Three cannabis products attenuated oxaliplatin-induced peripheral neuropathy by inhibiting proteins that mediate oxaliplatin transport.

doi:10.21203/rs.3.rs-3375771/v1

Download PDF

Research Article

Three cannabis products attenuated oxaliplatin-induced peripheral neuropathy by inhibiting proteins that mediate oxaliplatin transport.

https://doi.org/10.21203/rs.3.rs-3375771/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Oxaliplatin induced peripheral neuropathy (OIPN) has greatly limited its clinical application. The aim of this study was to investigate whether three plant cannabinoid products could reduce OXA-induced peripheral neurotoxicity by selectively inhibiting OXA uptake transporter expression. The results showed that the three cannabinoid products with CBD as the main component could effectively inhibit the expression of transporter OCT2/OCTN1/OCTN2, thereby reducing the platinum content in DRG and inhibiting OIPN. And promote the anti-tumor effect of OXA. Among them, full spectrum CBD containing 0.3%THC and other secondary cannabinoids has the most significant therapeutic effect, and the safe therapeutic dose range is wider. These results suggest that CBD down-regulates the expression of OXA transporter and inhibits the main component of OIPN. The addition of THC and other secondary cannabinoids can overcome the dose limitation of purified CBD and exert more significant therapeutic effect in synergy with CBD.

OIPN

Cannabinoid products

CBD

DRG

OCT2

OCTN1

OCTN2

1.CBD reduces platinum accumulation in DRG by inhibiting transporter expression

2.The secondary components of plant cannabinoids play a synergistic effect with CBD in the treatment of OIPN

3.The secondary component of phytocannabinoids breaks the therapeutic dose limit of purified CBD

4.THC plays a major role in inhibiting cold pain sensitivity

Oxaliplatin (OXA), a third-generation platinum drug, has been widely used in the clinical treatment of colorectal cancer, breast cancer, and other solid tumors. However, neurotoxicity is the main reason for dose reduction and limitation during cancer therapy. According to a survey, about 90% of patients treated with oxaliplatin have peripheral neuropathy (OIPN) of varying degrees¹. With the accumulation of doses, chronic neurotoxicity from OXA can be induced. After patients stop taking oxaliplatin, their neurological symptoms can continue to worsen for months or even years (the sliding phenomenon), which increases their pain, reduces their quality of life, and leads to a dilemma in their treatment decisions². Limiting the clinical use of OXA At present, there is a lack of effective prevention and treatment methods for OIPN in clinical practice³. Therefore, it is urgent to find effective drugs that maintain anti-tumor activity to effectively treat OIPN.

The mechanism of oxaliplatin-induced peripheral neuropathy is complex. The dorsal root ganglia (DRG) lacks blood-brain barrier protection in the nervous system and is highly vulnerable to toxic damage. OXA is distributed in the DRG in a dose-dependent manner in vivo⁴, damaging the nuclear DNA of neurons and inducing apoptosis. At the same time, due to the lack of nucleotide excision and repair pathways in mitochondria, OXA forms DNA-PT adjuncts with DNA in mitochondria, resulting in irreversible changes in mt-DNA epigenetics, mitochondrial vacuoles, and degeneration, resulting in mitochondrial dysfunction, abnormal inter-axonal transmission, and neuropathic pain. DRG neurons and mitochondria are the main target cells for the initiation and progression of OXA-induced peripheral neuropathy^5–7. Studies have shown that OXA content in DRG can still be maintained for several weeks after OXA treatment is stopped, and the severity of OIPN is related to drug levels⁸. Therefore, effective inhibition of OXA entering the DRG may be an effective measure to prevent or even reverse OIPN.

OXA as a carrier-dependent passive transport drug In DRG neurons⁹. OXA is effectively transported from extracellular to intracellular accumulation mainly through the cationic transporter OCT2 on the surface of the cell membrane and the zwitterion transporters OCTN1 and OCTN2 in mitochondria, and the transport efficiency of OCT2 is higher than that of OCTN1/OCTN2, which is the main reason for promoting the neurotoxicity of platinum drugs^9–11. Yaodong Yi et al. demonstrate that deletion of the OCT2 gene combined with the OCT2 receptor competitor cimetidine completely reverses the mechanical pain abnormalities and heat sensitivity of OIPN¹². At the same time, OCT2、OCTN2 expression is low in tumor cells, so selective inhibition of OCT2 、OCTN2expression without interfering with the anti-tumor effect of oxaliplatin may be the best target for the treatment of OIPN^13,14.

Cannabis, a plant of the genus Cannabis, has been used medicinally for thousands of years. The main components include cannabidiol (CBD), tetrahydrocannabinol (THC), cannabis terpene mushrooms, flavonoids, and other secondary components. With the increase in interest in the medical effects of cannabinoids, an agricultural cannabis extract with a THC content of less than 0.3% on a dry weight basis was introduced to break the traditional phytocannabinoid neurohallucinogenic, addictive, and other psychotoxic side effects in medical applications¹⁵. In recent years, high attention has been paid to the field of chemotherapy-induced peripheral neuropathy, in which THC, as the earliest active compound discovered from phytocannabinoids, has been demonstrated to effectively inhibit chemotherapy-induced peripheral neuropathy pain by agonizing the endogenous cannabinoid receptor in a number of rodent models^16,17.CBD, as the main non-psychoactive component of phytocannabinoids, has antagonized the neurotoxic side effects of THC and has been a research hotspot^18–20. A number of preclinical experiments have shown the anti-nociceptive effect of CBD on mechanical pathological pain caused by platinum drugs and paclitaxel or other pathological conditions, and CBD + OXA/PTX can synergistically inhibit the activity of tumor cells^21–25.Clinical trials have also shown that oral or transdermal topical application of CBD oil containing only CBD or CBD oil rich in less than 0.3% THC can prevent and attenuate peripheral nerve mechanical pain in patients receiving PTX or bortezomib adjuvant therapy to varying degrees without obvious toxic or side effects²⁶. Kirsten M. King et al. attenuated oxaliplatin-induced mechanical sensitivity by administering CBD alone and an ineffective dose of THC plus CBD, indicating a synergistic effect of the two in anti-peripheral neuropathy²⁷. Application of purified CBD has also been reported to elevate liver enzymes and increase the risk of hepatotoxicity¹⁷. The application of CBD-rich phytocannabinoid extracts (64.5% CBD, 4% THC, 4% other cannabis components, and a small amount of terpenes and flavonoids) can completely relieve thermal hyperalgesia, and the effect is significantly better than that of the CBD, THC, and THC + CBD groups, and the side effects are also less than those of the first three groups. These results suggest that cannabinoid terpenoids and flavonoids may have the ability to inhibit the metabolism of CBD and THC and improve the bioavailability of the drug, which confirms the synergistic effect of the active components of cannabinoids²⁸.In 2022, Diana E. et al. also showed that whole plant cannabis extract rich in high doses of CBD was more effective than THC and CBD alone in inhibiting the mechanical pain response induced by OXA in mice with peripheral neuropathy, which again confirmed that the internal components of whole plant cannabinoids synergistically played a more significant anti-peripheral nerve pain effect²⁹. Therefore, whole plant cannabinoids may be an effective treatment for antagonism and prevention of OIPN.

Many of the above studies have shown that cannabinoids can effectively inhibit nerve pain, but the mechanism of action is not very clear. Some researchers have proposed that a synthetic cannabis analogue, AB-FUBINACA, and cannabinoid extracts rich in different ratios of CBD and THC active components can inhibit OCT2 and OCTNS transporter activities to varying degrees^30,31. It affects drug (3-MMP) transport. OCT2/OCTNS, as the main membrane surface transporters involved in OXA transport, are highly expressed in DRG, which is significantly correlated with the occurrence and development of OIPN¹¹. At the same time, it has been found that OXA can promote the further expression of the OCT2 transporter and the accumulation of platinum in tissues³². Therefore, whether phytocannabinoids can prevent and reduce the occurrence of OIPN by inhibiting PT transport is worthy of further investigation and study.

The present experiment investigated whether plant cannabis extract reduces OXA accumulation in DRG neurons through selective inhibition of OCT2 and OCTN1 and OCTN2 expression and alleviates OXA-induced peripheral neurotoxicity without compromising its antitumor effect. By comparing the correlation between the differences in the effective components of three cannabinoids and the anti-neurotoxic effect, the main active components of cannabinoids against peripheral neuropathy are clarified, and basic experimental data and theoretical support are provided for the clinical application of cannabinoids.

2.1 Chemicals and reagents

(Fetal Bovine Serum, No. 10099141), 1640 (B0STER,PYG0006), DMEM (B0STER,PYG0095) were purchased from Wuhan Bode Biological Engineering Co., LTD. (Wuhan, China). OXA(O124003-100mg) from Aladdin Reagent Co., LTD. (Shanghai, China), and was dissolved in 5% glucose solution. OCTN1 (ab200641), OCTN2 (ab180757), OCT2 (ab170871), and Glyceradehyde-3-phosphate dehydrogenase (GAPDH) (GAPDH(D16H11)XP® Rabbit mAb #5174) were purchased from Cell Signaling Technology. Three phytocannabinoid products were purchased from Heilongjiang Zhongsheng Biotechnology Co., LTD. (Daqing, China): Full-spectrum CBD is an extract of cannabis flowers and leaves that has not been further purified and isolated and contains less than 0.3% THC in addition to 85% CBD, terpenes, other cannabinoids, vitamins, minerals, fatty acids, phytonutrients, and other substances extracted from the cannabis plant. Broad Spectrum CBD: THC is completely removed from the full spectrum CBD oil extraction process, and the other components are consistent with full spectrum CBD, which still contains 85% CBD; Purified CBD: CBD crystals that are isolated and purified from industrial hemp flowers and leaves can be up to 99% pure. and was dissolved in dimethyl sulfoxide or (Tween 80 – salinesolution 1:16 v/v³³for use in in vivo and in vitro studies.

2.2 Cell culture

Rat pheochromocytoma cell lines (PC12 (poorly differentiated TCR8)), human colon cancer cell lines (HCT-116), and human breast cancer cell lines (MCF-7) were purchased from the Center of Excellence in Molecular Cell Science, Chinese Academy of Sciences. PC12 cells were grown in RPMI 1640 medium enriched with 10% fetal bovine serum. HCT-116 was grown in a medium containing 10% fetal bovine serum (RPMI 1640) (Meilen). MCF-7 was grown in DMEM medium containing 0.5% insulin, 1% non-essential amino acids, and 10% fetal bovine serum. All cells were cultured in a constant-temperature incubator at 37°C in wet air with 5% carbon dioxide.

2.3 Cytotoxicity

2.3.1 Assessment of PC12 cytotoxicity and neural protrusion growth

PC12, a rat pheochromocytoma cell line, diferentiates in the presence of forskolin and is used as a model of neurodegeneration. We inoculated PC12 cells on 96/24-well plates with a density of 2.5 104 cells/cm2 for 24 hours, changed the solution every other day, and added 50 ng/ml growth factor (Sutaicheng (Beijing) Biological Co., LTD., China). was continued to be cultured in complete culture medium until PC12 was fully differentiated and pre-incubated in culture medium (full spectrum, broad spectrum, purified CBD 0.1–25 vg/L) for 24 hours, and then OXA (100 M 96-well plate) and OXA (3 M 24-well plate) were added for 24 hours. Construction of an OXA-induced neurotoxicity in vitro cell model³⁴. At the same time, a blank group and a control group treated with OXA alone were set up. After culture, the activity of 96-well plate cells was measured by the MTT method and phase contrast microscope (IX53 Olympus Corporation, Tokyo, Japan) analysis of cells in the 24-well plate. The neurite length was measured using the ImageJ 1.51 software (Wayne Rathband, National Institutes of Health, MD, USA).

2.3.2 Tumor cytotoxicity assay

To assess whether combined cannabinoid treatment would promote tumor growth, HCT116, MCF-7 cells were seeded in 96-well plates at a density of 1.0×104 cells/well. The next day, cells were treated with 0.1-25vg/L of each of the three cannabinoids for 24h. Cell viability was assessed using the MTT assay. Each experiment was performed in five Wells per group.

To assess whether cannabinoid combination treatment its affects the antitumor therapy of OXA, HCT116 cells were inoculated in 96-well plates at a density of 1.0×104 cells/well. The next day, cells were treated with 0.1–25 vg/L of the three cannabinoids for 24 h, followed by the addition of IC50uMOXA (HCT116) and incubated for 24 h. Cell viability was assessed using the MTT method. Each experiment was performed in 5 wells per group.

2.4 Animals

Male C57BL/6 mice (10-week-old, 25–32 grams) were used as models of peripheral neuropathy. All animal treatment procedures and study protocols were double-blind, with animals randomly assigned to treatment in groups of nine per cage on a 12-hour light-dark cycle. The animals have free access to water and food. All animal experiments were approved by the Jiamusi University Laboratory Animal Care and Use Committee and conducted in accordance with the guidelines of the National Institutes of Health and the International Association for the Study of Pain.The animals were used for experiments after one-week adaptive feeding.

To evaluate the neuroprotective effects of the three cannabinoids, mice were randomly divided into eight groups, with nine mice in each group: Control group, OXA alone group, OXA combined with purified CBD low dose group (10mg/kg OCBDL), OXA combined with purified CBD high dose group (30mg/kg OCBDH), OXA combined with broad spectrum CBD low dose group (10mg/kg OBSCL), OXA combined with broad spectrum CBD high dose group (30mg/kg OBSCH), OXA combined with full spectrum CBD low dose group (10mg/kg OFSCL), OXA combined with full spectrum CBD high dose group (30mg/kg OFSCH).The drug dosage was determined according to previous experimental reports^35,36. The establishment method of the oxaliplatin-induced chronic peripheral neuropathy animal model was referred to in the previous study of Sakurai M. et al.³⁷ Oxaliplatin (4mg/kg, ip) twice a week for four weeks (days 1, 4, 8, 11, 15, 18, 22, and 25) is equivalent to 130mg/m2 injected into the human body. In the combined group, OXA was intraperitoneally injected 30 minutes after the intraperitoneal injection of three cannabinoid products for four weeks (days 1, 4, 8, 11, 15, 18, 22, and 25). Animal behavior experiments were monitored and recorded 24 hours after each dose. After the last behavioral experiment, the animals were killed by OXA injection, and the CRG and sciatic nerve were collected for analysis.

2.4.1 Assessment for Mechanical Allodynia (von Frey Test)

Baseline sensitivity to mechanical stimuli was assessed prior to drug treatment in mice as described above. The remaining behavioral tests were performed 24 hours after dosing and at days 5, 9, 12, 16, 19, 23and 26.

A fiber-stimulated plantar withdrawal threshold (Von Frey) test was used to evaluate the efficacy of three cannabinoids on mechanical ectopic pain. Each mouse was adapted in a wire mesh box for 30 minutes before the test. The method of the von Frey test was described in a previous report³⁸. The plantar withdrawal thresholds for stimulations by von Frey flaments (0.02-2.0g, North Coast Medical, San Jose, CA, USA) were determined using a modified up-down method. Each experiment was performed on five to nine animals per group.

2.4.2 Assessment for Cold Hyperalgesia (Acetone Test)

The method was described in the previous report³⁷. Mice were adapted to the wire mesh box in the same manner as the von Frey test. Precooled acetone at 4°C was dropped on the pain-sensitive area of the hind foot of mice, and the number of drawal responses was observed to determine the sensitivity of mice to cold stimulation. From the beginning of the spray time, the reaction was observed within 40 seconds, and the Acetone Score (AS) was used as the cold irritation pain. Three trials were averaged for each assessment, separated by 5–10 min intervals

2.4.3 Assessment for thermal Hyperalgesia

Thermal hyperalgesia was measured using the hot plate method, as previously reported ³⁹. A ZH-6C hot plate (Anhui Zhenghua Biological Instrument Equipment Co., LTD.) was used to evaluate the licking, lifting, and grasping time thresholds. The temperature of the hot plate was maintained at 55 ± 0.5°C, the mice were placed on the hot plate, and the time when the mice appeared to lick their hind PAWS or lift their hind feet was recorded. The above experiments were repeated twice with an interval of more than 15 min.

2.4.4 The rotarod test

Motor impairment was measured using the rotarod. Mice were placed on a drum which increased rotation speed from 4 to 30 rpm over 300 s. The time at which mice stopped running on the drum or fell off was recorded as the rotarod latency.

2.5 Assessment of Sciatic Axonal Degeneration

After the last behavioral test, 3 mice in each group were randomly selected and sacrificed under anesthesia, and the sciatic nerve was fixed with 4% paraformaldehyde. Nerves were embedded in Epon and stained with Luxol Fast Blue (LFB) after sectioning. Each section was sectioned using a light microscope (Olympus Corporation) for observation, image acquisition, and analysis. Axon roundness was calculated as a quantitative index of axonal degeneration⁴⁰.

2.6 Electron microscope

The dorsal root ganglion was washed three times with PBS and quickly placed into a 1.5 mL EP tube with electron microscope fixative and fixed at 4°C for 2–4 h. It was rinsed with 0.1 M phosphate buffer PB (PH7.4). Tissues were fixed in 1% osmium oxide-0.1M phosphate buffer PB (PH7.4) for 2 h at room temperature (20°C) and rinsed in 0.1M phosphate buffer PB (PH7.4). Tissues dehydrated, configured according to the ratio of acetone: 812 embedding agent = 1: 1, treatment 2–4 h; acetone: 812 embedding agent = 2: 1 ratio of the solution, infiltration overnight; pure 812 embedding agent treatment 5–8 h; pure 812 embedding agent poured into the embedding plate, samples inserted into the embedding plate after the oven at 37°C overnight. 60°C oven polymerization for 48 h. Ultrathin slicers section 60–80 nm ultrathin sections. DRG using uranium-lead double staining (2% uranyl acetate-saturated alcoholic solution, lead citrate, each stained for 15 min), slices were dried at room temperature overnight. Sections were dried at room temperature overnight. The sections were observed under a transmission electron microscope, and images were collected for analysis.

2.7 Determination of OXA concentration by ICP-MS

200 µL of HNO3 and 400 µL of H2O2 were added into the cell lysate, mitochondria, DRGs, or tumours; the mixture was incubated for 2 h at 80 ℃. Then, 200 µL of 25% ammonia solution was added, and the volume of the solution was adjusted to 4.0 mL with ddH2O. Finally, the concentrations of OXA were determined by ICP-MS after the solution was filtered using a film filter (0.22 µm). The OXA content was determined relative to the total protein content for cells or relative to the weight for tissues.

2.8 Western blot

DRG neurons and mitochondria were lysed in RIPA Lysis Buffer (Dalian Meilun Biotechnology Co., LTD) which contained 1 mM PMSF and protein concentrations were measured by BCA protein assay kits (Beyotime Biotechnology). After boiled at 100 ◦C with 5 min, protein extracts (50mg) from each sample were separated by sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Dalian Meilun Biotechnology Co., LTD) and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA), with protein from rat kidney for positive control. The membranes were blocked with 5% skimmed milk, then incubated with primary antibodies, anti-OCT2 antibody (1: 1000), anti-OCTN1 antibody (1: 1000), anti-OCTN2 antibody (1: 1000), antiGAPDH antibody (1: 5000) for overnight at 4 ◦C. The next day, membranes were washed 6 times with Tris-buffered saline/Tween 20 and incubated with secondary antibodyfor 2 h at room temperature. After washed 6 times with Tris-buffered saline/Tween 20 again, membranes were visualized using the enhanced chemiluminescence (ECL) kit and G-BOX

2.9 Statistic analysis

Studies in cell models, and animal models were independently repeated at least three times. The data analysis was double-blind. The correlation curve of cell viability log10-OXA concentration was fitted to obtain the median lethal dose (LD50), and the histogram was drawn to describe the cell viability among different groups. Data are expressed as the standard error (SEM) of the mean. For in vitro data, each point was obtained from at least three separate experiments, each with five duplicate holes. Multigroup comparisons were performed using two-factor analysis of variance (ANOVA), followed by Bonferroni or Dunnett's post-event tests. A P value of 0.05 is considered to have statistical significance. All data were analyzed by GraphPad Prism 9.5.1.

3.1 Purified, broad-spectrum, and full-spectrum CBD can inhibit the neurotoxicity of OXA-induced PC12 cells.

To determine whether the application of cannabinoid products alone causes toxic damage to nerve cells, we evaluated the effects of three cannabinoid products on the viability of differentiated PC12 cells in vitro. The results are shown in Fig. 1: Compared with the blank control group, purified CBD in the concentration range of 0.1–2.5 mg/L and broad spectrum and full spectrum CBD in the concentration range of 0.1–10 mg/L had no toxic effect on PC12 (P > 0.05), and the application of broad spectrum and full spectrum CBD had a wider dose range and higher safety.

In order to evaluate whether the three cannabinoid products can inhibit OXA-induced peripheral nerve cell toxicity, we established an OXA-induced neurotoxicity cell model in vitro using induced differentiation PC12 and observed the activity of OXA combined with the three cannabinoid products and OXA alone. As shown in Fig. 1B: compared with the blank control group, the cell activity of PC12 after OXA treatment decreased to 44.79%, P < 0.0001. Compared with OXA alone, purified CBD, broad-spectrum CBD, and full-spectrum CBD pretreatment groups inhibited OXA neurocytotoxicity in a concentration-dependent manner, respectively, and increased the cell activity to 64.17%. 86.65%; 86.73%, P < 0.05, was statistically significant. Therefore, all three cannabinoids can effectively inhibit OXA cytotoxicity and promote cell growth.

3.2 Purified, broad-spectrum and full-spectrum CBD can inhibit OXA-induced neurodegeneration.

In order to further confirm the inhibitory effect of cannabinoids on OXA-induced nerve cell toxicity, PC12 was inoculated on 24-well plates to induce differentiation, and 3 uMOXA was added to construct an in vitro model of oxaliplatin-induced peripheral nerve cell subdeath. The axon growth of PC12 was observed under a phase contrast microscope⁴¹. Using Image analysis software (Image J 1.51), The axon length was measured, and the results are shown in Fig. 2. Compared with the blank control group, about 22% of the total cells in the visual field accounted for an axon length larger than the cell body in the OXA treatment group, and the outward growth of neurites was significantly inhibited (p < 0.0001), indicating that the in vitro model of OXA-induced cell subdeath was successfully established. In the pretreatment group of purified CBD, broad spectrum CBD, and full spectrum CBD, the number of cells with an axon length larger than the cell body accounted for about 60% of the total cells; 65%; and 74%, compared with the OXA alone treatment group, significantly (p < 0.0001) improved neurite outward growth, in which full-spectrum CBD had the best effect on improving axonal outward growth.

3.3 The increased sensitivity of OXA to induced peripheral neuromechanical stimulation and pain from cold and heat stimulation was attenuated by cannabinoids

The above cell experiments confirmed that cannabinoids can reduce nerve cell apoptosis and play a protective role in peripheral nerve cells. To further confirm the effect of three cannabinoid products at different doses on OIPN in vivo, we conducted behavioral tests in animal models of OIPN, along with statistical analysis and graphical representation of the data, calculated the baseline percentage sensitivity value for each mouse, and determined the mean baseline percentage sensitivity (SEM) for each treatment group. Each mouse's baseline sensitivity was calculated as their sensitivity threshold on the test day divided by their sensitivity threshold at baseline multiplied by 100. Thus, a baseline sensitivity of 100% represents no development of sensitivity to mechanical or thermal stimulation after treatment, and a lower % value corresponds to an increase in sensitivity to mechanical or thermal stimulation. Results Fig. 3AB: Neuropathy in animals treated with OXA alone (4mg/kg, ip) persisted until the last day of the study. At 24 hours after the last administration (day 26), the baseline sensitivity to mechanical stimulation and hot plate stimulation of mice in the OXA treatment group was reduced by 82.19% and 43.17% compared with the blank group, the mice had significant mechanical and thermal stimulation allergies (P < 0.05), indicating that OIPN modeling was successful. The three kinds of cannabinoid products combined with the OXA treatment group effectively increased the baseline sensitivity percentage value compared with the OXA treatment group alone and effectively reduced the pain sensitivity to mechanical and thermal stimulation in mice (P < 0.05). The mechanical and thermal sensitivity of broad-spectrum CBD (low and high dose groups) and full-spectrum CBD (low and high dose groups) were lower than those of purified CBD (low and high dose groups). The baseline sensitivity curve of purified CBD in the high-dose group (30 mg/kg, ip) was slightly higher than that in the low-dose group (10mg/kg, ip), the anti-hyperalgesia effect did not improve with the increase in dose. The percentage of baseline sensitivity to mechanical stimulation and hot plate stimulation in the broad spectrum CBD and full spectrum CBD high dose groups (30 mg/kg, ip) was higher than that in the low dose group (10mg/kg, ip), the anti-pain sensitivity was positively correlated with drug dose. In the thermal pain behavior experiment 24 hours after the last administration, the baseline sensitivity percentage of the full-spectrum CBD high-dose group of mice recovered to 100%, there was no development of thermal stimulation sensitivity, which had a significantly excellent anti-thermal hyperalgesia effect.

For OXA-induced hyperalgesia of cold stimulation, as shown in Fig. 3C: Compared with the blank control group, the OXA group alone significantly improved the cold stimulation pain score (P < 0.001), showing a significant difference. Compared with OXA alone, purified CBD (low and high dose groups), broad spectrum CBD (low and high dose groups), and full spectrum CBD (low dose group) had no significant difference in cold stimulation pain scores (P > 0.05). The cold stimulation pain score of the full-spectrum cannabinoid high-dose group was significantly different from that of the OXA treatment group alone (P < 0.05), which effectively weakened the OXA-induced cold stimulation pain hypersensitivity. Therefore, combined with the results of three kind of pain stimulation behavioral experiments, the three cannabinoid products can effectively alleviate OXA-induced abnormal peripheral nerve hyperalgesia, and the therapeutic effect of the full-spectrum cannabinoid high-dose group is more significant.

In order to further prove whether the three plant cannabinoid products have the side effects of central nervous system toxicity, we conducted a mouse rotating rod behavioral experiment. The results were shown in Fig. 3D: there was no significant difference in the rotation time of mouse rotating rod movement among all groups (P > 0.05), which did not have statistical significance. Therefore, the three plant cannabinoid products used in this experiment have no central toxic or side effects in mice and have no potential for central addiction.

3.4 Three cannabinoid products reduce the accumulation of platinum in the DRG in mice.

DRG neurons are the main target cells for the initiation and progression of OXA-induced peripheral neuropathy in animals and humans and are the main site of platinum accumulation⁴. The accumulation of platinum content in the DRG is a recognized pathological mechanism of chronic peripheral neuropathy. The content of Pt in DRG tissues was determined by ICP-MS. Results As shown in Fig. 4, Compared with the blank group, the platinum content in the dorsal root ganglia of OXA mice alone reached 3.6mg/kg. The platinum content in the DRG of mice was significantly increased(P < 0.0001), with a statistically significant difference. Compared with the OXA alone treatment group, OXA combined with three plant cannabinoid products (low dose and high dose) effectively reduced the platinum content in the DRG of mice (P < 0.0001). However, we found that the platinum content in DRG in the purified CBD high-dose treatment group was slightly higher than that in the low-dose treatment group, indicating the dose limit of effective therapeutic effect. However, the platinum content in DRG in OIPN mice gradually decreased with the increase in dose of broad spectrum and full spectrum CBD, and the platinum content in DRG in the different dose treatment groups of broad spectrum and full spectrum CBD was lower than that in the purified CBD dose treatment group. Therefore, combined with the results of behavioral experiments, the three plant cannabinoid products inhibited the accumulation of platinum in DRG. Thereby improving OXA-induced abnormal peripheral nerve pain. The ability of broad-spectrum and full-spectrum CBD to inhibit platinum accumulation in DRG is similar to but better than that of purified CBD.

3.5 Three cannabinoids inhibited the expression of OCT2, OCTN1 and OCTN2 transporters.

OXA has been proven to be the main substrate of OCTNS and OCT2。The application of platinum-based drugs will promote the overexpression of the cationic transporter OCT2 and the accumulation of OXA in cells. In order to clarify whether three cannabinoid products inhibit OXA accumulation in DRG by interfering with transporter expression. We established an animal model of chronic OIPN, dissected mouse DRG, and performed tissue immunohistochemistry and Westen Bolt experiments. The results showed that: As shown in Fig. 5 (A–G), compared with the blank group, OXA alone induced up-regulation of OCTN1/OCTN2/OCT2 expression (P < 0.0001). The upregulation of OCT2, OCTN1, and OCTN2 could be effectively inhibited after combined administration of three plant cannabinoid products (P < 0.01), with significant differences. The inhibitory effect of broad spectrum and full spectrum CBD on the upregulation of the above transporters was better than that of purified CBD, and the inhibitory effect of broad spectrum and full spectrum CBD on the transporters increased with the increase in drug dose. The inhibitory effect of purified CBD in the high-dose group was no better than that in the low-dose group. Western blot results, as shown in Fig. 5 (H–J), were consistent with immunohistochemical findings, OXA alone induced up-regulation of OCTN1/OCTN2/OCT2 expression (P < 0.0001). Compared with the OXA alone treatment group, the three plant cannabinoid products could effectively inhibit the up-regulation of 0CT2, OCTN1, and OCTN2 expression (P < 0.01). The inhibitory effect of broad-spectrum and full-spectrum CBD on the expression of transporters increased with the increase in dose, but the inhibitory effect of purified CBD on the up-regulated expression of transporters did not increase with the increase in dose. The expression of transporters in the treatment groups of broad spectrum (low and high dose) and full spectrum CBD (low and high dose) was lower than that of purified CBD (low and high dose). This is consistent with our previous experimental results. The three plant cannabinoid products can inhibit the expression of 0CT2, OCTN1, and OCTN2 transporters in DRG, and the inhibition effect of broad-spectrum and full-spectrum CBD is better.

3.6 Three plant cannabinoid products reduced OXA-induced mitochondrial swelling and vacuolation in DRG neurons.

Previous experimental results have shown that OCTN1/OCTN2/OCT2 is also expressed in DRG mitochondria, and OXA can also bind to mitochondrial DNA when entering DRG neurons. Since mitochondria lack DNA damage repair enzymes, mitochondrial damage may be an important mechanism of chronic OIPN persistence⁴². Our experiments showed that three cannabinoid products effectively reduced OXA accumulation in the DRG and inhibited peripheral nerve hyperalgesia. In order to further demonstrate the inhibitory effect of cannabinoids on OIPN.We conducted mitochondrial electron microscopy observations in DRG. Compared with the blank group, multiple vacuoles and swelling of mitochondria in DRG nerve cells, as well as mitochondrial shrinkage, were observed in the OXA group alone, indicating that the animal model of OIPN was successfully constructed. In DRG treated with OXA combined with three cannabinoids (low and high dose groups), mitochondrial vacuoles and swelling lesions were significantly improved compared with the OXA alone treatment group. The recovery of broad spectrum and full spectrum CBD group mitochondrial lesions was positively correlated with the increase in dose. The mitochondrial vacuolar and swelling lesions of broad spectrum and full spectrum cannabinoids in the high-dose combination group basically returned to normal, with no significant difference compared with the blank control group. The mitochondrial vacuole and swelling lesions in the purified CBD high-dose combination group were greater than those in the low-dose combination group, and the mitochondrial protection effect decreased with the increase in dose, showing effective dose restriction. At the same dose, the inhibition effect of broad-spectrum and full-spectrum CBD on mitochondrial swelling and vacuole was greater than that of purified CBD. Therefore, broad-spectrum and full-spectrum CBD have a more significant therapeutic effect in inhibiting OXA-induced mitochondrial vacuoles, swelling, and other pathological changes.

3.7 Reversal of OXA-induced sciatic neuropathy by three cannabinoid products

To further confirm the effect of cannabinoids on reducing platinum accumulation and inhibiting OXA-induced peripheral neuropathy. We performed myelin observations in pathological sections of sciatic neuropathology and calculated the axon circle as a quantitative indicator of degeneration in three animals per group. Results As shown in Fig. 7, the circular rate of the sciatic nerve in the OXA group alone (0.542 ± 0.005) was significantly different from that in the blank group (0.764 ± 0.012) (P < 0.0001). OXA could significantly cause sciatic nerve degeneration. The round rate of sciatic nerve was 0.644 ± 0.004 and 0.617 ± 0.01. The purified CBD (low dose and high dose) treatment groups, and 0.668 ± 0.007 and 0.701 ± 0.011 in the broad-spectrum CBD (low dose and high dose) treatment groups, respectively. The round rate of sciatic nerve was 0.661 ± 0.009 and 0.721 ± 0.008 in the full spectrum CBD (low dose and high dose) treatment groups, respectively.They are all significantly increased the round rate of sciatic nerve axons compared with 0.542 ± 0.005 in the OXA alone group (P < 0.0001). Three cannabinoid products can effectively reverse OXA-induced sciatic neurodegeneration. With the increase in the dose of purified CBD, the round rate of the sciatic nerve decreased, and the effect of inhibiting OXA-induced peripheral neurodegeneration was weakened, which manifested as dose limitation. However, with the increase in dose of broad-spectrum and full-spectrum CBD, the round rate of the sciatic nerve gradually increased, and the dose-dependent inhibition of OXA-induced peripheral neurodegeneration was observed. Breaking the dose limit for purified CBD. Meanwhile, in the same dose treatment group of three cannabinoid products, the sciatic nerve circular rate in the full spectrum CBD treatment group was > broad spectrum CBD > purified CBD. Therefore, full-spectrum CBD is better than broad-spectrum CBD or purified CBD in inhibiting OXA-induced peripheral neurodegeneration.The full-spectrum CBD is a better choice for inhibiting OXA-induced sciatic neurodegeneration.

3.8 Three cannabinoid products have a synergistic OXA anti-tumor effect.

Our experimental results show that all three plant cannabinoid products can effectively inhibit OXA-induced peripheral neuropathy,providing a new direction for the clinical treatment of OIPN. In order to explore the effects of three plant cannabinoid products on tumor cell growth.We observed the effects of three kinds of plant cannabinoid products with different concentrations on the viability of tumor cells after incubation with HCT116 and MCF-7 cells for 24 hours. The results show: Compared with the blank control group, the cell viability of HCT116 was reduced to 65.50%, 46.97%, and 25.6% after purified, broad-spectrum, and full-spectrum CBD treatment alone for 24 hours. Compared with the blank control group, the cell viability of MCF-7 was decreased after purified, broad-spectrum, and full-spectrum CBD treatment alone for 24 hours. The cell viability decreased to 58.37%, 47.46%, and 43.56%, showing significant anti-tumor activity.The full-spectrum CBD had the strongest inhibitory effect on the cell viability of HCT116 and MCF-7, with a statistically significant difference compared with the blank control group (P < 0.0001).Three cannabinoid products together with OXA acted on HCT116 cells. The results showed that compared with OXA alone, the tumor cell activity of the three kind of cannabinoid pretreatment groups was further reduced and the synergistic OXA promoted apoptosis of tumor cells. Therefore, cannabinoids have an inhibitory effect on the value-added of tumor cells.

OIPN has always been a dose-limited and irreversible neurotoxic side effect that has plagued tumor patients and clinicians, interfering with the anti-tumor efficacy of OXA, increasing patient pain, and reducing patient survival. At present, there are no effective drugs to treat or prevent OIPN. Considering that OXA is widely recommended for the treatment of solid tumors such as the rectum and breast⁴³, There is an urgent need to find drugs that can prevent and treat OIPN. Although previous studies have shown that CBD and THC can reduce peripheral nerve pain^{7,27,29,44–47}, the mechanism of action needs to be further explored. This time, we compared the protective effect of purified, broad-spectrum, and full-spectrum CBD on OXA-induced peripheral neuropathy and its relationship with platinum transport.

DRG is the main injury target organ of OIPN. OXA can accumulate in DRG through the transport of cell surface transporters to form PT-DNA adduction, resulting in cell cycle stop, neuron injury, neuron axon shortening and cell apoptosis, and the severity of OIPN is proportional to the platinum content in DRG⁴. Due to the limited number of effective neuronal cells obtained from the culture of mouse primary DRG neurons, we conducted in vitro cell studies on the effects of three cannabinoid products on OXA-induced neurotoxicity by differentiating rat pheochromocytoma cell line PC12 cells, which were widely used as an in vitro model of peripheral neuropathy. The results showed that: Compared with the control group, the proportion of PC12 cells with OXA treatment with cell activity and axon length larger than cell body decreased to 44.79% and 22%. In purified, broad-spectrum and full-spectrum CBD pretreatment groups, the cell activity increased to 64.17%, 86.65%; 86.73% respectively,and the proportion of cells whose axon length was longer than the cell body was increased to 60%; 65%; 74%.The three cannabinoid products can effectively reduce OXA-induced neuron apoptosis and axon length shortening and inhibit neurotoxicity. The purified, broad-spectrum, and full-spectrum CBD used in the experiment are rich in nearly 99% CBD, 85% CBD, and 85% CBD, respectively. As the main component of the three cannabinoids, CBD plays a major role in inhibiting the neurotoxicity of OXA. Compared with purified CBD, broad-spectrum and full-spectrum cannabinoids have a more significant inhibition effect on OXA toxicity, and full-spectrum CBD has a better effect. The broad-spectrum and full-spectrum cannabinoids contain terpenes, vitamins, minerals, and a small amount of other secondary cannabinoids in addition to the main component CBD. Combined with previous studies^48–50, it is considered that the main component of CBD in broad spectrum and full spectrum CBD can cooperate with other secondary components to play an anti-OXA neurotoxic effect. Compared with broad-spectrum CBD, full-spectrum CBD, rich in 0.3% THC, plays a better role in promoting axon growth. Considering that THC plays a synergistic role with CBD and other secondary components of cannabinoids to improve drug bioavailability and further inhibit OXA neurocytotoxicity.

To further demonstrate this view and determine whether a mouse model of chronic oxaliplatin treatment-induced neurotoxicity shows signs of pain.The inhibitory effects of cannabinoid products on OXA neurotoxicity in animals and the effects of different doses of cannabinoid on its effects were observed. The mice were randomly divided into a blank group, a combination group, and a model group, and their baseline injury response was tested. The OIPN animal model was established by intraperitoneal injection of 4mg/kg OXA twice a week for 4 weeks. In the combined group, low and high doses (10mg/kg and 30mg/kg) of three kinds of cannabinoids were intraperitoneally injected 30 minutes before OXA injection for 4 weeks. Testing the development of harsher responses to mechanical and hot and cold stimuli in mice.Compared to a baseline lasting four weeks, OXA-treated mice had significantly lower pain thresholds for mechanical, hot, and cold stimuli lasting from the first week of OXA treatment to four weeks later than the initial mice (p < 0.0001), as shown in Fig. 3ABC. Compared with the blank control group, the pain threshold of mechanical stimulation and thermal stimulation in the OXA treatment group was reduced by 82.19% and 43.17%, respectively. These results were consistent with previous reports, indicating that the OIPN model had been successfully established in mice. All three cannabinoids (low and high doses) effectively raised the threshold of OIPN mechanical stimulation and thermal stimulation pain and reduced the sensitivity of OXA-induced nerve pain. CBD is the main component of three cannabinoid products. So we believe that CBD plays a major role in inhibiting OIPN mechanical and thermal pain. However, it is worth noting that purified CBD with the highest CBD-rich content (99% CBD) has a lower threshold of OXA-induced peripheral nerve pain in the high-dose group than that in the low-dose group. The inhibitory effect of purified CBD on OXA peripheral nerve pain decreased with increasing dosage.Combined with previous studies, with the increasing dose of over-purified CBD, the medicinal efficacy of pain inhibition, anti-inflammatory, antiemetic, and other drugs decreased, showing a bell effect of dose restriction^51–53. Therefore, it is considered that purified CBD also has a dose-limiting bell effect in inhibiting nerve pain. The pain thresholds of thermal stimulation and mechanical stimulation of broad spectrum and full spectrum CBD (low and high dose groups) were higher than those of purified CBD, and the effect of both on nerve pain inhibition was more significant with the increase in dose. Combined with previous experimental reports, the active components of plant cannabinoid extract exert anti-inflammatory and nerve pain effects through different mechanisms of action^54,55.The effect of CBD and THC alone on inhibiting the pain of surgical ligation of the sciatic nerve was significantly weaker than that of the combined group, while the side effects were higher than those of the combined group²⁸. Therefore, we concluded that CBD in cannabinoids and the addition of other secondary components of cannabinoids, such as cannabis terpene compounds and THC, had a synergistic effect in the treatment of peripheral neuropathy. Overcoming the bell effect of purified CBD dose limitation, it can have a more significant therapeutic effect in the treatment of OIPN and provide a wider range of effective therapeutic doses.However, in the cold stimulation behavior experiment, high-dose full-spectrum CBD (30mg/kg) significantly improved the cold pain threshold of mice, and this effect was not observed in purified or broad-spectrum CBD. Since full-spectrum CBD contains 0.3% THC compared with broad-spectrum CBD, THC in full-spectrum cannabinoids may be the main component inhibiting cold pain. For the low-dose full-spectrum cannabinoid group, no obvious inhibition of cold stimulation pain was observed. Considering that the THC content in low-dose full-spectrum cannabinoids was too small, the effective therapeutic dose was not reached, so the therapeutic effect was not different. This study provides a new attempt for the rational combined use of chemotherapeutic drugs and plant cannabinoids.

It is considered that OXA accumulation in DRG-induced neuronal apoptosis, necrosis, and mitochondrial dysfunction may be an important mechanism of chronic OIPN. OXA is the main transport substrate of the SLC22A transporter OCT2. 80% of OCT2 is expressed in the kidney and the remaining 20% in DRG tissues. The OCT2 transporter on the surface of animal kidney cells can be overexpressed by intraperitoneal injection of first-generation platinum, which mediates the accumulation of platinum in the kidney^56,57. By transfecting 12 OXA-related transporters on the cell surface, OCTN1 and OCTN2 cationic transporters were screened to promote the accumulation of platinum in the cell, and OCTN1 had the best transport effect^9,11. Moreover, OCT2, OCTN1, and OCTN2 have been confirmed to be expressed in mouse DRG and mitochondria.Previous studies have reported that cannabis analogues have inhibitory OCTNS transport activity, and cannabinoids have high passive permeability. Therefore, we hypothesized that cannabinoids can partially inhibit OXA transport in DRG neurons and mitochondrial membranes by inhibiting SLC22A transporters (OCT2 and OCTN1/2), thereby reducing OXA neurotoxicity. After the last behavioral experiment, we dissected the animals and extracted DRG tissues for ICP-MS platinum content determination and western blot/immunohistochemistry. The experimental results are shown in Figs. 4 and 5. Compared with the OXA group alone, it was found that low and high doses of the three cannabinoids could effectively inhibit the expression of OXA-induced intra-tissue transporters OCT2, OCTN1, and OCTN2 in DRG while reducing the platinum content in DRG. Combined with the composition differences among the three cannabis products, CBD was the main component of the three cannabis products.Therefore, CBD is the main effective component in inhibiting the expression of transporters and reducing the accumulation of platinum in DRG. The effect of the high-dose purified CBD group (99% CBD) on inhibiting transporter expression and platinum accumulation was lower than that of the low-dose group. This is consistent with the results of our behavioral experiments, which show a bell effect of dose restriction on purified CBD. However, the effect of broad- and full-spectrum CBD on the expression of transporter proteins and the accumulation of platinum content is more significant with an increase in dose. It is considered that CBD and other secondary components in cannabinoid products cooperate with each other to break the dose limit of purified CBD.

Previous studies and experiments have shown that OCT2, OCTN1, and OCTN2 are also expressed in the mitochondria of the DRG in mice. After platinum enters mitochondria, it binds to mitochondrial DNA and interferes with mitochondrial DNA replication. Due to the lack of DNA repair enzymes in mitochondria, mitochondrial dysfunction and abnormal ATP production occur. Lead to peripheral nerve disorders such as sciatic nerve energy supply disorders, which cause neurodegeneration and other pathological changes, inducing nerve pain. After the last behavioral experiment, the DRG and sciatic nerve in the OIPN animal model were dissected. The mitochondria of the DRG were observed by electron microscopy, and the myelin sheath morphological changes of the sciatic nerve were observed by LFP staining. The results showed that low and high doses of three cannabinoid products could effectively inhibit OXA-induced mitochondrial edema, vacuolation, and degeneration of sciatic nerve myelin round rate. However, the effect of the high-dose purified CBD group on inhibiting the above OXA-induced pathological results was worse than that of low-dose CBD, again showing the dose limitation of effective therapeutic effect.In addition, CBD and other secondary cannabis in broad-spectrum and full-spectrum CBD cooperated with each other to produce a better therapeutic effect on the pathological apparent realization of mitochondrial and sciatic neuropathology, and the therapeutic effect increased with the increase in dose, breaking the dose-limiting bell effect of purified CBD. This is consistent with the results of animal behavior experiments and other pathological experiments. It was again confirmed that the three cannabinoid products can effectively treat OXA-induced peripheral neuropathy, and the main component of CBD is the main effective component for inhibiting OIPN. CBD and other secondary components of cannabinoids play a synergistic role in inhibiting OIPN.

In order to rule out the sedative effect of cannabinoids on the central nervous system, we used the rotating rod test to conduct behavioral tests on mice. The results showed that there was no significant difference in the rotating rod behavioral tests among the groups, and the mice showed no sedative or hypnotic effects. Therefore, the decreased sensitivity of cold stimulation and mechanical pain stimulation in mice co-administered with cannabinoids had nothing to do with the sedative/hypnotic effect. At the same time, it showed that the application of three cannabinoid products did not have central addictive and sedative effects, and their drug safety was further supported by data.

It is worth noting that when cannabinoids are used in combination with chemotherapy drugs for the prevention and treatment of OIPN, it is very important to determine whether they affect the anti-tumor effects of chemotherapy drugs. The results of our tumor cell experiment in vitro were consistent with our expected results, and the three cannabinoids promoted the apoptosis of colorectal cancer HCT116 and breast cancer MCF-7 cells

Full-spectrum CBD is the best cannabinoid among the three kinds of cannabinoid products for the treatment of OIPN. It not only has an anti-tumor proliferation effect but also has no toxic side effects on the central nervous system in mice. Combined with the safety of full-spectrum CBD in clinical use. Full-spectrum CBD may be a potential drug to mitigate OXA-induced peripheral neurotoxicity. However, there is little difference in therapeutic effect between full-spectrum CBD and broad-spectrum CBD. Considering that the THC content in full-spectrum cannabinoids is too low, it is speculated that appropriately increasing the THC ratio and inhibiting OIPN may play a greater synergistic role. In recent years, many experiments have shown that plant cannabinoids can block tumor migration and cell division and have anti-proliferation and anti-angiogenesis effects^58,59. CBD, the main component of cannabinoids, has been found to have antitumor effects in estrogen receptor-positive and triple-negative breast, thyroid, colorectal, and prostate cancers⁶⁰. And the secondary components of cannabinoids also have anti-cancer potential⁶¹. CBD has a mechanism of resistance to oxaliplatin in colon cancer⁶². CBD and THC also have the effects of being antiemetic, increasing appetite, relieving digestive tract discomfort symptoms in cancer patients, achieving multiple effects of one drug, and improving the treatment effect and quality of life of patients⁶³. Therefore, the full spectrum of cannabinoids for the treatment of OIPN has a good application prospect.

According to the results of the appeal experiment, we concluded that CBD, the main component of cannabinoids, inhibits platinum accumulation in DRG by inhibiting the expression of OCT2/OCTN1/OCTN2 transporters, reduces the mitochondrial destruction of DRG, reduces peripheral neurodegeneration, and inhibits the occurrence and development of OIPN. Other secondary components of cannabinoids, such as terpenoids and THC, have synergistic effects in inhibiting OIPN and anti-tumor proliferation and can break the dose limit of purified CBD applications.

Acknowledgements:

We gratefully acknowledge the support of the Heilongjiang Province key research and development plan project (JD22A016)，Heilongjiang Province "Double first-class" discipline collaborative innovation achievement construction project (LJGXCG2022-126)，Jiamusi University new medical education special project(2023XYK-11).Thanks to drawing site Figdraw and collaborative computing site SynergyFinder.

Funding: The research leading to these results received funding from Heilongjiang Province key research and development plan project (JD22A016), Heilongjiang Province "Double first-class" discipline collaborative innovation achievement construction project (LJGXCG2022-126),Jiamusi University new medical education special project(2023XYK-11)

CRediT taxonomy: Kaiyu Sun and Yuliu Wu : Conceptualization, Methodology, Resources, Investigation, Writing – original draft, Validation. Xiaoqi Yan: Visualization, Data curation, Formal analysis, Writing – review & editing. Yanping Song: Project administration, Supervision, Funding acquisition. Jinlian Li: Funding acquisition, Supervision. Yuanyuan Liu: Funding acquisition, Supervision. Dongmei Wu: Funding acquisition, Supervision, Writing – review & editing, Project administration.

Declaration of competing interes: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability:The data that support the fndings of this study are available from the corresponding author upon reasonable request

Gamelin, E., Gamelin, L., Bossi, L., and Quasthoff, S. (2002). Clinical aspects and molecular basis of oxaliplatin neurotoxicity: current management and development of preventive measures. Seminars in oncology 29, 21-33. 10.1053/sonc.2002.35525.
Resta, F., Micheli, L., Laurino, A., Spinelli, V., Mello, T., Sartiani, L., Di Cesare Mannelli, L., Cerbai, E., Ghelardini, C., Romanelli, M.N., et al. (2018). Selective HCN1 block as a strategy to control oxaliplatin-induced neuropathy. Neuropharmacology 131, 403-413. 10.1016/j.neuropharm.2018.01.014.
Loprinzi, C.L., Lacchetti, C., Bleeker, J., Cavaletti, G., Chauhan, C., Hertz, D.L., Kelley, M.R., Lavino, A., Lustberg, M.B., Paice, J.A., et al. (2020). Prevention and Management of Chemotherapy-Induced Peripheral Neuropathy in Survivors of Adult Cancers: ASCO Guideline Update. Journal of Clinical Oncology 38, 3325-3348. 10.1200/jco.20.01399.
Wang, M., Wang, J., Tsui, A.Y.P., Li, Z., Zhang, Y., Zhao, Q., Xing, H., and Wang, X. (2021). Mechanisms of peripheral neurotoxicity associated with four chemotherapy drugs using human induced pluripotent stem cell-derived peripheral neurons. Toxicology in vitro : an international journal published in association with BIBRA 77, 105233. 10.1016/j.tiv.2021.105233.
Bennett, G.J., Doyle, T., and Salvemini, D. (2014). Mitotoxicity in distal symmetrical sensory peripheral neuropathies. Nature reviews. Neurology 10, 326-336. 10.1038/nrneurol.2014.77.
Canta, A., Pozzi, E., and Carozzi, V.A. (2015). Mitochondrial Dysfunction in Chemotherapy-Induced Peripheral Neuropathy (CIPN). Toxics 3, 198-223. 10.3390/toxics3020198.
Trecarichi, A., and Flatters, S.J.L. (2019). Mitochondrial dysfunction in the pathogenesis of chemotherapy-induced peripheral neuropathy. International review of neurobiology 145, 83-126. 10.1016/bs.irn.2019.05.001.
Gregg, R.W., Molepo, J.M., Monpetit, V.J., Mikael, N.Z., Redmond, D., Gadia, M., and Stewart, D.J. (1992). Cisplatin neurotoxicity: the relationship between dosage, time, and platinum concentration in neurologic tissues, and morphologic evidence of toxicity. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 10, 795-803. 10.1200/jco.1992.10.5.795.
Huang, K.M., Leblanc, A.F., Uddin, M.E., Kim, J.Y., Chen, M., Eisenmann, E.D., Gibson, A.A., Li, Y., Hong, K.W., DiGiacomo, D., et al. (2020). Neuronal uptake transporters contribute to oxaliplatin neurotoxicity in mice. The Journal of clinical investigation 130, 4601-4606. 10.1172/jci136796.
Jong, N.N., Nakanishi, T., Liu, J.J., Tamai, I., and McKeage, M.J. (2011). Oxaliplatin transport mediated by organic cation/carnitine transporters OCTN1 and OCTN2 in overexpressing human embryonic kidney 293 cells and rat dorsal root ganglion neurons. The Journal of pharmacology and experimental therapeutics 338, 537-547. 10.1124/jpet.111.181297.
Yi, Y., Li, L., Song, F., Li, P., Chen, M., Ni, S., Zhang, H., Zhou, H., Zeng, S., and Jiang, H. (2021). L-tetrahydropalmatine reduces oxaliplatin accumulation in the dorsal root ganglion and mitochondria through selectively inhibiting the transporter-mediated uptake thereby attenuates peripheral neurotoxicity. Toxicology 459, 152853. 10.1016/j.tox.2021.152853.
Wei, G., Gu, Z., Gu, J., Yu, J., Huang, X., Qin, F., Li, L., Ding, R., and Huo, J. (2021). Platinum accumulation in oxaliplatin-induced peripheral neuropathy. Journal of the peripheral nervous system : JPNS 26, 35-42. 10.1111/jns.12432.
Chen, L., Chen, L., Qin, Z., Lei, J., Ye, S., Zeng, K., Wang, H., Ying, M., Gao, J., Zeng, S., and Yu, L. (2019). Upregulation of miR-489-3p and miR-630 inhibits oxaliplatin uptake in renal cell carcinoma by targeting OCT2. Acta pharmaceutica Sinica. B 9, 1008-1020. 10.1016/j.apsb.2019.01.002.
Sun, S., Chen, Z., Li, L., Sun, D., Tian, Y., Pan, H., Bi, H., Huang, M., Zeng, S., and Jiang, H. (2012). The two enantiomers of tetrahydropalmatine are inhibitors of P-gp, but not inhibitors of MRP1 or BCRP. Xenobiotica; the fate of foreign compounds in biological systems 42, 1197-1205. 10.3109/00498254.2012.702247.
Marinotti, O., and Sarill, M. (2020). Differentiating Full-Spectrum Hemp Extracts from CBD Isolates: Implications for Policy, Safety and Science. Journal of dietary supplements 17, 517-526. 10.1080/19390211.2020.1776806.
Masocha, W. (2018). Targeting the Endocannabinoid System for Prevention or Treatment of Chemotherapy-Induced Neuropathic Pain: Studies in Animal Models. Pain research & management 2018, 5234943. 10.1155/2018/5234943.
Solowij, N., Broyd, S., Greenwood, L.M., van Hell, H., Martelozzo, D., Rueb, K., Todd, J., Liu, Z., Galettis, P., Martin, J., et al. (2019). A randomised controlled trial of vaporised Δ(9)-tetrahydrocannabinol and cannabidiol alone and in combination in frequent and infrequent cannabis users: acute intoxication effects. European archives of psychiatry and clinical neuroscience 269, 17-35. 10.1007/s00406-019-00978-2.
Gottschling, S., Ayonrinde, O., Bhaskar, A., Blockman, M., D'Agnone, O., Schecter, D., Suárez Rodríguez, L.D., Yafai, S., and Cyr, C. (2020). Safety Considerations in Cannabinoid-Based Medicine. International journal of general medicine 13, 1317-1333. 10.2147/ijgm.S275049.
Morgan, C.J., Schafer, G., Freeman, T.P., and Curran, H.V. (2010). Impact of cannabidiol on the acute memory and psychotomimetic effects of smoked cannabis: naturalistic study: naturalistic study [corrected]. The British journal of psychiatry : the journal of mental science 197, 285-290. 10.1192/bjp.bp.110.077503.
Karniol, I.G., and Carlini, E.A. (1973). Pharmacological interaction between cannabidiol and δ9-tetrahydrocannabinol. Psychopharmacologia 33, 53-70. 10.1007/BF00428793.
Harris, H.M., Sufka, K.J., Gul, W., and ElSohly, M.A. (2016). Effects of Delta-9-Tetrahydrocannabinol and Cannabidiol on Cisplatin-Induced Neuropathy in Mice. Planta medica 82, 1169-1172. 10.1055/s-0042-106303.
Chan, J.Z., and Duncan, R.E. (2021). Regulatory Effects of Cannabidiol on Mitochondrial Functions: A Review. Cells 10. 10.3390/cells10051251.
Ward, S.J., McAllister, S.D., Kawamura, R., Murase, R., Neelakantan, H., and Walker, E.A. (2014). Cannabidiol inhibits paclitaxel‐induced neuropathic pain through 5‐HT1A receptors without diminishing nervous system function or chemotherapy efficacy. British Journal of Pharmacology 171, 636-645. 10.1111/bph.12439.
Blanton, H.L., Brelsfoard, J., DeTurk, N., Pruitt, K., Narasimhan, M., Morgan, D.J., and Guindon, J. (2019). Cannabinoids: Current and Future Options to Treat Chronic and Chemotherapy-Induced Neuropathic Pain. Drugs 79, 969-995. 10.1007/s40265-019-01132-x.
Xu, D.H., Cullen, B.D., Tang, M., and Fang, Y. (2020). The Effectiveness of Topical Cannabidiol Oil in Symptomatic Relief of Peripheral Neuropathy of the Lower Extremities. Current pharmaceutical biotechnology 21, 390-402. 10.2174/1389201020666191202111534.
D'Andre, S., McAllister, S., Nagi, J., Giridhar, K.V., Ruiz-Macias, E., and Loprinzi, C. (2021). Topical Cannabinoids for Treating Chemotherapy-Induced Neuropathy: A Case Series. Integrative cancer therapies 20, 15347354211061739. 10.1177/15347354211061739.
King, K.M., Myers, A.M., Soroka-Monzo, A.J., Tuma, R.F., Tallarida, R.J., Walker, E.A., and Ward, S.J. (2017). Single and combined effects of Δ(9) -tetrahydrocannabinol and cannabidiol in a mouse model of chemotherapy-induced neuropathic pain. Br J Pharmacol 174, 2832-2841. 10.1111/bph.13887.
Comelli, F., Giagnoni, G., Bettoni, I., Colleoni, M., and Costa, B. (2008). Antihyperalgesic effect of a Cannabis sativa extract in a rat model of neuropathic pain: mechanisms involved. Phytotherapy research : PTR 22, 1017-1024. 10.1002/ptr.2401.
Sepulveda, D.E., Vrana, K.E., Graziane, N.M., and Raup-Konsavage, W.M. (2022). Combinations of Cannabidiol and Δ(9)-Tetrahydrocannabinol in Reducing Chemotherapeutic Induced Neuropathic Pain. Biomedicines 10. 10.3390/biomedicines10102548.
Kim, S., Kim, D.K., Shin, Y., Jeon, J.H., Song, I.S., and Lee, H.S. (2020). In Vitro Interaction of AB-FUBINACA with Human Cytochrome P450, UDP-Glucuronosyltransferase Enzymes and Drug Transporters. Molecules (Basel, Switzerland) 25. 10.3390/molecules25194589.
Park, E.J., Park, R., Jeon, J.H., Cho, Y.Y., Lee, J.Y., Kang, H.C., Song, I.S., and Lee, H.S. (2020). Inhibitory Effect of AB-PINACA, Indazole Carboxamide Synthetic Cannabinoid, on Human Major Drug-Metabolizing Enzymes and Transporters. Pharmaceutics 12. 10.3390/pharmaceutics12111036.
曲晓宇, 翟., 高欢等. (2021). OCT2/MRP2在黄芪甲苷联合顺铂的减毒增效作用中的机制研究. 药学学报 56(09), 2536-2543. 10.16438/j.0513-4870.2021-0074.
da Silva, V.K., de Freitas, B.S., Garcia, R.C.L., Monteiro, R.T., Hallak, J.E., Zuardi, A.W., Crippa, J.A.S., and Schröder, N. (2018). Antiapoptotic effects of cannabidiol in an experimental model of cognitive decline induced by brain iron overload. Translational psychiatry 8, 176. 10.1038/s41398-018-0232-5.
Fujita, S., Hirota, T., Sakiyama, R., Baba, M., and Ieiri, I. (2019). Identification of drug transporters contributing to oxaliplatin-induced peripheral neuropathy. Journal of neurochemistry 148, 373-385. 10.1111/jnc.14607.
Ward, S.J., Ramirez, M.D., Neelakantan, H., and Walker, E.A. (2011). Cannabidiol Prevents the Development of Cold and Mechanical Allodynia in Paclitaxel-Treated Female C57Bl6 Mice. Anesthesia & Analgesia 113, 947-950. 10.1213/ANE.0b013e3182283486.
Bouchenaki, H., Danigo, A., Sturtz, F., Hajj, R., Magy, L., and Demiot, C. (2021). An overview of ongoing clinical trials assessing pharmacological therapeutic strategies to manage chemotherapy-induced peripheral neuropathy, based on preclinical studies in rodent models. Fundamental & clinical pharmacology 35, 506-523. 10.1111/fcp.12617.
Sakurai, M., Egashira, N., Kawashiri, T., Yano, T., Ikesue, H., and Oishi, R. (2009). Oxaliplatin-induced neuropathy in the rat: involvement of oxalate in cold hyperalgesia but not mechanical allodynia. Pain 147, 165-174. 10.1016/j.pain.2009.09.003.
Kawashiri, T., Egashira, N., Watanabe, H., Ikegami, Y., Hirakawa, S., Mihara, Y., Yano, T., Ikesue, H., and Oishi, R. (2011). Prevention of oxaliplatin-induced mechanical allodynia and neurodegeneration by neurotropin in the rat model. European journal of pain (London, England) 15, 344-350. 10.1016/j.ejpain.2010.08.006.
Yalcin, I., Charlet, A., Freund-Mercier, M.J., Barrot, M., and Poisbeau, P. (2009). Differentiating thermal allodynia and hyperalgesia using dynamic hot and cold plate in rodents. The journal of pain 10, 767-773. 10.1016/j.jpain.2009.01.325.
Yamamoto, S., Kawashiri, T., Higuchi, H., Tsutsumi, K., Ushio, S., Kaname, T., Shirahama, M., and Egashira, N. (2015). Behavioral and pharmacological characteristics of bortezomib-induced peripheral neuropathy in rats. Journal of pharmacological sciences 129, 43-50. 10.1016/j.jphs.2015.08.006.
Shigematsu, N., Kawashiri, T., Kobayashi, D., Shimizu, S., Mine, K., Hiromoto, S., Uchida, M., Egashira, N., and Shimazoe, T. (2020). Neuroprotective effect of alogliptin on oxaliplatin-induced peripheral neuropathy in vivo and in vitro. Scientific reports 10, 6734. 10.1038/s41598-020-62738-w.
Doyle, T.M., and Salvemini, D. (2021). Mini-Review: Mitochondrial dysfunction and chemotherapy-induced neuropathic pain. Neuroscience letters 760, 136087. 10.1016/j.neulet.2021.136087.
Staff, N.P., Grisold, A., Grisold, W., and Windebank, A.J. (2017). Chemotherapy-induced peripheral neuropathy: A current review. Annals of neurology 81, 772-781. 10.1002/ana.24951.
Mitchell, V.A., Harley, J., Casey, S.L., Vaughan, A.C., Winters, B.L., and Vaughan, C.W. (2021). Oral efficacy of Δ(9)-tetrahydrocannabinol and cannabidiol in a mouse neuropathic pain model. Neuropharmacology 189, 108529. 10.1016/j.neuropharm.2021.108529.
Hammell, D.C., Zhang, L.P., Ma, F., Abshire, S.M., McIlwrath, S.L., Stinchcomb, A.L., and Westlund, K.N. (2016). Transdermal cannabidiol reduces inflammation and pain-related behaviours in a rat model of arthritis. European journal of pain (London, England) 20, 936-948. 10.1002/ejp.818.
Kumar Kalvala, A., Bagde, A., Arthur, P., Kumar Surapaneni, S., Ramesh, N., Nathani, A., and Singh, M. (2022). Role of Cannabidiol and Tetrahydrocannabivarin on Paclitaxel-induced neuropathic pain in rodents. International immunopharmacology 107, 108693. 10.1016/j.intimp.2022.108693.
Nielsen, S.W., Hasselsteen, S.D., Dominiak, H.S.H., Labudovic, D., Reiter, L., Dalton, S.O., and Herrstedt, J. (2022). Oral cannabidiol for prevention of acute and transient chemotherapy-induced peripheral neuropathy. Supportive care in cancer : official journal of the Multinational Association of Supportive Care in Cancer 30, 9441-9451. 10.1007/s00520-022-07312-y.
Al-Ani, I., Zimmermann, S., Reichling, J., and Wink, M. (2018). Antimicrobial Activities of European Propolis Collected from Various Geographic Origins Alone and in Combination with Antibiotics. Medicines (Basel, Switzerland) 5. 10.3390/medicines5010002.
Pamplona, F.A., da Silva, L.R., and Coan, A.C. (2018). Potential Clinical Benefits of CBD-Rich Cannabis Extracts Over Purified CBD in Treatment-Resistant Epilepsy: Observational Data Meta-analysis. Frontiers in neurology 9, 759. 10.3389/fneur.2018.00759.
Russo, E.B. (2011). Taming THC: potential cannabis synergy and phytocannabinoid-terpenoid entourage effects. Br J Pharmacol 163, 1344-1364. 10.1111/j.1476-5381.2011.01238.x.
Bruni, N., Della Pepa, C., Oliaro-Bosso, S., Pessione, E., Gastaldi, D., and Dosio, F. (2018). Cannabinoid Delivery Systems for Pain and Inflammation Treatment. Molecules (Basel, Switzerland) 23. 10.3390/molecules23102478.
Badowski, M.E. (2017). A review of oral cannabinoids and medical marijuana for the treatment of chemotherapy-induced nausea and vomiting: a focus on pharmacokinetic variability and pharmacodynamics. Cancer chemotherapy and pharmacology 80, 441-449. 10.1007/s00280-017-3387-5.
Gallily, R., Yekhtin, Z., and Hanuš, L.O. (2015). Overcoming the bell‐shaped dose‐response of cannabidiol by using cannabis extract enriched in cannabidiol. Pharmacology Pharmacy 6, 75-85.
Maayah, Z.H., Takahara, S., Ferdaoussi, M., and Dyck, J.R.B. (2020). The molecular mechanisms that underpin the biological benefits of full-spectrum cannabis extract in the treatment of neuropathic pain and inflammation. Biochimica et biophysica acta. Molecular basis of disease 1866, 165771. 10.1016/j.bbadis.2020.165771.
Nahler, G. (2018). Pure Cannabidiol versus Cannabidiol-Containing Extracts: Distinctly Different Multi-Target Modulators. Alternative, Complementary & Integrative Medicine 4, 1-11. 10.24966/acim-7562/100048.
Ciarimboli, G., Deuster, D., Knief, A., Sperling, M., Holtkamp, M., Edemir, B., Pavenstädt, H., Lanvers-Kaminsky, C., am Zehnhoff-Dinnesen, A., Schinkel, A.H., et al. (2010). Organic cation transporter 2 mediates cisplatin-induced oto- and nephrotoxicity and is a target for protective interventions. The American journal of pathology 176, 1169-1180. 10.2353/ajpath.2010.090610.
Sprowl, J.A., Ciarimboli, G., Lancaster, C.S., Giovinazzo, H., Gibson, A.A., Du, G., Janke, L.J., Cavaletti, G., Shields, A.F., and Sparreboom, A. (2013). Oxaliplatin-induced neurotoxicity is dependent on the organic cation transporter OCT2. Proceedings of the National Academy of Sciences of the United States of America 110, 11199-11204. 10.1073/pnas.1305321110.
Davis, M.P. (2016). Cannabinoids for Symptom Management and Cancer Therapy: The Evidence. Journal of the National Comprehensive Cancer Network : JNCCN 14, 915-922. 10.6004/jnccn.2016.0094.
Guzmán, M., Duarte, M.J., Blázquez, C., Ravina, J., Rosa, M.C., Galve-Roperh, I., Sánchez, C., Velasco, G., and González-Feria, L. (2006). A pilot clinical study of Delta9-tetrahydrocannabinol in patients with recurrent glioblastoma multiforme. British journal of cancer 95, 197-203. 10.1038/sj.bjc.6603236.
Fraguas-Sánchez, A.I., and Torres-Suárez, A.I. (2018). Medical Use of Cannabinoids. Drugs 78, 1665-1703. 10.1007/s40265-018-0996-1.
Tomko, A.M., Whynot, E.G., Ellis, L.D., and Dupré, D.J. (2020). Anti-Cancer Potential of Cannabinoids, Terpenes, and Flavonoids Present in Cannabis. Cancers 12. 10.3390/cancers12071985.
Jeong, S., Kim, B.G., Kim, D.Y., Kim, B.R., Kim, J.L., Park, S.H., Na, Y.J., Jo, M.J., Yun, H.K., Jeong, Y.A., et al. (2019). Cannabidiol Overcomes Oxaliplatin Resistance by Enhancing NOS3- and SOD2-Induced Autophagy in Human Colorectal Cancer Cells. Cancers 11. 10.3390/cancers11060781.
Chow, R., Valdez, C., Chow, N., Zhang, D., Im, J., Sodhi, E., and Lock, M. (2020). Oral cannabinoid for the prophylaxis of chemotherapy-induced nausea and vomiting-a systematic review and meta-analysis. Supportive care in cancer : official journal of the Multinational Association of Supportive Care in Cancer 28, 2095-2103. 10.1007/s00520-019-05280-4.
Winklmayr, M., Gaisberger, M., Kittl, M., Fuchs, J., Ritter, M., and Jakab, M. (2019). Dose-Dependent Cannabidiol-Induced Elevation of Intracellular Calcium and Apoptosis in Human Articular Chondrocytes. Journal of orthopaedic research : official publication of the Orthopaedic Research Society 37, 2540-2549. 10.1002/jor.24430.

GA.png
Graphical Abstract

Download PDF

Editorial decision: Reject
23 Oct, 2023
Reviewers agreed at journal
18 Oct, 2023
Reviewers invited by journal
18 Oct, 2023
Editor assigned by journal
04 Oct, 2023
First submitted to journal
01 Oct, 2023

You are reading this latest preprint version

Three cannabis products attenuated oxaliplatin-induced peripheral neuropathy by inhibiting proteins that mediate oxaliplatin transport.

Status:

Version 1

Abstract

Figures

Highlights

Introduction

Material and method

2.1 Chemicals and reagents

2.2 Cell culture

2.3 Cytotoxicity

2.3.1 Assessment of PC12 cytotoxicity and neural protrusion growth

2.3.2 Tumor cytotoxicity assay

2.4 Animals

2.4.1 Assessment for Mechanical Allodynia (von Frey Test)

2.4.2 Assessment for Cold Hyperalgesia (Acetone Test)

2.4.3 Assessment for thermal Hyperalgesia

2.4.4 The rotarod test

2.5 Assessment of Sciatic Axonal Degeneration

2.6 Electron microscope

2.7 Determination of OXA concentration by ICP-MS

2.8 Western blot

2.9 Statistic analysis

Results

3.7 Reversal of OXA-induced sciatic neuropathy by three cannabinoid products

Discussion

Conclusion

Declarations

References

Supplementary Files

Status:

Version 1