Research Article |
Corresponding author: Abdelrahim Alqudah ( abdelrahim@hu.edu.jo ) Academic editor: Plamen Peikov
© 2023 Yousra Bseiso, Abdelrahim Alqudah, Esam Qnais, Mohammed Wedyan, Kayed Abu-Safieh, Omar Gammoh.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Bseiso Y, Alqudah A, Qnais E, Wedyan M, Abu-Safieh K, Gammoh O (2023) 2ʹ,3,3,5ʹ-Tetramethyl-4ʹ-nitro-2ʹH-1,3ʹ-bipyrazole exerts antinociceptive effect using various nociception models. Pharmacia 70(3): 509-517. https://doi.org/10.3897/pharmacia.70.e104828
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Background: 2ʹ,3,3,5ʹ-Tetramethyl-4ʹ-nitro-2ʹH-1,3ʹ-bipyrazole (TMNB) is a novel bipyrazole compound that exhibited antidiabetic and anti-inflammatory properties. However, its analgesic effect has not been investigated. This study aimed to assess the antinociceptive activity of TMNB using different nociception mouse models.
Methods: TMNB doses (50, 100, 150, and 200 µg/kg) were assessed in mice using the acetic acid-induced writhing test, hot plate test, and formalin-induced paw licking assay. The effects were compared to those of mice treated with acetylsalicylic acid or morphine in the presence or absence of naloxone. Capsaicin- and glutamate-induced paw-licking tests were also used to evaluate the involvement of the vanilloid and glutamatergic systems, respectively.
Results: TMNB produced significant dose-dependent inhibition of nociceptive behavior in the acetic acid-induced writhing test, showing 66% inhibition at a dose of 200 µg/kg. TMNB also caused a significant increase in the latency period in response to the hot plate test (68.2% at 200 µg/kg), and significantly inhibited both the neurogenic and inflammatory phases in the formalin-induced paw-licking test. Naloxone significantly reverses the effect of TMNB in both the hot plate test and formalin-induced paw-licking test. Moreover, TMNB significantly inhibited the neurogenic nociception induced by intraplantar injections of glutamate and capsaicin (53% and 77.1%, respectively at a dose of 200 µg/kg).
Conclusion: TMNB possesses antinociceptive activity in mice that is mediated through both central and peripheral pathways.
2ʹ,3,3,5ʹ-Tetramethyl-4ʹ-nitro-2ʹH-1,3ʹ-bipyrazole, nociception, naloxone, vanilloid, glutamate
Pain is a sensation or feeling that is uncomfortable and caused by injury to body tissues or associated with damage (
Non-steroidal anti-inflammatory drugs (NSAIDs) are considered one of the most frequently used therapeutics that can be used to treat inflammatory-related pain. Although NSAIDs, their long-term use is associated with several significant adverse effects such as bleeding, peptic ulcers, and gastrointestinal lesions (
Likewise, opioids are another major class of analgesics used in patients with severe pain which is prescribed for short-term post-operative/neurogenic pain. Opioids have significant side effects such as tolerance, respiratory depression, and constipation. Therefore, alternative therapeutic agents with mild action and fewer side effects are receiving increasing attention (
Azoles are monocyclic heteroarenes that contain one nitrogen atom and at least one non-carbon atom, such as nitrogen, oxygen, or sulfur, in their ring structure. They belong to a class of nitrogen-heterocycles and are significant compounds in the pharmaceutical and agrochemical industries (
Pyrazole groups are present in various pharmacological agents, including antimicrobials, hypoglycemic agents, and anti-inflammatory agents, due to their well-established biological properties that result in analgesic, anti-inflammatory, and anti-depressant effects (
A novel bipyrazole compound called 2ʹ,3,3,5ʹ-Tetramethyl-4ʹ-nitro-2ʹH-1,3ʹ-bipyrazole (TMNB) was synthesized through the classical Knorr pyrazole synthesis method. This involved combining 0.17 grams (1 mmol) of 5-hydrazino-1,3-dimethyl-4-nitro-1H-pyrazole with 0.11 grams (1.1 mmol) of acetyl-acetone. The structure of this compound was confirmed using various analytical techniques, including infrared (IR) spectroscopy, HNMR, CNMR, and mass spectrometry (
The biological properties of TMNB, specifically its anticancer activity, have only been investigated in human MCF-7 breast cancer cells and human K562 chronic myelogenous leukemia cells (
Despite the demonstrated antidiabetic, anti-inflammatory, and anticancer properties of TMNB, its potential antinociceptive effects have not been investigated. Therefore, the goal of this study was to explore the antinociceptive activity of TMNB on adult male mice using various nociception models.
Adult male Swiss albino mice weighing between 24–28 g, which were bred and raised at the Animal House Unit of the Hashemite University, were utilized for a study. The mice were kept under controlled temperature conditions of 21 ± 1 °C and a 12-hour light/dark schedule with the lights being on between 0600 and 1800 hours. The mice were given free access to food and water. To reduce stress, the mice were moved to the testing area one hour before the experiments to acclimate to the laboratory conditions. During the animal tests, two trained observers who were unaware of the experimental design monitored the mice to prevent bias. The tests were conducted in a soundproofed room.
In this experiment, six groups of mice were treated intraperitoneally (i.p.), as described previously (
In this test, the treatment was given to eight groups of mice during the test. The vehicle control group, group one, was administered 5% DMSO in distilled water. Groups two to five received TMNB doses of 50, 150, 150, and 200 µg/kg, respectively. Group seven was given Morphine (M8777, Sigma Aldrich, Merck) dissolved in sterile saline and administered i.p. at a dose of 5 mg/kg. To test for the involvement of the opioidergic system, groups six and eight received naloxone hydrochloride, a non-selective opioid receptor antagonist, at a dose of 5 mg/kg i.p., 15 minutes before treatment with TMNB (200 µg/kg) and morphine (5 mg/kg i.p.), respectively (
The first group (referred to as the vehicle control) was given distilled water containing 5% DMSO. The second to fifth groups received 50, 100, 150, and 200 µg/kg of TMNB, respectively. The sixth group was treated with morphine (5 mg/kg), and the seventh group received ASA (100 mg/kg). To investigate the involvement of the opioidergic system (
The purpose of this test was to evaluate the antinociceptive effect of TMNB on the vanilloid receptor, also known as the Transient Receptor Potential Vanilloid type-1 (TRPV1). As previously mentioned six groups of mice (n=6 mice/group) were treated through intraperitoneal injection as follows: The first group (the vehicle control) received distilled water with 5% DMSO, while groups two to five received TMNB at doses of 50, 100, 150, or 200 µg/kg, respectively. Group six was given the TRPV1 receptor antagonist, capsazepine (211280, Sigma-Aldrich, Merck), at a dose of 0.17 mmol/kg through intraperitoneal injection. Sixty minutes after this treatment, 20 µL (1.6 µmol/paw) of capsaicin (211275, Sigma Aldrich, Merck) was injected into the right hind paw of each mouse through the intraplantar route. The nociceptive response to capsaicin was measured as the time each mouse spent licking or biting the injection site, and these measurements were recorded 0–5 minutes after the injection of capsaicin.
This test was conducted to evaluate the antinociceptive effects of TMNB through the glutamatergic receptors. The experiment was conducted on five groups of mice, each consisting of six mice. The first group was given 5% DMSO intraperitoneally (i.p.) as vehicle control, while the remaining four groups received varying doses of TMNB (50, 100, 150, and 200 µg/kg) i.p. After 60 minutes, 20 µL of glutamate (1446600, Sigma-Aldrich, Merck) was injected into the ventral surface of the right hind paw of each animal. The mice were then observed for 15 minutes, and the time each mouse spent licking and/or biting the glutamate injection site was recorded (28, 30).
The Prism 5 software from GraphPad Software in the USA was used for data analysis. All analyzed parameters were tested for the normality of the data using Kolmogorov-Smirnov test. The data are presented as the mean ± standard error of the mean (SEM). Differences between groups were analyzed using one-way ANOVA, followed by Tukey’s posthoc test. Statistical significance was set at P<0.05.
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
The intraperitoneal administration of TMNB at doses of 100, 150, and 200 µg/kg resulted in significant reductions (P<0.001) in the number of acetic acid-induced writhing episodes in treated mice as compared to the control group (as shown in Fig.
Effect of TMNB on writhing in mice induced by 0.6% acetic acid. Six mice were included in the study and were injected with either 5% DMSO (vehicle), TMNB (at doses of 50, 100, 150, 200 µg/kg i.p.), or acetylsalicylic acid (ASA, 100 mg/kg). V *P<0.001 a significant reduction in the number of writhing compared to the vehicle. Similarly, the percent of inhibition caused by TMNB was also significantly different from the vehicle, with #P<0.001.
Administration of TMNB at doses of 50, 100, 150, and 200 µg/kg resulted in a significant (P<0.001) increase in the time it took for the animals to lick their posterior paw when placed on a hot plate (Fig.
Effect of TMNB on mice (n=6) in the hot plate test. The statistical analysis showed that TMNB caused a significant difference compared to the vehicle (5% DMSO) with ***P<0.001. Additionally, Nal blocked the effect of 200 μg/kg TMNB and 5 mg/kg morphine with ###P<0.001. (NAL: naloxone, Mor: morphine). $ denotes the difference between Nal + Mor compared to Mor alone.
To investigate the antinociceptive mechanism of TMNB, we administered naloxone (an opioid antagonist) to mice 15 minutes before administering either 200 µg/kg of TMNB or 5 mg/kg of morphine, followed by subjecting the mice to the hot plate test. Treatment with naloxone significantly blocked the morphine-induced increase in latency time (P<0.001) compared to morphine treatment alone. Similarly, naloxone significantly blocked the increase in latency time induced by TMNB (200 µg/kg) (P<0.001) compared to TMNB treatment alone (Fig.
In this study, mice were given different doses of TMNB (100, 150, and 200 µg/kg) and were observed for reductions in licking times after formalin injection. The results showed that all doses of TMNB caused significant reductions in both early and late phases of licking times (Fig.
The impact of TMNB on early (A) and late (B) phases of 2.5% formalin-induced paw-licking behavior in mice (n=6). The mice were given different injections: 5% DMSO (vehicle), TMNB (50, 100, 150, and 200 µg/kg, i.p), morphine (Mor) (5 mg/kg, i.p), or acetylsalicylic acid (ASA, 100 mg/kg, i.p). Naloxone (NAL, 5 mg/kg, i.p.) was administered 15 minutes before the injection with TMNB (200 µg/kg) or Mor (5 mg/kg, i.p.). **P<0.001 indicates a significant difference in the percentage of inhibition compared to the vehicle; @ P<0.001 indicates a significant difference in licking time compared to the vehicle. # # #P<0.001 indicates a significant difference between TMNB + Nal compared toTMNB (200 µg/kg) alone; $ indicates a significant difference between Nal + Mor compared to Morphine alone.
To investigate the role of the opioidergic system in the pain-relieving effects of TMNB and morphine, mice were given naloxone 15 minutes before receiving either TMNB (200 µg/kg) or morphine (5 mg/kg) before being subjected to the formalin-induced paw-licking test. The results showed that when naloxone was given before morphine, it significantly increased the licking time in both early and late phases compared to the group that received only morphine (P<0.001). In contrast, when naloxone was given before TMNB, the licking times were significantly reduced in both early and late phases to the same degree (P<0.001), indicating that the antinociceptive effects of TMNB are mediated by a mechanism of action of opioidergic system.
This experiment was conducted to examine the antinociceptive effect of TMNB on the vanilloid system. Administration of TMNB at doses of 50, 100, 150, and 200 µg/kg significantly (P<0.01) reduced the licking time in mice (Fig.
Effect of TMNB on capsaicin-induced paw licking in mice. The animals were divided into five groups, and each group contained six male mice. The mice were injected with either 5% DMSO (vehicle), TMNB at doses of 50, 100, 150, and 200 µg/kg (i.p.), or the capsaicin antagonist, capsazepine (Capsz, 0.17 mmol/kg, i.p.). After 60 minutes, the mice were challenged with capsaicin, and the licking time was recorded. **P<0.01 indicating a significant reduction of the percent of inhibition compared to the control group. ##P<0.01 indicating a significant reduction in licking time compared to the control group.
The administration of TMNB at doses of 100, 150, and 200 µg/kg resulted in a significant reduction (P<0.001) in the paw-licking time of mice injected with glutamate (Fig.
Effect of TMNB on glutamate-induced paw licking in mice. The animals were divided into five groups, and each group contained six male mice. The mice were injected intraperitoneally with either 5% DMSO (vehicle) or TMNB at doses of 50, 100, 150, and 200 µg/kg. After 60 minutes, 20 µL of glutamate was intraplantarly administered into the right hind paw, and the licking time was recorded. *P<0.001 indicating a significant reduction of the percent of inhibition compared to the control group. #P<0.001 indicating a significant reduction in licking time compared to the control group.
The findings of this study indicate that TMNB has antinociceptive effects, which have not been previously demonstrated. The results show that TMNB inhibits the nociceptive effects of acetic acid, which is a well-known inducer of pain. Acetic acid-induced writhing test, which works by directly activating a non-selective ion channel in peripheral nociceptive fibers (
The hot plate test is a method used to evaluate the supraspinal and spinal biological properties of novel drugs, without inputs from peripheral nociception neurons. hot plate test, in which paw licking and jumping behavior in response to hot plate stimulation occurs at the supraspinal level. The duration of the licking and jumping behavior can be used to assess animal responses. In this test, the latency time of mice can only be prolonged by centrally acting opioid-like drugs and not by peripherally acting drugs. A drug or substance that increases the latency of mice feeling discomfort on the hot plate indicates centrally mediated activity, similar to that of opioids (
We conducted the formalin-induced paw-licking test to confirm the antinociceptive activity of TMNB at both central and peripheral levels. The formalin-induced paw-licking has two distinct phases of nociceptive behavior that occur after formalin injection. The first phase begins immediately after injection and lasts for approximately 5 minutes which characterized by a direct effect of formalin on nociceptors, and prostaglandin does not have a role in this phase (
To investigate whether TMNB can modulate pain reception through vanilloid receptors and/or the glutamatergic system, we conducted capsaicin-induced and glutamate-induced paw-licking tests. Capsaicin activates TRPV1 receptors, which causes the influx of Ca2+ and Na+ (mostly Ca2+), and activates C- or Aδ-fibers in afferent neurons, leading to neurogenic pain. This test induces both hyperalgesia and analgesia (
Our findings showed that TMNB had a significant inhibitory effect on capsaicin-induced paw licking, as evidenced by the results in Fig.
Glutamate-induced paw-licking test is another test that is used in pain research. Glutamate is an important excitatory neurotransmitter present at high levels in the central nervous system, and its activation leads to nociceptive transmission. Glutamate receptors are distributed in both the central and peripheral nervous systems (
Our results show that the TMNB has antinociceptive effects through various physiological pathways in both the peripheral and central nervous systems. Specifically, it acts as an opioid receptor agonist and is capable of inhibiting the vanilloid and glutamatergic receptor systems.
All animal procedures were approved by the Animal Research Ethics Committee at the Hashemite University (IRB number:HU [[48/2021) and were in accordance with the guidelines of the U.S. National Institutes of Health on the use and care of laboratory animals and with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines (https://arriveguidelines.org).
The authors declare that there is no conflict of interest regarding the publication of this article.
The animal experiments for this study were funded by a grant from The Hashemite University, Jordan (Grant # 34/2020).
AA and YB contributed to the conception and design of the study AA, EYQ, MAW, and KAS carried out animal experiments and acquisition of data. YB & OG analyzed and interpreted data. AA & OG prepared the primary draft of the manuscript.