Extraction of Pure Ketamine Powder and Study their Analgesic Effect as a Gel on Mice Using a Hot – Plate Test

ABSTRACT

The present study was undertaken to extract ketamine powder from ketamine hydrochloride by precipitate ketamine. After that we examine the purity of this powder by infra-red (FTIR) and ultra-violet(UV) spectroscopy. ketamine gel in different concentrations was prepared ( 0.5 , 1 , 5 , 10 , 15 )% to evaluate the antinociceptive activity. ketamine powder was seen is pure and this show in infra-red and ultra-violet scanner. Ketamine gel at concentrations 0.5, 1, 5, 10,15) % produce antinociceptive in mice (5.6±2.2) (4.4±2.0) (8.2±4.3) (10.6±5.2) (8±2.1) second after 2 min respectively by using a hot plate test in comparison with control(2.4±2). The percentage of maximum possible effect (MPE) increased from (9.9) % in control group to (23.3) (18.3) (34.2) (44.2) (33.3)% respectively according to the concentrations of ketamine gel after 2 min . Purification of ketamine powder from ketamine solution and use as a gel to could be of value relief pain by topical application.

Key words: Purification of ketamine, Ketamine gel , analgesia , Infrared spectroscopy, Hot-plate test.

INTRODUCTION

Ketamine, a phencyclidine (PCP) analog, has been used for more than 30 years to produce “dissociative” anesthesia ⁽¹⁾. In this state the patient is awake and can respond to stimuli but has a diminished sense of awareness and an amnesia for events occurring while under the influence of ketamine. Early experience with ketamine revealed that it also produced analgesia that sometimes well outlasted its anesthetic effects. Although the mechanisms of ketamine’s analgesic effects remain the subject of debate, and are likely multiple(2-4). Antagonism at the NMDA-receptor site appears to be central to both its anesthetic and analgesic effects (5,6). Ketamine is available only I.M , I.V administration but it has been used orally. Ketamine is a dissociative anesthetic that is used to provide sedation and anesthesia in short surgical procedure, Patient may have adverse psychological effect including hallucinations, nightmares, delusion, dissociative reaction and schizophrenic form psychosis ⁽⁷⁾. Ketamine is primarily used for the induction and maintenance of general anesthesia, usually in combination with a sedative. Other uses include sedation in intensive care, analgesia (particularly in emergency medicine), and treatment of bronchospasm. It has been shown to be effective in treating depression in patients with bipolar disorder who have not responded to other anti-depressants ⁽⁸⁾. Pharmacologically, ketamine is classified as an N-Methyl D-Aspartat (NMDA) receptor antagonist ⁽⁹⁾. Ketamine is an antagonist of N-methyl 1-D-Aspartate (NMDA) class of glutamate receptors which is largely responsible for it's anesthetic and behavioral effect effects ⁽⁷⁾.NMDA inhibition produce catalepsy ,consistent with the effect of ketamine administration. Ketamine also produces profound analgesia which seen to be at least partially mediated by m- opioid receptor , in addition to it's binding to the phenylcyclidine binding site on the NMDA. ketamine is not frequently used for treatment of humans ,because it induces psychedelic episodes in patients ,especially adults, there are an increasing number of reports about patients that have become addicted to ketamine ⁽¹⁰⁾. Among the latest innovations of the pharmaceutical industry is the technology of drug delivery that overcomes the disadvantages of oral administration, these effects include first-pass metabolism and adverse drug side effects ⁽¹¹⁾. An ultimate route of administration that by pass these events would offer the patient drug delivery through skin has been a promising concept for a long time because skin is easy to access , has a large surface area with fast exposure to circulating and lymphatic net works and the rout is noninvasive ⁽¹²⁾.Therefore, the aim of the present study is to extract ketamine powder from ketamine hydrochloride solution and prepare ketamine gel with examine the analgesic effects of topical gel application in mice.

MATERIALS AND METHODS

Preparation of ketamine powder

10 ml of (1M) sodium bicarbonate was slowly added to 100 ml of the aqueous ketamine hydrochloride solution 5% (Alhukamma company) under continuous stirring until the pH of solution was close to 11, stirring was continued for one hour, and then the ketamine was precipitated. The solvent was eliminated by filtration and washed several times with distal water and then dried. The powder was studied by infra-red and ultra-violet spectroscopy in order to confirm the structure of the converted product.

Infrared Spectroscopy.

The infrared spectra recorded for prepared ketamine was examined by using Bruker Tensor 27 IR spectrophotometer (Germany) in the region (500-4000 cm) using KBr disc. This measurement was carried out in University of Mosul, College of Education, Iraq.

Electronic Spectra Measurement:

The measurement was carried out by using ethanol as a solvent with (1cm) diameter quartz cell by using Shimatsu-UV-Vis recording UV-1600 spectrophotometer (Japan). This measurement was carried out in Mosul University, Collage of Science, Department of Chemistry.

Preparation of ketamine gel

Ketamine gel was prepared (0.5, 1, 5, 10, 15)gm of ketamine powder in 100ml gel base to give a final concentration of (0.5%, 1%, 5%, 10%, 15%) with continuous mixing using Vortex device to prepare a homogenous gel. Gels were kept in plastic containers and store at room temperature.

Determination of Analgesic Activity of Ketamine Gel by Using a Hot –Plate Test:

Mice were divided into 2 main groups (A,B) each group were subdivided into six group with 5 animals per each group and the test was assessed by the hot plate method ( 13). The mice were treated topically with ketamine gel (0, 0.5, 1, 5, 10, 15) % respectively, on the planter area of the for and hind limb.

All the animals in group A were tested on hot plate after 1 minute while the mice in group B were tested after two minutes from topically application of the gel to determine the onset of action of gel. The mice were placed on top of hot plate of 55±1 0C. The time between placement and jumping or licking the hind paw was recorded as response latency.

The reaction time was recorded for control mice and for the animals treated with ketamine gel.

The percentage increase in reaction time was calculated thus using the following equation :- ⁽¹⁴⁾

% increase in reaction time (antinociceptive)=(T¹ -T⁰ / 30-T⁰) ×100

T⁰ = mean time for the control group (second)

T¹ = mean time for the test group (second)

30= cut off time (second)

Statistical analysis

The data were expressed as mean ± SD , difference between three experimental groups were statistically analyzed by one way analysis of variance ( ANOVA) followed by the least significant difference test. The level of significance was at p< 0.05 . ⁽¹⁵⁾

RESULTS

Figure (1) represents the vibrational response of pure Ketamine when passed via an infrared beam. The spectrum showed band at 3000-3200cm-1 which attributed to the N-H stretch from the amide group connected to the cyclohexanone. The spectrum also showing band at 2800-2900 cm-1 which assign to C-H stretch from an alkyl group. At this frequency the alkyl group is generally a non aromatic CH3 or CH2 stretch. The band at 1750 cm-1 due to R2 –C=O stretch which appear very precise and typical stretch for cyclic ketones. In Ketamine the carbonyl is connected to the cyclohexane ring. The band 1600 cm-1 assigned to C-N band (Generally expressed in C-NH2 and C-N=O compounds).The band at 1400-1500 cm-1 is attributed to C-H bend, this is another vibration mode of the CH2 or CH3 components of Ketamine. This is not the C-H bond from the aromatic carbons. The band at 1450cm-1 region is due to C-C stretch. This carbon to carbon stretch is not for the aromatic specie and hence characterizes the bonding involved in the cyclohexane ring. Pure ketamine extract measurement by UV (Ultra-Violet) spectroscopy Figure (2).

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