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Neuroprotective Propensity of 4-Allylpyrocatechol Derivatives against Oxaliplatin Induced Peripheral Neuropathy

Chemotherapy is used for the treatment of rapidly growing cell diseases in the body. It is most used for the treatment of different kinds of tumors. It can develop neuropathic pain due to damage of peripheral nerve cells and it is called Chemotherapy-Induced Peripheral Neuropathy (CIPN). In this study, we have reported the protective effects of 4-allyl pyrocatechol (4-APC) and its derivatives from biochemical and functional deficits associated with oxaliplatin (OP) induced neuropathy. The animals were submitted to mechanical and thermal hyperalgesia tests, after treatment with OP three times weekly at 0.20 mg/kg and 4-APC and derivatives (10 mg/kg & 30 mg/kg). The pain parameters were evaluated during the treatment period and at the end of treatment. 4-APC significantly prevented the mice from behavioural and biochemical alterations associated with OPinduced neuropathy. Thus, we conclude from this study, the use of 4-APC and its derivatives with OP might reduce the number of patients who develop painful peripheral neuropathy. Original Research Article Annavarapu et al.; JPRI, 33(24A): 92-100, 2021; Article no.JPRI.67786 93


INTRODUCTION
Platinum agents are a significant antitumor medication class, broadly utilized in the treatment of advanced tumors [1]. Oxaliplatin (OP) is a third-generation platinum-based anticancer drug widely used to treat advanced colorectal malignant growth, and furthermore as an adjuvant treatment in different forms of advanced solid tumors. The incorporation of OP in the FOLFOX regimen (5-fluorouracil and leucovorin and OP), the mainline treatment for metastatic colorectal cancer has improved the survival rate in patients with this disease [2]. However, peripheral neuropathy which is seen in 10-15% of patients after total infusion of OP is the major limitation of OP [3]. A compelling body of evidence suggests neuroinflammation and oxidative stress are the major causes of OPinduced peripheral neuropathy (OIPN). To prevent OIPN, different cancer prevention agentbased treatments have been recommended alongside calming interventions. They include acetyl-L-carnitine, Vitamin-E, Vitamin-C, glutathione, and amifostine. Nonetheless, they show no adequacy in clinical investigations, along these lines, it is necessary to identify an agent to effectively prevent OIPN without affecting its anticancer efficacy [4-6].
4-Allylpyrocatechol (4-APC) is the chief constituent responsible for the anti-inflammatory, antioxidant, wound healing, and cytoprotective properties of Piper betel (Piperaceae). Its antioxidant and anti-inflammatory properties are attributed to its COX-2, iNOS, NF-kappaB, IL-12 p40, and JNK inhibitory effects [7-8]. We hypothesize 4-APC could inhibit calpain and result in neuroprotection from oxidative stress and inflammation in CIPN. In the present study, we report the beneficial effects of 4-APC and its derivatives against OIPN.

MATERIALS AND METHODS
Tumour Necrosis Factor and ELISA kit were purchased from Himedia, Mumbai, Koma Biotech Inc., respectively. All other reagents and chemicals were analytical grade.

Synthesis of 4-APC Derivatives
4-APC derivatives were synthesized by monoallylation of catechol Scheme 1. The reaction of catechol with an equimolar amount of allyl bromide in presence of K 2 CO 3 and acetone at 65 °C for 4 h gave the mono allyl ether 1. The thermal rearrangement of 1 at 170°C for 1 h gave 2:1 regioisomeric mixture, i,e, 3-allyl catechol and 4-allylcatechol 2 respectively.
The structural components of Allylpyocatechol derivatives that is APC-1 and APC-2 were determined by 1 H NMR (400 MHz; CDCl3) and 13 C NMR (101 MHz; CDCl3). The spectrum graphical charts ( 1 H NMR, 13 C NMR) for APC-1 were shown in Fig. 1 and Fig. 2. Also, the Nuclear Magnetic Resonance spectrum for APC-2 is shown in Fig. 3

Cells and Culture Conditions
SH-SY5Y cells were maintained at 37 o C with 5% CO 2 in DMEM medium supplemented with 10% of FBS and 1% penicillin-streptomycin. ) were seeded in a 96 well plate and incubated at 37°C. The cells were exposed to varying concentrations of OP/4-APC/APC-1/APC-2 in DMSO. After incubation for 24h, the cell viability was assessed using MTT assay [9].

In vivo Neuroprotective Study in Mice
24 Swiss Albino mice were divided into 4 groups of 6 each. The mice in Group 1 (normal) & Group 2 (control) have received 5% w/v Glucose solution, 10 ml/kg, i.p.. Groups 3 and 4 received a daily dose of 4-APC, 10 and 30 mg/kg, i.p., respectively. All the mice except Group 1 received OP (1mg/kg. i.p., twice a week). The study was conducted for 6 weeks. The body weights, mechanical and thermal nociceptive thresholds were evaluated during the study period. Mice were sacrificed at the end of the study under deep ether anesthesia, to collect sciatic nerves and dorsal root ganglion (DRG) for estimation of biochemical parameters [10].

Hyperalgesia (Thermal)
The thermal hyperalgesia to both hot and cold, studied using tail immersion in hot (45 o C) and cold water (10 o C). In the tail immersion test, the tail-flick latency is the endpoint. The cutoff time is 15 s. Three consecutive readings were taken at 30 min intervals [11].

Hyperalgesia (Mechanical)
Mechanical hyperalgesia was studied by the pressure stimulation method as described by Randall and Selitto [12].

Biochemical Analysis
Biochemical analysis was done in sciatic nerve tissue homogenate.

Estimation of Tumor necrosis factor-alpha (TNF α)
Commercially available ELISA kits from Biovision(CA, USA) for assaying TNF-α and IL-6 proteins were used and levels expressed as pg/mg of protein in sciatic nerve homogenate [15].

Statistical analysis
Data are expressed as means ± SEM. Statistical significance was done using t-test and ANOVA. If p < 0.05 it is considered as significant. GraphPad Prism 6 software was used for statistical analysis.

Cell Viability
From

Effect of 4-APC and Derivatives on Pain Behaviour
OP treatment is associated with a significant decline in paw withdrawal pressure as compared to the normal group. OXA significantly prevented the lowering of paw withdrawal thresholds compared to the normal group on days 16, 20, and 28 (p<0.05). OP treatment at 10 and 30 mg/kg showed dose-dependent protection against OP-induced altered mechanical nociceptive threshold (p<0.05), Table 2.

Effect of 4-APC and Derivatives on Inflammatory Markers
OP resulted in a significant change in the inflammatory markers such as nitrate Fig. 6A,

CONCLUSION
A compelling body of evidence suggests the key role of neuroinflammation and oxidative stress in OP-induced CIPN. It was reported that calpain activation results in oxidative stress and neuroinflammation in CIPN. 4-APC is a polyphenol with antioxidant and antiinflammatory propensity [5, [16][17]. In the present study, 4-APC administration prevented OPinduced alterations in MDA level which means that its administration decreased the level of lipid peroxidation. OP administration significantly decreased the levels of SOD in sciatic nerves of mice. 4-APC treatment improved the SOD levels in the sciatic nerve. Hence, we can conclude that 4-APC can effectively combat chemotherapyinduced oxidative stress in rodents. The molecular mechanism of CIPN has also been reported to modulate TNF-alpha expression and myeloperoxidase (MPO) activation which plays a Crucial role in axonal degeneration, suggesting that oxidative stress and neuroinflammation play an important role in CIPN [18]. In the present study OP administration significantly increased the levels of TNF-α and MPO. The administration of 4-APC significantly reduced the levels of these pro-inflammatory mediators. Hence the 4-APC mediated neuroprotection may be partly attributed to its inhibitory activity against cytokines. In conclusion, the results of the present study demonstrated the prophylactic effect of 4-APC against OP-associated CIPN.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is approved by IAEC having the reference number of 1176/PO/Re/S/08/CPCSEA.