Synergistic antihypersensitive effects of pregabalin and tapentadol in a rat model ofneuropathic pain
Thomas Christoph a,⁎, Jean De Vry a, Klaus Schiene a, Ronald J. Tallarida b, Thomas M. Tzschentke a
a Grünenthal GmbH, Global Preclinical Research and Development, Department of Pharmacology, Zieglerstrasse 6, 52078 Aachen, Germanyb Department of Pharmacology, Temple University School of Medicine and Centre for Substance Abuse Research, 3420 N. Broad Street, Philadelphia, PA 19140, United States
Neuropathic pain is a clinical condition which remains poorly treated and combinations of pregabalin, anantagonist of the α2δ-subunit of Ca2+ channels, with tapentadol, a μ-opioid receptor agonist/noradrenalinereuptake inhibitor, or with classical opioids such as oxycodone and morphine might offer increased therapeuticpotential. In the rat spinal nerve ligationmodel, a dose dependent increase in ipsilateral pawwithdrawal thresholdswas obtained using an electronic von Frey filament after IV administration of pregabalin (1–10 mg/kg),tapentadol (0.316–10mg/kg), morphine (1–4.64 mg/kg) and oxycodone (0.316–3.16 mg/kg), with ED50 values(maximal efficacy) of 4.21 (67%), 1.65 (94%), 1.70 (96%) and 0.63 mg/kg (100%), respectively. Equianalgesic dosecombinations of pregabalin and tapentadol (dose ratio 2.5:1), morphine (2.5:1) or oxycodone (6.5:1) resulted inED50 values (maximal efficacy) of 0.83 (89%), 2.33 (97%) and 1.14 mg/kg (100%), respectively. The concept ofdose-equivalence suggested an additive interaction of pregabalin and either oxycodone or morphine, while asynergistic interaction was obtained with pregabalin and tapentadol (demonstrated by isobolographic analysis).There was no increase in contralateral paw withdrawal thresholds and no locomotor impairment, as measured inthe open field, for the combination of pregabalin and tapentadol; while a significant increase and impairmentwas demonstrated for the combinations of pregabalin and either morphine or oxycodone. Because combinationof pregabalin and tapentadol resulted in a synergistic antihypersensitive activity, it is suggested that, beside theuse of either drug alone, this drug combination may offer a beneficial treatment option for neuropathic pain.
Neuropathic pain may occur as a consequence of a lesion or diseaseof the peripheral or central nervous system. Patients suffer fromspontaneous and evoked pain, the treatment of which is inadequate inmany cases (Baron, 2009). Current treatment options comprise severalpharmacological classes, including anticonvulsants, antidepressants,local anesthetics and opioids. Combinations of different mechanisms ofaction targeting pain transmission at different levels of the paintransmission pathways may yield higher efficacy and may have fewerside effects in patients insufficiently treated with mono-therapy. Theanticonvulsants gabapentin and pregabalin bind to the α2δ subunit ofvoltage dependent Ca2+ channels in central nervous system neurons.Both compounds reduce tactile allodynia in spinal nerve-ligated rats(Field et al., 1999) and are used clinically for the treatment ofneuropathic pain conditions (Backonja and Glanzman, 2003). Opioids,the mainstay in the treatment of severe pain, inhibit nociceptivedischarges in pre- and postsynaptic neurons in the spinal cord.Moreover, they activate descending inhibitory pathways which origi-
nate in the brainstem and project to the spinal cord, leading toantinociception. However, in spinal nerve-ligated rats, the potency andefficacyof opioids, suchasmorphine, appear to be reduced, as comparedto sham controls (Ossipov et al., 1995), and this reflects the clinicalfinding that opioids have limited utility for the treatment of neuropathicpain (Benedetti et al., 1998). Noradrenaline is released in the spinal cordfrom descending inhibitory pathways and reduce nociceptive signalingbetween primary afferent and second order neurons through activationof α2-adrenoceptors. Noradrenaline reuptake inhibitors such asdesipramine prolong the availability of noradrenaline in the synapticcleft and thereby increase the antinociceptive efficacy of noradrenaline.Combination of morphine with desipramine or the α2-adrenoceptoragonist clonidine result in synergistic antiallodynic effect in spinalnerve-ligated rats (Ossipov et al., 1997). In addition, combinedadministration of morphine with gabapentin was reported to result ina supra-additive analgesic effect in the chronic constriction injury ratmodel of mononeuropathic pain (De la O-Arciniega et al., 2009).However, a recent clinical study failed to demonstrate that combinationwithpregabalin enhances the analgesic efficacy of the opioid oxycodonein neuropathic pain patients (Zin et al., 2010), questioning thetherapeutic value of this drug combination.
Tapentadol is a novel centrally acting analgesic that combines μ-opioid receptor agonism and noradrenaline reuptake inhibition in a
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single molecule (Tzschentke et al., 2006) and that shows highanalgesic potency and efficacy in animal models of acute and chronicpain (Tzschentke et al., 2007). It has been demonstrated previouslythat both mechanisms contribute to the analgesic effect of tapentadol,with a predominance of noradrenergic-based analgesia in spinalnerve-ligated rats and opioid-based analgesia in acute nociception,respectively (Schröder et al., 2010).
The aim of the present study was to further analyze the nature ofthe interaction between pregabalin and tapentadol, oxycodone ormorphine in spinal nerve-ligated rats, a model of mononeuropathicpain. Possible locomotor side effects of the drug combinations wereassessed in an open field to assess the behavioral specificity of theobtained effects.
2. Material and methods
2.1. Animals
Male Sprague–Dawley rats (Janvier, Le Genest St Isle, France) werehoused under a 12:12-h light–dark cycle (lights on at 06:00 h), withroom temperature 20 to 24 °C, relative air humidity 35 to 70%, 15 airchanges per h, and air movement less than 0.2 m/s. The animals hadfree access to standard laboratory food and tap water. There were atleast 5 days between the delivery of the animals and behavioraltesting or surgery. Average animal weights were 140–160 g at thetime of surgery and 200–400 g at the time of testing. All experimentswere conducted according to the guidelines of the InternationalAssociation for the Study of Pain (Zimmermann, 1983) and theGerman Animal Welfare Law.
2.2. Experimental procedures
Animals were assigned randomly to treatment groups. Differentdoses and vehicle were tested in a randomized fashion. Although theoperators performing the behavioral tests were not formally “blinded”with respect to the treatment, they were not aware of the studyhypothesis or the nature of the differences between drugs.
2.3. Spinal nerve ligation
The spinal nerve ligation model of neuropathic pain was adaptedfrom (Kim and Chung, 1992). Under pentobarbital anesthesia(Narcoren, 60 mg/kg intraperitoneally), the left L5 and L6 spinalnerves were exposed by removing a small piece of the paravertebralmuscle and a part of the left spinous process of the L5 lumbar vertebra.The L5 and L6 spinal nerves were then carefully isolated and tightlyligated with silk (NC-silk black, USP 5/0, metric 1, Braun Melsungen,Germany). After checking hemostasis, the muscle and the adjacentfascia were closed with sutures, and the skin was closed with metalclips. After surgery, animals were allowed to recover for 1 week.During this time mechanical hypersensitivity develops becomingstable one week after ligation. Sham animals underwent the sameprocedure without ligation of the spinal nerves.
2.4. Behavioral testing
2.4.1. Mechanical thresholdFor the assessment of mechanical hypersensitivity, which was stable
for at least 5 weeks, the ratswere placed on ametalmesh coveredwith aplastic dome and were allowed to habituate until exploratory behaviorceased. The threshold for mechanical hypersensitivity (i.e. decreasedipsilateral paw withdrawal thresholds) was determined with anelectronic von Frey anesthesiometer (Somedic AB, Malmö, Sweden)using themedianoffive consecutivemeasurements (inter-measurementinterval 1–2 min). Animals were tested before and 0.5, 1, and 3 h afteradministration of the test compounds on both hindpaws in a
randomized fashion. Withdrawal thresholds of the injured paws wereexpressed as the percentage ofmaximal possible effect (MPE) related toanti-hypersensitivity comparing thepre-drug threshold of spinal nerve-ligated animals (i.e., 0%MPE) and the control threshold of shamanimals(i.e., 100%MPE). Drugs or vehiclewere tested 2 to 5 weeks after surgery(1 test per week) in a counterbalanced within-group design. The sameanimal, randomly assigned to a treatment group received up to fourtreatments with a washout period of at least 7 days between thetreatments.
2.4.2. Open fieldExploratory locomotor activity wasmeasuredwith a computerized
video tracking system (EthoVision; Noldus Information Technology)in boxes with a size of 45×45 cm and 40 cm-high non-transparentwalls. Up to four animals could be measured simultaneously in fourdifferent boxes. Locomotor activity in the open field was determinedas covered distance of horizontal movements. After administration ofthe test compound, naive rats (i.e., rats which had no prior experiencewith the test apparatus) were placed individually in the center of thetest-box. Thereafter the behavior of the animals in the open field wasanalyzed for 5 min.
2.5. Drugs
Tapentadol HCl, (S)-pregabalin (both compounds synthesized bythe Chemistry Department of Grünenthal GmbH, Germany), oxyco-done HCl and morphine HCl (both purchased from Macfarlan SmithLtd., UK) were administered intravenously (IV) into the tail vein 5 cmdistal from the tail root. Saline (NaCl 0.9%) was used as a vehicle. Thevolume of administration was 5 mL/kg. All doses refer to therespective salt form as indicated above.
2.6. Statistical analysis
Group size was ten animals for all experiments. Data [antihyper-sensitive efficacy (maximal possible effect, % MPE) ipsilateral, andpaw withdrawal threshold (g) ipsi- and contralateral] were analyzedby means of two-factor analysis of variance (ANOVA) with repeatedmeasures. In case of a significant treatment effect, post hoc analysiswith Bonferroni adjustment was performed. Results were consideredstatistically significant if Pb0.05. ED50 values and 95% confidenceintervals (95% CI) were determined for antihypersensitive efficacy(% MPE) at the time of the peak effect for each drug, by regressionanalysis. Open field data [distance moved (m)] were analyzed byANOVA and post hoc analysis with Bonferroni adjustment. Theminimal effective dose was determined as the lowest dose whichresulted in a significant effect as compared to vehicle treatment andwas taken as an important indicator of a relevant effect size.
2.7. Dose-equivalence and isobolographic analysis
Analysis of the combination of two agonists proceeds from theconcept of dose-equivalence which follows from the dose effectcurves of the individual drugs (Tallarida, 2007; Tallarida and Raffa,2010). These curves allow a determination of equally effective dosesso that a dose a of drug A is equal to a dose of drug B which we denotebeq (a). Therefore dose combinations (a, b) acts like beq(a)+b, aquantity that is used in the dose effect equation of drug B to calculatethe expected effect. The expected effect is called “additive” because ofthe above addition and implies that there is no interaction. Thiscalculated additive effect is compared with the observed effect of the(a, b) combination that comes from experimentation. A significantdifference between the observed effect and the calculated additiveeffect indicates that an interaction has occurred. An interaction can beeither supra-additive (synergistic) or sub-additive. An alternate, butequivalent, way to assess a drug interaction uses a graphical method
74 T. Christoph et al. / European Journal of Pharmacology 666 (2011) 72–79
that constrains the sum beq(a)+b to a specified dose value such asthe ED50 of drug B. This constraint means that increasing values ofdose a lead to decreases in the dose b and therefore the plot of bagainst a, called an “isobole”, is a curve of negative slope in Cartesiancoordinates. When the parent dose-effect curves exhibit a constantpotency ratio (parallelism of log dose–effect lines) the isobole is astraight line segment with axial intercepts equal to the ED50 values(Tallarida, 2000; Loewe, 1953). Synergism is indicated by an observedpair (a, b) that plots below the isobole for the specified effect level,whereas sub-additivity is indicated when (a, b) plots above theisobole.
Tapentadol (Ipsilateral paw)
0 0.5 1.0 1.5 2.0 2.5 3.0
25
50
75
100
Sham vehicle
SNL vehicle
SNL 1
SNL 3.16
** **
*
SNL 10
* * SNL 0.316
*
(h)
(g)
Morphine (Ipsilateral paw)
0 0.5 1.0 1.5 2.0 2.5 3.00
25
50
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100
*
** Sham vehicle
SNL vehicle
SNL 1
SNL 2.15
SNL 4.64
(h)
(g)
Pregabalin (Ipsilateral paw)
0 0.5 1.0 1.5 2.0 2.5 3.0
25
50
75
100
Sham vehicleSNL vehicleSNL 1SNL 3.16
***
*
SNL 10
*
(h)
(g)
Oxycodon (Ipsilateral paw)
0 0.5 1.0 1.5 2.0 2.5 3.00
25
50
75
100
*
** Sham vehicle
SNL vehicle
SNL 0.316
SNL 1.0
SNL 3.16
*
(h)
(g)
A B
C D
E
G
Fig. 1. Effect of (A, B) tapentadol, (C, D) morphine, (E, F) oxycodone and (G, H) pregabalin (mthreshold in gram (mean±S.E.M.) measured with an electronic von Frey filament. * Pb0.0
3. Results
3.1. Effects on paw withdrawal thresholds
Sham operated animals received vehicle and showed withdrawalthresholds on both hind paws that were not different from thecontralateral withdrawal thresholds in vehicle treated neuropathicanimals (Figs. 1 and 2). Vehicle treated neuropathic animals showedclear hypersensitivity as indicated by comparison of ipsi- andcontralateral withdrawal thresholds (Figs. 1 and 2). During the testperiod of four weeks ipsi- and contralateral pawwithdrawal thresholds
Tapentadol (Contralateral paw)
0 0.5 1.0 1.5 2.0 2.5 3.0
25
50
75
100
Sham vehicle
SNL vehicle
SNL 1
SNL 3.16
SNL 10
SNL 0.316
*
(h)
(g)
Morphine (Contralateral paw)
0 0.5 1.0 1.5 2.0 2.5 3.00
25
50
75
100
*
*
*
Sham vehicle
SNL vehicle
SNL 1
SNL 2.15
SNL 4.64
(h)
(g)
Pregabalin (Contralateral paw)
0 0.5 1.0 1.5 2.0 2.5 3.0
25
50
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100
*
Sham vehicle
SNL vehicle
SNL 1
SNL 3.16
SNL 10
*
(h)
(g)
Oxycodon (Contralateral paw)
0 0.5 1.0 1.5 2.0 2.5 3.00
25
50
75
100 *
Sham vehicle
SNL vehicle
SNL 0.316
SNL 1.0
SNL 3.16
(h)
(g)
F
H
g/kg, IV) on ipsi- (left panel) and contralateral (right panel) hind paws on withdrawal5 versus vehicle.
Tapentadol + Pregabalin (Contralateral paw)
0 0.5 1.0 1.5 2.0 2.5 3.0
25
50
75
100
*Sham vehicleSNL vehicleSNL 0.1 T + 0.25 PSNL 0.3 T + 0.75 PSNL 1 T + 2.5 P
(h)
(g)
Morphine + Pregabalin (Contralateral paw)
0 0.5 1.0 1.5 2.0 2.5 3.0
25
50
75
100 **
*
Sham vehicle
SNL vehicle
SNL 3 M + 7.5 P
SNL 0.3 M + 0.75 P
SNL 1 M + 2.5 P
(h)(g
)
Oxycodon + Pregabalin (Contralateral paw)
0 0.5 1.0 1.5 2.0 2.5 3.0
25
50
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100
*
Sham vehicle
SNL vehicle
SNL 1.0 O + 6.5 P
SNL 0.1 O + 0.65 P
SNL 0.3 O + 1.95 P
(h)
(g)
B
D
F
Tapentadol + Pregabalin (Ipsilateral paw)
0 0.5 1.0 1.5 2.0 2.5 3.0
25
50
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100
Sham vehicle
SNL vehicle
SNL 0.1 T + 0.25 P
SNL 0.3 T + 0.75 P
***
SNL 1 T + 2.5 P
*
*
(h)
(g)
Morphine + Pregabalin (Ipsilateral paw)
0 0.5 1.0 1.5 2.0 2.5 3.0
25
50
75
100
Sham vehicle
SNL vehicle
SNL 0.3 M + 0.75 P**
*
SNL 1 M + 2.5 P
*
*
SNL 3 M + 7.5 P
*
(h)
(g)
Oxycodon + Pregabalin (Ipsilateral paw)
0 0.5 1.0 1.5 2.0 2.5 3.0
25
50
75
100
Sham vehicle
SNL vehicle
SNL 0.1 O + 0.65 P*
*
SNL 0.3 O + 1.95 P
*
*
SNL 1.0 O + 6.5 P
*
(h)
(g)
A
C
E
Fig. 2. Effect of equianalgesic dose combinations of (A, B) tapentadol (T), (C, D) morphine (M), (E, F) oxycodone (O) with pregabalin (P, mg/kg, IV) on ipsi- (left panel) andcontralateral (right panel) hind paws on withdrawal threshold in gram (mean±S.E.M.) measured with an electronic von Frey filament. * Pb0.05 versus vehicle.
75T. Christoph et al. / European Journal of Pharmacology 666 (2011) 72–79
were stable and did not show any time effect as determined based onthe baseline measures (P=0.661 ipsi; P=0.912 contra).
Tapentadol (0.316-10 mg/kg) showed a dose dependent increasein the withdrawal threshold of the ipsilateral hind paw (Fig. 1A). Thehighest dose tested showed full efficacy with 94% MPE. Potency wasquantified by an ED50-value (95% confidence interval) of 1.65 (1.20–2.35) mg/kg calculated from the peak effect versus control values at30 min after administration. Analysis of both ipsi- and contralateralhind paws revealed a specific antihypersensitive efficacy of tapenta-dol (Fig. 1A, B). 1 mg/kg tapentadol was the minimal effective dose(MED) which showed a significant effect on the ipsilateral pawwithdrawal threshold. At 10 mg/kg, ipsi- and contralateral pawwithdrawal thresholds were increased significantly. Thus, a specificantihypersensitive effect could be obtained at 1 and 3.16 mg/kg.[ANOVA ipsi: treatment F(5,54)=45.787, Pb0.001; time F(3,162)=90.044, Pb0.001; interaction F(15,162)=20.831, Pb0.001; ANOVAcontra: treatment F(5,54)=4.028, P=0.004; time F(3,162)=10.668,Pb0.001; interaction F(15,162)=5.518, Pb0.001)].
Morphine (1–4.64 mg/kg) showed a dose dependent increase in thewithdrawal threshold of the ipsilateral hind paw (Fig. 1C). The highestdose tested showed full efficacy with 96% MPE. Potency was quantifiedby an ED50-value (95% confidence interval) of 1.70 (1.53–1.87) mg/kg,calculated from the peak effect versus control values at 30 min afteradministration. Analysis of both ipsi- and contralateral hind paws did
not reveal a specific antihypersensitive efficacy ofmorphine (Fig. 1C, D).2.15 mg/kg morphine was the MED for both the ipsilateral andcontralateral paw withdrawal thresholds. Thus, no specific antihyper-sensitive effect could be obtained at any dose tested. [ANOVA ipsi:treatment F(3,36)=92.489, Pb0.0001; time F(3,108)=158.318,Pb0.0001; interaction F(9,108)=36.602, Pb0.0001; ANOVA contra:treatment F(3,36)=12.468, Pb0.0001; time F(3,108)=42.881,Pb0.0001; interaction F(9,108)=15.228, Pb0.0001)].
Oxycodone (0.316–3.16 mg/kg) showed a dose dependent increasein the withdrawal threshold of the ipsilateral hind paw (Fig. 1E). Thehighest dose tested showed full efficacy with 100% MPE. Potency wasquantified by an ED50-value (95% confidence interval) of 0.63 (0.47–0.81) mg/kg, calculated from the peak effect versus control values at30 min after administration. Analysis of both ipsi- and contralateral hindpaws revealed a specific antihypersensitive efficacy of oxycodone(Fig. 1E, F). A significant effect on the ipsilateral paw withdrawalthreshold was demonstrated at all doses tested, while a significantincrease in contralateral paw withdrawal threshold was seen onlyat 3.16 mg/kg. Thus, a specific antihypersensitive effect could beobtained at 0.316 and 1 mg/kg. [ANOVA ipsi: treatment F(3,36)=11.424, Pb0.0001; time F(3,108)=72.227, Pb0.0001; interaction F(9,108)=13.540, Pb0.0001; ANOVA contra: treatment F(3,36)=0.532, P=0.663; time F(3,108)=6.548, Pb0.0001; interaction F(9,108)=1.547, P=0.141)].
76 T. Christoph et al. / European Journal of Pharmacology 666 (2011) 72–79
Pregabalin (1–10 mg/kg) showed a dose dependent increase in thewithdrawal threshold of the ipsilateral hind paw (Fig. 1G). Thehighest dose tested showed efficacy of 67% MPE. Higher doses couldnot be tested due to side effects that interfered with the behavioralpain readout. Potency was quantified by an ED50-value (95%confidence interval) of 4.20 (3.37–5.43) mg/kg, calculated from thepeak effect versus control values at 30 min after administration.Analysis of both ipsi- and contralateral hind paws revealed a specificantihypersensitive efficacy of pregabalin (Fig. 1G, H). A significanteffect on the ipsilateral pawwithdrawal thresholdswas obtained at alldoses tested, while a significant increase in contralateral pawwithdrawal threshold was seen only at 10 mg/kg. Thus, a specificantihypersensitive effect (i.e. significant effect on the ipsi- but not onthe contralateral withdrawal threshold) could be obtained at 1 and3.16 mg/kg. [ANOVA ipsi: treatment F(3,36)=34.631, Pb0.0001;time F(3,108)=134.658, Pb0.0001; interaction F(9,108)=26.744,Pb0.0001; ANOVA contra: treatment F(3,36)=2.021, P=0.128;time F(3,108)=19.869, Pb0.0001; interaction F(9,108)=14.114,Pb0.0001)].
Tapentadol and pregabalin showed a potency difference whichamounts to a factor of 2.5, based on the ED50 values 30 min afteradministration. Combinations in a fixed ratio of 1:2.5 (tapentadol :pregabalin) were tested in doses of 0.1mg/kg+0.25 mg/kg, 0.3 mg/kg+0.75 mg/kg, 1 mg/kg+2.5 mg/kg tapentadol+pregabalin, re-spectively (Fig. 2A, B). These dose combinations showed a dosedependent increase in thewithdrawal threshold of the ipsilateral hindpaw. The highest dose combination tested showed full efficacy with89% MPE. Potency was quantified by an ED50 value (95% confidenceinterval) of 0.83 (0.74–0.92) mg/kg [0.24 (0.15–0.32) mg/kg tapenta-dol+0.59 (0.44–0.74) mg/kg pregabalin]; calculated from the peakeffect versus control values at 30 min after administration. Analysis ofboth ipsi- and contralateral hind paws revealed specific antihyper-sensitive efficacy of the drug combination. At 0.1+0.25 mg/kg,ipsilateral paw withdrawal thresholds were increased significantly,while contralateral paw withdrawal thresholds were decreasedslightly, although reaching statistical significance (P=0.027). Theother dose combinations were devoid of any effect on the contralat-eral hind paw. Thus, a specific antihypersensitive effect could beobtained for all dose combinations, including the dose combinationreaching full efficacy. [ANOVA ipsi: treatment F(3,36)=62.593,Pb0.0001; time F(3,108)=207.140, Pb0.0001; interaction F(9,108)=49.868, Pb0.0001; ANOVA contra: treatment F(3,36)=6.927,P=0.001; time F(3,108)=1.784, P=0.155; interaction F(9,108)=1.526, P=0.148)].
As in the case of tapentadol, morphine and pregabalin showed apotency difference which amounts to a factor of 2.5, based on the ED50
values30 min after administration. Combinations in afixed ratio of 1:2.5(morphine:pregabalin)were tested in doses of 0.3 mg/kg+0.75 mg/kg,
Table 1Summary of dose-equivalence calculations for the combinations of tapentadol, morphine aeffect in % MPE.
Combined dose Tapentadol Pregabalin
0.350 0.100 0.2501.05 0.300 0.7513.50 1.00 2.50
Combined dose Morphine Pregabalin
1.05 0.3 0.7503.5 1.00 2.5010.50 3.00 7.50
Combined dose Oxycodone Pregabalin
0.75 0.10 0.652.266 0.301 1.967.50 0.998 6.50
1 mg/kg+2.5 mg/kg, 3 mg/kg+7.5 mg/kg morphine+pregabalin, re-spectively (Fig. 2C, D). These dose combinations showed a dosedependent increase in the withdrawal threshold of the ipsilateral hindpaw. Thehighest dose combination tested showed full efficacywith 97%MPE. Potencywas quantifiedby anED50 value (95% confidence interval)of 2.33 (1.66–3.05) mg/kg [0.67 (0.60–0.73) mg/kg morphine+1.66(1.25–2.08) mg/kg pregabalin], calculated from the peak effect versuscontrol values at 30 min after administration. Analysis of both ipsi- andcontralateral hind paws did not indicate a specific antihypersensitiveeffect of the drug combination at any dose combination tested. [ANOVAipsi: treatment F(3,36)=28.039, Pb0.0001; time F(3,108)=53.349,Pb0.0001; interaction F(9,108)=15.776, Pb0.0001; ANOVA contra:treatment F(3,36)=16.984, Pb0.0001; time F(3,108)=24.1981,Pb0.0001; interaction F(9,108)=6.575, Pb0.0001)].
Oxycodoneandpregabalin showed apotency difference of factor 6.5,based on the ED50 values 30 min after administration. Combinations in afixed ratio of 1:6.5 (oxycodone:pregabalin) were tested in doses of0.1 mg/kg+0.65 mg/kg, 0.3 mg/kg+1.95 mg/kg, 1 mg/kg+6.5 mg/kgoxycodone+pregabalin, respectively (Fig. 2E, F). These dose combina-tions showed a dose dependent increase in thewithdrawal threshold ofthe ipsilateral hind paw. The highest dose combination tested showedfull efficacy with 100% MPE. Potency was quantified by an ED50 value(95% confidence interval) of 1.14 (0.72–1.56) mg/kg [0.15 (0.11–0.19)mg/kg oxycodone+0.99 (0.72–1.24) mg/kg pregabalin], calculatedfrom the peak effect versus control values at 30 min after administra-tion. Analysis of both ipsi- and contralateral hindpaws showed a specificantihypersensitive effect only at one dose combination tested (i.e.0.3 mg/kg oxycodone+1.95 mg/kg pregabalin). [ANOVA ipsi: treat-ment F(3,36)=30.689, Pb0.0001; time F(3,108)=89.739, Pb0.0001;interaction F(9,108)=19.703, Pb0.0001; ANOVA contra: treatmentF(3,36)=4.900, P=0.006; time F(3,108)=10.006, Pb0.0001; interac-tion F(9,108)=7.976, P=0.141)].
The combination data were analyzed by means of the dose-equivalence concept (Tallarida, 2007; Tallarida & Raffa, 2010) and thisanalysis revealed a significant synergistic interaction for the combi-nation of pregabalin with tapentadol but not with morphine oroxycodone (Table 1). Based on this result, the synergistic interactionof the combination of tapentadol with pregabalin is also seen on theisobologram (Fig. 3) which resulted in a linear isobole of additivitydue to the constant potency ratio (parallelism of log dose–effectregression lines). The experimental ED50 value (95% confidenceinterval) of 0.83 (0.74–0.92) mg/kg of the combination was belowthe theoretical additive ED50 value (95% confidence interval) of 2.91(2.28–3.54) mg/kg and was significantly different (Pb0.001) from theline of additivity. Thus, the interaction of tapentadol and pregabalincan be described as synergistic. Due to different regression slopes ofmorphine, oxycodone and pregabalin, isobolographic analysis was notsuitable for the combination of these drugs.
nd oxycodone with pregabalin. Dose and dose equivalents are expressed in mg/kg and
Fig. 3. Isobolographic analysis of the activity of tapentadol andpregabalin (mg/kg, IV), andof the interaction between a fixed combination of both compounds in spinal nerve ligatedrats. Calculations are based on %MPE values, 30 min after administration (mean±S.E.M.).The ED50 value on the line (gray symbol) indicates the theoretical additive ED50 value forthe 1:2.5 combination of both compounds (CI confidence interval).
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3.2. Effect on locomotion
Dose combinations which were effective in the neuropathic painmodel were tested in naive rats in the open field for the occurrence ofan effect on the distance moved (m) within the 5 min observationperiod.
The dose combinations of 0.3+0.75 and1+2.5 mg/kg tapentadol+pregabalin did not show any significant effect as compared tovehicle treatment. Mean (±S.E.M.) distance moved for vehicle, 0.3+0.75 and 1+2.5 mg/kg tapentadol+pregabalin was 13.73±0.85,12.36±0.68 (P=1.000 versus vehicle) and 12.29±0.60 m (P=1.000 versus vehicle), respectively. The dose combination of 3+7.5but not 1+2.5 mg/kg morphine+pregabalin showed a significanteffect, as compared to vehicle treatment, when tested in naïve rats inan open field paradigm. Mean (±S.E.M.) distance moved for vehicle,1+2.5 and 3+7.5 mg/kg morphine+pregabalin was 13.73±0.85,12.54±0.85 (P=1.000 versus vehicle) and 8.55±1.05 m (P=0.001versus vehicle), respectively.
The dose combination of 1+6.5 but not 0.3+1.95 mg/kg oxyco-done+pregabalin showed a significant effect as compared to vehicletreatment when tested in naïve rats in an open field paradigm. Mean(±S.E.M.) distance moved for vehicle, 0.3+1.95 and 1+6.5 mg/kgoxycodone+pregabalin was 16.79±0.84, 15.03±01.07 (P=0.670versus vehicle) and 12.66±0.92 m (P=0.021 versus vehicle),respectively.
4. Discussion
Pregabalin, tapentadol, morphine and oxycodone dose dependentlyincreased ipsilateral pawwithdrawal thresholds in spinal nerve-ligatedrats, a behavioral correlate for mechanical hypersensitivity in patientswith chronic neuropathic pain. Tapentadol, morphine and oxycodonewere superior to pregabalin both in terms of potency and efficacy.Equianalgesic combinations of pregabalin and tapentadol revealed asynergistic interaction while combination of pregabalin with morphineor oxycodone only resulted in an additive interaction. Differentmechanisms contribute to the analgesic effect of the tested drugs.Thus, morphine and oxycodone activate the μ-opioid receptor, whiletapentadol is both a μ-opioid receptor agonist and a noradrenalinereuptake inhibitior (Tzschentke et al., 2007). These two mechanismshave been shown to yield synergistic antiallodynia in this neuropathic
pain model, as demonstrated by combination experiments withmorphine and the α2-adrenergic agonist clonidine (Ossipov et al.,1997). Pregabalin binds to the α2δ subunit of voltage-gated Ca2+
channels and reduces the release of excitatory neurotransmitters, suchas glutamate, noradrenaline and substance P in the neocortex (Finket al., 2002; Dooley et al., 2000a; 2000b). Opioids inhibit GABAergicinterneurons in the rat periaqueductal gray, thereby disinhibitingdescending inhibitory projections and consequently increasing spinalnoradrenaline release (Fields et al., 1991; Osborne et al., 1996; Vaughanand Christie, 1997). This supraspinal effect, in combination with theactivation of pre- and postsynaptic spinal μ-opioid receptors, is likely tobe responsible for the supraspinal spinal synergy of opioids (Yeung andRudy, 1980), as well as the synergy between opioids and thenoradrenaline reuptake inhibitor desipramine (Ossipov et al., 1982) orthe α2-adrenoceptor agonist clonidine (Ossipov et al., 1990; Fairbanksand Wilcox, 1999). Likewise, the two mechanisms of action oftapentadol, i.e. μ-opioid receptor agonism and noradrenaline reuptakeinhibition in a single molecule (Tzschentke et al., 2006), were shown toresult in intrinsic synergistic analgesia both in an acute heat nociceptionmodel and a spinal nerve ligation model (Schröder et al., 2011). Whileopioidergic and noradrenergic mechanisms have been demonstrated tobe synergistic and this synergismcanbe explained by the functional andanatomical interaction between the opioid and the noradrenergicsystem, the addition of another unrelated mechanism (i.e. α2δ ligandbinding)might be expected, at best, to result in an additive rather than asynergistic interaction. Pregabalin has also been shown to modulatebrainstem-derived feedback loops such as descending serotonergicfacilitation (Bee and Dickenson, 2008), as well as descending norad-renergic inhibition, thereby leading to increased spinal levels ofnoradrenaline (Takeuchi et al., 2007), but a synergistic interaction ofpregabalin and opioids has not been demonstrated thus far. In a clinicalstudy it was found that neuropathic pain patients who were previouslydemonstrated to respond favorably to pregabalin treatment, showedimproved pain relief after combination therapy with pregabalin andoxycodone (Gatti et al., 2009). However, a recent study in patients withpostherpetic neuralgia and diabetic neuropathy suggested that acombination of oxycodone and pregabalin did not result in an additiveanalgesic effect (Zin et al., 2010). On the other hand, an opioid-sparingeffectwas shown for the combinationof the samedrugs in a randomizedcontrolled trial for pain after laparoscopic hysterectomy (Jokela et al.,2008). Hysterectomy is an acute pain condition and only a moderateopioid-sparing effect was to be expected due to the fact that pregabalinshows no clear antinociception in acute pain models (Moore, et al.,2009). Hence, while selective μ-opioid receptor agonists might not beable to increase the efficacy of pregabalin, a combination withtapentadol, because of its noradrenergic component as well as itsopioidergic component, might well be synergistic in clinical conditions,such as neuropathic pain, where plastic changes in receptors, ionchannels and transportes might occur. Indeed, all three above-mentioned mechanisms of action undergo differential regulationunder conditions of chronic neuropathic pain. Spinal nerve ligationresults in reduced expression of μ-opioid receptors in the spinal dorsalhorn, as well as in the ipsilateral dorsal root ganglion neurons (Porrecaet al., 1998; Kohno et al., 2005) and spinally administered morphineshows reduced efficacy and potency when compared with sham-operated animals (Ossipov et al., 1995). While immunohistochemistrystudies suggest decreased α2A-adrenoceptor and increased α2C-adrenoceptor expression (Stone et al., 1999), functional analysissuggests increased α2-adrenoceptor/G-protein coupling in the spinalcord of spinal nerve ligated rats (Bantel et al., 2005). Likewise,experiments with α2-adrenoceptor subtype-preferring antagonistssuggest a preferential role for α2C-adrenoceptors in the antihypersen-sitive efficacyof clonidine in the spinal nerve ligationmodel (Duflo et al.,2002). Finally, peripheral nerve injury results in increased descendinginhibitory noradrenergic innervation of the spinal cord (Ma andEisenach, 2003). This increased activity of the spinal adrenergic system
78 T. Christoph et al. / European Journal of Pharmacology 666 (2011) 72–79
might compensate for the functional down-regulation of μ-opioidreceptor in neuropathic pain conditions and allows a synergisticinteraction of drug combinations such as desipramine and morphine,or of drugs combining bothmechanisms such as tapentadol. Expressionof theα2δ subunit of voltage-gatedCa2+channels is increased followingspinal nerve ligation as evidenced by an increase of its mRNA in dorsalroot ganglia (Newton et al., 2001; Wang et al., 2002), as well as by anincrease of its protein levels in dorsal root ganglia and spinal cord dorsalhorn (Li et al., 2004). It has been suggested that impairment ofanterograde α2δ subunit trafficking might be a potential cellularmechanism of pregabalin in spinal nerve ligated rats (Bauer et al.,2009). Thus, nerve injury-induced differential expression and activity ofthe α2δ subunit, opioidergic and noradrenergic systems, together withpotential interactions of the Ca2+ channel-triggered increase indescending noradrenergic inhibitory tone, might form a basis for theobserved synergistic interaction of pregabalin and tapentadol in thepresent study.
Tapentadol, morphine, oxycodone and pregabalin, when testedalone, showed significant contralateral effects at high doses. At thesedoses, tapentadol, morphine and oxycodone show antinociceptiveeffects in various animal models (Tzschentke et al., 2006; B. Kögel,Personal communication). Pregabalin has no antinociceptive effects inacute pain models, such as the first phase of the formalin test (Fieldet al., 1997), or models of mechanical antinociception — an effect wasonly obtained at a high dose that also produced locomotorimpairment (Yokoyama et al., 2007). Thus, the increase in contralat-eral paw withdrawal detected in the current study for pregabalinmight reflect confounding locomotor side effects, rather thanantinociception. Likewise, the contralateral effects found for thecombination of pregabalin and either morphine or oxycodone, but notwith tapentadol, suggest that classical opioids in combination withpregabalin have an increased propensity for side effects, as indicatedby a reduction in locomotor activity seen in the open field.
In conclusion, when either drug was tested alone, tapentadol,morphine and oxycodone showed superior efficacy compared topregabalin in the spinal nerve ligation rat model of mononeuropathicpain. A combination of pregabalin and tapentadol resulted in asynergistic antihypersensitive interaction, while morphine and oxyco-done showed only an additive interaction with pregabalin, and theincreased potency coincidedwith a concomitant increase in side effects.Combined involvement of three different mechanisms, i.e. μ-opioidreceptor agonism and noradrenaline reuptake inhibition by tapentadoland α2δ subunit modulation by pregabalin, is suggested to be themolecular basis of the observed synergistic interaction. Thus, beside thetherapeutic potential of tapentadol or pregabalin when administeredalone, a combination of both drugs may offer a beneficial treatmentoption for neuropathic pain.
Acknowledgments
The authors would like to thank Simone Wigge, Reinhard Lerchand Hans-Josef Weber for excellent technical assistance.
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