+Modulation of Tramadol Antinociception by Ketamine and Baclofen in Mice

FARMACIA, 2008, Vol.LVI, 6

MODULATION OF TRAMADOL ANTINOCICEPTION BY KETAMINE AND BACLOFEN IN MICE

SORINA CUCUIET1*, G. DOGARU2, VERONICA NASTASA BILD3, MARIA T. DOGARU4

1Department of Pharmacology, Faculty of Medicine, University of Medicine and Pharmacy Tg.Mures, 38th Gh. Marinescu Street Tg. Mures

2Medical 3thClinic, University of Medicine and Pharmacy Tg. Mureş

3Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, University of Medicine and Pharmacy “Gr.T.Popa“ Iasi

4Department of Pharmacology, Faculty of Pharmacy, University of Medicine and Pharmacy Tg.Mures

*corresponding author:

Abstract

The paper presents the influence of two coanalgesics (ketamine and baclofen) on the antinociceptive effect of tramadol, a synthetic opioid with monoaminergic activity, mild efficacy, but low respiratory depression and reduced abuse potential in order to increase its efficacy. The study was performed on Albino mice using the hot plate test (the classical method) and also by a modern method of analyzing interactions - the method of fixed-ratio combination (composite additive line). The results showed that ketamine and baclofen exerted their own antinociceptive effect using the hot plate test. Ketamine (0.25 and 0.5 mg/kgbw) intensified significantly the analgesic effect of small doses of tramadol in simultaneous administration, and baclofen (1.25; 2.5 and 5 mg/kgbw) increased statistically significant the antinociceptive effect of tramadol.Using the method of fixed-ratio combination, there was determined the effective dose for each substance, respectively the interaction index. In conclusion, this study showed an aditive synergistic interaction between tramadol and these two coanalgesics. The authors suggest that such combinations could have therapeutical applications, but only after being tested in clinical studies.

Rezumat

Lucrarea de faţă studiază influenţa a două coanalgezice (ketamina şi baclofenul) asupra efectului antinociceptiv al tramadolului, un opioid sintetic atipic cu activitate monoaminergică, prezentând eficacitate moderată, dar un profil favorabil al efectelor adverse (deprimare respiratorie redusă şi potenţial scăzut de instalare a dependenţei), în scopul îmbunătăţirii eficacităţii acestuia. Studiul s-a efectuat pe şoareci albi masculi folosind testul plăcii încălzite (metoda clasică) precum şi metoda modernă de studiu a interacţiunilor - metoda combinaţiilor în proporţie fixă (metoda dreptei aditive compuse). Rezultatele au arătat că, atât ketamina cât şi baclofenul au exercitat efect antinociceptiv propriu, utilizând testul plăcii încălzite. Ketamina (0,25 şi 0,5 mg/kgc) a intensificat semnificativ efectul antialgic al dozelor mici de tramadol numai în cazul administrării concomitente, iar baclofenul (1,25; 2,5 şi 5 mg/kgc) a crescut statistic semnificativ intensitatea efectului antinociceptiv al tramadolului. Utilizând metoda combinaţiilor în proporţie fixă s-a determinat doza eficace pentru fiecare substanţă, respectiv indexul de interacţiune. În concluzie, acest studiu evidenţiază un sinergism aditiv între tramadol şi cele două coanalgezice. Autorii sugerează că asemenea asocieri ar putea avea perspective de utilizare practică, dar se impune realizarea unor studii clinico-farmacologice.

Tramadol
Ketamine
Baclofen
hot plate test

INTRODUCTION

Tramadol, a central synthetic opioid analgesic, was developed by the German Pharmaceutical Company Grünenthal GmbH in the last years of 1970s and marketed under the trade name Tramal®. In the last decade there was a true re-discovery of this drug, due to the recognition of its advantages, compared to other opioids. Tramadol possesses opioid agonist properties and activates spinal monoaminergic inhibition of pain by inhibiting of norepinephrine and serotonine re-uptake [13]. It exhibits analgesic efficacy similar to codeine, it is an effective agent for the treatment of mild to severe acute or chronic pain, including the neuropathic pain, with a good tolerability profile for the short term use, with antiinflammatory effect [7] and local anesthetic properties. It produces minor respiratory depression and has a low abuse potential. The main problem in the clinical use is its relative reduced efficacy. Our purpose was to find some combinations of tramadol with coanalgesics, in order to improve its efficacy and maintain the advantage of its favourable profile of adverse reactions. In this work we studied the influence of ketamine and baclofen on the antinociceptive effect of tramadol on Albino mice, using the hot plate test and also by a modern method of analyzing interactions - the method of fixed-ratio combination (composite additive line).

MATERIALS AND METHODS

The experiments were performed on 532 adult male Albino mice from the University of Medicine and Pharmacy Tg.Mures Biobase, using as analgesimetric method the hot plate test (53°C). The hot-plate test consisted of an electrically heated surface (Ugo Basile, Comerio, VA, Italy), kept at a constant temperature of 53°C. The latencies for paw licking were recorded for each animal. The analgesic efficacy of the drug was evaluated as the difference between mean test latency (at 30 and 60 minutes after drugs administration) and base-line latency, in seconds ± SEM and also as a % MPE, according to the formula: (TL-BL)/(60-BL)×100, where % MPE, percentage of maximum possible effect, TL= test latency; BL= base-line latency; 60 = cut-off time in seconds, SEM = standard error of the mean.

Animals were divided into several groups as follows:

- placebo groups of mice, treated with 0.1 mL saline solution/ 10 gbw s.c.);

- control groups of mice, receiving: tramadol 5, 10, 20, 40, 60, 80, mg/ kgbw s.c., ketamine (0.25 and 0.5 mg/ kgbw i.p.), baclofen 1.25, 2.5 and 5 mg/kgbw, i.p;

- group of mice receiving the simultaneous combination of tramadol (5, 10, 20, 40, 60, 80, mg/ kgbw s.c) + ketamine (0.25 and 0.5 mg/ kgbw i.p.);

- groupd of mice treated first with ketamine (0.25 and 0.5 mg/ kgbw i.p.) and after 2 hours with tramadol (5, 10, 20, 40, 60, 80, mg/ kgbw s.c.);

- groups of mice receiving the simultaneous combination of tramadol (5, 10, 20, 40, 60, 80, mg/ kgbw s.c.) + baclofen (1.25, 2.5 and 5 mg/kgbw, i.p.)

All experiments were performed in compliance with European Communities Council Directive 1986 (86/609/EEC) and Ordinance No. 37 of the Romanian Government from 2nd February 2002.

Statistical Analysis. In order to evaluate the differences between placebo and all treatments and also between tramadol and its combinations with ketamine or baclofen, a paired Student's t test was used. A probability level of p<0.05 was considered to be statistically significant.

RESULTS AND DISCUSSION

In experiments regarding the interaction of tramadol with ketamine (table I - VI), the results showed that ketamine alone had antinociceptive effect in both tested doses (0.25 and 0.5 mg/kgbw). Concomitant administration of ketamine 0.25 mg/kgbw with tramadol 5 mg/kgbw produced an antinociceptive effect, statistically significant compared to tramadol at 30 and 60 minutes after injection. The combination ketamine 0.25 mg/kgbw+ tramadol 10 mg/kgbw produced antinociceptive effect versus tramadol only, at 60' after the injection. The others associations of ketamine 0.25 mg/kgbw with tramadol (20, 40, 60 and 80 mg/kgbw) were statistically significant only versus placebo. Concomitant administration of ketamine 0.5 mg/kgbw with tramadol 5 mg/kgbw increased the antinociceptive effect of tramadol (p<0.01 at 30’, p<0.02 at 60’). Also, the combination ketamine 0.5 mg/kgbw + tramadol 10 mg/kgbw produced an analgesic effect, statistically significat versus tramadol (p<0.001 at 30’, p<0.02 at 60’). When ketamine 0.5 mg/kgbw was injected 2 hours before tramadol there was a statistically significant result versus tramadol only in the case of the combination: ketamine 0.5 mg/kgbw+tramadol 10 mg/kgbw at 30’.

In experiments performed with baclofen (table VII – VIII), the results indicate that baclofen alone exerted antinociceptive effect in all used doses (1.25; 2.5 and 5 mg/kgbw) at both moments of testing. Baclofen 1.25 mg/kgbw induced a statistically significant effect intensification of tramadol 5 mg/kgbw at 30 and 60 minutes after administration (p<0.05 at 30’, p<0.001 at 60’), increased the effect of tramadol 10 mg/kgbw only at 60’ (p<0.001) and potentiate the effect of tramadol 20 mg/kgbw (p<0.001 at 30’ and 60’). The combination baclofen 2.5 mg/kgbw+ tramadol 20 mg/kgbw was statistically significant versus tramadol only at 30 minutes after administration (p<0.01). Baclofen 5 mg/kgbw intensified the antinociceptive effect of tramadol 20 mg/kgbw at 30 and 60 minutes (p<0.001).

Table I

The influence of ketamine 0.25 mg/kgbw on the antinociceptive effect of tramadol (5, 10, 20, 40, 60, 80 mg/kgbw) in the hot plate test.

Simultaneous administration of the drugs tested

Treatment /Dose
(mg/kgbw) / n / BL
(sec)
M ± SEM / TL at 30’
(sec)
M ± SEM / TL at 60’ (sec)
M ± SEM / %
MPE
at 30’ / %
MPE
at 60’
Placebo / 38 / 14.8 ± 0.9 / 16.3 ± 0.9 / 18.2 ± 1.7 / 3.31 / 7.52
Tramadol 5 / 20 / 11.0 ± 0.5 / 16.2 ± 0.7 / 16.6 ± 1.0 / 10.61 / 11.42
Tramadol 10 / 20 / 11.3 ± 0.5 / 16.9 ± 0.9 / 18.0 ± 0.7 / 11.49 / 13.14
Tramadol 20 / 40 / 11.6 ± 0.5 / 28.5 ± 2.3 / 25.6 ± 1.6 / 34.91 / 28.92
Tramadol 40 / 20 / 15.1 ± 0.8 / 46.4 ± 3.4 / 40.7± 2.3 / 69.71 / 57.01
Tramadol 60 / 18 / 11.2 ± 0.5 / 47.8 ± 3.3 / 43.3 ± 3.4 / 75 / 65.77
Tramadol 80 / 20 / 11.4 ± 0.6 / 54.6 ± 1.9 / 48.9 ± 3.3 / 88.88 / 77.16
Ketamine 0.25 / 20 / 13.1 ± 1.1 / 25.3 ± 2.5 / 27.3 ± 3.1 / 26.01 / 30.27
Ketamine 0.25 +
Tramadol 5 / 20 / 12.5 ± 1.4 / 35.7 ± 2.6 / 29.3 ± 2.8 / 48.84 / 35.36
Ketamine 0.25 +
Tramadol 10 / 20 / 10.9 ± 0.6 / 15.1 ± 1.1 / 27.1 ± 2.5 / 8.55 / 32.99
Ketamine 0.25 +
Tramadol 20 / 20 / 11.2 ± 0.7 / 28.2 ± 2.8 / 30.4 ± 2.4 / 34.83 / 39.34
Ketamine 0.25 +
Tramadol 40 / 20 / 17.0 ± 0.8 / 32.9 ± 2.6 / 33.6 ± 3.1 / 36.97 / 38.6
Ketamine 0.25 +
Tramadol 60 / 18 / 16.2 ± 1.5 / 55.3 ± 2.6 / 55.8 ± 2.5 / 89.26 / 90.41
Ketamine 0.25 +
Tramadol 80 / 20 / 11.3 ± 0.8 / 55.7 ± 1.9 / 56.1 ± 2.5 / 91.17 / 91.99

Table II

The statistical analysis (Student t test) of the combination ketamine 0.25 mg/kgbw + tramadol (5, 10, 20, 40, 60, 80 mg/kgbw) in the hot plate test.

Simultaneous administration of the drugs tested

Treatment /Dose
(mg/kgw) / n / TL-BL at 30' (sec)
M ± SEM / TL-BL at 60’ (sec)
M ± SEM / p
at 30' / p
at 60'
Placebo / 38 / 1.5 ± 1.3 / 3.4 ± 1.9 / - / -
Tramadol 5 / 20 / 5.2 ± 0.9 / 5.6 ± 1.1 / < 0.05* / NS*
Tramadol 10 / 20 / 5.6 ± 1.0 / 6.4 ± 1.1 / <0.02* / NS*
Tramadol 20 / 40 / 16.9 ± 2.3 / 14.0 ±1.7 / <0.001* / <0.001*
Tramadol 40 / 20 / 31.3 ± 3.5 / 25.6± 2.4 / < 0.001* / <0.001*
Tramadol 60 / 18 / 36.6 ± 3.3 / 32.1± 3.4 / < 0.001* / < 0.001*
Tramadol 80 / 20 / 43.2 ± 2.0 / 37.5 ± 3.3 / <0.001* / <0.001*
Ketamine 0.25 / 20 / 12.2 ± 2.7 / 14.2 ± 3.3 / <0.001* / <0.01*
Ketamine 0.25 +
Tramadol 5 / 20 / 23.2 ± 2.9 / 16.8 ± 3.1 / <0.001*
<0.001** / <0.001*
<0.01**
Ketamine 0.25 +
Tramadol 10 / 20 / 4.2 ± 1.2 / 16.2 ±2.6 / NS*
NS** / <0.001*
<0.01**
Ketamine 0.25 +
Tramadol 20 / 20 / 17.0 ± 2.9 / 19.2 ± 2.5 / <0.001*
NS** / <0.001*
NS**
Ketamine 0.25 +
Tramadol 40 / 20 / 15.9 ± 2.7 / 16.6 ± 3.2 / <0.001*
NS** / <0.001*
NS**
Ketamine 0.25 +
Tramadol 60 / 18 / 39.1 ± 3.0 / 39.6 ± 2.9 / <0.001*
NS** / <0.001*
NS**
Ketamine 0.25 +
Tramadol 80 / 20 / 44.4 ± 2.1 / 44.8 ± 2.6 / <0.001*
NS** / <0.001*
NS**

Table III

The influence of ketamine 0.5 mg/kgbw on the antinociceptive effect of tramadol (5, 10, 20 mg/kgbw) in the hot plate test.

Simultaneous administration of the drugs tested

Table IV

The statistical analysis (Student t test) of the combination ketamine 0.5 mg/kgbw + tramadol (5, 10, 20 mg/kgbw) in the hot plate test

Treatment /Dose
(mg/kgbw) / n / TL-BL at 30' (sec)
M ± SEM / TL-BL at 60’ (sec)
M ± SEM / p
at 30' / p
at 60'
Placebo / 38 / 1.5 ± 1.3 / 3.4 ± 1.9 / - / -
Tramadol 5 / 20 / 5.2 ± 0.9 / 5.6 ± 1.1 / < 0.05* / NS*
Tramadol 10 / 20 / 5.6 ± 1.0 / 6.4 ± 1.1 / <0.02* / NS*
Tramadol 20 / 40 / 16.9 ± 2.3 / 14.0 ± 1.7 / <0.001* / <0.001*
Ketamine 0.5 / 20 / 13.3 ± 2.4 / 16.6 ± 2.6 / <0.001* / <0.001*
Ketamine 0.5 +
Tramadol 5 / 20 / 12.9 ± 2.2 / 11.8 ± 2.3 / <0.001*
<0.01** / <0.001*
<0.02**
Ketamine 0.5 +
Tramadol 10 / 20 / 13.1 ± 1.7 / 14.3 ± 2.9 / <0.001*
<0.001** / <0.001*
<0.02**
Ketamine 0.5 +
Tramadol 20 / 20 / 17.2 ± 2.7 / 16.4 ± 2.9 / <0.001*
NS** / <0.01*
NS**

Table V

The influence of ketamine 0.5 mg/kgbw (2 hours before testing) on the antinociceptive effect tramadol (10 and 20 mg/kgbw) in the hot plate test

Treatment /Dose
(mg/kgbw) / n / BL
(sec)
M ± SEM / TL at 30’
(sec)
M ± SEM / TL at 60’ (sec)
M ± SEM / %
MPE
at 30’ / %
MPE
at 60’
Placebo / 38 / 14.8 ± 0.9 / 16.3± 0.9 / 18.2 ± 1.7 / 3.31 / 7.52
Tramadol 10 / 20 / 11.3 ± 0.5 / 16.9± 0.9 / 18.0 ± 0.7 / 11.49 / 13.14
Tramadol 20 / 40 / 11.6 ± 0.5 / 28.5 ± 2.3 / 25.6 ± 1.6 / 34.91 / 28.92
Ketamine 0.5 (2 h before testing) / 20 / 10.0 ±0.3 / 13.4 ± 0.6 / 15.9 ± 1.0 / 6.8 / 11.8
Ketamine 0.5 (2 h before testing) +Tramadol 10 / 19 / 18.3 ± 1.0 / 30.4 ± 2.7 / 30.3 ± 2.7 / 29.01 / 28.77
Ketamine 0.5(2 h before testing) +Tramadol 20 / 20 / 16.3 ± 0.8 / 32.7 ± 2.8 / 35.5± 3.3 / 37.52 / 43.93

Table VI

The statistical analysis (Student t test) of the combination ketamine 0.5 mg/kgbw (2 hours before testing) + tramadol (10 and 20 mg/kgbw) in the hot plate test

Treatment /Dose
(mg/kgbw) / n / TL-BL
at 30' (sec)
M ± SEM / TL-BL
at 60’ (sec)
M ± SEM / p
at 30' / p
at 60'
Placebo (0.1mL/10gbw) / 38 / 1.5± 1.3 / 3.4 ± 1.9 / - / -
Tramadol 10 / 20 / 5.6 ± 1.0 / 6.4 ± 1.1 / <0.02* / NS*
Tramadol 20 / 40 / 16.9± 2.3 / 14.0 ± 1.7 / <0.001* / <0.001*
Ketamine 0.5 (2 h before testing) / 20 / 3.4 ± 0.7 / 5.9 ± 1.0 / NS* / NS*
Ketamine 0.5 (2 h before testing) +Tramadol 10 / 19 / 12.1± 2.9 / 12.0 ± 2.9 / < 0.01*
< 0.05** / < 0.02*
NS**
Ketamine 0.5(2 h before testing) +Tramadol 20 / 20 / 16.4 ±2.9 / 19.2 ± 3.4 / < 0.001*
NS** / < 0.001*
NS**

Table VII

The influence of baclofen (1.25; 2.5 and 5 mg/kgbw on the antinociceptive effect of tramadol (5, 10, 20 mg/kgbw) in the hot plate test.

Simultaneous administration of the drugs tested

Treatment/Dose
(mg/kgbw) / n / BL
(sec)
M ± SEM / TL at 30’
(sec)
M ± SEM / TL at 60’ (sec)
M ± SEM / %
MPE
at 30’ / %
MPE
at 60’
Placebo (0.1mL/10gbw) / 38 / 14.8 ± 0.9 / 16.3± 0.9 / 18.2 ± 1.7 / 3.31 / 7.52
Tramadol 5 / 20 / 11.0 ± 0.5 / 16.2 ± 0.7 / 16.6 ± 1.0 / 10.61 / 11.42
Tramadol 10 / 20 / 11.3 ± 0.5 / 16.9 ± 0.9 / 18.0 ± 0.7 / 11.49 / 13.14
Tramadol 20 / 40 / 11.6 ± 0.5 / 28.5 ± 2.3 / 25.6 ± 1.6 / 34.91 / 28.92
Baclofen 1.25 / 20 / 10.0 ± 0.5 / 23.6 ± 2.3 / 27.0 ± 3.0 / 27.2 / 34
Baclofen 1.25 +
Tramadol 5 / 20 / 10.3 ± 0.6 / 18.9 ± 1.2 / 22.6 ± 1.4 / 17.3 / 24.74
Baclofen 1.25 +
Tramadol 10 / 20 / 9.2 ± 0.6 / 16.9 ± 2.0 / 27.9 ± 2.3 / 15.15 / 36.81
Baclofen 1.25 +
Tramadol 20 / 20 / 10.0 ± 0.7 / 41.4 ± 2.9 / 45.5 ± 3.0 / 62.8 / 71
Baclofen 2.5 / 19 / 11.0 ± 0.6 / 31.8 ± 4.4 / 33.3 ± 3.7 / 44.44 / 45.5
Baclofen 2.5 +
Tramadol 20 / 18 / 11.2 ± 0.7 / 36.7 ± 3.4 / 32.1 ± 3.7 / 58.4 / 42.82
Baclofen 5 / 20 / 9.9 ± 0.6 / 32.2 ± 3.4 / 44.2 ± 3.3 / 44.51 / 68.46
Baclofen 5 +
Tramadol 20 / 18 / 9.2 ± 0.5 / 52.3± 3.2 / 54.1± 0.2 / 84.84 / 88.38

Table VIII

The statistical analysis(Student t test) of the combination baclofen (1.25; 2.5 and 5mg/kgbw) + tramadol (5, 10, 20 mg/kgbw) in the hot plate test.

Simultaneous administration of the drugs tested

Treatment /Dose
(mg/kgbw) / n / TL-BL at 30' (sec)
M ± SEM / TL-BL at 60’
(sec)
M ± SEM / p
at 30' / p
at 60'
Placebo 0,1ml/10g / 38 / 1.5 ± 1.3 / 3.4 ± 1.9 / - / -
Tramadol 5 / 20 / 5.2 ± 0.9 / 5.6 ± 1.1 / < 0.05* / NS*
Tramadol 10 / 20 / 5.6 ± 1.0 / 6.4 ± 1.1 / <0.02* / NS*
Tramadol 20 / 40 / 16.9 ± 2.3 / 14.0 ± 1.7 / <0.001* / <0,001*
Baclofen 1.25 / 20 / 13.6 ± 2.3 / 17.0 ± 3.0 / <0.001* / <0.001*
Baclofen 1.25 +Tramadol 5 / 20 / 8.6 ± 1.3 / 12.3 ± 1.5 / <0.001*
< 0.05** / <0.001*
< 0.001**
Baclofen 1.25 + Tramadol 10 / 20 / 7.7 ± 2.1 / 18.7 ± 2.4 / < 0.05*
NS** / <0.001*
< 0.001**
Baclofen 1,25 +Tramadol 20 / 20 / 31.4 ± 3.0 / 35.5 ± 3.1 / <0.001*
<0.001** / <0.001*
<0.001**
Baclofen 2.5 / 19 / 20.8 ± 4.4 / 22.3 ± 3.7 / <0.001* / <0.001*
Baclofen 2.5
+ Tramadol 20 / 18 / 28.5 ± 3.5 / 20.9 ± 3.8 / <0.001*
<0.01** / <0.001*
NS**
Baclofen 5 / 20 / 22.3 ± 3.4 / 34.3 ± 3.3 / <0.001* / <0.001*
Baclofen 5
+Tramadol 20 / 18 / 43.1± 3.2 / 44.9 ± 0.5 / <0.001*
<0.001** / <0.001*
<0.001**

Legend: BL= base-line latency, TL= test latency, % MPE = percentage of maximum possible effect, M = mean, SEM = standard error of the mean, TL-BL = difference between TL and BL, p = probability, * = Student t test, vs placebo, ** = Student t test vs treatment with tramadol alone; n= no. of animals group

Data analysis using the method of fixed-ratio combination (composite additive line).

The method presumes the determination of the Effective Dose 50 (ED50) for each substance taken into study, the determination of Zadd (total additive dose), Zmix (ED50 for the combination) and the interaction index (γ). The regression lines will be drawn and analyzed accordingly [15, 16, 17].

Tramadol-ketamine combination

The dose series used for the classical method were subjected to the protocol for the method of the composite additive line. Thus, the ED50 value for tramadol for the dose series comprised to the range of 5.0-80.0 mg/kgbw (Fig. 1A). In this situation, the regression line of the combination will be compared with the regression line for the compound for which the ED 50 was determined, using the nociception model taken into study.

Table IX

Dose series, ratios used and effects recorded for the tramadol-ketamine combination

Tramadol / ANTI-NOCICEPTION
% / Tramadol/ ketamine (1) (0.25mg/kgbw) / ANTI-NOCICEPTION
% / Tramadol/ ketamine (2) (0.5mg/kgbw) / ANTI-NOCICEPTION
%
30min / 60min / 30min / 60min / 30 min / 60 min
5.00 mg/ kgbw / 10.61 / 11.42 / 20:1 / 48.84 / 35.36 / 20:2 / 24.9 / 22.77
10.00 mg/ kgbw / 11.49 / 13.14 / 40:1 / 8.55 / 32.99 / 40:2 / 26.2 / 28.6
20.00 mg/ kgbw / 34.91 / 28.92 / 80:1 / 34.83 / 39.34 / 80:2 / 33.92 / 32.34
40.0 mg/ kgbw / 69.71 / 57.01 / 160:1 / 36.97 / 38.6
60.00 mg/ kgbw / 75.00 / 65.77 / 240:1 / 89.26 / 90.41
80.00 mg/ kgbw / 88.88 / 77.16 / 320:1 / 91.17 / 91.99

Figure 1

Analysis of the regression lines:

A. regression line for evaluating the ED50 for tramadol

B. regression line for the tramadol-ketamine combination (1) compared with the regression line for tramadol.

Table X

Estimation of ED50 for tramadol, Zadd and Zmix

for the tramadol-ketamine combination

Interaction Index γ
Tramadol / DE50
mg/kgbw / 25,279 ± 2,998
R = 0,972
(Y=-48,976 + 70,559*X) / γ ≈ 1 additive
γ > 1 sub-additive
Ketamine / DE50
mg/kgbw / 0,00
Combination
Tramadol-Ketamine / Zadd
mg/kgbw / 25,380 ± 3,010 (1)
25,927 ± 3,075 (2)
(Y=-48,976 + 70,559*X)
R = 0,972
Combination
Tramadol-Ketamine / Zmix mg/kgbw / 22,612 ± 11,722 (1)
(Y = -15,226 + 29,271*X)
R= 0,984 / γ = 0.9
24,612 ± 3,13 (2)
(Y = -12,104 + 15,848*X)
R = 0,929 / γ = 0.95

Although the regression line for the combination is slightly left-shifted compared to the regression line of tramadol, it shows an intersection point with the latter (Fig. 1B). Statistical parameters (Tables II, IV) and the numerical value of the interaction index demonstrate an additive effect (Table X).

Tramadol-baclofen combination

As the ED50 for tramadol is already known, we determined the ED50 for baclofen and we establish the combination ratios.

Table XI

Dose series used for determining the ED50 and

effect records for the tramadol-baclofen combination

Tramadol / ANTI-NOCICEPTION
% / BACLOFEN / ANTI-NOCICEPTION
% / COMBI-NATION
Tramadol/
Baclofen / ANTI-NOCICEPTION
%
30min / 60min / 30min / 60 min / 30 min / 60
min
5.00 mg/kgbw / 10.61 / 11.42 / 1.25 mg/kgbw / 27.2 / 34.00 / 6.25 mg/ kgbw / 17.3 / 24.74
10.00
mg/ kgbw / 11.49 / 13.14 / 2.5 mg/ kgbw / 44.44 / 45.5 / 11.25 mg/ kgbw / 15.15 / 36.81
20.00
mg/ kgbw / 34.91 / 28.92 / 5.00 mg/ kgbw / 44.51 / 68.46 / 21.25 mg/ kgbw / 62.8 / 71.0
40.0
mg/ kgbw / 69.71 / 57.01
60.00
mg/ kgbw / 75.00 / 65.77
80,00
mg/ kgbw / 88,88 / 77,16

Figure 2

Analysis of the regression lines:

A. composed additive line; B. regression line for the tramadol-baclofen combination compared with the additive composite line

Table XII

Estimation of ED50 for tramadol, baclofen,

and Zadd şi Zmix for the tramadol-baclofen combination

Interaction Index γ
Tramadol / DE50
mg/kgbw / 25,279 ± 2,998
R = 0,972
(Y=-48,976 + 70,559*X) / γ ≈ 1 addive
γ > 1 sub-additive
Baclofen / DE50
mg/kgbw / 6,171 ± 3,764
R= 0,868
(Y = 27,275 + 28,751*X )
Tramadol-Baclofen combination / Zadd2*
mg/kgbw / 15,725 ± 2,406
R= 0,967
(Y = 5,045 + 40,113*X )
ZaddC**
mg/kgbw / 13,204 ± 1,611
R = 0,967
(Y = 5,045 + 40,113*X)
Tramadol-Baclofen combination / Zmix mg/kgbw / 13,662 ± 6,727
R = 0,849
(Y = -35,791 + 75,553*X) / γ = 1,03

*Zadd2 exp. **ZaddC est.

As we were able to ascertain the ED50 for both substances, this aspect made possible the drawing of the composite additive line (Fig. 2A) and the establishing of the total additive line. According to the methodology, in this situation, a comparison will be made between the regression line of the combination and the composite additive line. The position of the regression line (Fig 2B) of the combination and the numerical value of the interaction index (Table XII) demonstrate an additive effect.

((±)-Tramadol is a synthetic analgesic formulated as a racemic mixture (1:1) of (-)- and (+)-tramadol, which differ in their potency to bind to μ-opioid receptors and to inhibit monoamine-reuptake. It is an effective analgesic in step 2 of the World Health Organization's guidelines for the treatment of patients with cancer pain. Tramadol can be considered as a therapeutic option for the control of neuropathic pain, in diabetic neuropathy, in patients with cancer. It presents also antiinflammatory activity [4] and local anesthetic properties. Compared to typical opioid analgesics, tramadol appears to have a low incidence of respiratory and cardiac depression, and a low abuse potential. Tramadol is well tolerated in short term use, having dizziness, nausea, sedation and sweating as main adverse effects. The current opinion is that tramadol is a useful alternative to the opioid analgesics currently available for the treatment of moderately acute or chronic pain. But, its analgesic efficacy remains moderate, compared to other opioid analgesics and there are many attempts to improve this feature. The main options are to associate tramadol with either non-opioid analgesics or with coanalgesics. Adjuvant analgesics or coanalgesics are a heterogenous group of drugs, with distinct main indication, but having antinociceptive properties. They could be used alone and in combination with opioid and nonopioid analgesics, mainly in the treatment of chronic pain. Their association with proper analgesics is justified because they potentiate antialgic effect, allowing reduced doses, with lower incidence of side effects and also because they could act as simptomatic medication, against anxiety, agitation, sleeplessness, nausea. The most prescribed coanalgesics are antidepressants, neuroleptics, anxiolytics, anticonvulsants, spasmolytic drugs, N-methyl-D-aspartate (NMDA) receptor antagonists, clonidine, calcitonin, glucocorticoids. Ketamine – an intravenous general anesthetic and baclofen – a spasmolytic, are well known and widely used drugs which demonstrated also analgesic properties. Therefore we investigated the influence of these two coanalgesics on the antinociceptive effect of tramadol in an acute model of pain (the hot plate test) on Albino mice.

The N-methyl-D-aspartate (NMDA) receptors for glutamate are involved in the development and maintainance of neuropathic pain and in the generation and maintenance of central (spinal) states of hypersensitivity. NMDA-receptor activation not only increases the cell's response to pain stimuli, but it also decreases neuronal sensitivity to opioid receptor agonists. In addition to preventing central sensitization, co-administration of NMDA-receptor antagonists with an opioid may prevent tolerance to opioid analgesia. Commercially available NMDA-receptor antagonists include ketamine, dextromethorphan, memantine and amantadine. The role of the NMDA receptor in processing of nociceptive input has led naturally to renewed clinical interest in NMDA receptor antagonists such as ketamine.

As it can be seen in table I-VI, ketamine administrated simultaneously with tramadol increased its antinociceptive action only in the case of small doses of tramadol, but when it was injected 2 hours before tramadol, the synergism could not be demonstrated. It comes out that there was a mild additive synergism between tramadol and ketamine. From the results presented in table VII-VIII it can be seen that baclofen exerted its own analgesic action and intensified the effect of small and moderate doses of tramadol. Also, these findings allow the conclusion that there was a synergic interaction between tramadol and baclofen. In order to explain the mechanisms producing this synergism, a review of the pharmacodynamic properties of these three drugs is necessary.

Tramadol-induced antinociception is mediated by opioid (μ) and non-opioid (inhibition of monoamine uptake) mechanisms. Its selectivity for μ receptors has been demonstrated, but the affinity for μ receptors of the CNS remains low, being 6000 times lower than that of morphine. The M1 metabolite of tramadol (O-demethyl tramadol), produced by liver O-demethylation, shows a higher affinity for opioid receptors than the parent drug. In contrast to other opioids, the analgesic action of tramadol is only partially inhibited by the opioid antagonist naloxone, which suggests the existence of another mechanism of action. The mechanism by which opioid analgesics inhibit pain perception involves, in part, activation of both descending serotoninergic and noradrenergic pathways. Many experimental studies have demonstrated the monoaminergic activity exerted by tramadol. Tramadol inhibits noradrenaline and serotonin (5-HT) reuptake, having a significant contribution to the analgesic action, by blocking nociceptive impulses at the spinal level. (+/-)-Tramadol beeing a racemic mixture of 2 enantiomers, each one displays different affinities for various receptors. In vitro receptor binding and synaptosomal uptake experiments showed that the (+)-enantiomer of tramadol is specific for the μ -opioid receptor site and for the serotonin (5-HT) carrier, whereas the (-)-enantiomer has a higher affinity to the noradrenaline (NA) transporter. Tramadol showed an inherent antidepressant-like (mood improving) activity, influencing the affective component of pain. The inhibitory effect on NMDA receptors may contribute to the antinociceptive effect of tramadol at relatively large concentrations [8]. At clinically relevant concentrations it inhibits muscarinic receptor, which could explain the anticholinergic effect of tramadol and it inhibits catecholamine secretion partly by inhibiting nicotinic AChR (acetylcholine receptors) functions in a naloxone-insensitive manner and alpha7 receptors [13]. In in vitro receptor binding experiments tramadol enhanced DA (dopamine) turnover via an opioid mechanism and in ex vivo neurotransmitter determinations enhanced DA release in definite brain areas [7].