Introduction
Neuropathic pain syndrome is a group of appalling pain disorders caused by injury to or disease of the central or peripheral nervous system [1]. It can emerge because of a lesion in the central neurons and somatosensory system, including peripheral fibres (Aβ, Aδ and C fibres) [2]. Neuropathic pain is a sequel of insult in the pain-conducting nervous system. Postherpetic neuralgia (PHN), complex regional pain syndrome (CPRS), trigeminal neuralgia, painful diabetic neuropathy (PDN), painful radiculopathy and the central poststroke pain syndrome are few examples of neuropathic pain syndrome [1, 2]. It is an intimidating therapeutic challenge since it poorly responds to conventional therapeutic approaches including analgesics and NSAIDs [2, 3]. Factors that inculpate neuropathic pain include imbalanced stimulative and repressive somatosensory signaling, permuted ion channels and variance in the modulation of pain messages in the CNS [4]. Pathogenesis of neuropathic pain shows the manifestation of many pathophysiological as well as biochemical changes varying from cellular to intra-nuclear level occurring in the nervous system in retaliation to an injury or insult [5].
A notable resemblance was reported between the biochemical and pathophysiological mechanisms of epilepsy and neuropathic pain. At pathophysiological level, similarity was noticed between kindling of hippocampal neurons in epilepsy and wind-up, documented at spinal cord dorsal horn neurons in models of neuropathic pain. Both kindling and wind-up seems to emerge from the activation of NMDA receptors [4, 6]. An observation showed that primary afferent and transmission neurons are susceptible to the action of sodium channel blockers in neuropathic pain models which is alike to their vulnerability seen in various epileptic models [3, 5]. Therefore, the rationale behind the repurposing of anticonvulsant agents in the symptomatic control of neuropathic pain was justified. Many drugs like oxcarbazepine [7], gabapentin [8], levitracetam [9], phenytoin [10] and pregabalin [11] have been repurposed for their anti-hyperalgesic activity based on observations in animal models of neuropathic pain. Oxcarbazepine and gabapentin have shown unprecedented success in their use as anti-hyperalgesic agents. Thus, anticonvulsants can not only relieve but possibly modify the pain perception in neuropathic pain disorders.
N’-[4-(4-fluorophenoxy)benzylidene]pyridine-4-carbohydrazide PCH 6 (Figure 1) was designed, synthesized and found as a prospective anticonvulsant agent in a concatenation of N’-[substituted]pyridine-4-carbohydrazide derivatives [12]. N-(4-methoxyphenyl)-2-[4-(4-methylphenoxy)benzylidene] hydrazinecarbothioamide PT 30 (fig. 1) was designed, synthesized and reported as a potent anticonvulsant in N-(substituted)-2-[4-(substituted)benzylidene]hydrazinecarbothioamides series of anticonvulsant compounds [13]. Three seizure models were used to evaluate the anticonvulsant activity of PCH 6 and PT 30, namely MES, scMET and 6 Hz seizure test. PCH 6 and PT 30 came up as the most promising derivative of N’-[substituted] pyridine-4-carbohydrazide series and N-(substituted)-2-[4-(substituted) benzylidene]hydrazinecarbothioamides series of compounds respectively.
Рис. 1. Структура N’-[4-(4-фторфенокси)бензилиден]пиридин-4-карбогидразида (ПКГ 6) и N-(4-метоксифенил)-2-[4-(4-метилфенокси)бензилиден]гидразинкарботиоамида (ГК 30).
Таблица 1. Продолжительность вылизывания в острой и/или воспалительной стадиях боли при проведении формалинового теста на мышах в контрольной группе для N’-[4-(4-фторфенокси)бензилиден]пиридин-4-карбогидразида (ПКГ 6)
| Dose (mg/kg) | Animal | Duration of Licking (sec) | ||||||||
| 0 min | 5 min | 10 min | 15 min | 20 min | 25 min | 30 min | 35 min | 40 min | ||
| 0.0 | 01 | 41.84 | 0.00 | 32.22 | 15.12 | 10.63 | 27.64 | 0.00 | 0.00 | 0.00 |
| 0.0 | 02 | 65.03 | 35.72 | 4.88 | 11.46 | 89.48 | 36.28 | 54.33 | 45.94 | 38.21 |
| 0.0 | 03 | 53.55 | 0.00 | 2.85 | 0.00 | 44.25 | 40.48 | 53.09 | 25.07 | 0.00 |
| 0.0 | 04 | 55.57 | 0.00 | 0.00 | 0.00 | 22.70 | 27.16 | 32.27 | 1.46 | 2.84 |
| 0.0 | 05 | 63.90 | 20.49 | 0.00 | 15.49 | 25.84 | 37.63 | 57.45 | 27.45 | 0.00 |
| 0.0 | 06 | 48.09 | 23.00 | 10.01 | 20.69 | 60.49 | 80.03 | 79.52 | 41.55 | 11.61 |
| 0.0 | 07 | 45.69 | 0.00 | 0.00 | 31.60 | 59.27 | 53.61 | 78.01 | 2.79 | 15.90 |
| 0.0 | 08 | 48.42 | 13.79 | 0.00 | 0.00 | 35.60 | 18.76 | 44.91 | 12.46 | 0.00 |
Keeping in view the fact that anti-seizure drugs (ASDs) can frequently reduce pain in various animal models of inflammatory and neuropathic pain, the potential of PCH 6 and PT 30 to alleviate the acute and/or inflammatory stages of pain was assessed in mouse formalin test of hyperalgesia.
Material and methods
PCH 6 and PT 30 were designed, synthesized and characterized by previously described procedure [12, 13]. The pharmacological evaluation of PCH 6 and PT 30 was done at the Epilepsy Branch, National Institute of Neurological Disorder and Stroke, National Institute of Health, Bethesda, USA under the Anticonvulsant Drug Development (ADD) program, presently known as Epilepsy Therapy Screening Program (ETSP). The pharmacological studies included anticonvulsant evaluation, neurotoxicity studies and assessment of the in vivo effect of PCH 6 and PT 30 on the alleviation of acute and chronic inflammatory pain in mouse.
Anticonvulsant evaluation was carried out by maximal electroshock (MES) and subcutaneous metrazole (scMET) test in mice. PCH 6 and PT 30 were administered intra-peritoneally at doses of 30, 100 and 300 mg/kg each at two dissimilar time intervals. Neurotoxicity was assessed by the rotarod test and observed as minimal motor impairment in mice. Oral activity in rat MES screen was also examined. Further, the compounds were evaluated in 6 Hz seizure model, identifying its anticonvulsant activity at five dissimilar time points. Finally, they put through quantification studies and corresponding ED50 and TD50 were determined. They were also screened in Pilocarpine Induced Status Prevention (PISP) Model and in vitro Hippocampal Slice Culture Neuroprotection Assay (NP) [12, 13].
PCH 6 exhibited noteworthy protection in MES and 6 Hz seizure tests. A MES ED50 value of 128.3 mg/kg and 6 Hz ED50 value of 53.3 mg/kg in mice was observed. The median toxic dose (TD50) of 343.6 mg/kg, with a protection index (PI) of 2.67 in the MES test and 6.44 in 6 Hz test was evaluated [12]. PT 30 showed considerable protection in MES and 6 Hz seizure tests with an ED50 value of >100 mg/kg in quantitative 6 Hz screen [13]. As neuropathic pain represent pathological states of abnormal neuronal discharges, at a dose near the mouse MES ED50, PCH 6 and PT 30 were tested in the acute and/or inflammatory stages of pain in mouse formalin test of hyperalgesia [14].
Male albino mice (18—25 g) were used for the study. The animals were accommodated in plastic cages, at constant room temperature (23±2 °C) and humidity (60—80%) under a 12 h light & dark cycle with water and food ad libitum. Study was performed in two groups of animal i.e. control and treatment group. Each group constituted eight mice; and animals were used only once in an experiment. They were euthanized immediately when the experiment winds-up. Prior to the administration of test drug or the vehicle, each mouse underwent a 15-min training period in 6» tall and 4» diameter plexiglass cages.
0.5% formalin was injected sub-dermally into the plantar region of the right hindpaw of a mouse. This elicited a recognizable observable response to the formalin injection distinguished by the mouse licking the affected paw. The licking behaviour was biphasic in nature designating the acute and inflammatory stages of pain in the mouse formalin test. The acute stage was identified by the mouse intensely licking the paw for approximately 5—10 min immediately following the injection. The acute stage is followed by a brief latent (usually <5 min) period wherein no behavioural activity is observed. A more lengthened inflammatory stage of about 20 to 30 min period of licking supervenes. Animals were observed for any of these effects of PCH 6 and PT 30 on both acute and inflammatory stages of persistent pain.
The mice (n=8/treatment group) were observed for licking activity for the duration of the experiment. Following the conditioning period, the vehicle (control group) or the titled compound PCH 6 (treatment group) was administered i.p. at a dose of 128 mg/kg approximating its MES ED50 of PCH 6. PT 30 was administered at a dose of 100 mg/kg approximating its 6 Hz seizure test ED50. The mice were kept back in its home cage. At the time of peak effect (TPE) of PCH 6 and PT 30, 0.5% formalin was injected sub-dermally into the plantar surface of the right hindpaw. Ensuing the formalin injection, each animal was observed for the first 2 minutes of each 5 min epoch until a total of 40 min has elapsed since the administration of the vehicle or test compound. The cumulative duration of licking (in seconds) during each 2 min recording period was measured for analysis across vehicle and drug pre-treated groups. Animals were immediately sacrificed after the conclusion of the experiment [14, 16].
Results
In the earlier study performed by our research group, PCH 6 exhibited protection against MES test, suggesting the compounds potential to impede the seizure outspread. PCH 6 exhibited 100% protection (1/1, 4.0 h) and 67% protection (2/3, 2.0 h) at a dose of 100 mg/kg. Oral activity in rat MES screen with 25% protection (1/4, 2.0 h) was observed at a dose of 30 mg/kg. 6 Hz seizure model, which suggests compounds potential to control partial seizures, also exhibited protection by PCH 6 at three dissimilar time points, i.e.100% (4/4, 0.5 h), 75% (3/4, 0.25 h) and 50% (2/4, 1.0 h) at a dose of 100 mg/kg. Although no protection was seen in scMET screen, the encouraging results in MES and 6 Hz seizure test, forwarded PCH 6 for quantification studies. In quantitative MES test PCH 6 exhibited an ED50 (95% confidence interval) of 128.3 mg/kg (95-155.9) at a time of peak effect (TPE) of 1h. In quantitative toxicity test, PCH 6 exhibited a TD50 (95% confidence interval) of 343.6 mg/kg (258.6—474.5) at a time of peak effect (TPE) of 8 h. In quantitative 6 Hz evaluation, PCH 6 exhibited an ED50 (95% confidence interval) of 53.3 mg/kg (28.4—81.4) at a time of peak effect (TPE) of 0.25 h. Neurotoxicity was not observed at the highest administered dose [12].
At a dose of 128.3 mg/kg, a value near the mouse MES ED50, PCH 6 showed significant alleviation in paw licking behaviour during the acute and/or inflammatory stages ensuing formalin injection into the paw. The results are shown in Table 1 and 2. At a dose of 128 mg/kg, PCH 6 exhibited 126.94 (AUC) in reference to control group with 205.67 (AUC) resulting in 61.71% control (SEM 7.525; p>0.05) in acute stage of pain. PCH 6 showed 133.6 (AUC) in reference to control group with 862.38 (AUC) resulting in 15.49% control (SEM 10.81; p>0.01) in inflammatory stage of pain in the mouse formalin test of hyperalgesia (table 3, fig. 2).
Таблица 2. Продолжительность вылизывания в острой и/или воспалительной стадиях боли при проведении формалинового теста на мышах в тестируемой группе для N’-[4-(4-фторфенокси)бензилиден]пиридин-4-карбогидразида (ПКГ 6)
| Dose (mg/kg) | Animal | Duration of Licking (sec) | ||||||||
| 0 min | 5 min | 10 min | 15 min | 20 min | 25 min | 30 min | 35 min | 40 min | ||
| 128.0 | 01 | 66.25 | 7.69 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
| 128.0 | 02 | 69.19 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 19.12 |
| 128.0 | 03 | 27.18 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 6.85 | 0.00 |
| 128.0 | 04 | 36.67 | 0.00 | 0.00 | 0.33 | 0.00 | 0.00 | 0.00 | 36.64 | 12.43 |
| 128.0 | 05 | 52.21 | 0.00 | 0.00 | 0.00 | 26.01 | 0.00 | 44.57 | 59.90 | 43.58 |
| 128.0 | 06 | 43.22 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
| 128.0 | 07 | 52.81 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 3.79 |
| 128.0 | 08 | 42.66 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
Таблица 3. Результаты формалинового теста на мышах в острой и/или воспалительной стадиях боли для N’-[4-(4-фторфенокси)бензилиден]пиридин-4-карбогидразида (ПКГ 6)
| Dose (mg/kg) | Test | Area Under the Curve | ||||
| Control | Drug Treated | % of Control | S.E.M | p Value | ||
| 128.0 | Acute | 205.67 | 126.94 | 61.719 | 7.525 | <0.05 |
| 128.0 | Inflammatory | 862.38 | 133.6 | 15.49 | 10.81 | <0.01 |
Рис. 2. График показывает результаты формалинового теста на мышах в острой и/или воспалительной стадиях боли для N’-[4-(4-фторфенокси)бензилиден]пиридин-4-карбогидразида (ПКГ 6).
In the earlier study performed by our research group, PT 30 exhibited protection against MES test, suggesting the compounds potential to impede the seizure outspread. PT 30 exhibited 100% protection (1/1, 4.0 h) at a dose of 300 mg/kg. It also showed protection in 6 Hz seizure test, which suggested compounds potential to control partial seizure. PT 30 exhibited protection at three dissimilar time points, i.e. 100% (4/4, 0.5 h) and 50% (2/4, 0.25 h; 2/4, 2.0 h) at a dose of 100 mg/kg. Although no protection was seen in scMET screen, the encouraging results in 6 Hz seizure test, forwarded PT 30 for quantification studies. In quantitative 6 Hz evaluation, PT 30 exhibited an ED50 (95% confidence interval) of >100 mg/kg at a time of peak effect (TPE) of 1.0 h. No neurotoxicity was observed at the highest administered dose [13].
At a dose of 100 mg/kg PT 30 showed significant alleviation in paw licking behavior during the acute and/or inflammatory phases ensuing formalin injection into the paw. The results are shown in Table 4 & 5. At a dose of 100 mg/kg, PT30 exhibited 147.61 (AUC) in reference to control group with 239.7 (AUC) resulting in 61.57% control (SEM 7.71; p>0.05) in the acute stage of pain. PT 30 showed 53.24 (AUC) in reference to control group with 882.07 (AUC) resulting in 6.03% control (SEM 2.98; p>0.01) in the inflammatory phase of pain in the mouse formalin test of hyperalgesia (table 6, fig. 3).
Таблица 4. Продолжительность вылизывания при острой и/или воспалительной стадиях боли при проведении формалинового теста на мышах в контрольной группе для N-(4-метоксифенил)-2-[4-(4-метилфенокси)бензилиден]гидразинкарботиоамида (ГК 30)
| Dose (mg/kg) | Animal | Duration of Licking (sec) | ||||||||
| 0 min | 5 min | 10 min | 15 min | 20 min | 25 min | 30 min | 35 min | 40 min | ||
| 0.0 | 01 | 80.69 | 15.51 | 49.27 | 23.76 | 44.09 | 25.58 | 61.31 | 17.22 | 12.52 |
| 0.0 | 02 | 41.00 | 50.01 | 22.34 | 6.88 | 36.49 | 45.06 | 13.75 | 24.73 | 38.21 |
| 0.0 | 03 | 53.55 | 0 | 2.85 | 0 | 44.25 | 40.48 | 53.09 | 25.07 | 0 |
| 0.0 | 04 | 55.57 | 0 | 0 | 0 | 22.70 | 27.16 | 32.27 | 1.46 | 2.84 |
| 0.0 | 05 | 63.90 | 20.49 | 0 | 15.49 | 25.84 | 37.63 | 57.45 | 27.45 | 0 |
| 0.0 | 06 | 48.09 | 23.00 | 10.10 | 20.69 | 60.49 | 80.03 | 79.52 | 41.55 | 11.61 |
| 0.0 | 07 | 45.69 | 0 | 0 | 31.60 | 59.27 | 53.61 | 78.01 | 2.79 | 15.90 |
| 0.0 | 08 | 48.42 | 13.79 | 0 | 0 | 35.60 | 18.76 | 44.91 | 12.46 | 0 |
Таблица 5. Продолжительность вылизывания при острой и/или воспалительной стадиях боли при проведении формалинового теста на мышах в тестируемой группе для N-(4-метоксифенил)-2-[4-(4-метилфенокси)бензилиден]гидразинкарботиоамида (ГК 30)
| Dose (mg/kg) | Animal | Duration of Licking (sec) | ||||||||
| 0 min | 5 min | 10 min | 15 min | 20 min | 25 min | 30 min | 35 min | 40 min | ||
| 100.0 | 01 | 55.93 | 6.6 | 5.01 | 0 | 20.36 | 0 | 0 | 0 | 0 |
| 100.0 | 02 | 63.06 | 0 | 0.93 | 0 | 0 | 0 | 0 | 0 | 0 |
| 100.0 | 03 | 51.90 | 0 | 0 | 0 | 5.54 | 0 | 0 | 0 | 0 |
| 100.0 | 04 | 42.63 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 100.0 | 05 | 76.49 | 0 | 26.03 | 29.24 | 0 | 0 | 0 | 0 | 0 |
| 100.0 | 06 | 39.24 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 7.54 |
| 100.0 | 07 | 46.88 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 20.60 |
| 100.0 | 08 | 51.06 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Таблица 6. Результаты формалинового теста на мышах в острой и/или воспалительной стадиях боли для N-(4-метоксифенил)-2-[4-(4-метилфенокси)бензилиден]гидразинкарботиоамида (ГК 30)
| Dose (mg/kg) | Test | Area Under the Curve | ||||
| control | drug treated | % of control | S.E.M | p value | ||
| 100.0 | Acute | 239.70 | 147.61 | 61.57 | 7.71 | > 0.05 |
| 100.0 | Inflammatory | 882.07 | 53.24 | 6.03 | 2.98 | < 0.01 |
Рис. 3. График показывает результаты формалинового теста на мышах в острой и/или воспалительной стадиях боли для N-(4-метоксифенил)-2-[4-(4-метилфенокси)бензилиден]гидразинкарботиоамида (ГК 30).
Discussion
Recent research works have demonstrated that anticonvulsants can frequently attenuate pain in various animal models of inflammatory and neuropathic pain. Therefore, PCH 6 and PT 30 were examined in mouse formalin test of hyperalgesia to ascertain its potential to alleviate pain response in animals. At a dose near the mouse MES ED50 of PCH 6 and mouse 6 Hz test ED50 of PT 30, they both showed noteworthy alleviation in paw licking behavior during the acute and/or inflammatory stages ensuing formalin injection into the mice paw. This observation can be explained on the fact that epilepsy and neuropathic pain both arise from pathological states of neuronal hyperexcitability. In epilepsy, this central neuron hyperexcitability reveals as seizures, while on the contrary, peripheral neuron hyperexcitability in neuropathic pain sequels in chronic repetitive firing recognized as pain. The potential of PCH 6 and PT 30 to alleviate the acute and/or inflammatory stages of pain in the mouse formalin test shown by significant lessening of paw licking behavior ensuing formalin injection into the paw is due to the reduction in peripheral neuronal hyperexcitabilty resulting in decreased chronic repetitive firing perceived as pain. Docking studies performed on PCH 6 and PT 30 in the previous study showed good binding properties with glutamate receptor, GABA (A) delta and alpha-1 receptor indicating their possible involvement as the molecular target for its anti-hyperalgesic behaviour [12, 13]. Nevertheless, further research is required to ascertain the exact molecular mechanism for the anti-hyperalgesic activity.
Conclusion
Thus, N’-[4-(4-fluorophenoxy)benzylidene]pyridine-4-carbohydrazide PCH 6 and N-(4-methoxyphenyl)-2-[4-(4-methylphenoxy)benzylidene] hydrazinecarbothioamide PT 30 exhibited significant anti-hyperalgesic activity in mouse pain model, besides anticonvulsant activity in animal models of epilepsy. Therefore, its exploration may lead to promising agent that will be clinically useful for the control of chronic pain in neuropathic pain syndrome.
Acknowledgment
The authors would like to express their gratitude to Epilepsy Branch, National Institute of Neurological Disorder and Stroke, National Institute of Health, Bethesda, USA for performing the anti-hyperalgesic activity of the titled compound.
Funding. The study had no sponsorship.
Author contribution:
Concept and design of the study — Praveen Kumar, Laxmi Tripathi
Collection and analysis of data — Praveen Kumar, Laxmi Tripathi
Statistical analysis — Praveen Kumar
Writing the text — Laxmi Tripathi
Editing — Laxmi Tripathi