cbd oil for arthritis nih studies

Cannabidiol (CBD): a killer for inflammatory rheumatoid arthritis synovial fibroblasts

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Abstract

Cannabidiol (CBD) is a non-intoxicating phytocannabinoid from cannabis sativa that has demonstrated anti-inflammatory effects in several inflammatory conditions including arthritis. However, CBD binds to several receptors and enzymes and, therefore, its mode of action remains elusive. In this study, we show that CBD increases intracellular calcium levels, reduces cell viability and IL-6/IL-8/MMP-3 production of rheumatoid arthritis synovial fibroblasts (RASF). These effects were pronounced under inflammatory conditions by activating transient receptor potential ankyrin (TRPA1), and by opening of the mitochondrial permeability transition pore. Changes in intracellular calcium and cell viability were determined by using the fluorescent dyes Cal-520/PoPo3 together with cell titer blue and the luminescent dye RealTime-glo. Cell-based impedance measurements were conducted with the XCELLigence system and TRPA1 protein was detected by flow cytometry. Cytokine production was evaluated by ELISA. CBD reduced cell viability, proliferation, and IL-6/IL-8 production of RASF. Moreover, CBD increased intracellular calcium and uptake of the cationic viability dye PoPo3 in RASF, which was enhanced by pre-treatment with TNF. Concomitant incubation of CBD with the TRPA1 antagonist A967079 but not the TRPV1 antagonist capsazepine reduced the effects of CBD on calcium and PoPo3 uptake. In addition, an inhibitor of the mitochondrial permeability transition pore, cyclosporin A, also blocked the effects of CBD on cell viability and IL-8 production. PoPo3 uptake was inhibited by the voltage-dependent anion-selective channel inhibitor DIDS and Decynium-22, an inhibitor for all organic cation transporter isoforms. CBD increases intracellular calcium levels, reduces cell viability, and IL-6/IL-8/MMP-3 production of RASF by activating TRPA1 and mitochondrial targets. This effect was enhanced by pre-treatment with TNF suggesting that CBD preferentially targets activated, pro-inflammatory RASF. Thus, CBD possesses anti-arthritic activity and might ameliorate arthritis via targeting synovial fibroblasts under inflammatory conditions.

Introduction

Cannabidiol (CBD) is a non-intoxicating cannabinoid found in cannabis sativa 1 . In contrast to the psychoactive constituent tetrahydrocannabinol (THC), CBD demonstrates no direct effect at cannabinoid receptors 1 and 2 (CB1 and CB2) but modulates the effect of agonists suggesting an allosteric function 2 . In addition, CBD binds to PPARγ, GPR3/6/12/18/55, TRPV1/2, TRPA1, 5-hydroxytryptamine receptor, and mitochondrial proteins 3–11 . Despite its promiscuous pharmacology, CBD is well tolerated even when given in high concentrations 12,13 . Side effects of CBD in humans include diarrhea and fatigue and, more importantly, CBD interacts with other drugs since it is metabolized by CYP enzymes in the liver thereby inhibiting the degradation of other therapeutic compounds 14,15 . While the therapeutic benefits of CBD in childhood epilepsy are well documented, its effects on inflammation have only been investigated in animal models 13,16 . Studies in rodents with osteoarthritis or collagen-induced arthritis demonstrated anti-inflammatory and analgesic effects of CBD, but these studies did not identify the mechanism of action 17–19 . Here, we investigate the effect of CBD on intracellular calcium, cell viability, and cytokine production in rheumatoid arthritis synovial fibroblasts (RASF). RASF are one major contributor of joint destruction in RA as they secrete pro-inflammatory cytokines and matrix degrading enzymes 20 . In fact, subsets of RASF selectively mediate joint destruction or the inflammatory response, emphasizing their important role in the pathogenesis of RA 21 . In previous studies, we already identified TRPA1 as a therapeutic target since the TRPA1 agonist Polygodial selectively deleted TNF-activated RASF 22 . CBD also binds TRPA1 7,23 , and therefore we hypothesized that CBD has detrimental effects on cell viability, which might explain in part its mechanism of action at sites of inflammation.

Results

CBD reduces cell viability and proliferation of RASF

Over the course of 6 h we found that CBD (≥5 µM) decreases cell viability (Fig. 1a, b ), but a stimulatory effect was detected for 1 µM CBD in TNF pre-incubated RASF (Fig. ​ (Fig.1b). 1b ). CBD combined with the TRPA1 antagonist, A967079, recovered cell viability (Fig. ​ (Fig.1h). 1h ). TRPA1 is upregulated by TNF (Fig. ​ (Fig.1f) 1f ) and we also detected an increase of TRPA1 mRNA by real-time PCR after 24 h under the influence of TNF (data not shown). Ruthenium Red (RR) also reduced the detrimental effects of CBD (Fig. ​ (Fig.1i). 1i ). Surprisingly, 4,4′-Diisothiocyanatostilbene-2,2′-disulfonate (DIDS), supported cell viability at low CBD concentrations but enhanced its cytotoxic effects at concentrations ≥1 µM (Fig. ​ (Fig.1j), 1j ), while Cyclosporin A (CsA), blocked the effects of CBD (Fig. ​ (Fig.1k). 1k ). Since RealTime-Glo assays were conducted at 37 °C in serum-free medium but without CO2 and humidity control, we also confirmed these results in cell titer blue endpoint assays (Fig. ​ (Fig.5d). 5d ). In vivo, CBD is bound to lipoproteins/albumin lowering the available concentration of free CBD 24 . Therefore, we investigated the effect of fetal calf serum content with CBD on proliferation. CBD in concentrations ≥5 µM reduced proliferation of RASF in medium without or 2% FCS (Fig. 1c, d ). TNF pre-stimulation enhanced proliferation at 5 µM CBD in 0% FCS (Fig. ​ (Fig.1c) 1c ) while the opposite was true using 2% FCS (Fig. ​ (Fig.1d). 1d ). With 10% FCS, CBD inhibited proliferation of RASF only at 20 µM (Fig. ​ (Fig.1e). 1e ). DMSO (vehicle control) alone had a stimulatory effect on proliferation (Fig. ​ (Fig.1e, 1e , green line). These findings underline the need for relatively high concentrations of CBD when used in in vivo settings 13 .

a, b Mean cell viability of unstimulated (a) or TNF pre-stimulated (b) RASF after CBD challenge monitored in real-time over the course of 375 min. ce Mean proliferation of RASF with (red bars) and without (white bars) TNF pre-stimulation in response to CBD in medium containing 0% FCS (c), 2% FCS (d), and 10% FCS. The dotted line represents the unstimulated control, which was set to 100%. f Flow cytometric detection of TRPA1 protein in RASF with or without TNF stimulation for 72 h. gk Mean cell viability of TNF pre-stimulated RASF after CBD challenge and concomitant addition of inhibitors over the course of 20 h. n is the number of replicates and patient samples investigated. ANOVA with Dunnett’s T3 post-hoc test was used for comparisons in a, b, gk. ANOVA with Bonferroni post-hoc test was used for comparisons in ce. Two-tailed t-test was used for comparisons in f. *p < 0.05; **p < 0.01, ***p < 0.001 vs control. The error bars in cf represent the standard error of mean (sem).

RASF were incubated for 72 h with TNF. After wash-off, RASF were challenged with antagonists for 30 min followed by CBD addition for 72 h. ANOVA was used for all comparisons vs control w/o CBD. a, c, e, g IL-6, IL-8, MMP-3 production, and cell number after 72 h challenge with CBD. b, d, f, h IL-6, IL-8, MMP-3 production, and cell number after 72 h challenge with CBD (10 µM and 20 µM) with concomitant addition of the TRPA1 antagonist A967079 and the mPTP inhibitor CsA. Significant differences between CBD in different concentrations are depicted as *p < 0.05, **p < 0.01, and ***p < 0.001, and CBD versus CBD/antagonist treatment are depicted as #p < 0.05, ##p < 0.01, and ###p < 0.001. ANOVA with Bonferroni post-hoc test was used for all comparisons. The dotted line in g, h represents the control value which was set to 100%. n is the number of replicates out of four different patient samples. The error bars represent the standard error of mean.

CBD increases intracellular calcium and its effects are enhanced by TNF

CBD influences calcium mobilization 9,23,25 and we found that concentrations ≥5 µM increased intracellular calcium (Fig. ​ (Fig.2a). 2a ). The potency of CBD was enhanced by pre-incubation with TNF for 72 h (Fig. ​ (Fig.2b). 2b ). Under these conditions, intracellular calcium levels were significantly increased compared to untreated RASF (Fig. ​ (Fig.2d). 2d ). When extracellular calcium was omitted by using PBS, CBD still increased intracellular calcium although to a smaller extent (Fig. ​ (Fig.2c) 2c ) Besides calcium, we also analyzed the uptake of the cell viability dye PoPo3 iodide under CBD stimulation. PoPo3 uptake increases when membrane integrity is compromised during apoptosis or necrosis. CBD dose-dependently increased the uptake of PoPo3 which was enhanced by extracellular calcium and TNF pre-stimulation (Fig. 2f, g ). Basal uptake of PoPo3 and intracellular calcium levels were increased by TNF pre-treatment (Supplementary Fig. 3A, B). The detrimental effect of CBD on cell viability was also confirmed in the XCELLigence system using untreated RASF (Supplementary Fig. 1).

ac Intracellular calcium mobilization of RASF after CBD challenge in HBSS (a, b) or without extracellular Ca 2+ (PBS; c). The EC50 values obtained for the increase of intracellular calcium were significantly different (p < 0.001) between unstimulated and TNF-pre-stimulated RASF. eg PoPo3 uptake by RASF after CBD challenge in HBSS (e, f) or without extracellular Ca 2+ (PBS; f). RASF were pre-stimulated with TNF (b, c, f, g) or untreated (a, e). d, h Comparison between unstimulated and TNF pre-treated RASF regarding intracellular calcium levels (d) and PoPo3 uptake (h). n is the number of experiment replicates from 29 different patient samples (a, e), 38 patient samples (b, f), and 13 patient samples (c, g). ***p < 0.001, *p < 0.05 for differences between [c] of CBD. ANOVA with Dunnett’s T3 post-hoc test was used for all comparisons.

Calcium mobilization and PoPo3 uptake partly depend on TRPA1 activation

Since CBD binds TRPV1, TRPV2, and TRPA1 7 we investigated the involvement of these ion channels. RR, a general inhibitor for several TRP channels 26–28 , reduced intracellular calcium levels (Fig. 3e, f ) and PoPo3 uptake (Fig. 3g, h ) but the magnitude of inhibiton was small. RR did not change basal calcium levels or PoPo3 uptake in unstimulated but did so in TNF pre-stimulated RASF (Supplementary Fig. 3). Next, we combined CBD with the TRPV1 antagonist Capsazepine (CPZ) 29 . CPZ had only a minor influence on CBD-induced calcium levels and PoPo3 uptake (Fig. 3i–l ). Of note, CPZ also modulated basal calcium and PoPo3 levels (Supplementary Fig. 3) With TRPA1 inhibition we found that without TNF pre-stimulation, the antagonist A967079 (10 µM) increased intracellular calcium (Fig. ​ (Fig.3m) 3m ) but decreased it when RASF were pre-incubated with TNF (Fig. ​ (Fig.3n). 3n ). A967079 increased PoPo3 uptake under basal conditions (Fig. ​ (Fig.3o) 3o ) but decreased it after TNF pre-incubation (Fig. ​ (Fig.3p). 3p ). Furthermore, A967079 enhanced basal calcium levels and PoPo3 uptake (Supplementary Fig. 3A–D).

Mean intracellular calcium mobilization (a, b, e, f, i, j, m, n, q, r, u, v) and mean PoPo3 uptake (c, d, g, h, k, l, o, p, s, t, w, x) of RASF after CBD challenge and concomitant inhibition with the antagonists given in the figure. n is the number of experiment replicates from 24 patient samples (a, c), 33 patient samples (b, d), and 8 patient samples (n, p). ex (except n, p) n number equals number of replicates and different patient samples. ANOVA with Dunnett’s T3 post-hoc test was used for all comparisons versus (ad). *p < 0.05, ***p < 0.001.

Mitochondrial targets mediate the effects of CBD

We investigated four proposed mitochondrial targets for CBD: the mitochondrial calcium uniporter (MCU), the sodium/calcium exchanger (NCLX) 30 , the voltage-gated anion channel (VDAC1) 8 and the mitochondrial membrane permeability transition pore (mPTP) which initiates apoptotic and necrotic events 31 .While we detected a minor influence of NCLX and MCU (Supplementary results and Supplementary Fig. 2) on intracellular calcium levels and PoPo3 uptake, inhibition of mPTP exerted the strongest influence (Fig. 3q–t ). CBD-induced changes in intracellular calcium were reduced by the mPTP inhibitor CsA (Fig. ​ (Fig.3q). 3q ). In TNF pre-stimulated RASF, CsA reduced calcium levels over all CBD concentrations (Fig. ​ (Fig.3r). 3r ). In addition, CsA accelerated PoPo3 uptake (Fig. 3s, t ) but did not alter basal calcium or PoPo3 levels (Supplementary Fig. 3A–D). Next, we combined CBD with DIDS, which is an inhibitor of VDAC in the outer mitochondrial and the plasma membrane 32,33 . CBD stabilizes a closed conformation of VDAC, which excludes the exchange of metabolites from the cytosol into mitochondria, but enhances its calcium transport function 8,34 . DIDS increased CBD-induced calcium mobilization regardless of TNF pre-stimulation (Fig. 3u, v ) and it completely abolished PoPo3 uptake under all conditions (Fig. 3w, x ). DIDS also increased basal calcium levels in TNF pre-stimulated RASF (Supplementary Fig. 3B) and decreased PoPo3 levels (Supplementary Fig. 3C, D). Since we found TRPA1 to be involved in the effects of CBD (Figs. ​ (Figs.1h 1 h and 3m–p ), we were interested in the cellular localization of this receptor. Since we assumed this to be an intracellular site, we used Thapsigargin to deplete calcium stores in the endoplasmatic reticulum (ER) 35,36 and Gly-Phe-β-naphthylamide (GPN), which disrupts lysosomes and releases calcium stored in this organelle 37,38 . We found that GPN reduced the elevation of intracellular calcium by CBD (p < 0.001, Fig. 4e, f ), while PoPo3 uptake was significantly enhanced (Fig. 4g, h ). Of note, GPN per se induced an increase of intracellular calcium and PoPo3 uptake (Supplementary Fig. 3A–D). Similarly, the inhibitor of the endoplasmic reticulum Ca 2+ -ATPase, Thapsigargin, reduced the CBD-induced increase in intracellular calcium levels (Fig. 4i, j ) and slightly attenuated PoPo3 uptake (Fig. 4k, l ) but increased basal calcium and PoPo3 levels (Supplementary Fig. 3A–D). Cationic and uncharged compounds are taken up by organic cation transporters (OCT) 39,40 and therefore we assessed the effects of Decynium-22 (D22), an inhibitor of all OCT isoforms on intracellular calcium and the uptake of PoPo3. Under all conditions, we found that D22 prevented the increase of intracellular calcium/PoPo3 uptake induced by CBD (Fig. 4m–p ) and it reduced basal intracellular calcium (Supplementary Fig. 3A, B) but slightly elevated PoPo3 levels (Fig. 3c, d ). Lastly, we determined the influence of CBD on the production of IL-6, IL-8, and MMP-3 by RASF. CBD (10 µM and 20 µM) significantly decreased the production of IL-6 (Fig. ​ (Fig.5a), 5a ), which was inhibited by the addition of CsA (Fig. ​ (Fig.5b). 5b ). IL-8 production was modified by CBD (20 µM) alone (Fig. ​ (Fig.5c) 5c ) and the addition of CsA increased IL-8 levels significantly (Fig. ​ (Fig.5d). 5d ). MMP-3 levels were reduced by 20 µM CBD (Fig. ​ (Fig.5e). 5e ). The cytokine-reducing effects of CBD might be related to the reduction in viable cells, since we detected a reduction in cell number at 10 µM and 20 µM CBD which was inhibited by the addition of CsA (Fig. 5g, h ).

Mean intracellular calcium mobilization (a, b, e, f, i, j, m, n) and mean PoPo3 uptake (c, d, g, h, k, l, o, p) of RASF after CBD challenge and concomitant inhibition with the antagonists given in the figure. ANOVA with Dunnett’s T3 post-hoc test was used for all comparisons. ***p < 0.001.

Discussion

In this study, we demonstrated that CBD decreases cell viability, proliferation, and cytokine production but increases intracellular calcium and PoPo3 levels of RASF and all effects were enhanced by TNF pre-stimulation. These effects were mediated by TRPA1 and by the assembly of the mPTP under pro-inflammatory conditions, whereas under unstimulated conditions, TRPA1 was not involved.

We demonstrated that CBD reduces cell viability, but RealTime-Glo assays were conducted in serum-free medium without carrier protein. Therefore, we assessed whether CBD influences RASF proliferation in medium containing FCS, since in vivo, CBD is bound to serum albumin, which lowers its free concentration available for receptor binding 24,41 . We confirmed that the anti-proliferative effect of CBD is dependent on FCS content. Consequently, for in vivo applications, CBD needs to be administered in high concentrations to elicit beneficial effects as shown in the treatment of Dravet syndrome 13 . In order to identify the cellular targets for CBD, we used the TRPA1 antagonist A967079 and the pan-TRP antagonist RR to inhibit the effects of CBD on cell viability as CBD has already been identified as ligand for TRPA1 23 . Moreover, we found that CsA reversed the detrimental effects of CBD on cell viability, which confirms results from Olivas-Aguirre et al. 25 that showed mitochondrial calcium overload correlates with assembly of the mPTP by CBD. It has been demonstrated that CBD influences calcium homeostasis 25 and we also found CBD to elevate intracellular calcium in RASF. This confirms own previous results demonstrating calcium mobilization in response to TRPA1 ligation 22 . Moreover, we showed that TNF up-regulates TRPA1 protein in RASF, which translates into increased sensitivity to TRPA1 ligands 22 . CBD also increased calcium levels without extracellular calcium by using PBS instead of HBSS, suggesting mobilization form intracellular stores. In fact, in dorsal root ganglia neurons it has been shown that TRPA1 is located in lysosomes, where its activation fosters neurotransmitter release 42 . Although we do not provide direct evidence regarding the localization of TRPA1, the use of the cell-impermeable pan-TRP inhibitor RR 43 , which was only able to slightly attenuate the effects of CBD suggests an intracellular target protein. In line with this, the lipophilic antagonist A967079 decreased calcium mobilization and PoPo3 uptake after CBD challenge. Moreover, we used Thapsigargin to deplete ER calcium stores and GPN to disrupt lysosomes and both compounds reduced the elevation of intracellular calcium after CBD exposure. This shows that ER calcium stores are involved and it has been shown that even if calcium originates from lysosomes, the signal is amplified by depletion of ER stores 44 . This is important, because although GPN has been reported to mobilize lysosomal calcium, a recent study claimed that GPN increases calcium through an ER-dependent mechanism 45 . CBD is also an agonist at TRPV1/2 ion channels 7,23,46,47 , but neither CPZ nor RR inhibited the effects of CBD, ruling out these receptors as target molecules. CPZ is also an agonist at TRPA1 48 , and we did detect a small increase in basal intracellular calcium in response to this ligand alone. Accordingly, CPZ slightly elevated the calcium response of RASF to CBD suggesting a sensitizing effect on TRPA1. TRPA1 inhibition with A967079 reduced calcium mobilization and PoPo3 uptake in TNF pre-stimulated but not naïve RASF, where we found the opposite, suggesting that CBD exerts additional effects via different cellular targets besides TRPA1. This demonstrates that TRPA1 contributes to the rise in intracellular calcium only in TNF pre-stimulated RASF, in which TRPA1 is upregulated. Besides binding to TRPs, it has been demonstrated that CBD ligates several proteins 11,49 with mitochondrial targets being the most prominent 8–10,25,30 . In mitochondria, CBD targets VDAC1, NCLX, MCU, and controls assembly of the mPTP 8–10,25 . Although protective effects of CBD against mitochondrial toxins have been shown 10,50 , the majority of studies demonstrated that CBD induces cell death by disturbing calcium homeostasis 8,9,25 . This confirms our results, since CBD augmented calcium levels with a concomitant increase in cell death. Excess cytosolic calcium is taken up by mitochondria which are depolarized in this process 51 . If mitochondrial calcium levels exceed a certain threshold, mPTP is assembled leading to cell death 52 . In fact, we demonstrated that only CsA prevented cell death, suggesting that mitochondrial calcium overload occurs in CBD-stimulated RASF. In line with this, CsA reduced intracellular calcium, which is another indicator that mitochondria provide a significant contribution to the increase in calcium by CBD. Calcium is increased by mPTP formation as mitochondria are permeabilized releasing stored calcium into the cytosol 53 . Thus, inhibiting mPTP formation by CsA decreases calcium leakage from mitochondria and subsequent cell death. In addition, we found that the NCLX inhibitor CGP reduced cytosolic calcium levels. CGP blocks calcium transport from the mitochondrial matrix into the intermembrane space, thus increasing mitochondrial and reducing cytosolic calcium levels 54 . We also used the reverse mode inhibitor of the NCLX, KB-R7943, but results with this inhibitor are difficult to interpret due to its interaction with the MCU and NCLX. In the latter it can act as forward or reverse mode antagonist dependent on cell type, NCLX isoform, and concentration 55,56 . Using the specific MCU inhibitor DS16570511 we found increased cytosolic calcium levels in TNF pre-stimulated but not in unstimulated RASF. This might be explained by TRPA1, which only contributed to calcium level alterations in TNF pre-treated RASF. For the inhibition of VDAC1 we used DIDS which increased intracellular calcium in unstimulated, but decreased calcium in TNF pre-treated RASF. This might depend on the initial calcium signal generated by CBD, because DIDS can permeabilize the inner mitochondrial membrane depending on calcium concentration leading to formation of mPTP 57 . Besides calcium, PoPo3 uptake served as readout for changes in cell viability as it is supposed to enter cells with a compromised plasma membrane only. However, several studies showed that the uptake of PoPo3 related compounds also occurs via specific receptors/ion channels 58,59 . In addition, it has been demonstrated that the family of organic cation transporters (OCT) mediates the uptake of many charged but also electroneutral compounds into the cell 39 . Therefore, it is quite possible that PoPo3 is also taken up by OCT and indeed we show that decynium-22, which inhibits all OCT isoforms 39 strongly reduced PoPo3 uptake and it also blunted the increase of intracellular calcium, which might be due to the electrogenic properties of D22 40 . Another possibility is that OCT mediates the uptake of CBD and D22 would limit the access of CBD to intracellular compartments. D22 did not influence basal uptake of PoPo3, but reduced the CBD-induced uptake and this might be related to changes in intracellular calcium since the activity of OCT is regulated by calcium-dependent proteins 39 . DIDS completely blocked PoPo3 uptake but these results are difficult to interpret since DIDS does not inhibit OCT but membrane anion channels, which should not mediate the uptake of the cationic dye PoPo3. It might be that the negatively charged DIDS binds PoPo3 directly, thereby inhibiting binding to DNA and the increase in fluorescence. PoPo3 might be suitable as a surrogate marker for the uptake of chemical compounds/drugs, which is enhanced by CBD. Since RASF produce high amounts of IL-6, IL-8, and MMP-3 60 , we also investigated the impact of CBD on production of these mediators. CBD dose-dependently reduced IL-6, IL-8, and MMP-3 with concomitant reduction in cell viability. CsA was able to rescue RASF from cell death and increased Il-6 and IL-8 production confirming that cell death is the influencing factor on cytokine production.

From our data, we propose a mechanism of how CBD influences RASF function and induces cell death under pro-inflammatory conditions (Fig. ​ (Fig.6). 6 ). TNF sensitizes RASF to the action of CBD by up-regulating TRPA1 22 . CBD increases intracellular calcium by activating TRPA1 but it also binds several mitochondrial targets like VDAC1, MCU, and NCLX, which on their part influence cytosolic calcium. Eventually, mitochondrial calcium overload occurs and mPTP is assembled leading to cell death.

TNF increases TRPA1 protein, which is located in intracellular compartments. CBD activates TRPA1 and Ca 2+ is released into the cytosol. Elevations in cytosolic Ca 2+ are reduced through uptake into mitochondria. In addition, increased cytosolic Ca 2+ might enhance the activity of organic cation transporters (OCT) which might mediate the uptake of the fluorescent dye PoPo3. Additionally, OCT might mediate the uptake of CBD itself. By binding to VDAC1, CBD increases Ca 2+ flux through the outer mitochondrial membrane. Ca 2+ is then taken up into the matrix by the mitochondrial Ca 2+ uniporter (MCU) and, if mitochondria are depolarized, by the Na + /Ca 2+ exchanger (NCLX), which operates in reverse mode under these conditions. Ca 2+ overload occurs, the mitochondrial permeability transition pore (mPTP) assembles and cell death occurs.

CBD has been used in animal model of RA, demonstrating anti-inflammatory and analgesic effects but the mechanism of action has not been identified 17,19 . Here, we demonstrate that CBD reduces cell viability preferentially in TNF-activated RASF via TRPA1 and mitochondrial targets. CBD might decrease chronic inflammation since RA is characterized by a hypoxic environment in the joint with concomitant mitochondrial dysfunction 61 . In this setting, immune cells and RASF might be specifically vulnerable to a “second hit” induced by CBD, leading to deletion of pro-inflammatory immune cells and fibroblasts thereby resolving inflammation. In addition, CBD might also synergize with anti-rheumatic drugs like methotrexate or JAK inhibitors, since it has been reported in tumor cell lines that CBD works in synergy with e.g. the chemotherapeutic drug doxorubicin 62 . Furthermore, CBD also targets TRPV2, which not only increases the uptake of cytotoxic chemotherapeutic agents but also reduces RASF invasion and matrix metalloproteinase production 47,63 . In conclusion, CBD might be beneficial as an adjuvant treatment in rheumatoid arthritis that might support the action of currently used disease-modifying anti-rheumatic drugs.

Materials and methods

Biochemicals

Patients

In total, 40 patients with long-standing RA fulfilling the American College of Rheumatology revised criteria for RA (24) were included in this study. The RA group comprised of 32 females and 8 males with a mean age of 67.8 years ±10.5 years and 66.9 years ±8.2 years, respectively. C-reactive protein was 47.9 mg/dl ± 186.3 mg/dL for females and 28.7 mg/dL ± 43.2 mg/dL for males and rheumatoid factor was 184.4 iU/mL ± 280.4 iU/mL for females and 31.8 iU/mL ± 37.6 iU/mL for males. IN all, 11 out of 40 patients received glucocorticoids, 7 out of 40 methotrexate, 3 out of 40 biologicals, and 1 out of 40 a JAK inhibitor. All patients underwent elective knee joint replacement surgery, and they were informed about the purpose of the study and gave written consent. The study was approved by the Ethics Committees of the University of Düsseldorf (approval number 2018-87-KFogU) and Regensburg (approval number 15-1 01-021). We confirm that all experiments were performed in accordance with relevant guidelines and regulations (Table ​ (Table1 1 ).

Table 1

Biochemicals used in this study.

order # vendor solvent [c] used in experiments Reference
DIDS 4523 Tocris DMSO 50 µM 32,64
Cannabidiol 1570 Tocris DMSO 0.5 µM, 1 µM, 5 µM, 10 µm, 20 µM 7,8,23
<"type":"entrez-protein","attrs":<"text":"CGP37157","term_id":"875406365","term_text":"CGP37157">> CGP37157 1114 Tocris DMSO 1 µM, 10 µM 30,65
KB-R7943 1244 Tocris DMSO 2.5 µM, 25 µM 55,56
A967079 4716 Tocris DMSO 10 µM 22,66,67
Ruthenium red 1439 Tocris H2O 10 µM 68,69
Cyclosporin A 30024 Sigma DMSO 10 µM 9
Capsazepine 464 Tocris DMSO 10 µM 29,70
GPN 14634 Cayman DMSO 250 µM 29,70,71
Thapsigargin 1138/1 Tocris DMSO 10 µM 72
Decynium-22 4722 Tocris DMSO 10 µM 39,40

The concentrations used in this study are based on values found in the literature (reference).

Synovial fibroblast and tissue preparation

Samples from RA synovial tissue were isolated and prepared as described previously 22 (for details see also Supplementary methods).

Proliferation of RASF

Proliferation was assessed by the cell titer blue viability assay (Promega, Madison, USA, # G8080) according to manufacturer’s instructions.

Intracellular calcium and PoPo3 uptake

In black 96-well plates, RASF were incubated with 4 µM of calcium dye Cal-520 (ab171868, abcam, Cambridge, UK) in Hanks buffered salt solution (1 mM Ca 2+ ; HBSS, sigma, # 55037 C) or PBS (no Ca 2+ ) with 0.02% Pluoronic F127 (Thermo fisher scientific, Waltham, USA, # P6866) for 60 min at 37 °C followed by 30 min at room temperature. After washing, HBSS or PBS containing 1 µM PoPo3 iodide (Thermo fisher scientific, # P3584) and respective antagonists/ligands/inhibitors were added for 30 min at room temperature. After that, CBD was added and the intracellular Ca 2+ concentration as well as PoPo3 uptake were evaluated with a TECAN multimode reader over 90 min.

Flow cytometry

RASF were trypsinized, washed and fixed for 20 min with 3.7% formaldehyde (F8775, Sigma Aldrich). Cells were permeabilized with 0.1% Triton X-100 (X100, Sigma) in PBS for 10 min. Then, 0.2 µg/50 µl primary antibody (Proteintech, 19124-1-AP) was added for 2 h. The secondary antibody (Abcam, goat anti-rabbit IgG H&L (Alexa Fluor® 488), ab150077) was incubated for 1 h. Cells were analyzed using a MACS Quant 9 analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany).

RealTime-Glo cell viability assay

Cell viability was assessed according to manufacturer’s instructions (Promega, # G9711).

Statistical analysis

Statistical analysis was performed with SPSS 25 (IBM, Armonk, USA). The statistic tests used are given in the figure legends. Normal distribution was determined using the Shapiro–Wilk test, equal variance was determined by Levene’s test. In the case of equal variance, the Bonferroni post-hoc test was used, otherwise the Dunnet’s post-hoc test was employed. When data are presented as box plots, the boxes represent the 25th to 75th percentiles, the lines within the boxes represent the median, and the lines outside the boxes represent the 10th and 90th percentiles. When data are presented as line plots, the line represents the mean. When data are presented as bar charts, the top of the bar represents the mean and error bars depict the standard error of the mean (sem). The level of significance was p < 0.05.

CBD (Cannabidiol)

In addition to the items below, for more information on CBD you should check out Project CBD.

Page last updated by Dec. 16, 2020 by Doug McVay, Editor.

“Since several years, other pharmacologically relevant constituents of the Cannabis plant, apart from Δ9-THC, have come into the focus of research and legislation. The most prominent of those is cannabidiol (CBD). In contrast to Δ9-THC, it is nonintoxicating, but exerts a number of beneficial pharmacological effects. For instance, it is anxiolytic, anti-inflammatory, antiemetic, and antipsychotic. Moreover, neuroprotective properties have been shown.1,2 Consequently, it could be used at high doses for the treatment of a variety of conditions ranging in psychiatric disorders such as schizophrenia and dementia, as well as diabetes and nausea.1,2

“At lower doses, it has physiological effects that promote and maintain health, including antioxidative, anti-inflammatory, and neuroprotection effects. For instance, CBD is more effective than vitamin C and E as a neuroprotective antioxidant and can ameliorate skin conditions such as acne.3,4

“The comprehensive review of 132 original studies by Bergamaschi et al. describes the safety profile of CBD, mentioning several properties: catalepsy is not induced and physiological parameters are not altered (heart rate, blood pressure, and body temperature). Moreover, psychological and psychomotor functions are not adversely affected. The same holds true for gastrointestinal transit, food intake, and absence of toxicity for nontransformed cells. Chronic use and high doses of up to 1500 mg per day have been repeatedly shown to be well tolerated by humans.1

“Nonetheless, some side effects have been reported for CBD, but mainly in vitro or in animal studies. They include alterations of cell viability, reduced fertilization capacity, and inhibition of hepatic drug metabolism and drug transporters (e.g., p-glycoprotein).1 Consequently, more human studies have to be conducted to see if these effects also occur in humans. In these studies, a large enough number of subjects have to be enrolled to analyze long-term safety aspects and CBD possible interactions with other substances.”

Iffland, Kerstin, and Franjo Grotenhermen. “An Update on Safety and Side Effects of Cannabidiol: A Review of Clinical Data and Relevant Animal Studies.” Cannabis and cannabinoid research vol. 2,1 139-154. 1 Jun. 2017, doi:10.1089/can.2016.0034
https://www.ncbi.nlm.nih.gov/p.

“Cannabidiol (CBD) oil is essentially a concentrated solvent extract made from cannabis flowers or leaves that is dissolved in an edible oil such as sunflower, hemp, or olive oil. Solvents used can vary from relatively innocuous organic solvents (ethanol, isopropyl alcohol) to more harmful ones (petroleum-ether, naphtha), or even supercritical fluids (butane, CO2). The exact conditions and solvents applied have a great impact on, for example, the taste, color, and viscosity of the final product. Because many other plant components are co-extracted with the desired cannabinoids present in the herbal material, these are sometimes removed by a treatment known as “winterization.” By placing the extract in a freezer (–20 to –80°C) for 24–48 h, components with a higher melting point such as waxes and triglycerides, as well as chlorophyll will precipitate, so they can be removed by filtration or centrifugation [1]. This treatment can significantly improve the taste and color of the final product.

“Cannabis oils may contain various concentrations of CBD, tetrahydrocannabinol (THC), and minor cannabinoids, mainly depending on the cannabis variety used for extraction. The most popular product currently is CBD oil, but for example cannabigerol (CBG)-rich oil has been spotted as well [2], and others will very likely follow soon. The THC-rich type of cannabis oil has already been known for some years, and is generally known under the name “Simpson oil” [3]. Terpenes may or may not be present in these products, depending on the preparation method used [4]. Because they are highly volatile, elevated temperatures (such as those applied during drying of plant materials, or during the evaporation of solvents) may result in a significant loss of terpene components [5]. However, it is possible to capture evaporated terpenes by condensation, and reintroduce them back into the final oil. Additional ingredients may be added to further adjust properties such as color, viscosity, taste, or shelf-life stability.”

Hazekamp A: The Trouble with CBD Oil. Med Cannabis Cannabinoids 2018;1:65-72. doi: 10.1159/000489287
https://www.karger.com/Article.

“Recent developments suggest that non-psychotropic phytocannabinoids exert a wide range of pharmacological effects (Figure 1), many of which are of potential therapeutic interest. The most studied among these compounds is CBD, the pharmacological effects of which might be explained, at least in part, by a combination of mechanisms of action (Table 1, Figure 1). CBD has an extremely safe profile in humans, and it has been clinically evaluated (albeit in a preliminary fashion) for the treatment of anxiety, psychosis, and movement disorders. There is good pre-clinical evidence to warrant clinical studies into its use for the treatment of diabetes, ischemia and cancer. The design of further clinical trials should: i) consider the bell-shaped pattern of the dose–response curve that has been observed in pre-clinical pharmacology, and ii) establish if CBD is more effective or has fewer unwanted effects than other medicines. A sublingual spray that is a standardized Cannabis extract containing approximately equal quantities of CBD and D9-THC (Sativex ® ), has been shown to be effective in treating neuropathic pain in multiple sclerosis patients [76].
“The pharmacology of D9-THCV (i.e. CB1 antagonism associated with CB2 agonist effects) is also intriguing because it has the potential of application in diseases such as chronic liver disease or obesity—when it is associated with inflammation—in which CB1 blockade together with some CB2 activation is beneficial.
“The plant Cannabis is a source of several other neglected phytocannabinoids such as CBC and CBG. Although the spectrum of pharmacological effects of these compounds is largely unexplored, their potent action at TRPA1 and TRPM8 might make these compounds new and attractive tools for pain management.”

Izzo, Angelo A.; Borrelli, Francesca; Capasso, Raffaele; Di Marzo, Vincenzo; and Mechoulam, Raphael, “Non-psychotropic plant cannabinoids: new therapeutic opportunities from an ancient herb,” Trends in Pharmacological Sciences (London, United Kingdom: October 2009) Vol. 30, Issue 10, pp. 525-526.
http://www.ncbi.nlm.nih.gov/pu.
http://cannabisinternational.o.

“We synthesised available evidence on the safety and efficacy of cannabinoids as an adjunctive treatment to conventional AEDs [Antiepileptic Drugs] in treating drug-resistant epilepsy. In many cases, there was qualitative evidence that cannabinoids reduced seizure frequency in some patients, improved other aspects of the patients’ quality of life and were generally well tolerated with mild-to-moderate AEs [Adverse Events]. We can be much more confident about this statement in the case of children than adults, because the recent, larger, well-conducted RCTs [Randomized Controlled Trials] were performed in children and adolescents.

“In studies where there was greater experimental control over the type and dosage of cannabinoid used, there was evidence that adjuvant use of CBD reduced the frequency of seizures, particularly in treatment-resistant children and adolescents, and that patients were more likely to achieve complete seizure freedom. There was a suggestion that the benefits of adding CBD may be greater when patients were also using clobazam. 11 12 However because clobazam and CBD are both metabolised in the cytochrome P450 pathway, the pharmacokinetic interactions of these two drugs still need to be fully determined 56 Further randomised, double-blind studies with a placebo or active control are needed to strengthen this conclusion.

“Non-RCT evidence was consistent with RCT evidence that suggested cannabinoids may reduce the frequency of seizures. In most of these studies, cannabinoid products and dosages were less well-controlled, and outcomes were based on self-report (often by parents). These studies provide lower quality evidence compared with RCTs due to the potential for selection bias in the study populations, and other weaknesses in study design. There was also some evidence that studies at very high risk of bias had higher reported proportions of participants reporting reductions in seizures and lower proportions reporting AEs. In RCTs, and most of the non-RCTs, cannabinoids were used as an adjunctive therapy rather than as a standalone intervention, so at present there is little evidence to support any recommendation that cannabinoids can be recommended as a replacement for current standard AEDs.”

Stockings, Emily & Zagic, Dino & Campbell, Gabrielle & Weier, Megan & Hall, Wayne & Nielsen, Suzanne & K Herkes, Geoffrey & Farrell, Michael & Degenhardt, Louisa. (2018). Evidence for cannabis and cannabinoids for epilepsy: a systematic review of controlled and observational evidence. Journal of Neurology, Neurosurgery & Psychiatry. jnnp-2017. 10.1136/jnnp-2017-317168.
https://www.ncbi.nlm.nih.gov/p.
http://jnnp.bmj.com/content/ea.

“Some studies indicate that under certain circumstances, CBD acute anxiolytic effects in rats were reversed after repeated 14-day administration of CBD.2 However, this finding might depend on the used animal model of anxiety or depression. This is supported by a study, where CBD was administered in an acute and “chronic” (2 weeks) regimen, which measured anxiolytic/antidepressant effects, using behavioral and operative models (OBX=olfactory bulbectomy as model for depression).18 The only observed side effects were reduced sucrose preference, reduced food consumption and body weight in the nonoperated animals treated with CBD (50 mg/kg). Nonetheless, the behavioral tests (for OBX-induced hyperactivity and anhedonia related to depression and open field test for anxiety) in the CBD-treated OBX animals showed an improved emotional response. Using microdialysis, the researchers could also show elevated 5-HT and glutamate levels in the prefrontal cortex of OBX animals only. This area was previously described to be involved in maladaptive behavioral regulation in depressed patients and is a feature of the OBX animal model of depression. The fact that serotonin levels were only elevated in the OBX mice is similar to CBD differential action under physiological and pathological conditions.

“A similar effect was previously described in anxiety experiments, where CBD proved to be only anxiolytic in subjects where stress had been induced before CBD administration. Elevated glutamate levels have been proposed to be responsible for ketamine’s fast antidepressant function and its dysregulation has been described in OBX mice and depressed patients. Chronic CBD treatment did not elicit behavioral changes in the nonoperated mice. In contrast, CBD was able to alleviate the affected functionality of 5HT1A receptors in limbic brain areas of OBX mice.18 and references therein

“Schiavon et al. cite three studies that used chronic CBD administration to demonstrate its anxiolytic effects in chronically stressed rats, which were mostly mediated via hippocampal neurogenesis.19 and references therein For instance, animals received daily i.p. injections of 5 mg/kg CBD. Applying a 5HT1A receptor antagonist in the DPAG (dorsal periaqueductal gray area), it was implied that CBD exerts its antipanic effects via these serotonin receptors. No adverse effects were reported in this study.”

Iffland, Kerstin, and Franjo Grotenhermen. “An Update on Safety and Side Effects of Cannabidiol: A Review of Clinical Data and Relevant Animal Studies.” Cannabis and cannabinoid research vol. 2,1 139-154. 1 Jun. 2017, doi:10.1089/can.2016.0034
https://www.ncbi.nlm.nih.gov/p.

“Today, CBD is used for the treatment of a wide range of medical conditions. This started with the somewhat serendipitous discovery (by parents experimenting with self-medication for their children) that CBD had a therapeutic effect on a serious form of epilepsy in children, called Dravet syndrome [8]. This effect is now under clinical investigation with the pharmaceutical CBD product Epidiolex®, which is currently in phase 3 trials with encouraging results [9, 10]. The media attention generated by its effect on severely ill children gave CBD the push needed to become a much desired medicine almost overnight [11]. Other medical indications that may be treated with CBD, and are supported to some extent by clinical proof, include Parkinson’s disease [12], schizophrenia [13], and anxiety disorder [14]. However, although research into the therapeutic effects of CBD is rapidly increasing, most current uses of CBD are not (yet) supported by clinical data. The popular use of these products means that physicians may be confronted with the effects of CBD oil even when they do not prescribe it themselves.

“An excellent example is the use of CBD (and also THC) products for the self-medicating of cancer, with the intention of fully curing it [15]. This is based on an increasing body of preclinical evidence showing cannabinoids to be capable, under some conditions, of inhibiting the development of cancer cells in vitro or in vivo by various mechanisms of action, including induction of apoptosis, inhibition of angiogenesis, and arresting the cell cycle [16]. This is certainly exciting news, and research is ongoing around the world, but there is no solid clinical evidence yet to support that cannabinoids – whether natural or synthetic – can effectively and safely treat cancer in actual humans [17]. In fact, there are indications that certain types of cancer may even accelerate when exposed to cannabinoids [18]. This becomes problematic when patients choose to refuse chemotherapy treatment because they firmly believe in the rumored curative properties of cannabinoids. As a result, recommendation of cannabinoids for treating cancer should be done with great care, and with distinction as to the type of cancer being treated [19].”

Hazekamp A: The Trouble with CBD Oil. Med Cannabis Cannabinoids 2018;1:65-72. doi: 10.1159/000489287
https://www.karger.com/Article.

“Various studies on CBD and psychosis have been conducted. 20 For instance, an animal model of psychosis can be created in mice by using the NMDAR antagonist MK-801. The behavioral changes (tested with the prepulse inhibition [PPI] test) were concomitant with decreased mRNA expression of the NMDAR GluN1 subunit gene (GRN1) in the hippocampus, decreased parvalbumin expression (=a calcium-binding protein expressed in a subclass of GABAergic interneurons), and higher FosB/ΔFosB expression (=markers for neuronal activity). After 6 days of MK-801 treatment, various CBD doses were injected intraperitoneally (15, 30, 60 mg/kg) for 22 days. The two higher CBD doses had beneficial effects comparable to the atypical antipsychotic drug clozapine and also attenuated the MK-801 effects on the three markers mentioned above. The publication did not record any side effects. 21

“One of the theories trying to explain the etiology of bipolar disorder (BD) is that oxidative stress is crucial in its development. Valvassori et al. therefore used an animal model of amphetamine-induced hyperactivity to model one of the symptoms of mania. Rats were treated for 14 days with various CBD concentrations (15, 30, 60 mg/kg daily i.p.). Whereas CBD did not have an effect on locomotion, it did increase brain-derived neurotrophic factor (BDNF) levels and could protect against amphetamine-induced oxidative damage in proteins of the hippocampus and striatum. No adverse effects were recorded in this study. 22

“Another model for BD and schizophrenia is PPI of the startle reflex both in humans and animals, which is disrupted in these diseases. Peres et al., list five animal studies, where mostly 30 mg/kg CBD was administered and had a positive effect on PPI.20 Nonetheless, some inconsistencies in explaining CBD effects on PPI as model for BD exist. For example, CBD sometimes did not alter MK-801-induced PPI disruption, but disrupted PPI on its own. 20 If this effect can be observed in future experiments, it could be considered to be a possible side effect.”

Iffland, Kerstin, and Franjo Grotenhermen. “An Update on Safety and Side Effects of Cannabidiol: A Review of Clinical Data and Relevant Animal Studies.” Cannabis and cannabinoid research vol. 2,1 139-154. 1 Jun. 2017, doi:10.1089/can.2016.0034
https://www.ncbi.nlm.nih.gov/p.

“CBD, which is nonhedonic, can reduce heroin-seeking behavior after, for example, cue-induced reinstatement. This was shown in an animal heroin self-administration study, where mice received 5 mg/kg CBD i.p. injections. The observed effect lasted for 2 weeks after CBD administration and could normalize the changes seen after stimulus cue-induced heroin seeking (expression of AMPA, GluR1, and CB1R). In addition, the described study was able to replicate previous findings showing no CBD side effects on locomotor behavior. 23 “

Iffland, Kerstin, and Franjo Grotenhermen. “An Update on Safety and Side Effects of Cannabidiol: A Review of Clinical Data and Relevant Animal Studies.” Cannabis and cannabinoid research vol. 2,1 139-154. 1 Jun. 2017, doi:10.1089/can.2016.0034
https://www.ncbi.nlm.nih.gov/p.

“There are various mechanisms underlying neuroprotection, for example, energy metabolism (whose alteration has been implied in several psychiatric disorders) and proper mitochondrial functioning. 24 An early study from 1976 found no side effects and no effect of 0.3–300 μg/mg protein CBD after 1 h of incubation on mitochondrial monoamine oxidase activity in porcine brains. 25 In hypoischemic newborn pigs, CBD elicited a neuroprotective effect, caused no side effects, and even led to beneficial effects on ventilatory, cardiac, and hemodynamic functions. 26

“A study comparing acute and chronic CBD administration in rats suggests an additional mechanism of CBD neuroprotection: Animals received i.p. CBD (15, 30, 60 mg/kg b.w.) or vehicle daily, for 14 days. Mitochondrial activity was measured in the striatum, hippocampus, and the prefrontal cortex. 27 Acute and chronic CBD injections led to increased mitochondrial activity (complexes I-V) and creatine kinase, whereas no side effects were documented. Chronic CBD treatment and the higher CBD doses tended to affect more brain regions. The authors hypothesized that CBD changed the intracellular Ca2+ flux to cause these effects. Since the mitochondrial complexes I and II have been implied in various neurodegenerative diseases and also altered ROS (reactive oxygen species) levels, which have also been shown to be altered by CBD, this might be an additional mechanism of CBD-mediated neuroprotection. 1,27

“Interestingly, it has recently been shown that the higher ROS levels observed after CBD treatment were concomitant with higher mRNA and protein levels of heat shock proteins (HSPs). In healthy cells, this can be interpreted as a way to protect against the higher ROS levels resulting from more mitochondrial activity. In addition, it was shown that HSP inhibitors increase the CBD anticancer effect in vitro. 28 This is in line with the studies described by Bergamaschi et al., which also imply ROS in CBD effect on (cancer) cell viability in addition to, for example, proapoptotic pathways such as via caspase-8/9 and inhibition of the procarcinogenic lipoxygenase pathway. 1

“Another publication studied the difference of acute and chronic administration of two doses of CBD in nonstressed mice on anxiety. Already an acute i.p. administration of 3 mg/kg was anxiolytic to a degree comparable to 20 mg/kg imipramine (an selective serotonin reuptake inhibitor [SSRI] commonly prescribed for anxiety and depression). Fifteen days of repeated i.p. administration of 3 mg/kg CBD also increased cell proliferation and neurogenesis (using three different markers) in the subventricular zone and the hippocampal dentate gyrus. Interestingly, the repeated administration of 30 mg/kg also led to anxiolytic effects. However, the higher dose caused a decrease in neurogenesis and cell proliferation, indicating dissociation of behavioral and proliferative effects of chronic CBD treatment. The study does not mention adverse effects. 19 “

Iffland, Kerstin, and Franjo Grotenhermen. “An Update on Safety and Side Effects of Cannabidiol: A Review of Clinical Data and Relevant Animal Studies.” Cannabis and cannabinoid research vol. 2,1 139-154. 1 Jun. 2017, doi:10.1089/can.2016.0034
https://www.ncbi.nlm.nih.gov/p.

“Numerous studies show the CBD immunomodulatory role in various diseases such as multiple sclerosis, arthritis, and diabetes. These animal and human ex vivo studies have been reviewed extensively elsewhere, but studies with pure CBD are still lacking. Often combinations of THC and CBD were used. It would be especially interesting to study when CBD is proinflammatory and under which circumstances it is anti-inflammatory and whether this leads to side effects (Burstein, 2015: Table 1 shows a summary of its anti-inflammatory actions; McAllister et al. give an extensive overview in Table 1 of the interplay between CBD anticancer effects and inflammation signaling). 29,30

“In case of Alzheimer’s disease (AD), studies in mice and rats showed reduced amyloid beta neuroinflammation (linked to reduced interleukin [IL]-6 and microglial activation) after CBD treatment. This led to amelioration of learning effects in a pharmacological model of AD. The chronic study we want to describe in more detail here used a transgenic mouse model of AD, where 2.5-month-old mice were treated with either placebo or daily oral CBD doses of 20 mg/kg for 8 months (mice are relatively old at this point). CBD was able to prevent the development of a social recognition deficit in the AD transgenic mice.

“Moreover, the elevated IL-1 beta and TNF alpha levels observed in the transgenic mice could be reduced to WT (wild-type) levels with CBD treatment. Using statistical analysis by analysis of variance, this was shown to be only a trend. This might have been caused by the high variation in the transgenic mouse group, though. Also, CBD increased cholesterol levels in WT mice but not in CBD-treated transgenic mice. This was probably due to already elevated cholesterol in the transgenic mice. The study observed no side effects. 31 and references within

“In nonobese diabetes-prone female mice (NOD), CBD was administered i.p. for 4 weeks (5 days a week) at a dose of 5 mg/kg per day. After CBD treatment was stopped, observation continued until the mice were 24 weeks old. CBD treatment lead to considerable reduction of diabetes development (32% developed glucosuria in the CBD group compared to 100% in untreated controls) and to more intact islet of Langerhans cells. CBD increased IL-10 levels, which is thought to act as an anti-inflammatory cytokine in this context. The IL-12 production of splenocytes was reduced in the CBD group and no side effects were recorded. 32

“After inducing arthritis in rats using Freund’s adjuvant, various CBD doses (0.6, 3.1, 6.2, or 62.3 mg/day) were applied daily in a gel for transdermal administration for 4 days. CBD reduced joint swelling, immune cell infiltration. thickening of the synovial membrane, and nociceptive sensitization/spontaneous pain in a dose-dependent manner, after four consecutive days of CBD treatment. Proinflammatory biomarkers were also reduced in a dose-dependent manner in the dorsal root ganglia (TNF alpha) and spinal cord (CGRP, OX42). No side effects were evident and exploratory behavior was not altered (in contrast to Δ9-THC, which caused hypolocomotion). 33 “

Iffland, Kerstin, and Franjo Grotenhermen. “An Update on Safety and Side Effects of Cannabidiol: A Review of Clinical Data and Relevant Animal Studies.” Cannabis and cannabinoid research vol. 2,1 139-154. 1 Jun. 2017, doi:10.1089/can.2016.0034
https://www.ncbi.nlm.nih.gov/p.

“Another chemical shared by both industrial hemp and marijuana is Cannabidiol (CBD). 48 CBD is unique because it is not intoxicating and it also moderates the euphoric effect of THC. 49 Marijuana, which has disproportionately higher levels of THC than industrial hemp, also contains lower levels of CBD. 50 The higher THC and lower CBD concentration gives marijuana its psychoactive effect. 51 Conversely, industrial hemp’s low THC levels and comparatively high CBD levels produce none of the intoxicating effects of marijuana. 52 “

Cannabidiol for the Treatment of Aromatase Inhibitor-Associated Arthralgias

I. To determine the effect of 15 weeks of CBD on aromatase inhibitor (AI)-associated worst joint pain, as assessed with the Brief Pain Inventory (BPI).

I. To determine the effect of 15 weeks of CBD on other AI-associated pain symptoms, including average pain and pain interference, as assessed with the BPI.

II. To determine the effect of 15 weeks of CBD on other AI-associated symptoms, including anxiety, depression, insomnia, and cognitive function, as assessed with the Patient Reported Outcomes Measurement Information System (PROMIS-29+2) Profile.

III. To examine the safety and tolerability of 15 weeks of CBD in patients with breast cancer taking aromatase inhibitor therapy.

IV. To examine the effect of 15 weeks of CBD on circulating estrogen concentrations.

I. To examine the impact of 15 weeks of CBD therapy on inflammatory biomarkers in patients with breast cancer taking aromatase inhibitor therapy.

II. To examine demographic, clinical, psychosocial, and inflammatory predictors of response to CBD therapy.

Patients receive CBD orally (PO) twice daily (BID) for 15 weeks in the absence of unacceptable toxicity.

After completion of study treatment, patients are followed up at 30 days.

Trial Phase & Type

Trial Phase Phase II

Trial Type Supportive care

Lead Organization

Lead Organization
University of Michigan Comprehensive Cancer Center

Principal Investigator
N. Lynn Henry

Trial IDs

  • Primary ID UMCC 2020.041
  • Secondary IDs NCI-2021-01667, HUM00182109
  • Clinicaltrials.gov ID NCT04754399
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