Evaluation of the antinociceptive effect of xylazine and it’s interaction with metoclopramide in the acute pain model in mice

Evaluation of the antinociceptive effect of xylazine and it’s interaction with metoclopramide in the acute pain model in mice

Kh.A. Shaban¹, M.H. Ibrahim² and G.A. Faris³

Department of Physiology, Biochemistry and Pharmacology, College of Veterinary Medicine, Mosul University, Mosul, Iraq

Email: ¹akhalid18@yahoo.com, ²munaalzubaidy77@ yahoo.com, ³ghadafaris2018@gmail.com

Abstract

The study was designed to qualitative and quantitative evaluation of the antinociceptive effect of metoclopramide and xylazine each alone or as a concomitant administration in mice. Adult albino Swiss mice weighing 20-30 mg used in all experiments. By using the hot plate test, the individual analgesic dose (ED₅₀) of metoclopramide and xylazine detected depending on the up and down method. Isobolographic analysis used to evaluate the type of interaction between two drugs at the ratio 0.5:0.5 of individual ED₅₀ for each drug at the level of antinociception effect. Simultaneously administration of the double dose of individual ED₅₀ and low doses (sedative, non-analgesic doses) of both drugs, also evaluated at the level of central and visceral analgesia using a hot plate and writhing response test respectively. The individual ED₅₀ of xylazine and metoclopramide was 10.8 and 25.6mg/kg IP respectively. A synergistic interaction at the level of analgesia explored between two drugs at ratio 0.5:0.5 which represented as decreased in ED₅₀ of metoclopramide and xylazine by 58.75 and 58.15% respectively. The animal suffered from only slight sedation and docile. Simultaneously IP administration of xylazine and metoclopramide at double dose of ED₅₀ for each drug-induced significant increase in latency time of thermal response, as well as a significant decrease in writhes number, which induced by acetic acid in comparison with control groups. The percentage of analgesia at sub analgesic doses of a concomitant administration of both drugs was 100% in comparison with each drug alone. These results suggested safe and good use of both drugs in veterinary medicine.

Keywords: Xylazine, Metoclopramide, Analgesia, Hot plate, Acetic acid

DOI: 10.33899/ijvs.2019.126070.1226, ©2020, College of Veterinary Medicine, University of Mosul.

This is an open access article under the CC BY 4.0 license (http://creativecommons.org/licenses/by/4.0/).

تقییم التأثیر المسکن للألم للزیلازین وتداخله مع المیتوکلوبروماید فی نموذج الألم الحاد فی الفئران

خالد احمد شعبان، منى حازم البراهیم و غادة عبدالمنعم فارس

فرع الفسلجة والکیمیاء الحیاتیة والأدویة، کلیة الطب البیطری، جامعة الموصل، الموصل، العراق

الخلاصة

صُمِمَتْ الدراسة لتقییم الفعل المسکن لعقار المیتوکلوبروماید والزیلازین تقیما کمیا ونوعیا عند إعطائهما کل لوحده أو معا للحیوان نفسه (الفئران). استخدمت فئران بیضاء سویسریة بالغة تزن 20-30 غم فی کل التجارب. باستخدام اختبار الصفیحة الساخنة تم تحدید الجرعة الفاعلة المسکنة الوسطیة جم 50 لعقار الزیلازین والمیتوکلوبروماید کل لوحده اعتمادا على طریقة الصعود والنزول فی الجرعة واستخدم تحلیل الایزوبولوکرافی لتقییم نوع التداخل الدوائی بین العقارین عند نسبة 0.5:0.5 من الجرعة الفاعلة المسکنة الوسطیة لکل عقار على مستوى التسکین من الألم. فضلا عن تقییم إعطاء العقارین بالجرع المضاعفة للجرعة المسکنة الوسطیة لکل عقار وبالجرع المسدرة (غیر المسکنة) على مستوى التسکین من الألم المرکزی والحشوی باستخدام اختبار الصفیحة الساخنة واختبار التلوی على التوالی. کانت الجرعة الفاعلة المسکنة الوسطیة للمیتوکلوبروماید والزیلازین 25.6 و 10.8 ملغم/کغم من وزن الجسم بالحقن داخل الخلب على التوالی. کان التداخل بین العقارین تأزرا عند نسبة 0.5:0.5 من الجرعة الفاعلة الوسطیة لکل عقار والذی تمثل بالانخفاض المعنوی فی قیمة الجرعة لعقار الزیلازین والمیتوکلوبروماید وبنسبة 58.15 و 58.75% على التوالی. ولم تعانی الحیوانات المعاملة بالعقارین معا بهذه الجرعة أی أثار جانبیة فیما عدا التسدیر البسیط وقلة الحرکة. أدى حقن العقارین معا فی الخلب بالجرع المضاعفة لکل عقار الى زیادة معنویة فی مدة بقاء الحیوان على الصفیحة الساخنة فضلا عن الانخفاض المعنوی فی عدد مرات التلوی المحدث بحامض الخلیک مقارنة مع مجموعة السیطرة. کانت النسبة المئویة للتسکین عند الجرع تحت المسکنة 100% مقارنة مع إعطاء کل عقار لوحده. اثبتت النتائج استخدام جید وأمین لهذین العقارین فی مجال الطب البیطری.

Introduction

Metoclopramide is a benzamine derivative (1), used in humans and animals (2,3), as centrally acting antiemetic and reduces stomach movement. These effects related to its centrally antagonizing of dopamine D₂-receptors antagonist, and serotonergic effects 5HT₃ (4). Indirect cholinergic action also involved in this mechanism (5). The analgesic effect of metoclopramide was reported recently in humans (6,7), dogs (8), rats and mice (9). The mechanism of antinociception effect by metoclopramide remain is unknown yet. Xylazine is one of alpha-2 adrenergic receptor agonists, widely used in veterinary practice as sedative, analgesic and muscle relaxant (10,11). Xylazine also can be used as pre-anesthetic with ketamine to produce balance anesthetic (12,13). Different combinations of xylazine with other analgesic were evaluated in different animal models to improve the quality of analgesic properties and minimizing the side effects. Combination of xylazine with dipyrone (14) or tramadol (15) in mice and in chicks (16) or with chlorpromazine in sheep (17)and chicks(18), produced safe and effective analgesia at low doses of each drug (synergistic antinociceptive effect).

However, the combination of xylazine and metoclopramide was not evaluated in previous studies, so the purpose of current study was to qualitative and quantitative evaluation of the analgesic effect of metoclopramide and xylazine each alone or as a combination. the type of interaction between two drugs also evaluated in mice by using two models of acute pain induction, hot plate (central) and writhing response (peripheral) (19,20).

Materials and methods

Animals

All experiments were carried out by using male and female albino Swiss mice weighing 20-30 g. The animals were housed in the animal house of the college of veterinary medicine, University of Mosul. They were maintained under standard conditions with 10/14 hrs. light/dark cycle, at 22±2ºC room temperature. Food and water available at the laboratory. The scientific committee of the department of physiology, biochemistry and pharmacology at the college of veterinary medicine has reviewed and approved the protocol of this study. The doses of metoclopramide (pure powder Vainkunth Co., India) and xylazine (20 mg/ml injectable solution, Holland) were dissolved in physiological saline solution at volume of injection 5ml/kg body weight, IP

Methods

By using the up-and-down method (21) the median effective analgesic doses of metoclopramide and xylazine (alone or as a combination) were detected in mice by using a hot plate (thermal method). The initial dose was determined based on experience and previous studies (22,23). The increasing and decreasing in the later dose of each drug was at a constant value. The analgesic effect was assessed by placing the mice on a hot plate (Heidolph Me, Germany, Hei-standard Co. at 56ºC), the time between placing the mice on the hot plate and licking of fore or hind paw or/and jumping off from the surface was recorded as the response in seconds. Only mice that response to nociceptive response within 30 seconds was used in this study, the cut off time was 30 seconds to avoid any thermal injury to the paw (19), animals were used only once. The response was measured before (baseline) and at 15 minutes after injected of each drug doses.

Analgesic interaction between metoclopramide and xylazine

In order to evaluate the type of interaction between metoclopramide and xylazine. The first animal was simultaneously injected with initial dose of metoclopramide and xylazine at 12.8 mg/kg i.p and 5.4 mg/kg i.p respectively at ratio 0.5:0.5 of ED₅₀for each drug. The type of interaction was detected by isobolographic analysis and Y interaction index (24-26). A straight line drawled between the ED₅₀of metoclopramide (pointed on x axis) and that for xylazine (pointed on y axis). The ED₅₀value of combination (0.5:0.5) also pointed, when this value detects under the straight diagonal line, the interaction is synergistic, while falling this value on/and above the line, the interaction are additive and antagonized respectively. The addition interaction index was calculated by using equation: Y= da/Da +db/Db.

Where: (Da and Db) are individual ED₅₀ of metoclopramide and xylazine respectively, while the (da and db) represent the combination doses that produce the same effect (analgesic). When the value of Y =1 its additive (no interaction), Y<1 synergism (supra additive) and Y>1 its antagonism (sub additive), this is an interaction index.

Acetic acid inducing writhing reflex (chemical test) and hot plate (thermal test) at double doses of ED₅₀ of metoclopramide and xylazine

Mice were used in this experimental divided into four groups (5 animals for each group), group Ι and Ш injected with normal saline while group Π and ΙV injected with metoclopramide and xylazine at 51.2mg/kg b.w i.p and 21.6mg/kg b.w i.p respectively (double dose of ED₅₀).

Animals in group Ι and Π placed on hot plate before and 15 minute after injection of normal saline and drugs for calculation of latency time, while group Ш and ΙV were received normal saline and drugs 15 minute before injected with 1% acetic acid at volume 0.1 ml/10g b.w i.p to induce writhing response and calculate number of writhing which recorded within 30minute after injection of acetic acid (27).

Estimation percentage of analgesia of pain in subanalgesic doses of metoclopramide and xylazine each alone or in combination by thermal method (hot plate)

In this experimental, mice divided into three groups, each group consist of 5 animals, group I injected with metoclopramide 10 mg/kg b.w i.p, group Π injected with xylazine 2mg/kg b.w i.p and group Ш injected with metoclopramide and xylazine at 10 mg/kg b.w i.p and

2mg/kg b.w i.p respectively. animals placed on hot plate before and 15 minutes after injection of drugs for calculate of latency time (19).

Data analysis

The data for latency time (thermal test) were statistically subjected to unpaired t-test, while number of writhing were analyzed by Mann Whitney u-test, and for percentage of analgesia (hot plate) by fisher test (28). The level of significant was P < 0.05.

Results

In the present study the dose that produce 50% of analgesic effect in mice for metoclopramide and xylazine induced was 25.6 and 10.8 mg/kg, respectively by using up and down method (Table 1).while concomitant injected mice with a combination of metoclopramide and xylazine at ratio 0.5:0.5 of ED₅₀ for each, was markedly reduced the value of ED₅₀ to 10.56 and 4.52 mg/kg, respectively (Table 1). The mice that injected with metoclopramide and xylazine were suffered from signs of slight sedation and docile.

Table1: Determination of median effective doses of metoclopramide and xylazine

^ax - analgesia; o - no analgesia. The ED₅₀ were determined by the up and down method.

By using Isobolographic analysis, pointed the value of ED_50s for each alone, then detected the situation of ED₅₀ value for a combination, this value was fallen down under the diagonal line that connect the ED_50s for each drug alone that revealed a synergistic antinociceptive interaction in mice (Figure 1).

Furthermore, calculate the Y value from equation of interaction index, it was lower than 1 (0.83) that indicated the synergism interaction (Y<1).

In second experiment we evaluated the peripheral (chemical test of acetic acid) and central (thermal test) antinociception effect for further examine the synergistic

interaction between metoclopramide and xylazine given as combination (doubling of ED₅₀ for each drug), whereas significantly increased the latency time of thermal response and significant decreased in writhing number of acetic acids in compares with control groups (Table 2).

Evaluation of analgesia percentage of pain by administration of sub analgesic (sedated) dosages of metoclopramide (10 mg/kg i.p) and xylazine (2 mg/ kg i.p) each alone or in combination in mice. whereby was analgesia percentage of pain 0% when administered each drug alone while analgesia percentage of pain 100% when administered two drugs in combined form (Table 3).

Figure 1: Isobolographic analysis of interaction between metoclopramide and xylazine at ratio 0.5:0.5 in mice. The diagonal line joints the individual ED_50sof metoclopramide and that of xylazine, the triangle points which represent 0.5:0.5 of ED₅₀ combination of two drugs, located below the diagonal line, indicate synergism interaction. xylazine at dose 10.8mg/kg IP, metoclopramide at dose 25.6 mg/kg IP, xylazine and metoclopramide at ratio 0.5:0.5.

Table 2: Metoclopramide and xylazine analgesia following combined administration in mice

Values are Mean ± SE of 5 mice / group. Analgesia evaluated in mice 15 minutes after administration of both drugs.

* Significantly different from the respective control value at P < 0.05.

Discussion

In this study we explored the antinociceptive effect of xylazine alone or as a combination with metoclopramide in mice. Xylazine is an alpha-2 agonist which induced analgesia by inhibit NE and dopamine reuptake in CNS, which in turn produce CNS depression (10,28). Metoclopramide is well known as antiemetic, but recently reported as analgesic in man (6,7), dog (8), rat (9) and mice, alone or as a combination with other drugs as ketamine (3), tramadol (29) and diphenhydramine (30).

Table 3: Percentage of analgesia for each drug alone or as a combination by using hot plate test

* Significantly different from each drug alone (P < 0.05).