Neuroprotective Effect of Phloroglucinol in Cisplatin Induced Peripheral Neuropathy

INTRODUCTION

Cisplatin is most widely-used anti-neoplastic drugs over the last 40 years. It displays therapeutic efficacy in a broad range of solid tumors especially against testicular, ovarian and bladder cancers. It exerts its antitumor activity by binding to DNA and distorting the helical structure in a way that inhibits transcription and induces apoptotic cell death through DNA damage recognition pathways.¹ Peripheral neuropathy is a dose - limiting and disabling side effect of several important chemotherapeutic agents, including, vincristine, cisplatin, oxaliplatin, paclitaxel, docetaxel etc. Its prevalence in patients treated with chemotherapeutic agents is 30-40%. However, upto 60% incidences have been also reported with cisplatin.² Chemotherapy induced peripheral neuropathy (CIPN) is often causing weakness, numbness and pain, usually in hands and feet, but it may also occur in other areas of body. It is thought to result when sensory neurons generate impulse at abnormal (ectopic) locations, for example at sites of nerve injury or demyelination. The prevalence of neuropathic pain seems to be increasing to the aging population as well as the increasing use of neurotoxic agents.

The dorsal root ganglia (DRG) are the main target of platinum drug–induced CIPN. Although most of the presented results were obtained in cisplatin models, it is likely that the pathophysiology of chronic CIPN induced by carboplatin and oxaliplatin is similar. The body of the experimental evidence points toward 2 different putative mechanisms, not necessarily mutually exclusive, because both can eventually produce DRG neuron apoptosis: (i) the formation of platinum intrastrand adducts and inter-strand crosslinks, which influence the tertiary structure of the nuclear DNA, alterating cell-cycle kinetics and (ii) the interaction with mitochondrial DNA, leading to oxidative stress and possibly to p53 increased activity and mitochondrial release of cytocrome-c pathway, independent of fas receptor activation, as well as activation of p38 and ERK1/2. These mechanisms do not explain the acute, transient symptoms induced by oxaliplatin that have been reported to be secondary to oxalate-induced dysfunction of nodal axonal voltage-gated Na⁺ channels.

Most of the studies reported were based on the hypothesis that the same genes relevant in the treatment of cancer cells might be similarly relevant also for neurotoxicity. This is an unproven assumption that led to focus the investigation mostly of key genes for intracellular detoxication, DNA repair and drug cellular influx or efflux. Glutathione S-transferase-1 (GST1) is one of the most important oxidative stress agent scavengers acting by catalysing the conjugation of many hydrophobic and electrophilic compounds with reduced glutathione and has been examined in oxaliplatin-treated patients. GSTP1 Ile105Val single nucleotide polymorphism (SNP) has been targeted by 20 trials which reported a positive correlation with CIPN in about half of the studies.³

Phloroglucinol has been reported to have vast activities such as anti-spasmodic⁴ , neuroregenerative⁵ , anti-inflammatory⁶ , anti- cancer⁷ ,anti-oxidant8 , anti- depressant⁹ , anti-proliferative¹⁰, anti-stress¹¹ and anti –ulcer¹². Phloroglucinol, being an antioxidant might reduce oxidative stress caused by ROS in cisplatin induced neurotoxicity. It is also hypothesized that the phloroglucinol can regenerate nerve fibres in peripheral neuropathy. So in our study we hypothesize that phloroglucinol, when used as supportive therapy might protect the CIPN in rats.

MATERIALS AND METHOD

Chemical reagents

Cisplatin, Phloroglucinol and other chemicals were purchased from Cipla Pvt. Ltd, Himedia and Sigma Pvt. Ltd. Respectively. Other reagents and chemicals were of analytical grade.

Animals

Adult male Wistar rats weighing160-200 g were used in the study. The uses of animals in our experiment were approved by IAEC (Institutional Animal Ethical Committee) and CPCSEA guidelines were followed for handling of animals. Animals were maintained under controlled temperature at 20±20 °C and relative humidity of 50-60 % with an alternating 12 h light/dark cycle (light on 6:00-18:00 h) and water provided ad libitum. The animals were fed with commercially available standard pellet chow (Amrut Feeds, Bangalore).

Induction of peripheral neuropathy and groupings

Cisplatin (2 mg/kg, i.p. twice weekly) was administered for 6 weeks to induce peripheral neuropathy in rats.²⁰ The dose of cisplatin to induce neuropathy was standardized in our laboratory. Then animals were divided into 4 groups. Group I served as normal control (Distilled water p.o for 8 weeks), Group II as Cisplatin control (2 mg/kg i.p for 6 weeks), Group III and Group IV served as treatment group where Cisplatin control (2 mg/kg i.p for 6 weeks) rats were treated with phloroglucinol (250 mg/kg & 400 mg/kg p.o for 8 weeks) respectively. The dose of phloroglucinol was decided based on LD₅₀ studies¹³ reporting that LD₅₀ (oral) in rats of > 4000 mg/kg bw. After 8weeks of the study blood was withdrawn from retro orbital sinus under light anesthesia for measurement of biochemical parameters. Behavioral studies were carried out before starting day 0 and after completion of 8 weeks to know the preventive effect of phloroglucinol after stopping of the cisplatin. Rats were sacrificed by overdose of anesthesia and sciatic nerves were isolated for nerve conduction velocity and for measuring antioxidant enzyme in tissue homogenates.

Behavioral studies

Tail immersion test

Cold hyperalgesia and thermal hyperalgesia were assessed by immersing terminal part of the tail (1 cm) in water. Temperature was maintained at 46 °C in thermal hyperalgesia and 4 °C Cold hyperalgesia. Tail withdrawal response was recorded as response to heat and cold sensation with a cut-off time of 20s.¹⁴

Mechanical allodynia

Mechanical allodynia was measured using electronic von-Frey (Fabricated electronic von frey instrument) as per the published literature.¹⁵

Mechanical hyperalgesia

Mechanical hyperalgesia was quantified using a fabricated electronic Randall-Selittodevice as per the published literature.¹⁶

Measurement sciatic of nerve conduction velocity

A sciatic nerve conduction velocity test (SNCV) measured by using A-D instrument, Australia. Rat sciatic nerve having a length of 3 – 5 cm was prepared and mounted into the nerve chamber consisting of small quantity of phosphate buffer pH 7 to humidify the compartment. First pair of 5 mm spaced electrodes is connected via the stimulator cable (MLA 270) to output 1 and 2 of the power lab unit. The recording cable (MLA 285) was connected to the input 1 of the power lab and microhooks of the same cable to the desired electrode wires depending upon the length of the sciatic nerve. After recording a set of responses latency was calculated using lab chart 7 software. Distance between the stimulating electrode and recording electrode was measured. Nerve conduction velocity (m/sec) was calculated using the following formula: NCV = Distance (mm) / latency.¹⁷

Haematological estimation

After completion of experimental period, approximately 0.5 ml of blood was drawn from the retro-orbital plexus of the rats under light anaesthesia. Each blood sample was collected in clean, labelled and dry micro-centrifuge tubes containing 10% EDTA solution (0.5 ml blood). The blood samples were analyzed within 6 h for the estimation of haemoglobin, WBC, RBC and platelet count.¹⁸

Biochemical estimation

All the experimental animals were sacrificed by overdose of anesthesia after the experimental period. For the estimation of biochemical parameters sciatic nerve was rinsed and homogenized (10% w/v) in 0.1 M phosphate buffer (pH 7.0) and centrifuged for 10 min and the resulting supernatant was used for estimation of lipid peroxide (malondialdehyde (MDA))¹⁹, catalase activity²⁰ and superoxide dismutase (SOD).²¹ The remaining supernatant was centifused at 3200 rpm for 20 m and used for the estimation of reduced glutathione (GSH).²²

Statistical analysis

Values are expressed as Mean±SEM (n=6). The data were statistically analyzed by One way ANOVA followed by Dunnet’s Multiple Comparison Test. Cisplatin treated group *P< 0.05 were considered as significant, **P<0.01 were considered as highly significant and ***P<0.001were considered as very highly significant when compared with cisplatin control group.

RESULTS

Effect on behavioural studies 

Cold and thermal hyperalgesia

Cispaltin treated rats exhibited significantly lower tail withdrawal latency when compared to that of normal rats, which indicates development of hyperalgesia in cisplatin control rats. Treatment with phloroglucinol (250 mg/kg) significantly increased the tail withdrawal latency when compared to cisplatin control rats while phloroglucinol (400mg/kg) did not exhibit any significant effect when compared to cisplatin control rats (Fig No. 1a & 1b).

Mechano-tactile allodynia

Cispaltin treated rats exhibited significantly lower paw withdrawal threshold when compared to that of normal rats, which indicates development of allodynia in cisplatin control rats. Treatment with phloroglucinol (250 mg/kg) significantly increased the paw withdrawal threshold when compared to cisplatin control rats while phloroglucinol (400 mg/kg) did not exhibit significant effect when compared to cisplatin control (Fig No.2).

Mechanical hyperalgesia

Cispaltin treated rats exhibited significantly lower mechanical threshold when compared to that of normal rats, which indicates development of hyperalgesia in cisplatin control rats. Treatment with phloroglucinol (250 mg/kg) significantly increased the mechanical threshold when compared to cisplatin control. Phloroglucinol (400 mg/kg) did not exhibit significant effect when compared to cisplatin control (Fig No.3).

Effect on sciatic nerve conduction velocity

Sciatic nerve conduction velocity of cisplatin control rats was significantly lower than that of normal rats. Sciatic nerve conduction velocity in rats treated with phloroglucinol (250, 400 mg/kg oral) was significantly higher than the cisplatin treated rats (Fig No.4).

Effect on haematological parameters

Cispaltin treated rats exhibited significantly lower haematological parameters when compared to that of normal rats. WBC count in rats treated with phloroglucinol (250, 400 mg/kg oral) showed no significant changes as compared with cisplatin treated rats. RBC count, haemoglobin content and platelet count in rats treated with phloroglucinol (250, 400 mg/kg oral) are significantly higher than the cisplatin treated rats (Table No.1).

Effect on biochemical parameters

Cispaltin treated rats exhibited significantly lower Catalase activity, superoxide dismutase activity and reduced glutathione levels when compared to that of normal rats. Catalase activity, superoxide dismutase activity and reduced glutathione levels in rats treated with phloroglucinol (250 mg/kg oral) was significantly higher than the cisplatin treated rats. Cispaltin treated rats exhibited significantly higher Malonaldehyde level when compared to that of normal rats. Malonaldehyde level in rats treated with phloroglucinol (250 g/kg oral) were significantly lower than the cisplatin treated rats (TableNo.2).

DISCUSSION

Most chemotherapeutic drugs penetrate the blood–brain barrier (BBB) poorly, but readily penetrate the blood–nerve-barrier (BNB) and bind to the dorsal root ganglia (DRG) and peripheral axons. Experimental studies reveal that chemotherapeutic drugs preferentially accumulate and bind in the DRG and peripheral nerves. The blood-nerve barrier is less efficient than the BBB, specifically deficient at the areas of the DRG and nerve terminals, which allows easier access for potential neurotoxins into the periphery.²³

Cisplatin is an important component of chemotherapy used against various tumour types such as pancreatic cancer, breast cancer, osteosarcoma and metastatic melanoma however their clinical use is severe by dose-limiting neurotoxicity. Along with this mechanism cisplatin has also been reported to cause ROS generation and causes lipid peroxidation. Studies suggest that cisplatin reduces peripheral nerve blood supply through potent angiogenic effect which may predispose the nerve damage. In this study administration of cisplatin (2 mg/kg i.p weekly twice for 6 weeks) in rats exhibited decreased bodyweight, thermal hyperalgesia, decreased mechanical allodynia, mechanical hyperalgesia, nerve conduction velocity deficits and decreased antioxidant levels all of which are symptoms frequently occurring in patients with peripheral neuropathy. All these explain that peripheral neuropathy was successfully induced in rats and this model can be used for the screening of neuroprotective drugs for the treatment of cisplatin induced peripheral neuropathy. In the present study treatment with phloroglucinol significantly alleviated cisplatin-induced peripheral neuropathy in rats. Rats treated with these drugs exhibited increased cold and thermal hyperalgesia, improved nerve conduction velocity, improved mechanotactile sensitivity and mechanical hyperalgesia when compared with cisplatin control rats.

Cisplatin induces oxidative stress by the autoxidation of monosaccharides, which leads to production of superoxide and hydroxyl radicals. It is well documented that pain transmission requires production of reactive oxygen species. In the present study results a significantly higher level of lipid peroxidation marker, MDA, in sciatic nerve of cisplatin control animals was observed. Glutathione (GSH), a potent endogenous antioxidant is a first line of defense against free radicals. The GSH levels were significantly lowered in the sciatic nerve of cisplatin rats compared to control group of rats. The treatments with phloroglucinol significantly reduced lipid peroxidation and increased intracellular antioxidant enzymes content in the sciatic nerve; this is probably because of their free radical scavenging activities.

Further, reports suggest that neurotoxic chemotherapy agents can damage autonomic nerves in the bone marrow and compromise hematopoietic stem cells mobilization and hematopoietic regeneration. This study results provide evidence that Phloroglucinol improves the haematological status by showing significant increase in haemoglobin, platelet and RBC as compared to cisplatin treated rats and the WBC count in treated groups remained normal without any significant difference.

CONCLUSION

This study has demonstrated that cisplatin induced neuropathy can be suppressed by treatment with phloroglucinol. The neuroprotective effect of phloroglucinol is due to its anti-nociceptive and anti-oxidant mechanism. This shows phloroglucinol when administered as supportive treatment along with cisplatin presents a promise for preventing and/or delaying cisplatin induced neuropathy.

ACKNOWLEDGEMENTS

The authors are thankful to the management and Principal of Al-Ameen College of Pharmacy, Bangalore, India, for providing financial assistance and facilities.

Values are expressed as Mean±SEM(n=6). The data were statistically analyzed by One way ANOVA followed by Dunnet’s Multiple Comparison Test. Cisplatin treated group *P< 0.05 were considered as significant, **P<0.01 were considered as highly significant and ***P<0.001were considered as very highly significant when compared with cisplatin control group.

Values are expressed as Mean±SEM (n=6). The data were statistically analyzed by One way ANOVA followed by Dunnet’s Multiple Comparison Test. Cisplatin treated group *P< 0.05 were considered as significant, **P<0.01 were considered as highly significant and ***P<0.001were considered as very highly significant when compared with cisplatin control group.