**3. Results**

#### *3.1. THC, but Not CBD, Reverses Mechanical Hypersensitivity in Neuropathic Male Mice*

To assess the ability of pure CBD or THC to reduce cisplatin-induced neuropathic pain, von Frey tests were conducted to measure mechanical sensitivity in neuropathic male mice treated with varying doses (5, 10, or 20 mg/kg) of pure CBD or THC. Acute CBD treatment, administered 1 h prior to von Frey testing, had no significant impact on mechanical hypersensitivity in neuropathic male mice (Figure 1A). In contrast, there was a dose dependent effect of THC on pain in these animals; with only the lowest dose (5 mg/kg) being unable to reduce pain compared to vehicle treated animals (Figure 1B). Based on these findings, in order to investigate the potential interaction between these two compounds, we used a standard dose of 6 mg/kg of total cannabinoid (THC alone, CBD alone, or a combination of various THC:CBD ratios) for all subsequent experiments, with the knowledge that, at this dose, the THC (THC:CBD ratio of 1:0) would serve as a positive control, based upon previous work [13]. The dose of 6 mg/kg was selected for two reasons, first we wanted to be able to have a dose that limited the cataleptic effects of THC on mice and, second, we wanted a dose of THC that would be responsive to any additive effects of CBD on reducing hyperalgesia.

**Figure 1.** Dose response of cannabinoids on mechanical sensitivity in neuropathic male mice. (**A**) Mice were treated with CBD administered i.p. at 5, 10, or 20 mg/kg 1 h prior to measuring mechanical sensitivity. (**B**) Same as panel A except THC was administered. *n* = 5 mice per group. \* *p* < 0.05, \*\*\*\* *p* < 0.001.

#### *3.2. CBD Does Influence THC-Induced Decreases in Mechanical Hypersensitivity*

We next investigated the anti-nociceptive effects of five THC and CBD combinations while maintaining the total cannabinoid administered at 6 mg/kg as shown in Table 1. In neuropathic male mice, we observed a statistically significant decrease in mechanical hypersensitivity after administration of THC:CBD only at the 2:1 ratio (Figure 2A). In contrast, in neuropathic female animals we observed a statistically significant decrease in mechanical hypersensitivity after administration of THC:CBD at ratios of 1:2 and 2:1 (Figure 2B). While the addition of CBD reduces the concentration of THC necessary to reduce hyperalgesia (note that 5 mg/kg pure THC was without effect in Figure 1), no combination of CBD and THC is greater at reducing sensitivity than THC alone.

**Figure 2.** THC reduces mechanical sensitivity in neuropathic mice. (**A**) Neuropathic male mice were treated with 6 mg/kg of total cannabinoid at varying ratios of THC:CBD and mechanical sensitivity was measured by von Frey filament. (**B**) Same as panel A except in female neuropathic mice. *n* = 10 mice per group. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.005, \*\*\*\* *p* < 0.001.

#### *3.3. CBD and THC Extracts Are Effective at Attenuating Mechanical Hypersensitivity in Neuropathic Mice*

We next compared the anti-nociceptive effects of THC and CBD ratios using CBDdominant or THC-dominant botanical extracts. Extracts were mixed at varying ratios of THC: CBD. The composition of the extracts (as delivered to the mice) for cannabinoid and most abundant terpene content (Tables S1 and S2), are shown in Tables 2 and 3, respectively.

For full composition of the undiluted extracts please see Supplemental Tables S1 and S2. Using a CBD-dominant botanical extract (containing 40 μg/mL THC; labeled "0:1"), we found a statistical difference between vehicle and treated animals for both sexes (Figure 3A,B).

In neuropathic male mice, we saw a further reduction in mechanical hypersensitivity when THC was added (1:2, 1:1, and 2:1 ratios compared to 0:1, CBD extract), but no change in hypersensitivity as THC concentration increased (Figure 3A). In contrast, in neuropathic female mice, we did not see any further reduction in mechanical hypersensitivity when THC was included in the treatment. That is, the reduction in hypersensitivity was consistent across the tested ratios with what was observed for the CBD extract (0:1 ratio). However, the THC extract alone (1:0 ratio) was significantly better than the CBD extract (Figure 3B). Because the CBD extract contains trace amounts of THC and vice versa; it was not possible to have any extract ratio where the other cannabinoid was exactly zero. Table 4 shows the concentration of THC and CBD delivered at each of the ratios.


**Table 2.** Cannabinoid composition of extracts as administered to mice; concentrations are in mg/mL. Cells with no values represent assay results below the level of detection.

**Table 3.** Terpene composition of extracts as administered to mice; concentrations are in ppm. Cells with no values represent assay results below the level of detection.


**Table 4.** Dose of cannabinoid administered at each ratio for THC and CBD extracts.


**Figure 3.** Cannabinoid extracts containing either CBD or THC reduce mechanical sensitivity in neuropathic mice. (**A**) Neuropathic male mice were treated with 6 mg/kg of total cannabinoid at varying ratios of THC:CBD and mechanical sensitivity was measured by von Frey filament. (**B**) Same as panel A except in female neuropathic mice. *n* = 10 mice per group. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\*\* *p* < 0.001.
