DRUG DEVELOPMENT RESEARCH 78 : 105–115 (2017) DDR Research Article Antiallodynic Activity of Ceftriaxone and Clavulanic Acid in Acute Administration is Associated with Serum TNF-a Modulation and Activation of Dopaminergic and Opioidergic Systems A. Ochoa-Aguilar,1 M.A. Sotomayor-Sobrino,1 R. Jaimez,1 R. Rodrıguez,1 R. Plancarte-Sanchez, 2 and R. Ventura-Martinez 1* 1Departmento de Farmacologıa, Facultad de Medicina, Universidad Nacional Autonoma de Mexico, C.P. 04510, Delegacion Coyoacan, Ciudad de Mexico, Mexico 2Clınica del Dolor, Instituto Nacional de Cancerologıa Strategy, Management and Health Policy Enabling Preclinical Development Clinical Development Technology, Preclinical Toxicology, Formulation Phases I-III Postmarketing Genomics, Research Drug Delivery, Regulatory, Quality, Phase IV Proteomics Pharmacokinetics Manufacturing ABSTRACT The aim of this study was to determine the antiallodynic effect of acute administration of the b-lactam antimicrobials, ceftriaxone (CFX) and clavulanic acid (CLAV), for the control of estab- lished pain on a model of neuropathic pain (NP). We also investigated the involvement of dopaminer- gic and opioidergic pathways as well as alterations in serum concentrations of TNF-a in the antiallodynic actions of these drugs. CFX, CLAV, or gabapentin (GAP), a reference drug, were adminis- tered i.p. twelve days after constriction of the sciatic nerve in rats. Mechanic and cold allodynia were evaluated for 3 h and alterations in serum concentration of TNF-a determined. Both CFX and CLAV had antiallodynic effects in response to mechanical and cold stimulation, similar to GAP. The antiallo- dynic effects of CFX and CLAV were blocked by haloperidol (HAL), a D2 receptor antagonist, and by naloxone (NLX), an opioid receptor antagonist. Additionally, serum TNF-a levels were attenuated fol- lowing CFX and CLAV administration. These results suggest that acute administration of CFX and CLAV may represent a promising approach for treating the acute allodynia of NP, and that the mechanisms involved in these effects involve activation of dopaminergic and opioidergic pathways as well as modu- lation of TNF-a production. Drug Dev Res 78 : 105–115, 2017. VC 2017 Wiley Periodicals, Inc. Key words: ceftriaxone; clavulanic acid; neuropathic pain INTRODUCTION Neuropathic pain (NP) is a chronic disorder that affects up to 10% of the general population, with *Correspondence to: R. Ventura-Martinez, Av. Universi- dad No. 3000, Col. Ciudad Universitaria, Delegacion Coy- affected patients presenting a wide range of disabil- oacan, P.O. 04510, Mexico City, Mexico. E-mail: rventuram7@ ities [van Hecke et al., 2013]. NP syndromes are hotmail.com characterized by allodynia, an exaggerated response Grant sponsor: CONACYT; Grant number: 294713; Grant to non-noxious stimuli; although numerous pharma- sponsor: UNAM-DGAPA-PAPIIT-IN204416; Grant number: IN204416 cological treatments have been studied, only a mod- Received 23 February 2017; Accepted 24 February 2017 est efficacy has been reported in the control of this Published online in Wiley Online Library (wileyonlinelibrary. type of pain [Finnerup et al., 2015]. com). DOI: 10.1002/ddr.21381 VC 2017 Wiley Periodicals, Inc. 106 OCHOA-AGUILAR ET AL. The difficulty in achieving adequate pain con- determine the antiallodynic effects of acute adminis- trol stems from the complexity of the pathophysiology tration of CFX and CLAV in established NP induced of this disorder [Nickel et al., 2012]. Indeed, interac- by chronic constriction injury (CCI) of the sciatic tions between both neuronal [Costigan et al., 2009] nerve in rats. We also investigated the involvement of and immunological [Clark et al., 2013] systems the dopaminergic and opioidergic systems as well as underpin the numerous changes in different sensory alterations in concentrations of TNF-a, a proinflam- pain transduction, perception, and integration sys- matory cytokine, in the antiallodynic actions of these tems that ultimately lead to a state of chronic pain drugs. [Price and Prescott, 2015]. The neuronal aspects of NP involve changes in both excitatory and inhibitory METHODS AND MATERIALS neurotransmitter systems. However, increased gluta- Animals matergic neurotransmission is one of the main changes related to NP induction [Nickel et al., 2012]; Male Wistar rats weighing 200–250 g were on the other, decreased levels of neurotransmitters in housed in plastic cages at a room with controlled descending pathways, as dopamine (DA) and endoge- temperature (228C) and maintained in a 12:12 h nous opioids, have been implicated in NP induction light/dark cycle with food and water available ad libi- [Coffeen et al., 2010]. Proinflammatory cytokines, for tum. All experiments were performed between 8:00 example, tumor necrosis factor-a (TNF-a), can also and 13:00. Experiments were conducted in accor- stimulate spontaneous nociceptor activity and con- dance with the regulations of the Ethics Committee tribute to axonal damage. Increased levels of TNF-a of the International Association for the Study of Pain occur in various chronic pain states [Sacerdote et al., [Zimmermann, 1983], and of the Mexican Official 2013]. Norm for Animal Care and Handling (NOM-062- Ceftriaxone (CFX), a b-lactam antimicrobial ZOO-1999) and approved by our institutional Ethics molecule (BLM) [Donnelly et al., 2017] has shown Commission. The number of experimental animals antinociceptive effects after repeated administration was kept to a minimum (n 5 8 per group) and these in NP models [Hu et al., 2010; Gunduz et al., 2011; were euthanized by cervical dislocation at the end of Hajhashemi et al., 2013; Shi et al., 2016] and can the experiments. enhance the analgesic effect of nonsteroidal anti- inflammatory drugs (NSAIDs) in inflammatory and Compounds and Drugs visceral models [Stepanovic-Petrovic et al., 2014]. CFX (Pisa, Mexico City, Mexico), potassium CFX also has neuroprotective [Cui et al., 2014] and clavulanate (CLAV; Sigma, St. Louis, MO), gabapen- antiepileptic properties [Uyanikgil et al., 2016], inhib- tin (GAP; Sigma, St. Louis, MO), and naloxone its drug-seeking reinstatement [Sari et al., 2009], and (NLX; Sigma, St. Louis, MO) were dissolved in saline can release DA from the nucleus accumbens [Altho- solution (0.9% NaCl); while haloperidol (HAL; Sig- baiti et al., 2016]. ma, St. Louis, MO) was dissolved in a drop of acetic Clavulanic acid (CLAV), is another BLM that acid, diluted with distilled water, and adjusted to pH lacks antibiotic effects but is widely used as a BLM of 4.5–5. All the compounds were prepared immedi- adjuvant due to its ability to inhibit the b-Lactamase ately before their intraperitoneal (i.p.) administration secreted by bacteria preventing the degradation of and administered at a final volume of 1 ml/kg of BLM antibiotics thus enhancing their efficacy [Sau- body weight. dagar et al., 2008]. It has neuroprotective [Kost et al., 2012] and anxiolytic activities [Kim et al., 2009] and CCI Procedure to Induce Neurophatic Pain produces antinociceptive effects [Hajhashemi and Dehdashti, 2014]. Like CFX, it can enhance DA To induce NP, the CCI model was used [Ben- release in the central nervous system (CNS) [Kost nett and Xie, 1988]. Briefly, rats were anesthetized et al., 2011]. with ketamine/xylazine (50:7.5 mg/kg, i.p.) and the The antinociceptive effects of both BLMs have area over the right pelvic joint was shaved and disin- been principally attributed to their capacity to fected. An incision of 10 mm was made over the increase GLT-1 expression, the major glutamate pelvic joint and all muscles were separated to expose transporter that removes the high concentrations of the sciatic nerve. Four ligatures where applied sur- this neurotransmitter from extracellular space [Roth- rounding the sciatic nerve with a spacing of 1mm stein et al., 2005; Hu et al., 2010; Shi et al., 2016], between each with chromic gut sutures (3-0) and although other mechanisms could be involved in their muscle and skin were separately sutured with 4-0 antinociceptive effects. The aim of this study was to silk. The sciatic nerves of sham-operated rats were Drug Dev. Res. ACUTE ANTIALLODYNIC EFFECTS OF CFX AND CLAV 107 identically exposed but not ligated. Rats were TNF-a Assessment returned to their home cages and monitored daily After performing the behavioral tests, a blood until the day of the experiment. All surgical proce- sample was obtained from each animal to determine dures were performed by the same person under concentration of TNF-a. The blood samples were normal sterile conditions. taken under deep pentobarbital anesthesia (36 mg/kg, i.p.) from the abdominal aorta with a vacutainer Behavioral Assessment device through direct puncture, after which the ani- Behavioral testing with mechanical and cold mals were sacrificed. Blood samples were centrifuged stimuli was conducted twelve days postsurgery. For at 3000 rpm for 20 min; serum was separated and this, animals were placed in transparent acrylic cages stored at 2208C. The levels of TNF-a in samples of with a wire mesh floor, which allowed access to the serum were quantified using an indirect enzyme- plantar surface of the hind paw. They were allowed linked immunosorbent assay kit (TNF-a Abcam, to habituate to this environment until exploratory ab46070; Cambridge, Mass) according to the instruc- behavior diminished (a minimum of 20 min) before tion of the manufacturer. The TNF-a concentration stimulation was initiated. Baseline levels of each type in each sample was expressed as picograms of antigen of stimulation were obtained prior to treatment per milliliter of serum (pg/ml). administration to ensure consistent behavioral responses. Experimental Design The protocol consisted of two groups of experi- Mechanical Allodynia (Von Frey Test) ments. In the first, antiallodynic effects produced by acute administration of CFX and CLAV as well as To evaluate mechanical allodynia, withdrawal the changes in TNF-a concentration were evaluated; threshold to mechanical stimuli was measured using in the second, the possible participation of dopami- a series of calibrated nylon monofilament fibers (Von nergic or opioidergic pathways on the antiallodynic Frey filaments; Stoelting, Wood Dale, IL).