upsurge of dopamine caused by cisplatin vii(~ 13.43 → 0.007 ng/mg tissue wet weight, P < 0.001) in the brain area of AP & BS, while a significant reduction in 5HT (~ 0.588 → 0.017 ng/mg tissue wet weight, P < 0.001) was observed in the intestine. Furthermore, CS-HexFr (10 mg), ZO-ActFr (50 mg) and combination of CS-HexFr (10 mg) with BM-ButFr (5 mg) significantly decreased dopamine (~ 7.36 → 0.098 ng/mg tissue wet weight, P < 0.001) in AP, while a significant decrease in 5HT (~ 0.292 → 0.002 ng/mg tissue wet weight, P < 0.001) was observed in the brain area of BS and at the level of intestine. None of the treatment (CS, BM & ZO extract) and combination (CS-HexFr 10 mg + BM-ButFr 5 mg) altered the basal neurotransmitter level except the decrease in the concentration of 5HIAA in the brain area of BS, which was found to be statistically significant. In the S. murinus, cisplatin treatment induced C-fos protein expression in the hind brain areas including area postrema, nucleus tractus solitarius, and dorsal motor nucleus of vagus nerve and in the forebrain area of hypothalamus including dorsomedial and ventromedial nucleus of hypothalamus. Treatment with BM-MetFr (10, 20 & 40 mg), BM-ButFr (5, 10 & 20 mg) & combination of WIN 55, 212-2 (10 mg) with BM-ButFr (5 mg) significantly attenuated (P < 0.001) cisplatin induced C-fos activity in all the brain areas. These findings highlight the intrinsic anti-emetic activity of CS hexane extract (rich in cannabinoids), ZO acetone extract (rich in gingerols) and BM extract (rich in bacosides); against cisplatin induced R + V in vomit models (pigeon & S. murinus). The significant findings to suppress the behavioral signs of CIV, reduction in the upsurge of serotonin and dopamine neurotransmitters caused by cisplatin and attenuation of C-fos immunoreactivity in vomit model of S. murinus, especially the BM extracts that we found for the first time in viiithis study, is an additional avenue to explore further for its anti-emetic potentials in other animal models. Moreover, combination of BM extract with CS extract has shown promising synergistic anti-emetic effect in vomit model of pigeon. Furthermore, no alteration observed in the basal neurotransmitter level by these treatments is encouraging. The CS, BM & ZO plant extracts and combination need to be further explored in gold standard vomit models of ferret and dog and in clinics as well, keeping in view the safe and tolerable profile of these extracts.