Drug–Drug Interactions of Cannabidiol with Standard-of-Care Chemotherapeutics

Cannabidiol (CBD) is an easily accessible and affordable Marijuana (Cannabis sativa L.) plant derivative with an extensive history of medical use spanning thousands of years. Interest in the therapeutic potential of CBD has increased in recent years, including its anti-tumour properties in various cancer models. In addition to the direct anticancer effects of CBD, preclinical research on numerous cannabinoids, including CBD, has highlighted their potential use in: (i) attenuating chemotherapy-induced adverse effects and (ii) enhancing the efficacy of some anticancer drugs. Therefore, CBD is gaining popularity as a supportive therapy during cancer treatment, often in combination with standard-of-care cancer chemotherapeutics. However, CBD is a biologically active substance that modulates various cellular targets, thereby possibly resulting in unpredictable outcomes, especially in combinations with other medications and therapeutic modalities. In this review, we summarize the current knowledge of CBD interactions with selected anticancer chemotherapeutics, discuss the emerging mechanistic basis for the observed biological effects, and highlight both the potential benefits and risks of such combined treatments. Apart from the experimental and preclinical results, we also indicate the planned or ongoing clinical trials aiming to evaluate the impact of CBD combinations in oncology. The results of these and future trials are essential to provide better guidance for oncologists to judge the benefit-versus-risk ratio of these exciting treatment strategies. We hope that our present overview of this rapidly advancing field of biomedicine will inspire more preclinical and clinical studies to further our understanding of the underlying biology and optimize the benefits for cancer patients.


Introduction
Understanding drug interactions is a fundamental yet challenging pharmacological issue that is especially problematic in oncology due to the usually narrow therapeutic window and potential serious toxicity profiles of drugs that are often applied to vulnerable patients and those with a long history of pre-treatment. Drug interactions may occur as a result of pharmacokinetic, pharmacodynamic, or biological factors, resulting in various outcomes, including decreased or increased therapeutic or adverse responses [1]. Despite extensive research and development work, most anticancer therapies have severe adverse effects. To mitigate such therapy-associated adverse effects, patients frequently use vitamins, dietary supplements, or herbal products, with Cannabis plant-based products occupying a prominent position. Indeed, a recent survey indicated that approximately two-thirds of patients with cancer had used Cannabis products during their ongoing therapy to alleviate adverse effects [2,3]. In these studies, cannabis products were mainly taken via inhalation, ingestion, or topically. These products were predominantly in the form of

CBD Interactions with Antimetabolites
Antimetabolites (Table 1) are small molecules resembling standard nucleotides-the building blocks of DNA. Because of this resemblance, they can be incorrectly incorporated into DNA or may inhibit DNA synthesis. Antimetabolites were among the first anticancer chemotherapeutics introduced in the late 1940s; a notable example is aminopterin, which was used to treat paediatric acute lymphoblastic leukaemia. Currently, antimetabolites represent a cornerstone of treatment for various cancer types [31,32].

5-Fluorouracil
5-fluorouracil is an uracil analogue that inhibits thymidylate synthetase. The inhibited enzyme is incapable of deoxythymidine monophosphate conversion, which results in the depletion of deoxythymidine triphosphate used for DNA synthesis [32,33]. 5-fluorouracil is commonly used to treat a range of diverse solid tumours, including those of the gastrointestinal tract and head-and-neck, with common adverse effects such as gastrointestinal and oral mucositis [32,33]. Cuba et al. (2020) [33] evaluated the effect of CBD on oral mucositis inflicted by 5-fluorouracil in mice. Interestingly, CBD treatment reduced oral mucositis, probably as a consequence of its antioxidant properties, which were documented by the increased catalase and glutathione levels in CBD-cotreated mice. CBD with 5-fluorouracil and some other chemotherapeutics is planned to be tested for efficacy and safety in a randomized clinical trial on colon and rectal cancers (NCT03607643) [34].

Gemcitabine
Gemcitabine is a nucleoside analogue-specifically, a deoxycytidine. After intake, the drug is metabolized and incorporated into the DNA strand instead of the original nucleoside, which leads to inhibition of DNA polymerase and ribonucleotide reductase [32,35]. Gemcitabine is commonly used in patients with non-small cell lung, pancreatic, urinary bladder, and breast cancer, with haematological toxicity, oedema, and pulmonary, cutaneous, and gastrointestinal toxicity as common adverse effects [35]. Although the impact of CBD on gemcitabine-induced adverse effects is unknown, preclinical studies suggest an interesting potentiation of its anticancer toxicity. For example, combined treatment with CBD and gemcitabine significantly extended animal survival in the pancreatic ductal adenocarcinoma (PDAC) mouse model and also enhanced the inhibition of the proliferation of PDAC cell lines. This effect can be explained by CBD's antagonism of the GRP55 protein, a receptor of lysophosphatidylinositol, which is known to have a role in tumour progression [36][37][38]. GRP55 inhibition suppresses cell cycle progression and hence cell proliferation, reduces MAPK kinase activation, and lowers the overall abundance of ribonucleotide reductases in PDAC cells, all of which are associated with gemcitabine resistance [36,38]. The potentiation of gemcitabine toxicity by CBD was also confirmed in another PDAC cell line (MiaPaCa-2) [39]. Combined treatment with CBD and gemcitabine will be assessed for pancreatic cancer in a randomized clinical trial (NCT03607643) [34].

Methotrexate
Folates, compounds related to the vitamin D family, function via one-carbon metabolism, which is essential for purine, thymidylate, and polyamine synthesis. Folates are involved in the synthesis of S-adenosyl methionine, which is critical for the methylation of DNA, histones, lipids, and neurotransmitters [40]. Methotrexate is an antifolate antagonist targeting dihydrofolate reductase (DHFR), a key component of the folate pathway. Due to its structural similarity to folic acid, methotrexate can block the binding pocket of DHFR, leading to secondary inhibition of downstream enzymes in the folate pathway [32,[40][41][42]. Methotrexate is used in combination with other chemotherapeutics to treat several types of cancer, including acute lymphocytic leukaemia, lymphoma, carcinomas of the breast and urinary bladder, and osteosarcoma [40,41]. Brown and Winterstein (2019) [6] suggested that CBD may interact with methotrexate treatment because it inhibits the ABCG2/BCRP transporter [24], which contributes to cellular efflux of methotrexate [43].
CBD cotreatment could thus lead to methotrexate accumulation, increasing the drug's efficacy but also potentially strengthening its adverse effects. However, this hypothesis awaits experimental confirmation.

Interactions of CBD with Alkylating Agents and Platinum-Based Drugs
Alkylating agents (Table 2) are reactive chemical substances that target DNA and proteins and belong to the oldest chemotherapeutics. Their ability to alter biological molecules is based on the transfer of alkyl groups to target molecules such as DNA, which often alters their structure or disrupts their function [44,45]. The alkylating agent mustard gas was first used as a chemotherapeutic in the early 1930s to treat skin cancer [45,46]. Mechlorethamine, an analogue of mustard gas, was approved as a chemotherapeutic in the late 1940s, followed by a number of related drugs [45].
Platinum-based drugs are commonly classified as alkylating agents even though they do not directly alkylate DNA; instead, they form covalent crosslinks between DNA strands and covalent DNA-protein adducts [45].
It has been hypothesized that CBD interferes with carmustine via the transient potential vanilloid 2 receptor (TRPV2), a nonselective cation channel that is usually activated by heat, altered osmolarity, or membrane stretching. As a TRPV2 agonist, CBD increases Ca 2+ influx [19], possibly affecting the uptake and retention of some anticancer drugs. This theory was experimentally confirmed in glioblastoma cells, where CBD coadministration caused sensitization against carmustine. Interestingly, this effect was not observed in normal human astrocytes [49,50]. TRPV2 expression is also upregulated by a spliced variant of the AML1a (acute myeloid leukaemia) protein, induced by CBD treatment. As a transcription factor, AML1a impacts the expression of genes whose products are implicated in various biological processes, including hematopoietic self-renewal, cell proliferation, and differentiation [51,52]. Although the function of AML1a in GBM (glioblastoma multiforme) remains poorly understood, AML1a is upregulated during GBM stem-like cell (GSC) differentiation, and downregulation of AML1a is associated with GBM chemoresistance. Thus, upregulation of AML1a by CBD may lead to GSC differentiation, resulting in restoration of carmustine sensitivity. This concept was experimentally validated at the level of GSC cell lines [52]. The potentiating effect of CBD on carmustine cytotoxicity was further examined by Deng et al. (2017) [53] in human GBMs, mouse primary GBMs, and mouse neural progenitor cells. Although CBD sensitized to carmustin both the murine and human GBM cells, a subsequent analysis of drug interactions between carmustine and CBD, their concentration dependence, and their effects on efficacy revealed a range of concentration-dependent synergistic, additive, or antagonistic effects on cell viability and proliferation among the three analysed model systems, overall indicating the complexity of such drug interactions [53].

Temozolomide
Temozolomide is an imidazotetrazine lipophilic prodrug that is activated and metabolized under physiological pH conditions. The resulting methyldiazonium salt is a lipophilic alkylating agent that passes through the blood-brain barrier. Consequently, temozolomide is particularly suitable for treating brain tumours [54,55]. Reported adverse effects of this drug include haematological, gastrointestinal, and neurological toxicities [56]. Experiments using the human U87MG glioma cell line, primary glioblastoma cells, and normal human astrocytes (NHAs) conducted by Nabissi et al. (2013) [49] suggested that temozolomide's efficacy is potentiated by CBD, again via TRPV2 activation. As in the case of carmustine, temozolomide cytotoxicity was potentiated in glioblastoma cells but not in NHAs.
The CBD-promoted potentiation of temozolomide's effects against GBM may also partly reflect effects on extracellular vesicles (EVs). Cells use EVs for drug efflux, prooncogenic signalling, invasion, and immunosuppression [57], and there is clear evidence that CBD is an effective modulator of EV properties. For example, CBD treatment of chemoresistant and chemosensitive patient-derived GBM cells led to the formation of EVs with reduced levels of pro-oncogenic miR21 and an elevated anti-oncogenic miR126 [58]. The same study suggested that, apart from its effects on EV properties, CBD treatment may reduce levels of prohibitin, a protein that protects mitochondrial function and may contribute to temozolomide chemoresistance.
In addition to the additive/synergistic effects observed between CBD and temozolomide toxicity, concentration-dependent antagonistic effects have been reported in human GBMs, mouse primary GBM cells, and mouse neural progenitor cells, suggesting a rather complex relationship [53]. In one study, the combination of CBD with temozolomide increased tumour growth in a mouse GBM model compared to temozolomide treatment alone [59]. However, the opposite effect was observed in another study using the same mouse model [60]. It is unclear why these two studies reached such highly discrepant results.
Notably, there are also limited clinical data. Likar et al. (2019) [61] evaluated brain tumours in patients who took CBD concomitantly with radiochemotherapy, including temozolomide. At the time of publication, patients taking CBD survived longer than expected. Currently, two clinical trials evaluate the effects of CBD and temozolomide combinations in patients with GBM (NCT03607643 and NCT03687034) [34,62].

Cisplatin
Cisplatin ((SP-4-2)-diamminedichloridoplatinum (II)) interacts with purines incorporated into DNA and causes DNA lesions. It is widely used as a chemotherapeutic agent for treating solid tumours despite having severe side effects, including renal toxicity, ototoxicity, hepatotoxicity, and gastrointestinal toxicity [45,63]. Several lines of evidence indicate that CBD is an effective attenuator of these cisplatin-induced adverse effects. For example, renal toxicity, a frequent limiting factor for cisplatin treatment, was effectively suppressed by CBD in mice [64]. Indeed, CBD was shown to protect kidneys from cisplatin-induced effects by reducing oxidative and nitrosative stress, inflammation, and cell death, while improving renal function. Moreover, studies on cisplatin-induced emesis in shrews reported by Kwiatkowska et al. (2004) [65] showed that treatment with low doses of CBD attenuated cisplatin-induced vomiting, whereas high doses potentiated this adverse effect. Similar findings were obtained in another study on shrews by Rock et al. (2012) [66], which in a mouse model showed that the anti-emetic and anti-nausea effect of CBD was mediated by indirect activation of the somatodendritic serotonin autoreceptor 5-HT1A in the dorsal raphe nucleus. Activation of 5-HT1A reduces the excitability of serotoninergic neurons and thus reduces serotonin release in terminal forebrain regions [66,67]. The same mechanism was confirmed in shrews and rats after treatment with cannabidiolic acid (CBDA), a compound similar to CBD that is effective at substantially lower concentrations [68].
Besides the ability to reduce adverse effects, CBD also seems to potentiate cisplatin toxicity in endometrial cancer cell lines. The enhanced response to cisplatin appeared to result from TRPV2 receptor activation and was strengthened in TRPV2 overexpression models [69]. Conversely, in the ovarian cancer cell line SKOV-3, CBD did not affect cisplatin-induced cytotoxicity, and a cell protective effect was observed at higher CBD concentrations [70]. Similarly, in GBM cell lines, combined treatment with CBD and cisplatin had mixed results; additive, synergistic, and antagonistic effects were all observed, depending on the drug concentrations used [53].

Oxaliplatin
Oxaliplatin is a third-generation platinum drug based on diaminocyclohexane (DACH) that can overcome cisplatin-induced resistance. Oxaliplatin is less reactive toward DNA than cisplatin, forming fewer protein-DNA adducts and DNA-DNA crosslinks. Despite its lower reactivity, oxaliplatin displays a similar cytotoxicity profile to cisplatin. It is believed that the DACH molecule causes specific DNA lesions that are difficult to detect and/or repair. Moreover, oxaliplatin interacts more strongly with proteins than cisplatin [71,72]. In addition to generating DNA lesions, oxaliplatin induces nucleolar and ribosomal stress, which may contribute significantly to its overall toxic effects [73]. Oxaliplatin is primarily used to treat colon cancer [63,72] and has less severe side effects than cisplatin; nephrotoxicity and ototoxicity are relatively rare. Moreover, while oxaliplatin treatment can cause gastrointestinal toxicity and hepatotoxicity, the main dose-limiting adverse effect is neurotoxicity [72] accompanied by mechanical allodynia. Importantly, CBD is a potent attenuator of oxaliplatin-induced neuropathy-associated pain in mice [74]. Pereira et al. (2021) [75] recently investigated the role of the endocannabinoid system in oxaliplatin-induced peripheral sensory neuropathy and showed that CBD has an analgesic effect that was partly attributed to interactions with the CB1 receptor. CBD treatment also attenuated mechanical hyperalgesia and c-Fos expression in the spinal cord's dorsal root ganglion and dorsal horn without cannabimimetic effects.
In another study on the potentiation of oxaliplatin by CBD, experiments using parental and oxaliplatin-resistant human colorectal cancer cell lines and their mouse xenografts conducted by Jeong et al. (2019) [76] showed that CBD restored oxaliplatin sensitivity tested in the resistant cancer cells. Mechanistically, this was attributed to a CBD-promoted decrease in NOS3 phosphorylation, which is otherwise elevated in oxaliplatin-resistant tumour cell lines. Reducing NOS3 phosphorylation lowered the level of NO and superoxide dismutase 2, resulting in ROS induction and mitochondrial dysfunction. Oxaliplatin combined with CBD is under evaluation in a randomized clinical trial of colorectal cancer (NCT03607643) [34].

Carboplatin
Carboplatin, or cis-diamine-1,1 -cyclobutane dicarboxylate platinum (II), is a secondgeneration platinum-based drug. Its mechanism of action resembles that of cisplatin, and its therapeutic effect is weaker than or equal to that of cisplatin itself [77,78]. Carboplatin is used to treat testicular germ cell tumours as well as gynaecological, head-and-neck, thoracic, and urinary bladder cancers [79]. It has fewer adverse effects than cisplatin because of its different pharmacodynamics; its main adverse effects are myelosuppression, nausea, and vomiting [80]. Current knowledge of the interaction between CBD and carboplatin is limited because the combination of both drugs has only been tested in cellular models of canine urothelial carcinoma, in which the effect was antagonistic [81].

Interactions of CBD with Microtubule-Targeting Agents
Microtubules (Table 3) are components of the cytoskeleton that are dynamically polymerized and depolymerized. Microtubule-targeting agents inhibit one of these two processes, which has a profound impact on cytoskeleton morphology, cellular transport, motility, and mitosis, and thereby exert anticancer and anti-angiogenic effects [82,83]. The first microtubule-targeting agent used in the clinic was vinblastine, which was approved for the treatment of lymphomas and various solid tumours in the early 1960s. Its success led to the discovery and development of a wider spectrum of agents with similar activity, with several of these drugs currently used to treat various types of malignancies [83].

Vinblastine
Vinblastine is an indole-containing alkaloid isolated from Catharanthus roseus and its endophytes [84] that shows microtubule-destabilizing properties. As an inhibitor of microtubule polymerization [82], it is widely used to treat Hodgkin's lymphoma, lymphosarcoma, choriocarcinoma, neuroblastoma, various carcinomas, leukaemia, Wilkins's tumour, and reticulum cell sarcoma [85]. Its known adverse effects include gastrointestinal, genitourinary, neurologic, haematological, and hepatic toxicities and patients treated with vinblastine are also prone to infections [86].
The effect of CBD on vinblastine-induced adverse effects is not yet known, but some studies suggest potentiation of the curative effect. For example, in leukaemia cells, CBD can help overcome vinblastine resistance that is caused by p-glycoprotein (p-gp) transporter overexpression. P-gp overexpression is responsible for multidrug resistance, and vinblastine is among its known substrates. CBD was shown to reduce the transporter's expression, reducing the efflux of vinblastine from cells. CBD cotreatment also increased vinblastine toxicity, but only in cells overexpressing the transporter. Notably, the effect was relatively mild, and verapamil, an established inhibitor of the p-gp transporter, was considerably more effective [25]. The synergistic effect of CBD and vinblastine manifested by reduced viability and increased apoptosis was further confirmed in canine urothelial carcinoma cells [81].

Paclitaxel
Paclitaxel, a member of the taxane family isolated from Taxus brevifolia [87], is a microtubule-stabilizing agent used to treat various solid tumours. Its common adverse effects include myelosuppression and peripheral neuropathy [88]. Ward et al. (2011;2014) [89,90] used CBD to attenuate paclitaxel-induced peripheral neuropathy and showed that CBD cotreatment prevented this side effect in mice [89,90]. The neuroprotective activity of CBD was partially explained by its agonistic effect on the 5-HT1A receptor, which is involved in central nervous system sensitization [89][90][91]. The same study also reported additive and synergistic effects in murine and human breast cancer cell lines [90]. The attenuating effect on paclitaxel-induced mechanical allodynia was also confirmed by King et al. (2017) [74] in mice. Apart from 5-HT1A, the protective effect of CBD on paclitaxel-induced peripheral neuropathy might reflect the impact on mitochondrial Na + Ca 2+ exchanger-1 (mNCX-1), which is primarily responsible for calcium homeostasis. Indeed, the knockdown of mNCX-1 attenuated the protection conferred by CBD in dissociated dorsal root ganglia [92]. Another study confirmed CBD as an attenuator of paclitaxel-induced mechanical sensitivity and showed that CBD could not reverse previously established mechanical sensitivity in mice [93].
Other studies have examined the capacity of CBD to modulate the efficacy of paclitaxel. Synergistic or additive effects with CBD pre-/co-treatment on cytotoxicity were observed in breast cancer and ovarian cancer cell lines. These studies tested two CBD preparations-CBD in solution and CBD in polymeric microparticles-both of which were effective in the human MCF7, MDA-MB-231, and SKOV-3 cell lines and reduced tumour growth during in ovo studies on the chorioallantoic membrane of chicken embryos [70,94].
According to Luongo et al. (2020) [39], CBD and paclitaxel cotreatment had synergistic and additive effects in pancreatic cancer cell lines but only at relatively high CBD concentrations. Synergy was also observed in the MCF7 breast cancer cell line model [95]. CBD and paclitaxel cotreatment has also been investigated in human colorectal and gastric adenocarcinoma cell lines [96]. While CBD did not reduce viability after paclitaxel treatment in some cell lines, an additive effect on the inhibition of DNA replication was observed. Interestingly, the authors highlighted the importance of the foetal calf serum content in cell culture media, leading to different results after CBD application, suggesting the potential influence of growth factors on the observed effects of CBD. In addition, CBD enhanced the cytotoxic effect of paclitaxel in endometrial cancer cell lines regardless of TRPV2 overexpression [69].
Finally, Brown and Winterstein (2019) [6] demonstrated that paclitaxel is a substrate of the ABCB11/BSEP transporter, which is another CBD target. Combined treatment with CBD may thus increase the efficacy of paclitaxel, albeit at the cost of a simultaneous increase in adverse effects.

Docetaxel
Docetaxel is a semisynthetic agent consisting of 10-diacetyl baccatin III derived from Taxus baccata that has been esterified with a synthetic side chain. Docetaxel is a microtubule stabilizer [97] that is used to treat metastatic breast, lung, prostate, gastric, and head-and-neck cancers; it seems to be more potent than paclitaxel. However, its poor solubility in water necessitates administration as a solution in ethanol and polysorbate 80, which introduces additional solvent-induced side effects. Docetaxel also has more severe adverse effects than paclitaxel, including neutropenia, musculoskeletal toxicity, and neurotoxicity [98].
De Petrocellis et al. (2013) [99] studied the pro-apoptotic effects of cannabinoids when combined with docetaxel in prostate carcinoma. Both CBD and CBD-DBS (extracts from Cannabis sativa L. strains enriched in particular cannabinoids) were examined. Interestingly, these researchers highlighted the importance of media composition (with/without sera) in experiments using CBD by showing that certain media can potentiate docetaxel toxicity in human LNCaP (androgen receptor AR-positive) cells. In the same study, CBD also potentiated the effect of docetaxel in AR-negative human DU-145 cells at lower concentrations but showed a tendency towards a protective effect at higher concentrations. In experiments using the DU-145 mouse xenograft model, CBD-BDS potentiated the effects of docetaxel, but a mild protective effect was observed following cotreatment with CBD and docetaxel in an LNCaP xenograft model. Combined treatment with docetaxel and CBD has also been examined in the MCF7 breast adenocarcinoma cell line, revealing that synergistic effects can be obtained at specific molar ratios [95].

Vincristine
Vincristine is a vinca alkaloid isolated from Catharanthus roseus that interferes with microtubule polymerization [100] and is used to treat cancers including leukaemia, lymphoma, central nervous system cancers, and sarcomas. Its common adverse effects include neuropathy and constipation [101].
While mechanical allodynia, a neurological condition induced by vincristine, was not attenuated by CBD pre-treatment [74], multiple lines of evidence suggest that CBD potentiates the anticancer effect of vincristine. First, as mentioned above, CBD targets the ATP-binding cassette (ABC) transporter (ABCC1/MRP1) responsible for multidrug resistance, including resistance to vincristine. In ovarian cancer cell lines overexpressing ABCC1/MRP1, CBD treatment attenuated the ABCC1/MRP1-mediated drug transport and increased the accumulation of vincristine inside the cells [102]. Second, combined CBD and vincristine treatment reduced cell proliferation in a synergic or additive manner in canine neoplastic cell lines. The authors suggested that this effect could be related to CBDmediated induction of ERK and JNK kinase-mediated phosphorylation signalling leading to autophagy and apoptosis [103]. Third, a case report suggested a favourable impact of CBD on patients with high-grade gliomas treated with radiotherapy accompanied by a combination of vincristine, lomustine, and procarabine. Importantly, two patients cotreated with CBD showed an improved health condition that exceeded expectations [104].

CBD Interactions with Anthracyclines
The anthracyclines (Table 4) are a class of compounds that intercalate into DNA, causing inhibition of DNA synthesis and interference with topoisomerase II [105]. The proto-typic anthracyclines, doxorubicin and daunorubicin, were isolated from Streptomyces peucetius in the 1960s. Since then, several other compounds of this class have received clinical approval [106].

Doxorubicin
Doxorubicin has many therapeutic applications, including in the treatment of haematological malignancies, diverse types of carcinomas, and sarcomas. Its main adverse effects include cardiotoxicity, neurotoxicity, nephrotoxicity, and hepatotoxicity [107]. Doxorubicin has a complex molecular mechanism of action that involves both free radical induction and DNA intercalation leading to DNA breaks and chromosomal aberrations, often resulting in cell senescence or death [108].
Cardiomyopathy is the most severe doxorubicin-evoked adverse effect, which is manifested by myocardial injury, oxidative and nitrative stress, cardiac dysfunction, reduced mitochondrial biogenesis and function and decreased expression of medium-chain acyl-CoA dehydrogenase and uncoupling proteins 2 and 3 [109]. Preclinical studies have indicated that CBD may effectively attenuate these effects. For example, Fouad et al. (2013) [110] examined the effects of CBD on doxorubicin-mediated cardiotoxicity in rats, revealing that rats co-treated with CBD had attenuated cardiac injury, along with reduced levels of serum creatine kinase-MB, troponin T, lipid peroxidation, cardiac malondialdehyde, tumour necrosis factor-α, nitric oxide, calcium ions, nitric oxide synthase, nuclear factor-κB, Fas ligand, and caspase-3. These CBD-mediated effects were accompanied by increased levels of cardiac glutathione (GSH), selenium, zinc ions, and survivin expression in cardiac tissue, all features that likely contributed to improved histological and biomarker readouts. A cardioprotective effect of CBD in doxorubicin-treated mice was also reported by Hao et al. (2015) [109], who observed reduced levels of cardiac oxidative and nitrative stress markers, inflammation, and cell death together with enhanced expression of matrix metalloproteinases and improvements in cardiac biogenesis and mitochondrial function.
The potentiation of doxorubicin's anticancer activity by CBD has also been widely studied. Increased doxorubicin accumulation was observed after CBD treatment of Caco-2 and LLC-PK1/MDR1 cancer cell lines; this effect is probably attributable to the blocking of doxorubicin efflux by CBD-induced inhibition of the p-gp transporter [26]. CBD is also an agonist of the TRPV2 channel, which increases doxorubicin uptake, as shown in the U87MG glioma cell line [49] and hepatocellular carcinoma cell lines [111]. Positive modulation of TRPV2 by CBD and improved efficacy of doxorubicin were also confirmed in human triple-negative breast cancer cell lines, including their mouse xenografts [112]. Consistently, CBD enhanced the cytotoxic effect of doxorubicin in an endometrial cancer cell line overexpressing TRPV2 to a greater extent than was observed in parental cells with regular TRPV2 expression [69]. Synergic effects of CBD and doxorubicin were also confirmed in the MCF7 breast adenocarcinoma cell line [95]. Patel et al. (2021) [113] tested combined treatment with doxorubicin and free CBD or CBD encapsulated in EVs as a new delivery system in a model of triple-negative breast cancer (TNBC). Both formulations showed comparable potency in terms of sensitizing cancer cells to doxorubicin, which was manifested by the accumulation of cells in the G1 phase, and affected markers of inflammation, metastasis, and apoptosis. In the xenograft model, the combined treatment led to a reduction in tumour volume that was comparable for both CBD formulations.
CBD formulated in microparticles has also been tested with doxorubicin as a pre-/coadministration strategy, with several preclinical studies showing an enhanced cytotoxic effect in human breast cancer and ovarian cell lines, but only when using a pre-plus cotreatment strategy [70,94].
CBD-mediated potentiation of doxorubicin efficacy has recently been investigated in two TNBC cell lines [114]. The authors observed variable effects (without pre-treatment) as CBD cotreatment resulted in synergism or antagonism depending on the cell line and drug concentration used. Moreover, CBD was shown to effectively sensitize cells against doxorubicin in 3D cultures. Combining the two substances also potentiated the antimigratory effect in human TNBC MDA-MB-231 cells. The authors suggested that this effect may reflect mechanisms involving integrins, LOX, ATG5, autophagy and ABCA2, all of which are downregulated by CBD.
Combined CBD and doxorubicin treatment have also been tested for potential veterinary cancer therapy applications, suggesting that they synergistically or additively reduce cell proliferation in canine neoplastic cell lines [103]. Additionally, an antagonistic effect was described for lower concentrations of CBD and doxorubicin [103].

Interactions of CBD with Proteotoxic Stress-Inducing Drugs
Protein homeostasis involves protein synthesis, folding, and degradation in cells. Protein turnover is generally high in cancer cells due to uncontrolled cell divisions and growth as well as numerous genetic alterations that may give rise to proteins with altered structures or alter the composition of multimeric complexes [115]. Most cancer cells, therefore, experience elevated proteotoxic stress making them highly dependent on the proper function of protein-degradation machinery such as the ubiquitin-proteasome system (UPS). Protein homeostasis is therefore seen as a promising target for cancer therapy and has been studied extensively [116]. The first drug targeting this process, bortezomib, was approved for clinical use in 2003 [117].

Bortezomib
Bortezomib ( Table 5) is one of the clinically used proteasome inhibitors and is commonly used to treat multiple myeloma (MM). It has a broad spectrum of adverse effects, including haematological and gastrointestinal toxicity and neurotoxicity [118]. CBD was shown to synergistically potentiate the anticancer effect of bortezomib, leading to increased growth inhibition, cell cycle arrest, and cell death through the ERK/AKT/NFκB pathway in human MM cell lines. The strongest cytotoxic response to bortezomib in combination with CBD was observed in TRPV2-overexpressing MM cells [119]. Bortezomib combined with CBD is about to be tested in phase II clinical trial of patients diagnosed with MM, GBM, and GI malignancies (NCT03607643) [34].

Disulfiram
Proteasome inhibitors aside, there are currently no approved anticancer drugs that directly target the UPS or protein homeostasis. However, some FDA-approved medicines targeting UPS that were initially approved for other indications are now being repurposed for cancer treatment. Disulfiram, which has been used for over 60 years to treat alcoholism, is an ideal candidate for repurposing as it is a well-tolerated drug with a substantial anticancer effect supported by numerous preclinical studies, case reports, and clinical trials, including several ongoing clinical trials (NCT04521335; NCT03323346; NCT03950830) [120][121][122][123][124]. The UPS-targeting effect of disulfiram is due to its metabolite, a copper-diethyldithiocarbamate complex (CuET), which targets the NPL4 protein, a cofactor of p97 segregase [125]. P97, with its cofactors, is a vital component of the UPS acting upstream of the proteasome through p97's ATPase activity that enables the segregation from diverse subcellular structures, unfolding, and translocation of ubiquitinated proteins for proteasome-mediated degradation [126]. CuET causes NPL4 aggregation leading to p97/NPL4 complex malfunction, triggering suprathreshold, irresolvable proteotoxic stress and consequently cancer cell death [125]. We recently showed that CBD effectively blocks CuET-mediated proteotoxic stress and toxicity in cancer cells by upregulating metallothioneins MT-1E and MT-2A, small proteins that are responsible for metal homeostasis and heavy metal detoxification, including copper. Because CuET is a copper complex, increased metallothionein expression reduces its activity against its primary target, NPL4, and thus reduces its toxicity [127]. These results indicate that the promising anticancer effect of the disulfiram/copper combination may be severely compromised by the simultaneous use of CBD products as supportive care.

CBD Interactions with Topoisomerase Inhibitors
Topoisomerases are essential modulators of DNA replication, recombination, repair, and transcription because of their role in releasing topological DNA stress. Topoisomerases catalyse single-or double-strand cleavage of DNA, and their proper function is crucial for cellular differentiation, proliferation, and survival. These enzymes were first identified as potential anticancer targets in the mid-1980s, and several topoisomerase inhibitors have since then been discovered and established in oncological praxis [128].

Topotecan
Topotecan (Table 5) selectively inhibits topoisomerase I, which catalyses single-strand DNA cleavage. Topotecan binds to the enzyme and prevents the re-ligation step, causing the formation of a single-strand DNA gap and simultaneously trapping the enzyme [129]. Topotecan is used as part of second-line therapy for metastatic ovarian cancers and relapsed small cell lung cancers. The most limiting adverse effect of topotecan is haematological toxicity; less severe effects include fatigue, nausea, vomiting, hypokalaemia, and increased γ-glutamyltransferase activity [130].
While the impact of CBD on adverse effects of topotecan is unknown, it seems that CBD may overcome resistance to topotecan mediated by the ABCG2/BCRP efflux transporter, which is directly targeted by CBD, as has been shown in the ABCG2/BCRP transporteroverexpressing MEF3.8 cell line [24].

Discussion
Currently, up to two-thirds of oncology practitioners say that they have discussed Cannabis product use with their patients but acknowledge that they do not have sufficient information to provide solid recommendations [131]. Indeed, recent surveys suggest con-siderable use of CBD and Cannabis-derived products among patients with cancer [2,3]. This review aims to provide oncologists and cancer patients with an overview of the potential benefits and drawbacks, as well as some uncertainties, associated with concomitant CBD use during ongoing chemotherapy. We have focused on summarizing the available preclinical data concerning the drug-drug interactions of CBD with commonly used chemotherapeutics in cell cultures and animal models (Tables 1-5). However, the list of anticancer chemotherapeutics discussed herein is not exhaustive, and the effects of CBD in combination with other cannabinoids, which are discussed in several publications, were excluded due to the length restrictions and the focus of our present review.
Patients have two primary motivations for using CBD during ongoing anticancer therapy: (i) attenuation of adverse effects and (ii) enhancement of the therapeutic efficacy. Adverse effects are often limiting factors of chemotherapy treatment, and CBD, in combination with certain chemotherapeutics, can enable patients to withstand therapy for a longer time and/or at a higher dosage. According to available surveys [2,3], adverse effects are the main reason why patients with cancer use Cannabis products. Among the notable preclinical examples discussed in this review, CBD can alleviate 5-fluorouracil-induced oral mucositis, cisplatin-induced renal and gastric toxicities, oxaliplatin-induced neuropathic pain, paclitaxel-induced neuropathy, and doxorubicin-induced cardiotoxicity, as well as show some desirable anti-emetic and anti-nausea effects during chemotherapy. There is a consensus in most of the cited studies that CBD has good potential to improve the quality of life of patients with cancer undergoing standard chemotherapy.
The modulation of treatment efficacy by CBD is another fascinating issue that warrants further investigation. CBD has been proven to have a multi-target impact on various cellular processes, some of which are necessary for cancer-cell survival or modulate the toxic effects of various anticancer drugs. We collected and highlighted preclinical data illustrating these impacts. For example, CBD was shown to potentiate the effects of gemcitabine, carmustine, cisplatin, temozolomide, paclitaxel, and vincristine in various preclinical models. The mechanistic explanations for these combinatorial effects mainly involve effects on drug influx/efflux resulting from interactions with transporters and/or channels such as TRPV2, which is activated by CBD and increases the influx and retention of carmustine, cisplatin, temozolomide, doxorubicin, and bortezomib. The well-known p-gp efflux transporter, which is responsible for multidrug resistance, is yet another very relevant target of CBD in this context. CBD-mediated inhibition of the p-gp transporter leads to increased accumulation of vinblastine and doxorubicin in p-gp-overexpressing cell lines. CBD also inhibits the ABCC1/MRP1 and ABCG2/BCRP transporters and thereby facilitates the accumulation of methotrexate, vincristine, and topotecan, at least in cell lines overexpressing these transporters. Importantly, the ability of CBD to increase the accumulation of these drugs is not always cancer cell-/tissue-specific, a challenging aspect that raises the risk of unwanted increased adverse chemotherapy effects and overdose in patients using CBD in conjunction with these anticancer agents.
Other modes of chemotherapy potentiation by CBD that does not involve transporters have also been proposed. These include regulation of EV trafficking of anti-and prooncogenic miRNAs in temozolomide-treated glioblastoma [56]; induction of ERK and JNK kinase pathways, which promotes autophagy in vincristine-and doxorubicin-treated canine neoplastic cells [103]; GRP55 inhibition, which reduces growth and cell cycle progression in gemcitabine-treated PDAC cells [36]; and ROS induction in oxaliplatin-treated colorectal cell lines [76].
Importantly, there is also preclinical evidence that CBD might significantly reduce the efficacy of anticancer drugs in some cases. This serious issue remains relatively unexplored and has been mentioned in some studies as an unexpected finding without being actively studied mechanistically. However, one mechanistic explanation has been provided recently when it was shown that CBD efficiently protects cancer cells against certain metalcontaining drugs by inducing the expression of specific detoxifying proteins known as metallothioneins [127]. Indeed, metallothioneins are involved in cellular defence against multiple chemicals, and their effects are not limited to heavy metal complexes [132,133]. Consequently, this effect could be relevant for numerous anticancer drugs. Accordingly, antagonistic effects of CBD have been reported for carmustine, temozolomide, cisplatin, carboplatin, and doxorubicin. This suggests that concomitant use of CBD during anticancer therapy may be unsuitable for some drug combinations because it may appear to reduce adverse effects while actually reducing the likelihood of successful treatment.
Overall, based on the collected data here, it can be concluded that CBD shows exciting potential for improving cancer chemotherapy outcomes when combined with various standard-of-care drugs and not only in terms of side effect attenuation. For example, cancers with developed treatment resistance might benefit from concomitant CBD application due to its reported effects on transporters. In addition, combinations including carmustine, paclitaxel, and doxorubicin seem promising. However, some of the data are less conclusive, with conflicting findings involving, for example, temozolomide and cisplatin. Finally, CBD combined with carboplatin or disulfiram appears to be a potentially dangerous combination (decreasing the drug efficacy). The last two examples even raise an essential concern that some of the CBD-promoted side effect attenuation might reflect a reduced amount of available active drugs. Thus, given the lack of clinical data and only scheduled or ongoing clinical trials, the reported promising preclinical results need to be carefully evaluated and translated into meaningful clinical benefits.

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