International Journal of Research in Pharmacology & Pharmacotherapeutics

Transcription

1 279 International Journal of Research in Pharmacology & Pharmacotherapeutics Available online at Print ISSN: Online ISSN: IJRPP Volume 2 Issue Research article Protective effect of an adenosine receptor agonist on neuropathic pain using chemotherapy induced neuropathy in mice. * Vishweswar Rao.V, Sumadhuri Sreerama, Sowjanya Kumar Reddy.R, Santosh.M. Vision College of Pharmaceutical sciences & Research, R.N.S.Colony, Boduppal, Ghatkesar (M), R.R(Dist), Andhra Pradesh, India. ABSTRACT Neuropathy is defined as the pain condition that results from damage affecting peripheral nerves, posterior roots, spinal cord or certain regions of brain. Increased neuronal excitability is thought to be the underlying mechanism for all forms of painful neuropathies. The work was framed to study the adenosine based treatment for chemotherapy induced neuropathy. Involvement of adenosine in nociception created an interest to work for chemotherapy induced neuropathy. Tricyclic antidepressants (TCAs), though often the first choice in most patients causes sedation and cardiovascular issues, Anticonvulsants, like the TCAs, are only partially effective in the majority of patients, Opioids, though often prescribed for moderate to severe pain, are sometimes avoided because of their potential for dependence and tolerance, scheduling issues and side effects. Hence in order to overcome these side effects we made an attempt to develop a newer drug for the treatment. Mice were first treated with vincristine sulphate (100 µg/kg, i.v). A single intravenous dose of vincristine causes painful peripheral neuropathy. Then we administer drugs for five days after inducing neuropathic pain. Baseline and 5 days after induction of neuropathic pain hyperalgesia (mechanical) and allodynia (mechanical) of all groups were measured in order to confirm the development of neuropathic pain. Hyperalgesia and allodynia of drug treated group was compared on 5 th day with vehicle treated group in order to confirm the effectiveness of drug in neuropathy pain. Hence adenosine was found to be significant against neuropathic pain in lower and also in higher dose. Hence adenosine may provide a better insight in the development of the newer drug for neuropathic pain. Key words: Neuropathy, Adenosine, Chemotherapy. INTRODUCTION Pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage 1. Pain is of two types nociceptive pain and neuropathic pain. Nociceptive pain is the neural processes of encoding and processing noxious stimuli. It is the afferent activity produced in central * Corresponding author: Vishweswar Rao.V address: visu_rx@yahoo.com and peripheral nervous system by stimuli that have the potential to damage tissue. Neuropathic pain is defined as the pain condition that results from damage affecting peripheral nerves, posterior roots, spinal cord or certain regions of brain. Accumulation of novel expression of sodium channels in periphery, increased activity at glutamate receptor sub

2 280 Vishweswar Rao.V et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-2(1) 2013 [ ] population, reduction of GABA inhibition and changes in penetration of calcium in the cell are reported as possible mechanism for the development of neuropathic pain. Symptoms of neuropathic pain 2 are 1. Stimulus dependent pain (can be shooting, lancinating or burning) 2. Stimulus evoked pain is a common component of peripheral nerve injury or damage and has two key features. Hyperalgesia and Allodynia. Hyperalgesia is an increased pain response to a suprathreshold noxious stimulus and is the result of abnormal processing of nociceptor input. Allodynia is the sensation of pain elicited by a non noxious stimulus and can be produced by two ways 1. By the action of low threshold myelinated A fibres on an altered central nervous system. 2. By a reduction in the threshold of nociceptor terminals in the periphery. Stimulus evoked hyperalgesia are classified into subgroups on the basis of modality mechanical, thermal and chemical. Mechanism involved in pain is complex and involve both peripheral and central pathophysiological phenomenon. Neuropathic pain is a severe debilitating condition which affects approximately six million people in US alone 3. Increased neuronal excitability is thought to be the underlying mechanism of all forms of painful neuropathies. Therefore current pharmacotherapy of neuropathic pain generally involves the use of drugs that either reduces neuronal discharge or increases endogenous antinociception system. Sodium channel blockers, antiepileptic agents, opioids, tricyclic antidepressents, gabapentin etc have been employed to treat the painful symptoms of different forms of neuropathy. Mainly adenosine play a major role in the pathophysiology of neuropathic pain for several reasons at the spinal cord, intrathecal selective A 1 adenosine receptor agonists inhibited C- fiber evoked responses in the dorsal horn neurons in rats 4. These results suggest a role of adenosine A 1 receptor in the modulation of both acute and chronic pain in autonomic nervous system is particularly involved in neuropathic pain. Adenosine binds to presynaptic A 1 receptor and via a G1 protein leads to a reduction in the camp concentration, in turn leading to a decreased release of glutamate by damaged nerve fibers which results in decrease in neuropathic pain. Hence in our present investigation Granisetron an adenosine A 1 receptor agonist (class of purinergic receptors, G-protein coupled receptor with adenosine as ligand) is used to know its protective effect on neuropathic pain using chemotherapy (vincristine) induced neuropathic pain. MATERIALS AND METHODS Drugs and chemicals Sodium chloride was obtained from Sisco research laboratories Pvt. Ltd, Mumbai. Gabapentin, was purchased from Bava medicals, Pallavaram. Vincristine and Granisetron were purchased from Muthu pharmacy, Purasawalkam. All chemicals and reagents used were of analytical grade. Experimental animals Adult male Swiss albino mice weighing g were used in the pharmacological Studies. The inbred animals were taken from the animal house in Vel s College of Pharmacy, Pallavaram, Chennai-117. The animals were housed in groups of 6 per cage. They were maintained in well-ventilated room temperature with relative humidity of 45-55% and natural 12h: 12h day-night cycle in propylene cages. They were fed balanced rodent pellet diet from Poultry Research Station, Nandanam, Chennai-35 and tap water adlibitum throughout the experimental period. All the experiments were carried out between 10:00 am to 5:00 pm. The animals were housed for one week, prior to the experiments to acclimatize laboratory temperature. Food, not water, was withdrawn 3 hrs before and during experiment. The experiment protocol was approved by the Institutional Animal Ethics Committee IAEC Ref. No. 290/CPCSEA/2009-PH/PCOL-02. Preparation of drug solution Drug was dissolved in saline and administered to animals through i.p. route. Assessment of effectiveness of Granisetron for neuropathic pain (Chemotherapy induced neuropathic pain model) Chemotherapy induced neuropathic pain was performed following the method of 5 Joseph et al., Mice were first treated with vincristine sulphate (100 µg/kg, i.v). A single intravenous dose of vincristine causes painful peripheral neuropathy.

3 Vishweswar Rao.V et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-2(1) 2013 [ ] 281 Then we administer drugs for five days after inducing neuropathic pain. Baseline and 5 days after induction of neuropathic pain hyperalgesia (mechanical) and allodynia (mechanical) of all groups were measured in order to confirm the development of neuropathic pain. Hyperalgesia and allodynia of drug treated group was compared on 5 th day with vehicle treated group in order to confirm the effectiveness of drug in neuropathic pain Experimental groups The mice were divided into four groups consisting of 6 per each group Group I -Vehicle- Saline (10 ml/kg, i.p.) for 5 days. Group II- Granisetron (1 mg/kg, i.p) for 5 days. Group III- Granisetron (10 mg/kg, i.p) for 5 days. Group IV- Gabapentin (100 mg/kg, p.o) for 5 days. Behavioral tests Mechanical hyperalgesia 6 Mechanical hyperalgesia was tested by using the pin prick test. Animals were placed on the elevated grid; a pin prick test was performed using a safety pin. The lateral plantar surface of the right hind paw was briefly stimulated at intensity sufficient to indent but not penetrate the skin (pin prick test). The duration of paw withdrawal was recorded, with an arbitrary minimal time of 0.5 (sec) and a maximal cut off 15 (sec). Mechanical Allodynia (Touch Evoked Tactile Allodynia) 7 Tactile allodynia was assessed by lightly stroking the injured leg with a paintbrush. Allodynia response was ranked as described by Minami et al No response. 1. Mild squeaking with attempts to move away from the stroking probe. 2. Vigorous squeaking, biting the stroking probe and strong efforts to escape from the stroking probe. Assessment of effectiveness of Granisetron on motor coordination In order to assess whether the protection of neuropathic pain of granisetron was via impaired motor activity, motor coordination studies of Granisetron was performed using the following animal models. Experimental groups Mice were divided into three groups each group having 6 animals. Group I -Vehicle- 0.9% Saline (10 ml/kg, i.p.) Group II- Granisetron (1 mg/kg, i.p) Group III- Granisetron (10 mg/kg, i.p) Behavioral tests Rota rod Test 8 Mice were placed on the rota-rod. Appropriate speed of rotation was selected. Time taken by the animal to fall off from the rotating rod was observed. A normal (untreated) mouse generally falls off within 3 mins. Locomotor Activity 8 The locomotor activity can be easily measured using actophotometer which operates on photoelectric cells which are connected in a circuit with a counter. When a beam of light falling on photocell is cut off by the animal, a count is recorded. Mice were individually placed in the activity cage for 10 mins and the scores were recorded. Statistical analysis The data are represented as mean ±S.E.M and statistical significance between groups were analysed by means of student paired t-test, ANOVA followed by Dunnet s t-test, as applicable. P<0.05 implies significance. All the statistical analysis was carried out using graph pad prism 5.0 version software. RESULTS Vincristine induced neuropathic pain model Development of signs of neuropathic pain All the vincristine treated animals developed qualitative signs indicative of neuropathic pain. This was clearly present on the 5 th day after administration of vincristine at a dose of 100 µg/kg, i.v. Table 1, Table 2 showed vincristine treated group paw withdrawal latency was reduced on 5 th day in comparison to basal reading of the same animals. The paw withdrawal latency of the animal was decreased and allodynia score on 5 th day was significantly increased in comparison to baseline measurements indicating the development of hyperalgesia and allodynia.

4 282 Vishweswar Rao.V et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-2(1) 2013 [ ] Effect of Granisetron on Mechanical Hyperalgesia In the vehicle treated animals, the response duration decreased at 5 th day in comparison to baseline measurements (Table 1) from 14.33±0.33 to 4.00±0.36 ** on the 5 th day. Whereas the other two groups treated with granisetron at a dose of 1mg/kg and 10 mg/kg from 14.33±0.33 to 7.23±0.42*and 9.26±0.28** the response duration was significantly increased at both the dose levels. Thus administration of Granisetron at both dose levels shows the increased the paw withdrawal latency in comparison to the vehicle treated group (Figure 1). The results are dose dependent and comparable with that of standard drug gabapentin. Table 1: Development of mechanical hyperalgesia in chemotherapy induced neuropathic pain model. S.No Treatment Paw withdrawal latency(sec) Baseline 5 th day 1 Vehicle-Saline(10ml/kg, i.p) 14.33± ±0.36 ** Statistical significance test was done by paired t test. Values are mean± S.E.M of 6 animals per group. Comparison was made between base line Vs 5 th day of the vehicle treated groups. **P<0.01 Effect of Granisetron on Mechanical allodynia The baseline paw withdrawal frequencies (0.5 (sec)) determined by mechanical stimulation with paint brush was enhanced at 5th day in vehicle treated mice compared to baseline responses of the same animals. Granisetron treated group at a dose level of 1mg/kg and 10mg/kg decreased the allodynia score from the value of 3.16±0.16** to 2.50±0.14** for 1mg/kg and 2.20±0.18** for 10mg/kg. The values are statistically significant and the results were comparable with that of standard drug gabapentin 100 mg/kg, p.o (Figure 2). Table 2: Development of mechanical allodynia in chemotherapy induced neuropathic pain model S.No Treatment Allodynia Score Baseline 5 th day 1 Vehicle-Saline(10 ml/kg, i.p) 0.00± ±0.16 ** Statistical significance test was done by paired t test. Values are mean± S.E.M of 6 animals per group. Comparison was made between base line Vs 5th day of the vehicle treated groups. **P<0.01

5 Vishweswar Rao.V et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-2(1) 2013 [ ] 283 Figure 1: Effect of Granisetron on mechanical hyperalgesia in chemotherapy induced neuropathic pain model paw withdrawal latency (sec) 20 0 Vehicle Mechanical hyperalgesia *** *** *** Granisetron 1 mg/kg Granisetron 10 mg/kg Gabapentin 100 mg/kg 5th day Statistical significance test was done by one way ANOVA followed by Dunnet s t test. Values are mean± S.E.M of 6 animals per group. Comparison was made between vehicle Vs 5th day of the all groups. ***P<0.001 Allodynia Score Figure 2: Effect of Granisetron on mechanical allodynia in chemotherapy induced neuropathic pain model Mechanical allodynia 4 * * ** 2 0 Vehicle Granisetron 1 mg/kg Granisetron 10 mg/kg Gabapentin 100 mg/kg 5th day Statistical significance test was done by one way ANOVA followed by Dunnet s t test Values are mean± S.E.M of 6 animals per group. Comparison was made between vehicle Vs 5 th day of the all groups. *P<0.05 **P<0.01 DISCUSSION The mice chemotherapy-induced neuropathic pain model utilized in the present study is one of many; diverse animal models that have been used to investigate the pharmacological attenuation of neuropathic pain 9. Rodent models of neuropathy have been developed for three of the most widely used and most neurotoxic cancer drugs cisplatin, paclitaxel and vincristine. These models have all been used to determine the efficacy of neuroprotective agents 10 and to study the mechanisms of sensory dysfunction that underlie neuropathy 11. However, the vincristine and paclitaxel models have several advantages over cisplatin models. General health effects are an issue for cisplatin models. Low cumulative doses of either paclitaxel or vincristine produce quantifiable changes in sensory thresholds withoutt decreasing motor function or significantly impairing general health 12. To date, no drug or drug class is considered to be both a safe and effective analgesic in the treatment of chemotherapy induced pain. Tricyclic antidepressants (TCAs), though often the first choice in most patients, have significant side effects including sedation and various cardiovascular issues, and they often require several days of treatment prior

6 284 Vishweswar Rao.V et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-2(1) 2013 [ ] to producing positive effects 13, 14. Anticonvulsants, like the TCAs, are only partially effective in the majority of patients suffering from chemotherapy induced pain 14, 15. Opioids, though often prescribed for moderate to severe pain, are sometimes avoided because of their potential for dependence and tolerance, scheduling issues and side effects 16. Other therapeutic agents are prescribed for the treatment of cancer pain, in general. For example, non-steroidal anti-inflammatory drugs (NSAID) have been reported as having some analgesic efficacy in cancer pain 2,17,18,19,20. However, prolonged NSAID usage can be associated with significant toxicity, including that to the gastrointestinal system and liver 17. Epidural clonidine, an α 2 -adrenoceptor agonist, is indicated (in combination with opioids) for the treatment of severe cancer pain, however, profound hypotension can be a side effect. Hence in order to overcome these side effects we made an attempt to study the vincristine induced neuropathy. Vincristine is basically a mitotic inhibitor. They bind to tubulin, prevents its polymerization and assembly into microtubule, causing disruption of mitotic spindle thereby interferering with cytoskeletal function. Vincristine also inhibits cellular activity of microtubules like axonal transport in neurons. Thus they disrupt the normal axonal transport and lead to change in excitability of neurons thus producing chronic pain. A single intravenous dose of vincristine (50, 100, or 200 mg kg -1 ) causes a painful peripheral neuropathy in rats verified by mechanical hyperalgesia and mechanical allodynia. A single administration of vincristine is well tolerated at all of the doses studied (50, 100 or 200 mg /kg) Rats treated with vincristine gain weight at a slower rate than control rats but there are no marked differences in the general appearance of control versus vincristine-treated rats. There are also no motor deficits in rats treated with a single dose of vincristine. Models in which neuropathy is induced by multiple doses of vincristine produce a rapid-onset painful neuropathy but cumulative doses more than 500 mg/kg also produce moderate to severe effects on general health and motor deficits in most studies 21. At cumulative doses more than 750 mg/kg vincristine causes significant mortality 22. So in our present study we have studied the role of Granisetron against the single dose vincristine induced neuropathy pain. Results showed that Granisetron at lower and higher dose level of 5 days administration attenuated mechanical and mechanical allodynia. REFERENCE [1] Merskey H, Bogduk N. Classification of Chronic Pain, 2nd edn. Seattle: IASP Pres, [2] Jenkins CA, Bruera E. Nonsteroidal anti-inflammatory drugs as adjuvant analgesics in cancer patients. Palliat Med 1999; 13: [3] Berger A et al. Clinical characteristics and economic costs of patients with painful neuropathic disorders. J. Pain 2004;5: [4] Bjorn A Meyerson and Bengt Linderoth. Mode of Action of Spinal Cord Stimulation in Neuropathic Pain. Journal of Pain and Symptom Management,2006. [5] Chiang Siau and Gary J Bennett. Dysregulation of Cellular Calcium Homeostasis in Chemotherapy-Evoked Painful Peripheral Neuropathy. Anesth Analg 2006;102: [6] Chris Pasero. Pathophysiology of Neuropathic Pain. Pain Management Nursing,2004 Vol 5, No 4:3-8. [7] Annika B Malmberg and Sandra R Chaplan. Mechanisms and mediators of neuropathic pain. 2002, pp: [8] Kulkarni SK, Dandiya PC. Indian J. Med. Res 1975; 63: [9] Seltzer Z. The relevance of animal neuropathy models for chronic pain in humans. Semin Neurosci 1995; 7: [10] Flatters SJL, Bennett GJ. Ethosuximide reverses paclitaxel and vincristine-induced painful peripheral neuropathy. Pain 2004; 109: [11] Dina OA. Integrin signaling in inflammatory and neuropathic pain in the rat. Eur. J. Neurosci 2004; 19: [12] Authier N.Pain related behaviour during vincristine-induced neuropathy in rats. Neuroreport, 1999;10:

7 Vishweswar Rao.V et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-2(1) 2013 [ ] 285 [13] Wolfe GI, Barohn RJ. Painful peripheral neuropathy. Curr Treat Options Neurol 2002; 4: [14] Uhm JH, Yung WKA. Neurological complications of cancer therapy. Curr Treat Options Neurol 1999; 1: [15] Bosnjak S et al. Gabapentin for relief of neuropathic pain related to anticancer treatment: a preliminary study. J Chemother 2002;14: [16] Dahl JL. Working with regulators to improve the standard of care in pain management: the U.S. experience. J Pain Symptom Manage 2002; 24: [17] Mercadante S. The use of anti-inflammatory drugs in cancer pain. Cancer Treat Rev 2001; 27: [18] Pellegrini A et al. Effect of IV indoprofen on cancer pain and serum prolactin and growth hormone levels. A controlled pharmacological study versus IM morphine in placebo. Int J Clin Pharmacol Ther Toxicol 1983; 21: [19] Stambaugh JE. Analgesic efficacy, safety and acceptability of zomepirac sodium in comparison to morphine sulphate in the treatment of pain secondary to malignancy. Curr Ther Res 1982; 31: [20] Sunshine A, Olson NZ. Analgesic efficacy of ketoprofen in post partum, general surgery and chronic cancer pain. J Clin Pharmacol 1988; 28: S47 S54. [21] Aley KO. Vincristine hyperalgesia in the rat: a model of painful vincristine neuropathy in humans. Neuroscience, 1996;73: [22] Authier N. A new animal model of vincristine-induced nociceptive peripheral neuropathy. Neurotoxicology, 2003;24: *******************************