Pain Medicine: Basic Science
Antihyperalgesic/Antinociceptive Effects of Ceftriaxone and Its Synergistic Interactions with Different Analgesics in Inflammatory Pain in Rodents
Stepanovic`-Petrovic`, Radica M. Ph.D.; Micov, Ana M. B.Pharm.; Tomic`, Maja A. Ph.D.; Kovacevic`, Jovana M. B.Pharm.ˇ; Boškovic`, Bogdan D. Ph.D.
Background: The β-lactam antibiotic ceftriaxone stimulates glutamate transporter GLT-1 expression and is effective in neuropathic and visceral pain models. This study examined the effects of ceftriaxone and its interactions with different analgesics (ibuprofen, celecoxib, paracetamol, and levetiracetam) in somatic and visceral pain models in rodents.
Methods: The effects of ceftriaxone (intraperitoneally/intraplantarly), analgesics (orally), and their combinations were examined in the carrageenan-induced paw inflammatory hyperalgesia model in rats (n = 6–12) and in the acetic acid-induced writhing test in mice (n = 6–10). The type of interaction between ceftriaxone and analgesics was determined by isobolographic analysis.
Results: Pretreatment with intraperitoneally administered ceftriaxone (10–200 mg/kg per day) for 7 days produced a significant dose-dependent antihyperalgesia in the somatic inflammatory model. Acute administration of ceftriaxone, via either intraperitoneal (10–200 mg/kg) or intraplantar (0.05–0.2 mg per paw) routes, produced a significant and dose-dependent but less efficacious antihyperalgesia. In the visceral pain model, significant dose-dependent antinociception of ceftriaxone (25–200 mg/kg per day) was observed only after the 7-day pretreatment. Isobolographic analysis in the inflammatory hyperalgesia model revealed approximately 10-fold reduction of doses of both drugs in all examined combinations. In the visceral nociception model, more than 7- and 17-fold reduction of doses of both drugs was observed in combinations of ceftriaxone with ibuprofen/paracetamol and celecoxib/levetiracetam, respectively.
Conclusions: Ceftriaxone exerts antihyperalgesia/antinociception in both somatic and visceral inflammatory pain. Its efficacy is higher after a 7-day pretreatment than after acute administration. The two-drug combinations of ceftriaxone and the nonsteroidal analgesics/levetiracetam have synergistic interactions in both pain models. These results suggest that ceftriaxone, particularly in combinations with ibuprofen, celecoxib, paracetamol, or levetiracetam, may provide useful approach to the clinical treatment of inflammation-related pain.
What We Already Know about This Topic
* Ceftriaxone is a third-generation cephalosporin antibiotic capable of increasing the expression of the GLT-1 glutamate transporter. This action may underlie ceftriaxone's analgesic properties.
What This Article Tells Us That Is New
* Pretreatment of rodents with ceftriaxone provided antinociceptive effects in inflammatory pain models.
* By using inflammatory somatic and visceral pain models, synergistic interactions of ceftriaxone and several commonly used analgesics were demonstrated.
IN inflammatory somatic and visceral pain, tissue damage or inflammation sensitizes peripheral afferents and dorsal horn neurons.1
Synaptic input from primary afferents onto second-order neurons in the dorsal horn is excitatory. This excitation is mostly mediated by the release of glutamate, which is the major excitatory neurotransmitter in the central nervous system.2
There are several reports that glutamate elicits hyperalgesia via
the direct excitation of peripheral afferent fiber terminals.4
Because glutamate is critical for neuroplasticity in nociceptive network in inflammatory pain, there have been considerable efforts to develop therapeutic approaches that suppress glutamatergic activity. There is no enzyme that metabolizes the glutamate in extracellular space, but there is high-affinity, efficient, high-capacity glutamate transporter system within the central and peripheral nervous systems that removes glutamate from the extracellular space. The glutamate transporter GLT-1 is distributed primarily in astrocytes and plays a crucial role in glutamate clearance in the synaptic cleft in the central nervous system,6–8
as well as in satellite and Schwann cells in the peripheral nervous system.9
A novel strategy to decrease synaptic glutamate by upregulating the glutamate transporter GLT-1 might be effective in mitigating inflammatory pain.10
Evidence suggests that the β-lactam antibiotic, ceftriaxone, is a potent stimulator of GLT-1 expression10
and that it can attenuate neuropathic11
and visceral pain.13
There are no data about its efficacy in inflammatory somatic pain.
As there is evidence that inflammatory somatic as well as visceral pain processes are associated not only with glutamate but also with prostaglandins and some other released mediators, combination therapy is of value in this context.1
Moreover, when combinations of suitable drugs are used, submaximal doses can be applied, with fewer adverse effects and greater efficacy.17
Nonsteroidal antiinflammatory drugs and paracetamol are useful in the management of both somatic and visceral pain. The analgesic effect of nonsteroidal antiinflammatory drugs has traditionally been related to the reduction of prostaglandin synthesis, by inhibition of cyclooxygenases (COX)-1/2. The mechanism of analgesic effect of paracetamol is still a matter of debate. Recent evidence suggests that the reduction of prostaglandin synthesis caused by the inhibition of COX-2 could be the main mechanism of its action.19
Our research group has shown that levetiracetam, a novel antiepileptic drug, induces antihyperalgesia in the somatic inflammatory model of pain via
adrenergic, opioidergic, 5-hydroxytryptaminergic, γ-aminobutyric acid (GABA)ergic, and adenosine receptors.21
It seems that levetiracetam is favorable for coadministration with other drugs because unlike other antiepileptics, it does not induce and is not a high-affinity substrate for cytochrome P-450 isoforms or glucuronidation enzymes and thus it is devoid of pharmacokinetic (metabolic) interactions with other drugs.23
The current study was undertaken to determine the efficacy of ceftriaxone, as well as the types of interactions (additivity, synergism, or antagonism) of ceftriaxone with different analgesics (ibuprofen, celecoxib, paracetamol, and levetiracetam) in somatic and visceral inflammatory pain models in rodents.
Materials and Methods
All experiments were approved by the Institutional Animal Care and Use Committee of the Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia, and were carried out in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.24
Experiments were performed on male Wistar rats (weighing 180–220 g) and Swiss Webster mice (weighing 25–30 g) obtained from the Military Medical Academy Breeding Farm, Belgrade, Serbia. The animals were housed under a 12 h light/dark cycle, with food and water available ad libitum
. For all measurements, the experimenter was blinded to the treatment group.
Drugs and Their Administration
Ceftriaxone (Longaceph; Galenika, Belgrade, Serbia) was dissolved in saline. Ibuprofen (Pharmagen GmbH, Frankfurt, Germany), celecoxib (Celebrex; Pfizer Manufacturing Deutschland GmbH, Illertissen, Germany), paracetamol (Panadol; GlaxoSmithKline Dungarvan Ltd., Dungarvan, Ireland), and levetiracetam (Keppra; UCB Pharma AG, Bruselles, Belgium) were suspended in distilled water.
Two distinct protocols were used for ceftriaxone administration. In the pretreatment protocol, ceftriaxone was intraperitoneally administered once daily for 7 consecutive days, and the experiments were performed on day 8 (starting 24 h after the last ceftriaxone injection). In the acute treatment protocol, ceftriaxone was administered either via intraperitoneal or intraplantar routes only once, at the time of performing experiments. Analgesics were administered by oral gavage and always as an acute treatment. The volumes of the intraperitoneal/oral drugs administration were 2 ml/kg body weight for rats and 10 ml/kg body weight for mice.
Carrageenan λ (Sigma-Aldrich Chemie GmbH, Munich, Germany) was dispersed in saline (1% m/v) and injected intraplantarly. Intraplantar injection of ceftriaxone was either coadministered with the carrageenan into the rat right hind paw or administered alone into the contralateral hind paw. All intraplantar treatments were administered in a final volume of 0.1 ml per paw using a 1 ml syringe with a 26-gauge needle. Diluted acetic acid (0.75%; Zorka Pharma, Šabac, Serbia) was injected intraperitoneally into mice in a volume of 10 ml/kg body weight.
Model of Somatic Inflammatory Hyperalgesia
The difference between the forces (df, expressed in grams) that were applied to the left, healthy, and right, inflamed (carrageenan-injected) hind paws was measured with an analgesimeter (Hugo Sachs Elektronik, March-Hugstetten, Germany) as previously described.21
The rat was placed with its hind paws on two transducer platforms and gently pushed downward by the investigator’s hand so that the force (pressure) was applied simultaneously to both paws until one of the paws exceeded the trigger of 100 g. This pressure represents a mild nociceptive stimulus that is required to detect nociceptive hypersensitivity (hyperalgesia).
Basal df was obtained before the induction of inflammation and administration of the drugs. The time points when the drugs were administered were chosen according to data about the time course of carrageenan hyperalgesia28
and the time course of the antihyperalgesic effects and/or pharmacokinetic properties of the examined drugs.11
Ceftriaxone was administered in both pretreatment and acute treatment protocols. The analgesics were administered only as an acute treatment. In the acute treatment protocols, ceftriaxone was either administered intraperitoneally 1 h after carrageenan or coadministered intraplantarly with carrageenan. To exclude a potential systemic effect of intraplantarly administered ceftriaxone, the highest tested dose of the drug was given contralaterally (into the left hind paw) to a separate group of rats. Ibuprofen and celecoxib were administered at the same time as carrageenan. Paracetamol and levetiracetam were administered 1 h after carrageenan. Posttreatment df values were measured at six time points during a 300 min period after the induction of inflammation.
Model of Visceral Nociception
The writhing test described previously26
was used. Mice were injected intraperitoneally with acetic acid solution. The number of writhes (N) was counted during a 15 min period, starting 5 min after the administration of the acetic acid. Ceftriaxone was administered in both pretreatment and acute treatment protocols. The analgesics were administered only as an acute treatment. In the acute treatment protocols, ceftriaxone and analgesics were administered 25 and 55 min before the acetic acid administration, respectively.
Calculations in the Hyperalgesia/Nociception Models
To calculate the effective dose that produces 50% of the antihyperalgesic/antinociceptive effect (ED50) of each drug, the df or N values were converted to percentages of antihyperalgesic (%AH) or antinociceptive (%AN) activities, according to the following formulas:
Equation (Uncited)Image Tools
Equation (Uncited)Image Tools
values were estimated from corresponding log dose–response curves by linear regression.31
Analysis of Interactions between Ceftriaxone and Analgesics
Interactions between ceftriaxone and the analgesics were evaluated by isobolographic analysis at the ED50
level of the effect as described previously.26
In the experiments in which the drugs were combined, the two drugs were administered at fixed-dose fractions of their respective ED50
. Ceftriaxone was applied as a pretreatment and the analgesic as an acute treatment. The experimental ED50
for the drug mixture (ED50 mix
) was calculated from the corresponding log dose–response curve. When the drug combination produced an ED50 mix
that was significantly lower than the calculated theoretical ED50
), it was interpreted as there is a supraadditive (synergistic) interaction between the drugs.31
In addition, an interaction index was used to describe the magnitude of the interaction.26
Analysis of Duration of the Effect of Drugs/Drug Combinations in a Model of Somatic Inflammatory Hyperalgesia
To compare the duration of the effect of the drug when applied alone with the duration of the effect produced by the same drug when applied in combination, the slopes of the %AH–ΔAUC regression lines were calculated as previously described.26
In brief, the areas under the time–df curves (AUC) were calculated for the control (AUCC
) and the drug/drug combination (AUCD
) groups, and the differences were calculated as ΔAUC = AUCC
The ΔAUC for each drug/drug combination group is presented as a function of its peak %AH (ΔAUC = slope × %AH + intercept). In this regression line, the slope is the relative measure of the duration of the drug/drug combination effect; the treatment with a significantly greater slope has a longer effect than the treatment with a lesser slope.26
A high correlation coefficient of the %AH–ΔAUC regression line indicates that the duration of the effect of the drug/drug combination treatment is dose dependent.26
Rotarod performance was assessed to evaluate the effects of ceftriaxone and levetiracetam on motor coordination or sedation.35
The test was performed using a rotarod apparatus (Treadmill for rats 47700 or Treadmill for mice 7600; Ugo Basile, Milano, Italy), consisting of a rod rotating at a constant speed of 15 rpm.30
The animals were trained to drive the rotarod four times a day for 2 days. Only those animals that could stay on the rod for 180 s on two consecutive trials were used in the experiments. Ceftriaxone was administered in both pretreatment and acute treatment protocols, and levetiracetam was administered only as an acute treatment. In the acute treatment protocols, ceftriaxone and levetiracetam were administered 30 or 60 min before the testing, respectively. The posttreatment latency to remain on the rotating rod (with a 180 s cutoff) was recorded at four time points, during 240 min.
All pharmacological computations were performed using Pharm PCS (Micro-Computer Specialists, Philadelphia, PA) and Pharm Tools Pro (The McCary Group, Schnecksville, PA). The statistical analysis was conducted using SigmaPlot 11 (Systat Software Inc., Richmond, CA). The data were normally distributed (Shapiro–Wilk test) and are presented as the means ± SEM. Time-course data in the inflammatory hyperalgesia model were analyzed using a two-way repeated-measures ANOVA, followed by Tukey honestly significant difference test for between-group comparisons and comparisons at each time point. Two-way ANOVA was used to compare the peak effects of each ceftriaxone dose that was achieved after the pretreatment and the acute treatment. Data from the visceral nociception model were analyzed by one-way ANOVA. Tukey honestly significant difference test was used for post hoc
comparisons. Data from the rotarod test were analyzed by independent sample Student t
test. The differences between ED50 mix
and ED50 add
were examined by modified t
The slopes of the %AH–ΔAUC regression lines were compared by the test for parallelism.33
value less than 0.05 was considered statistically significant.
Effects of Ceftriaxone on Somatic Inflammatory Hyperalgesia
Seven-day pretreatment with intraperitoneally administered ceftriaxone (10–200 mg/kg per day) produced a dose-dependent reduction of hyperalgesia (P
< 0.001 by two-way repeated-measures ANOVA; fig. 1A
). The maximal effects were observed 120–240 min after the induction of inflammation. The corresponding ED50
± SEM calculated from the peak effect values was 31.96 ± 12.02 mg/kg per day (table 1
Acute treatment with intraperitoneally administered ceftriaxone (10–200 mg/kg) also produced a dose-dependent antihyperalgesia (P
< 0.001 by two-way repeated-measures ANOVA; fig. 1B
), which peaked 300 min after the induction of inflammation (240 min after ceftriaxone administration). The peak effect produced with each tested dose of ceftriaxone in the acute treatment was significantly lower than the peak effect of the same dose produced after the 7-day pretreatment (P
< 0.001 by two-way ANOVA; fig. 1C
Acute treatment with intraplantar ceftriaxone (0.05–0.2 mg per paw) reduced hyperalgesia in a dose-dependent manner (P
< 0.001 by two-way repeated-measures ANOVA; fig. 2A
). The effect of ceftriaxone was local because it was not observed after the injection of the highest dose into the contralateral paw (P
> 0.05 by Tukey honestly significant difference post hoc
test; fig. 2A
). The peak antihyperalgesic effects of the intraplantarly administered ceftriaxone (fig. 2B
) occurred 180–300 min after administration.
Effects of Ceftriaxone on Visceral Nociception
Intraperitoneal pretreatment with ceftriaxone (25–200 mg/kg per day) for 7 days produced a dose-dependent antinociception, as observed in the writhing test (P
< 0.001 by one-way ANOVA; fig. 3A
). The corresponding ED50
± SEM was 69.32 ± 2.76 mg/kg per day (table 2
Acute treatment with intraperitoneally administered ceftriaxone (25–200 mg/kg) did not produce significant antinociception (P
= 0.089 by one-way ANOVA; fig. 3B
) although a tendency for a reduction of the number of writhes in a dose-dependent manner could be observed. When the effects of each acutely administered dose of ceftriaxone were compared with the effects of the same dose when applied as a 7-day pretreatment, it was significantly less for 50, 100, and 200 mg/kg (P
< 0.001 by two-way ANOVA; fig. 3C
Interactions between Ceftriaxone and Analgesics in a Model of Somatic Inflammatory Hyperalgesia
Orally administered ibuprofen (12.5–100 mg/kg), celecoxib (3.75–30 mg/kg), paracetamol (50–200 mg/kg), and levetiracetam (10–200 mg/kg) caused dose-dependent antihyperalgesia (P
< 0.001 by two-way repeated-measures ANOVA, not shown). Their ED50
values were calculated from the corresponding log dose–response curves (fig. 4
) and are summarized in table 1
Two-drug combinations of intraperitoneally administered ceftriaxone (7-day pretreatment) with oral analgesics also caused a dose-dependent reduction of hyperalgesia (P
< 0.001 by two-way repeated-measures ANOVA; fig. 5
). For all of the examined combinations, the ED50 mix
was significantly lower than the ED50 add
< 0.05 by t
test) and the interaction index was less than 1 which indicates a synergistic interaction (fig. 6
and table 1
). According to the interaction index values, at all of the examined combinations, there was almost the same degree of potentiation with approximately 10-fold reduction of doses of both drugs when the drugs were applied in combination compared with the doses that produced the same level of effect after individual administration.
Analysis of the Duration of the Effects of Ceftriaxone, Analgesics, and Ceftriaxone–Analgesic Combinations in a Model of Somatic Inflammatory Hyperalgesia
Slope of the %AH–ΔAUC regression line for ceftriaxone (7-day pretreatment) was greater than the slope for each analgesic (P
< 0.05 by test for parallelism; table 3
). This result indicates that the effects produced by individually administered ceftriaxone lasted longer than the effects produced by individually administered analgesics. The slopes for all two-drug combinations of ceftriaxone were not different from the slope for ceftriaxone per se
> 0.05 by test for parallelism; table 3
). This indicates that the duration of the effects of ceftriaxone was unchanged when it was applied alone and when it was applied with analgesics. However, the slopes for ceftriaxone–ibuprofen and ceftriaxone–celecoxib combinations were greater than the slopes for the corresponding analgesics when applied individually (P
< 0.05 by test for parallelism; table 3
). This indicates that the actions of ibuprofen and celecoxib were longer when combined with ceftriaxone. The high correlation coefficients of the %AH–ΔAUC line for all of the treatments indicate that the durations of the effects were dose dependent (table 3
Interactions between Ceftriaxone and Analgesics in a Visceral Nociception Model
Oral ibuprofen (12.5–100 mg/kg), celecoxib (3.75–30 mg/kg), paracetamol (5–150 mg/kg), and levetiracetam (1–25 mg/kg) caused a dose-dependent reduction of writhes induced by acetic acid (P
< 0.001 by one-way ANOVA, not shown). The corresponding log dose–response curves are shown in fig. 7
, and the ED50
values are summarized in table 2
Two-drug combinations of intraperitoneally administered ceftriaxone (7-day pretreatment) with oral analgesics also reduced nociception in a dose-dependent manner (P
< 0.001 by one-way ANOVA; fig. 8
). Isobolographic analysis revealed synergistic interactions for all of the examined combinations (table 2
and fig. 9
). Interaction index values indicated a different degree of potentiation with the following rank: ceftriaxone–celecoxib = ceftriaxone–levetiracetam > ceftriaxone–ibuprofen = ceftriaxone–paracetamol. A more than 17-fold reduction of doses of both drugs in the ceftriaxone–celecoxib and ceftriaxone–levetiracetam combinations and a more than seven-fold reduction of doses in the ceftriaxone–ibuprofen and ceftriaxone–paracetamol combinations are observed when compared with the corresponding doses after individual administration.
Effects of Ceftriaxone and Levetiracetam in a Rotarod Test
Intraperitoneally administered ceftriaxone either after 7-day pretreatment (200 mg/kg per day) or an acute treatment (200 mg/kg) did not influence the rotarod performance in both rats and mice (P > 0.05 by Student t test, not shown). Oral levetiracetam (200 mg/kg) was also without significant effect in both species (P > 0.05 by Student t test, not shown).
Antihyperalgesic/Antinociceptive Effects of Ceftriaxone in Somatic and Visceral Inflammatory Pain Models
The current study shows that the administration of ceftriaxone for 7 days caused a significant and dose-dependent reduction of carrageenan-induced mechanical hyperalgesia in rats and acetic acid-induced nociception in mice. Our results widen the findings by Lin et al.
Hu et al.
and Yang et al.38
who observed that ceftriaxone, in preventive 7-day administration, is effective not only in chronic neuropathic and visceral pain but also in somatic inflammatory pain. The animal model of carrageenan-induced hyperalgesia, as a model of somatic pain, mimics clinical inflammatory conditions.39
Our study shows for the first time that the acute treatment with ceftriaxone in the same dose range produces a significant and dose-dependent antihyperalgesic effect which is significantly lower than that produced by 7-day treatments with all doses. As ceftriaxone did not produce a significant impairment of motor performance in the rotarod test in rats and mice at the highest used doses, it would appear that the antihyperalgesic/antinociceptive effects of ceftriaxone were not due to motor impairment or sedation. The current study also demonstrates a modest efficacy of ceftriaxone as a local peripheral antihyperalgesic drug in the carrageenan-induced pain inflammatory model. There is no literature data regarding ceftriaxone’s local peripheral antinociceptive efficacy. The local nature of this action was verified by the absence of effect of ceftriaxone after it was injected into the contralateral hind paw. A significant local effect of ceftriaxone (0.05–0.2 mg per paw) was obtained with much lower doses (up to 40 times lower) than with the lowest effective systemic dose (10 mg/kg). This finding suggests that when ceftriaxone is given systemically (either as an acute dose or a 7-day pretreatment), it achieves effective concentrations at the periphery. It also points to the contribution of a peripheral antihyperalgesic effect to the net effect of systemically administered ceftriaxone.
Peripheral inflammation induced by carrageenan causes glutamate release from primary sensory afferents and excitatory interneurons in the dorsal horn of the spinal cord, producing inflammatory hyperalgesia through the activation of peripheral and spinal glutamate receptors.1
Ceftriaxone can selectively upregulate the expression of glutamate transporter GLT-1 and accordingly reduce intrasynaptic glutamate.10
GLT-1 is considered to be the glutamate transporter in the central8
nervous systems. Because ceftriaxone produced analgesic activity in the paw pressure test after systemic and local peripheral application, it could be suggested that the reduction of intrasynaptic glutamate in the spinal cord and peripheral sensory terminals would contribute to the antihyperalgesic effect of ceftriaxone in somatic inflammatory pain.
Both peripheral and central mechanisms contribute to visceral hypersensitivity in the writhing test in mice. It seems that visceral hyperalgesia is due to lowering the threshold of “high threshold” receptors and activation of previously unresponsive receptors, and subsequent neuroplastic changes in terms of increased release of the excitatory glutamate, principally in the spinal cord.44
Therefore, the antinociceptive effect of ceftriaxone in the writhing test could be explained by enhanced glutamate uptake primarily in the spinal cord.
Moreover, it has been shown that the hyperalgesic/nociceptive activity of carrageenan/acetic acid in the paw pressure/writhing test is due to the release of proinflammatory cytokines, tumor necrosis factor-α, and interleukins from immune and glial cells.45
Amin et al.17
observed that a 1-week treatment with ceftriaxone significantly attenuates these cytokines in the spinal cord. Several previous studies have described the synergistic interaction of inflammatory cytokines and glutamate in neuronal sensitization.17
Therefore, ceftriaxone might produce antihyperalgesic/antinociceptive effects after 7 days of systemic administration at least by decreasing cytokine levels. This could also explain the observed antihyperalgesia after acute systemic administration of ceftriaxone. However, the synergistic interactions of inflammatory cytokines and glutamate and the observation that ceftriaxone reduces intrasynaptic glutamate through increased transcription of the GLT-1 gene10
(hours and days are needed for its full effect) could explain the significantly lower antihyperalgesia/antinociception after an acute systemic administration of ceftriaxone than that after its systemic administration for 7 days in both models.
There is evidence that both somatic and visceral components, as well as inflammation, contribute to many kinds of postoperative pain.47
The available data suggest that ceftriaxone could also work as an adjunctive treatment in certain types of postoperative pain because ceftriaxone is often given for perioperative prophylaxis (acute administration) and treatment (7-day administration) of postoperative infections. Unless the metabolism of ceftriaxone differs in rodents and humans, the lowest and one above the lowest dose of intraperitoneally administered ceftriaxone that produced significant antihyperalgesic effects after both acute and 7-day administration as well as the lowest dose that produced a significant antinociceptive effect after 7 day administration are comparable with the doses used in surgical prophylaxis (1–2 g/day, intramuscular/intravenous) and in severe infections (2–4 g/day, intramuscular/intravenous) in humans.
Effects of Two-drug Combinations of Ceftriaxone and Different Analgesics in Somatic and Visceral Inflammatory Pain
Our results reveal that ceftriaxone exerts a synergistic interaction with ibuprofen, celecoxib, paracetamol, or levetiracetam in reducing carrageenan-induced mechanical hyperalgesia in rats and acetic acid-induced writhing in mice. Synergistic pharmacodynamic interactions could be ascribed to the activation of different complementary pathways of the observed actions.49
The inflammation and injury induced by carrageenan and acetic acid are associated not only with release of glutamate and cytokines (tumor necrosis factor-α and interleukins) but also with local release of proinflammatory prostaglandins, particularly E series, resulting in activation and sensitization of somatic/visceral peripheral nociceptive afferents.1
The hyperactivity and sensitization of afferent fibers stimulate the release of glutamate, cytokines, and prostaglandins in the spinal cord, contributing to the central sensitization in dorsal horn neurons.1
Ibuprofen, a nonselective COX-1/2 inhibitor, and celecoxib, a selective COX-2 inhibitor, exert their antihyperalgesic/antinociceptive actions by blocking prostaglandin synthesis at peripheral and spinal sites.51
A satisfactory mechanism of action of paracetamol still remains to be established. The analgesic effect of paracetamol could be ascribed to its inhibitory action on peripheral and central COX-2,19
as well to its activation of descending opioid and serotonergic pathways.56
Our research group has shown in the somatic inflammatory pain model that levetiracetam produces an antihyperalgesic effect. This effect is at least in part mediated by central γ-aminobutyric acid type A receptors, peripheral adenosine, and both central and peripheral opioidergic, 5-hydroxytryptaminergic, and α2
Although we have shown for the first time that levetiracetam is effective in the visceral pain model, our results do not clarify the mechanism of the antinociceptive effect of levetiracetam in this model. However, it could be suggested that the inflammatory nature of nociception in writhing test and carrageenan-induced hyperalgesia in paw pressure test leading to a facilitated state may be similar. Enhanced glutamate uptake and attenuated cytokine levels, at least at spinal sites, contribute to the antihyperalgesia/antinociception by ceftriaxone in inflammatory pain models in rodents.11
Therefore, synergistic interactions between ceftriaxone and ibuprofen, celecoxib, paracetamol, or levetiracetam could be explained by the involvement of multiple different targets and sites in their antihyperalgesic/antinociceptive effects in these inflammatory pain models.
The pharmacokinetic interactions between ceftriaxone and the examined analgesics were not within the scope of our work. However, they cannot be excluded in the combinations of ceftriaxone with ibuprofen and celecoxib that displayed prolonged actions. A prolongation of the effect of drug combination is likely to be expected in the case of pharmacokinetic interaction which would result in potentiation of pharmacological effects. Thus, it seems that there could be more than one possible mechanism which could explain the synergism observed between ceftriaxone and ibuprofen/celecoxib. This prolongation of the drug’s effects, in addition to their potentiation, could be beneficial in the potential clinical use of these combinations. A pharmacokinetic interaction between ceftriaxone and paracetamol/levetiracetam appears to be less likely (unchanged duration of effect).
The occurrence of side effects with all of these combinations is less likely because the doses of the individual components are markedly lower. Also, because the components have different side effects, the addition/potentiation of their individual effects is not expected.
In conclusion, the major findings of our study are (1) ceftriaxone exerts antihyperalgesia/antinociception in both somatic and visceral inflammatory pain, with higher efficacy after 7-day than after acute administration; (2) ceftriaxone exerts synergistic interactions with different analgesics (ibuprofen, celecoxib, paracetamol, levetiracetam) and is superior to monotherapy for both somatic and visceral inflammatory pain. Our results suggest that ceftriaxone, particularly in combination with certain analgesics, may provide a useful approach to the clinical treatment of inflammation-related pain.
The authors thank Ronald Tallarida, Ph.D. (Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania), for his helpful advice concerning the experimental design and analysis of the type of interaction between the drugs that were applied in combination.
This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Belgrade, Serbia), Grant no. 175045.
The authors declare no competing interests.
1. Millan MJ. The induction of pain: An integrative review. Prog Neurobiol. 1999;57:1–164
2. Salter MW. Cellular signalling pathways of spinal pain neuroplasticity as targets for analgesic development. Curr Top Med Chem. 2005;5:557–67
3. Woolf CJ, Salter MW. Neuronal plasticity: Increasing the gain in pain. Science. 2000;288:1765–9
4. Carlton SM, Hargett GL, Coggeshall RE. Localization and activation of glutamate receptors in unmyelinated axons of rat glabrous skin. Neurosci Lett. 1995;197:25–8
5. Wang H, Liu RJ, Zhang RX, Qiao JT. Peripheral NMDA receptors contribute to activation of nociceptors: A c-fos expression study in rats. Neurosci Lett. 1997;221:101–4
6. Beart PM, O’Shea RD. Transporters for. L. -glutamate: An update on their molecular pharmacology and pathological involvement. Br J Pharmacol. 2007;150:5–17
7. Robinson MB. The family of sodium-dependent glutamate transporters: A focus on the GLT-1/EAAT2 subtype. Neurochem Int. 1998;33:479–91
8. Rothstein JD, Dykes-Hoberg M, Pardo CA, Bristol LA, Jin L, Kuncl RW, Kanai Y, Hediger MA, Wang Y, Schielke JP, Welty DF. Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron. 1996;16:675–86
9. Carozzi VA, Canta A, Oggioni N, Ceresa C, Marmiroli P, Konvalinka J, Zoia C, Bossi M, Ferrarese C, Tredici G, Cavaletti G. Expression and distribution of ‘high affinity’ glutamate transporters GLT1, GLAST, EAAC1 and of GCPII in the rat peripheral nervous system. J Anat. 2008;213:539–46
10. Rothstein JD, Patel S, Regan MR, Haenggeli C, Huang YH, Bergles DE, Jin L, Dykes Hoberg M, Vidensky S, Chung DS, Toan SV, Bruijn LI, Su ZZ, Gupta P, Fisher PB. Beta-lactam antibiotics offer neuroprotection by increasing glutamate transporter expression. Nature. 2005;433:73–7
11. Hu Y, Li W, Lu L, Cai J, Xian X, Zhang M, Li Q, Li L. An anti-nociceptive role for ceftriaxone in chronic neuropathic pain in rats. Pain. 2010;148:284–301
12. Gunduz O, Oltulu C, Buldum D, Guven R, Ulugol A. Anti-allodynic and anti-hyperalgesic effects of ceftriaxone in streptozocin-induced diabetic rats. Neurosci Lett. 2011;491:23–5
13. Lin Y, Roman K, Foust KD, Kaspar BK, Bailey MT, Stephens RL. Glutamate transporter GLT-1 upregulation attenuates visceral nociception and hyperalgesia via spinal mechanisms not related to anti-inflammatory or probiotic effects. Pain Res Treat. 2011;2011:507029
14. Dray A. Inflammatory mediators of pain. Br J Anaesth. 1995;75:125–31
15. Robinson DR, Gebhart GF. Inside information: The unique features of visceral sensation. Mol Interv. 2008;8:242–53
16. Ferreira SH, Prado WA, Ferrari LFBeaulieu P, Lusser D, Porreca F, Dickenson A. Potassium and calcium channels in pain pharmacology Pharmacology of Pain. 2010 Seattle IASP Press:163–84 . Edited by
17. Amin B, Hajhashemi V, Hosseinzadeh H, Abnous Kh. Antinociceptive evaluation of ceftriaxone and minocycline alone and in combination in a neuropathic pain model in rat. Neuroscience. 2012;224:15–25
18. Horvath G, Kekesi G. Interaction of endogenous ligands mediating antinociception. Brain Res Rev. 2006;52:69–92
19. Hinz B, Cheremina O, Brune K. Acetaminophen (paracetamol) is a selective cyclooxygenase-2 inhibitor in man. FASEB J. 2008;22:383–90
20. Hinz B, Brune K. Paracetamol and cyclooxygenase inhibition: Is there a cause for concern? Ann Rheum Dis. 2012;71:20–5
21. Micov A, Tomić M, Popović B, Stepanović-Petrović R. The antihyperalgesic effect of levetiracetam in an inflammatory model of pain in rats: Mechanism of action. Br J Pharmacol. 2010;161:384–92
22. Stepanović-Petrović RM, Micov AM, Tomić MA, Ugrešić ND. The local peripheral antihyperalgesic effect of levetiracetam and its mechanism of action in an inflammatory pain model. Anesth Analg. 2012;115:1457–66
23. De Smedt T, Raedt R, Vonck K, Boon P. Levetiracetam: The profile of a novel anticonvulsant drug-part I: Preclinical data. CNS Drug Rev. 2007;13:43–56
24. National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals. Guide for the Care and Use of Laboratory Animals. 20118th edition Washington National Academies Press (US)
25. Tomić MA, Vučković SM, Stepanović-Petrović RM, Ugrešić N, Prostran MŠ, Bošković B. The anti-hyperalgesic effects of carbamazepine and oxcarbazepine are attenuated by treatment with adenosine receptor antagonists. Pain. 2004;111:253–60
26. Tomić MA, Vučković SM, Stepanović-Petrović RM, Ugrešić ND, Prostran MŠ, Bošković B. Synergistic interactions between paracetamol and oxcarbazepine in somatic and visceral pain models in rodents. Anesth Analg. 2010;110:1198–205
27. Stepanović-Petrović RM, Tomić MA, Vučković SM, Poznanović G, Ugrešić ND, Prostran MŠ, Bošković B. Pharmacological interaction between oxcarbazepine and two COX inhibitors in a rat model of inflammatory hyperalgesia. Pharmacol Biochem Behav. 2011;97:611–8
28. Morris CJWinyard PG, Willoughby DA. Carrageenan-induced paw edema in the rat and mouse Inflammation Protocols (Methods in Molecular Biology). 2003;Vol 225 Totowa Humana Press Inc.:115–23 . Edited by
29. Francischi JN, Chaves CT, Moura AC, Lima AS, Rocha OA, Ferreira-Alves DL, Bakhle YS. Selective inhibitors of cyclo-oxygenase-2 (COX-2) induce hypoalgesia in a rat paw model of inflammation. Br J Pharmacol. 2002;137:837–44
30. Stepanović-Petrović RM, Tomić MA, Vučković SM, Paranos S, Ugrešić ND, Prostran MŠ, Milovanović S, Bošković B. The antinociceptive effects of anticonvulsants in a mouse visceral pain model. Anesth Analg. 2008;106:1897–903
31. Tallarida RJ, Stone DJ Jr, Raffa RB. Efficient designs for studying synergistic drug combinations. Life Sci. 1997;61:PL 417–25
32. Tallarida RJ. The interaction index: A measure of drug synergism. Pain. 2002;98:163–8
33. Manual of Pharmacologic Calculations with Computer Programs. 19862nd edition New York, Berlin, Heidelberg, London, Paris, Tokyo Springer Verlag
34. Yaksh TL, Noueihed RY, Durant PA. Studies of the pharmacology and pathology of intrathecally administered 4-anilinopiperidine analogues and morphine in the rat and cat. ANESTHESIOLOGY. 1986;64:54–66
35. Kimura M, Hayashida K, Eisenach JC, Saito S, Obata H. Relief of hypersensitivity after nerve injury from systemic donepezil involves spinal cholinergic and γ-aminobutyric acid mechanisms. ANESTHESIOLOGY. 2013;118:173–80
36. Rylander D, Recchia A, Mela F, Dekundy A, Danysz W, Cenci MA. Pharmacological modulation of glutamate transmission in a rat model of. L. -DOPA-induced dyskinesia: Effects on motor behavior and striatal nuclear signaling. J Pharmacol Exp Ther. 2009;330:227–35
37. Lin Y, Tian G, Roman K, Handy C, Travers JB, Lin CL, Stephens RL Jr. Increased glial glutamate transporter EAAT2 expression reduces visceral nociceptive response in mice. Am J Physiol Gastrointest Liver Physiol. 2009;296:G129–34
38. Yang M, Roman K, Chen DF, Wang ZG, Lin Y, Stephens RL Jr. GLT-1 overexpression attenuates bladder nociception and local/cross-organ sensitization of bladder nociception. Am J Physiol Renal Physiol. 2011;300:F1353–9
39. Suyama H, Kawamoto M, Gaus S, Yuge O. Effect of etodolac, a COX-2 inhibitor, on neuropathic pain in a rat model. Brain Res. 2004;1010:144–50
40. Basbaum AI, Bautista DM, Scherrer G, Julius D. Cellular and molecular mechanisms of pain. Cell. 2009;139:267–84
41. Carlton SM. Peripheral excitatory amino acids. Curr Opin Pharmacol. 2001;1:52–6
42. Okano K, Kuraishi Y, Satoh M. Involvement of spinal substance P and excitatory amino acids in inflammatory hyperalgesia in rats. Jpn J Pharmacol. 1998;76:15–22
43. Chen W, Mahadomrongkul V, Berger UV, Bassan M, DeSilva T, Tanaka K, Irwin N, Aoki C, Rosenberg PA. The glutamate transporter GLT1a is expressed in excitatory axon terminals of mature hippocampal neurons. J Neurosci. 2004;24:1136–48
44. Giamberardino MA. Recent and forgotten aspects of visceral pain. Eur J Pain. 1999;3:77–92
45. Ribeiro RA, Vale ML, Thomazzi SM, Paschoalato AB, Poole S, Ferreira SH, Cunha FQ. Involvement of resident macrophages and mast cells in the writhing nociceptive response induced by zymosan and acetic acid in mice. Eur J Pharmacol. 2000;387:111–8
46. Watkins LR, Maier SF. Beyond neurons: Evidence that immune and glial cells contribute to pathological pain states. Physiol Rev. 2002;82:981–1011
47. Tong C, Conklin D, Eisenach JC. A pain model after gynecologic surgery: The effect of intrathecal and systemic morphine. Anesth Analg. 2006;103:1288–93
48. Kfoury T, Mazoit JX, Schumacher M, Benhamou D, Beloeil H. A comparison of different dosages of a continuous preperitoneal infusion and systemic administration of ropivacaine after laparotomy in rats. Anesth Analg. 2011;113:617–25
49. Yoon MH, Yaksh TL. Evaluation of interaction between gabapentin and ibuprofen on the formalin test in rats. ANESTHESIOLOGY. 1999;91:1006–13
50. Baba H, Kohno T, Moore KA, Woolf CJ. Direct activation of rat spinal dorsal horn neurons by prostaglandin E2. J Neurosci. 2001;21:1750–6
51. Yaksh TL, Dirig DM, Conway CM, Svensson C, Luo ZD, Isakson PC. The acute antihyperalgesic action of nonsteroidal, anti-inflammatory drugs and release of spinal prostaglandin E2 is mediated by the inhibition of constitutive spinal cyclooxygenase-2 (COX-2) but not COX-1. J Neurosci. 2001;21:5847–53
52. Feng Y, Cui M, Willis WD. Gabapentin markedly reduces acetic acid-induced visceral nociception. ANESTHESIOLOGY. 2003;98:729–33
53. McCormack K. Non-steroidal anti-inflammatory drugs and spinal nociceptive processing. Pain. 1994;59:9–43
54. Dirig DM, Isakson PC, Yaksh TL. Effect of COX-1 and COX-2 inhibition on induction and maintenance of carrageenan-evoked thermal hyperalgesia in rats. J Pharmacol Exp Ther. 1998;285:1031–8
55. Yoshino T, Kimoto A, Kobayashi S, Noguchi M, Fukunaga M, Hayashi A, Miyata K, Sasamata M. Pharmacological profile of celecoxib, a specific cyclooxygenase-2 inhibitor. Arzneimittelforschung. 2005;55:394–402
56. Bonnefont J, Chapuy E, Clottes E, Alloui A, Eschalier A. Spinal 5-HT1A receptors differentially influence nociceptive processing according to the nature of the noxious stimulus in rats: Effect of WAY-100635 on the antinociceptive activities of paracetamol, venlafaxine and 5-HT. Pain. 2005;114:482–90
57. Rezende RM, França DS, Menezes GB, dos Reis WG, Bakhle YS, Francischi JN. Different mechanisms underlie the analgesic actions of paracetamol and dipyrone in a rat model of inflammatory pain. Br J Pharmacol. 2008;153:760–8
© 2014 American Society of Anesthesiologists, Inc.
Publication of an advertisement in Anesthesiology Online does not constitute endorsement by the American Society of Anesthesiologists, Inc. or Lippincott Williams & Wilkins, Inc. of the product or service being advertised.