EXPERIMENTAL RESEARCHES ON THE EFFECTS OF TRAMADOL SOFT MATTER VESICLES IN NOCICEPTIVE PROCESSES IN MICE
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Annals of RSCB Vol. XVI, Issue 1
EXPERIMENTAL RESEARCHES ON THE EFFECTS OF TRAMADOL
SOFT MATTER VESICLES IN NOCICEPTIVE PROCESSES IN MICE
Liliana Tartau1, R. V. Lupusoru2, C. Andritoiu1, V. Melnig3
1
DEPARTMENT OF PHARMACOLOGY-ALGESIOLOGY, 2DEPARTMENT OF
PATHOPHYSIOLOGY, FACULTY OF MEDICINE, “GR.T. POPA” UNIVERSITY OF
MEDICINE AND PHARMACY, IASI, ROMANIA; 3UNIVERSITY OF AGRICULTURAL
SCIENCES AND VETERINARY MEDICINE, IASI, ROMANIA; 3DEPARTMENT OF
PHYSICS - COMB LABORATORY, FACULTY OF PHYSICS,
“AL. I. CUZA” UNIVERSITY, IASI, ROMANIA
Summary
Nanoparticles designed for drug delivery are defined as submicrometer-sized colloidal
particles. Among the carriers for drug delivery, vesicles are self-assembled colloidal particles
used to encapsulate different substances within their core, with broad usage in controlled
drug-release. Tramadol is a synthetic 4-phenyl-piperidine analogue of codeine, a central
acting analgesic, with a low agonistic affinity for opioid receptors. The objective of this study
consists of experimental researches on the effects of tramadol loaded nanoparticulate
formulations, in somatic and visceral pain models in mice. Using an original methodology we
realized the incorporation of tramadol in lipid vesicles. The vesicles were physicochemical
and structural analyzed using a Malvern Zetasizer Nano ZS, ZEN-3500 model and a
Shimadzu UV-1700 PharmaSpec spectrophotometer. The colloidal solution stability is
defined according to the average value of Zeta potential.
The experiments were carried out on white Swiss mice (20-25 g), divided into 3 groups
of 7 animals each, treated orally with the same volume of solution: Group I (Control):
distilled water 0,3ml, Group II (TMD): 10mg/kbw tramadol, Group III (TMD-ves):
10mg/kbw tramadol entrapped in lipid vesicles. The model of somatic pain used consists of
hot plate test. The latency time reaction to thermal stimulus applied on the paws was
measured before the experiment and 15, 30, 60, 90, 120 minutes, 4, 6, 8, 10, 12 hours, after
the substances administration. The nociceptive visceral testing was performed using mouse
model of writhing test with acetic acid 0,6%. Antinociception was recorded by counting the
number of writhes every 5 minutes, for period of 20 minutes after intraperitoneal
administration of irritant stimulus. The behavior modifications (contractions of the abdomen,
writhes) were observed scored and data were statistically analyzed with SPSS software for
Windows version 17.0 and ANOVA method. Experimental protocol was implemented
according to recommendations of the Gr.T. Popa University Committee for Research and
Ethical Issues, and to the guidelines of IASP Committee for Research and Ethical Issues.
The administration of tramadol entrapped in soft matter vesicles resulted in a
prolongation of the latency time responses to thermal noxious stimulation, 6 hours after
substance administration, in hot plate test. Tramadol lipid vesicles induced a decrease of
writhes number, effect that started after 3 hours and lasted for around 6 hours in writhing test.
Our study proved that tramadol loaded nanoparticulate formulation possesses analgesic
properties in somatic and visceral pain model used. Tramadol entrapped in nano-vesicles
determined a prolongation of analgesic effects compared with non entrapped substance in
both somatic and visceral pain tests.
Keywords: tramadol entrapped, vesicles, hot plate, writhing test.
lylytartau@yahoo.com
194Annals of RSCB Vol. XVI, Issue 1
Introduction
Nanoparticle drug carriers consists of Vesicles preparation and morphologic
solid biodegradable particles in size ranging analysis
from 10 to 1000 nm in which the active Using an original methodology we
principle is dissolved, entrapped or realized the incorporation of tramadol in
encapsulated, and to which the active lipid vesicles.
principle is absorbed or attached. The soft matter vesicles designed
Nanoparticles have been studied after tramadol immobilization inside lipid
extensively as particulate carriers in vesicles, were obtained by dissolving the
different pathophysiologic conditions. lipid in chloroform and removing the
Generally, nanoparticles can be used to solvent by evaporation, which leaded to a
provide targeted delivery of active dry lipid film. The film was then hydrated,
substances, to sustain drug effect in target by adding distilled water with tramadol. In
tissue, to decrease its adverse effects. the end, the vesicles were stabilized with a
(Zhang et al., 2008) The important 0.5% biodegradable polymer chitosan
technological advantages of vesicles used solution. (Garlea et al., 2007;
as drug carriers are the high stability and Tiyaboonchai, 2003; Discher & Eisenberg,
carrier capacity, feasibility of incorporation 2002). The N-deacetylation degree of
of both hydrophilic and hydrophobic utilized chitosan was 79.7%, the average
substances, and feasibility of variable molecular weight was Mw = 310,000g/mol
routes of administration. (Morrow et al., and the polydispersity index was 3.26.
2007; Barauskas et al., 2005) The tramadol vesicles were
Tramadol is a synthetic (±) cis -2- physicochemical and structural analyzed
[(dimethylamino)methyl]-1-(3- using a Malvern Zetasizer Nano ZS, ZEN-
methoxyphenyl) cyclohexanol 3500 model and a Shimadzu UV-1700
hydrochloride derivative. It is a centrally PharmaSpec spectrophotometer. The
acting analgesic, with a low agonistic colloidal solution stability is defined
affinity for opioid receptors and weak according to the average value of Zeta
inhibitory action on reuptake of potential.
norepinephrine and serotonin. (+)-O- Animals
Desmethyltramadol is the most important Male white Swiss mice (20-25g) were
metabolite of tramadol produced in the used. Lighting was on a 12 hours light/dark
liver. cycle (lights on at 6:00 a.m.), with standard
In this study we aimed to investigate laboratory food and tap water freely
the effects of tramadol loaded available, except during the time of the
nanoparticulate formulations, in somatic experiments. Before the experiment, mice
and visceral pain models in mice. were placed on a raised wire mesh, under a
clear plastic box and allowed 2 hours to
Material and methods acclimate in the testing room.
Substances: Procedure
The lipid used, Egg Yolk The experiments were carried out on
L-α-phosphatidylcholine (L-α-lecithin), white Swiss mice (20-25 g), divided into 3
approximately 99% (TLC) pure, chitosan groups of 7 animals each, treated orally
(biocompatible and biodegradable polymer) with the same volume of solution:
and tramadol, were obtained from Sigma- Group I (Control): distilled water
Aldrich Company. The 0.5 (w/w) chitosan 0,3ml,
solutions were prepared in a 0.5% (v/v) Group II (TMD): 10mg/kbw tramadol,
acetic acid. All solutions were made using Group III (TMD-ves): 10mg/kbw
distilled water, purchased from Sicomed, tramadol entrapped in lipid vesicles.
Romania.
195Annals of RSCB Vol. XVI, Issue 1
The model of somatic pain used Results and discussions
consists of hot plate test. (Kreuter, 2007; Le The tramadol vesicles were found to
Bars, 2001) have a mean size of 699nm and mean Zeta
The baseline latency (before drug potential of +16.6mV, value that suggest a
injection) in the hot plate test was 4,2 ± 0.2 moderate stability of the solution.
seconds (mean ± standard error of mean -
SEM). The recommended cut-off time of 10
seconds was used to prevent tissue damage.
Differences between the experimental
and baseline latencies are interpreted as an
index of analgesia. Increases in the latency
for the mouse to react to thermal noxious
stimulus, are indicative of analgesia, while
decreases in latency response are indicative
of hyperalgesia. (Keefe, 1991)
The latency time reaction to thermal
stimulus applied on the paws was measured
before the experiment and 15, 30, 60, 90,
120 minutes, 4, 6, 8, 10, 12 hours, after the
substances administration.
The nociceptive visceral testing was Figure 1. The latency time period of mice
performed using mouse model of writhing treated with the tramadol 10mg/kbw, and
tramadol 10mg/kbw entrapped in soft vesicles
test with acetic acid 0,6%. Antinociception
response to thermal noxious stimulus in hot
was recorded by counting the number of plate test. Each point is the mean ± SEM of
writhes every 5 minutes, for period of 20 latency time (seconds) for seven mice.
minutes after intraperitoneal administration *p< 0.05, ** p< 0.01 vs control.
of irritant stimulus. The behavior
modifications (contractions of the abdomen,
writhes) were observed and scored. (Ma, Statistical analysis of the results obtained in
2007; Mogil, 2009) hot plate test shows that:
Experimental protocol was
implemented according to - tramadol 10mg/kbw determined a
recommendations of the University rapidly and statistically significant
Committee for Research and Ethical Issues, (pAnnals of RSCB Vol. XVI, Issue 1
Conclusions
We developed new carrier system
formulations that entrapped tramadol in soft
vesicles.
The results of our experimental study
are congruent with the literature
communicated data, regarding the dose-
dependent antinociceptive effects of the
opioid drug tramadol in this experimental
cutaneous pain model in mice.
Our study proved that both tramadol
and tramadol loaded nanoparticulate
Figure 2. The number of writhes of mice formulation possesses analgesic properties
treated with tramadol 10mg/kbw, and tramadol in somatic and visceral pain model used.
10mg/kbw entrapped in soft vesicles as a Tramadol entrapped in nano-vesicles
response to chemical noxious stimulus in determined a prolongation of analgesic
writhing test. Each point is the mean ± SEM of effects compared with non entrapped
latency time (seconds) for seven mice. *p< substance in hot plate test and in writhing
0.05, ** p< 0.01 vs control. test.
Our experimental study proved that,
Statistical analysis of the results obtained in
the use of soft matter vesicles as carrier for
writhing test shows that:
tramadol presented the advantage of a dose
dependent sustained release of drug,
- oral administration of tramadol
comparing with non entrapped substance, in
10mg/kbw resulted in a decrease of
both somatic and visceral pain tests.
the writhes number (pAnnals of RSCB Vol. XVI, Issue 1
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