**4. Discussion**

In this study, we examined the effects of CBD and THC on pain associated with chemotherapeutic-induced peripheral neuropathy (CIPN), both as pure compounds and botanical extracts at varying ratios of THC:CBD. These data are important because patients taking medical cannabis or cannabis-based products do not typically take pure CBD or pure THC, but rather are using botanical or botanically derived products. These products typically have varying ratios of THC:CBD but there are few studies that have investigated the optimal ratios of the two primary cannabinoids for treating medical conditions. Most studies that have investigated the interactions between these two cannabinoids have limited their scope to the 1:1 ratio typically found in nabixmols (Sativex®) [14–17]. While our data sugges<sup>t</sup> that a 1:1 ratio of CBD to THC may not be optimal, there is a benefit of including CBD in combination with THC to reduce neuropathic pain, as lower levels of THC produced a reduction in sensitivity when CBD was included, this is especially true for the botanical extracts.

Here, we found that pure CBD alone had little impact on acute pain associated with CIPN. This is in contrast to several other studies that have found that CBD can reduce neuropathic pain in animal models [10,12,19]. An important difference between our study and the previous work is that we are looking at the acute effects of CBD administration as compared to those studies that looked at more prolonged effects of CBD treatment. Interestingly, we did observe that botanically derived CBD mixtures were able to reduce pain in neuropathic animals at a dose where CBD alone was ineffective. While there was a small amount of THC in this extract, the dose administered (0.13 mg/kg) would be too low to account for the observed reduction in pain. A number of the terpenes present in the CBD extract, such as β-caryophyllene and α-humulene, have been found to have antinociceptive properties in their own rights [20,21]. Further studies will need to be conducted to determine which other constituents of the CBD extract contribute to the improved pain tolerance observed in neuropathic mice or if CBD contributes at all.

In contrast, we found that THC, when administered at a dose as low at 2 mg/kg, was able to reduce neuropathic pain when combined with CBD, and the level to which pain was

reduced was fairly consistent regardless of the ratio of THC:CBD tested (particularly for botanical extracts). Although, in male mice there is a significantly greater reduction in mechanical sensitivity by pure THC when CBD was excluded. These data are consistent with a recent meta-analysis that found that high THC:CBD ratios were better at reducing pain severity across a wide range of conditions, including diabetic neuropathy, in patients [22]. These data are also consistent with reports that CBD is an antagonist at cannabinoid receptors 1 and 2, and can blunt the effects of THC [23–25]. Based upon the large number of studies that have reported that nabiximols (mixtures of approximately 1:1 THC:CBD) have antinociceptive properties, it was unexpected that we did not see a larger difference in anti-nociceptive potential with different ratios [14–17]. Our data sugges<sup>t</sup> that there is little effect of CBD on reducing neuropathic pain, and that most of the reduction in pain can be attributed to THC, although CBD may help to augmen<sup>t</sup> the impact of THC. This is true for not only the pure compounds, but also the botanically derived compounds, particularly in male mice (although the highest concentration of THC also was more effective than the highest concentration of CBD in female mice).

The data comparing the botanical extracts to pure compounds also allows us to examine the controversial "entourage" effect, the idea that the whole plant provides an additive benefit over individual pure compounds [26]. While we are not able to directly compare the responses between the animals that received pure compounds and botanical extracts, the animals that received botanical extracts did show a trend towards increased levels of force compared to those receiving pure compounds. Furthermore, animals, particularly males, receiving the botanical extracts exhibited a greater response at much lower levels of THC when in a botanical extract compared to pure THC.

Another potential reason for looking at combinations of THC:CBD is that the addition of CBD may alter the metabolism or prolong the effectiveness of THC at reducing pain. While our current studies did not directly address this, a recent study in a rat model of neuropathic pain actually found that CBD, when co-administered with THC, reduced the therapeutic window in which THC had an effect [11]. This is in contrast to recent data in humans that has reported that co-administration of THC and CBD can prolong the effects of THC [27]. For this reason, studies in mice or rats may not be optimal for examining the interaction of THC and CBD on the metabolism of cannabinoids, but instead such pharmacokinetic studies might best be conducted in human derived microsomes and ultimately patients.

The mechanism by which THC and CBD reduce neuropathic pain in our model was not examined, but will be the work of future studies. One potential mechanism would be that THC is acting through CB1 receptor to reduce the release of neurotransmitters and neuronal excitability [28]. Both CBD and THC are also known agonists of the TRPV1 receptor and have been shown to reduce pain in murine models through this receptor [29]. CBD and THC have also recently been shown to reduce neuropathic pain through both CB1 and CB2 [30]. Our own recent work on cannabigerol (CBG) and neuropathic pain suggests that it is likely to be a complicated interaction between multiple receptors [31], including α2-adrenergic receptors, of which CBG is a known agonist; however, the role of CBD and THC at this receptor are unknown.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/biomedicines10102548/s1, The full composition for each extract tested can be found in supplemental Tables S1: Full Cannabinoid Content of Extracts and Table S2: Full Terpene Content of Extracts. Supplemental Figure S1: Pre and Post Cisplatin von Frey assessment for the Dose Response Experiment. Supplemental Figure S2: Pre and Post Cisplatin von Frey Assessment for the Pure Compound Experiment and Supplemental Figure S3: Pre and Post Cisplatin Responses for Extract Experiment.

**Author Contributions:** Conceptualization, K.E.V., N.M.G. and W.M.R.-K.; Formal analysis, D.E.S., N.M.G. and W.M.R.-K.; Investigation, D.E.S.; Resources, K.E.V.; Data curation, W.M.R.-K.; Writing— original draft preparation, D.E.S. and W.M.R.-K.; Writing—review and editing, K.E.V., N.M.G. and W.M.R.-K.; Visualization, W.M.R.-K.; Project administration, W.M.R.-K.; Funding acquisition, K.E.V. and N.M.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This project was supported by a NARSAD Young Investigator Award (27364; NMG) and by the Pennsylvania Department of Health using Tobacco CURE Funds (NMG). KEV (and the Penn State College of Medicine) is the recipient of research support from PA Options for Wellness (a state-approved medical marijuana clinical registrant). The funding sources were not involved in: study design, providing any experimental materials, data collection, analysis and interpretation; writing of the report; or the decision to submit the article for publication.

**Institutional Review Board Statement:** The animal study protocol was approved by the Institutional Review Board of Penn State College of Medicine (protocol #01327, approved on 5/5/2020).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors would like to acknowledge members of the state-approved medical marijuana academic clinical research center at Penn State for insights and comments on the data and study design, along with the Drug Discovery, Development, and Delivery (D4) core for assistance with the preparation of the compounds.

**Conflicts of Interest:** The authors declare no conflict of interest.
