Analgesic Effects On Neuropathic Pain And Mechanisms Of Ammoxetine As A Novel Potent Serotonin And Norepinephrine Reuptake Inhibitor

Backgrounds &AimNeuropathic pain is a common chronic pain syndrome associated with high prevalence and complicated with depression, anxiety and sleep disorders, which seriously reduces the quality of life in patients. The conventional analgesics or even the narcotics are often ineffective for the treatment of neuropathic pain due to the complex pathogenesis. Thus, to clarify its pathogenesis and develop new drugs with specific targets are currently the focus of attention. Basic and clinical studies have shown that antidepressants can relieve pain and improve mood and sleep which have long been applied for the treatment of neuropathic pain. The selective serotonin and norepinephrine reuptake inhibitors (SNRIs) duloxetine has been approved by the FDA for the treatment of diabetic peripheral neuropathic pain, fibromyalgia and chronic musculoskeletal pain. Duloxetine, which has a huge and increasing market share, has been the first choice for the treatment of several neuropathic pain disorders. Unfortunately, duloxetine has been banned for for patients with liver damage because of its hepatotoxicity, which restricts its clinical application. Thus, the development of new potent and low toxicity SNRIs with own property for the treatment of depression and neuropathic pain is the goal of our research group.In our preliminary studies, we synthesized and screened a new powerful and balanced SNRI named as ammoxetine used duloxetine as a precursor compound and replaced naphthalene ring structure which cause liver toxicity in order to reduce liver damage. Our further research indicated that ammoxetine displayed lower hepatotoxicity than duloxetine and exhibits signidicant analgesic effect in acute and inflammatory pain in animal models.In the present study, we evaluate the analgesic effects of ammoxetine using a variety of pain animal models. Moreover, we examined the involvement of monoamines, activation of neuroglial cells and the release of inflammatory cytokines in the mechanisms of the analgesic effects of ammoxetine. This study will provide experimental evidence for the future applications of ammoxetine in neuropathic pain and also provide useful clues to the mechanism involved in the analgesic effect of antidepressants.Methods:The analgesic effects of ammoxetine were assayed using models of acetic acid induced visceral inflammatory pain and formalin-induced acute and persistent pain in mice. Sciatic nerve injury (SNI) and chronic constriction injury (CCI) in rats are used to evaluate the pain-relieving effect of ammoxetine on neuropathic pain induced by nerve injury. Diabetes model in rats induced by streptozotocin (streptozocin, STZ,60 mg/kg) was used to evaluate the analgesic effect of ammoxeitne on diabetic neuropathic pain (DNP). The analgesic effect of ammoxetine on fibromyalgia was evaluated in rats subcutaneously administered with reserpine (1 mg/kg) for three consecutive days.Tryptophan hydroxylase enzyme inhibitor p-chlorophenylalanine (PCPA) and tyrosine hydroxylase inhibitora-methyl-para-tyrosine methylester (AMPT) were used to investigate the effect of 5-HT and NE depletion on the pain-relieving effect of ammoxetine in CCI rats. The contents of monoamine including 5-HT and its metabolite 5-HIAA, NE, DA and its metabolites DOPAC and HVA in the spinal cord and brain in fibromyalgia rats were analysed using HPLC-ECD.The expression levels of inflammatory cytokines in the spinal cord in CCI and DNP rats were assayed by ELISA. The expression of the microglial marker ionized calcium binding receptor molecule 1 (iba-1), astrocyte marker glial fibrillary acidic protein (GFAP), mitogen-activated protein kinases (MAPK, including p38, JNK, ERK) and NF-κB p65 were determined by western blot.Results1. Orally administered ammoxetine significantly reduced the visceral inflammatory pain induced by acetic acid with ED50 (±95% CL) at 2.01(0-7.06) mg/kg, which was lower than the ED50 (±95% CL) of duloxetine at 10.03(6.94-13.10) mg/kg.2. Ammoxetine reduced the formalin-induced acute and persistent pain with ED50(±95% CL) as 9.89(6.55-13.23) mg/kg and 4.74 (2.84-6.64). The ED50(±95% CL) values for duloxetine in this modle were 19.65(14.52-24.79) mg/kg for acute pain and 10.06 (9.13-11.00) mg/kg for persistent pain.3. Ammoxetine (2.5-10 mg/kg) demonstrated dose-dependently analgesic effects on mechanical allodynia in SNI rats.4. Both single and successively for two weeks given ammoxetine reduced the mechanical allodynia as well as thermal hyperalgesia in CCI rats. While duloxetine 10mg/kg displayed pain-relieving effect only on thermal hyperalgesia in CCI rats.5. Administration of ammoxetine (2.5-10 mg/kg) for 4 weeks dose-dependently reduced the mechanical allodynia in DNP rats.6. Ammoxetine (30 mg/kg) or duloxetine (50 mg/kg) significantly attenuated the tactile allodynia in rats with reserpine-induced fibromyalgia.7. The antiallodynic effect of ammoxetine was abolished by pretreatment with para-chlorophenylalanine methyl ester hydrochloride (PCPA) or a-methyl-para-tyrosine methylester (AMPT) in CCI rats.8. Ammoxetine 30 mg/kg or duloxetine 50 mg/kg increased the levels of 5-HT and NE and decreased the 5-HIAA (5-hydroxyindole-3-acetic acid)/5-HT ratios without effect on the levels DA and DOPAC/DA in the spinal cord, hypothalamus, thalamus and prefrontal cortex in the fibromyalgia rats.9. In CCI rats, the levels of pro-inflammatory cytokines TNF-α、IL-1β、IL-6 and the expression of ibal and GFAP in the spinal cord were increased significantly. Ammoxetine decreased the the levels of TNF-α、IL-1β、IL-6 and the expression of both ibal and GFAP. In addition, ammoxetine reduced theexpression of p-p38 and p-ERK and NE-κB p65.10. In DNP rats, the levels of pro-inflammatory cytokines TNF-α、IL-1βand the expression of ibal increased significantly but not GFAP in the spinal cord Ammoxetine decreased the levels of TNF-α、IL-1βand the expression of ibal. Besides, ammoxetine reduced the expression of p-p38 and p-ERK in the spinal cord in DNP rats.ConclusionAmmoxetine produces analgesia against visceral inflammatory pain, acute and continuous pain, neuropathic pain induced by nerve injury, diabetic neuropathic pain and fibromyalgia-related pain. The pain relieving effects of ammoxetine are associated with enhancing the transmission of both 5-HT and NE. On the other hand, the suppression of spinal cord glial cell activation and inflammatory reactions may also participate in the mechanism for the analgesic effect of ammoxetine. Ammoxetine may play anti-inflammatory effects by inhibiting phosphorylation of p38 and JNK in the MAPK signaling pathways and the activation of NF-κB. In summary, ammoxetine is expected to be developed as an effective drug for neuropathic pain with less adverse effects.