Cannabinoids-Promising Antimicrobial Drugs or Intoxicants with Benefits?

Novel antimicrobial drugs are urgently needed to counteract the increasing occurrence of bacterial resistance. Extracts of Cannabis sativa have been used for the treatment of several diseases since ancient times. However, its phytocannabinoid constituents are predominantly associated with psychotropic effects and medical applications far beyond the treatment of infections. It has been demonstrated that several cannabinoids show potent antimicrobial activity against primarily Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA). As first in vivo efficacy has been demonstrated recently, it is time to discuss whether cannabinoids are promising antimicrobial drug candidates or overhyped intoxicants with benefits.


Introduction
The worldwide spread of bacterial resistance against market antibiotics has been identified as one of the major threats to public health by scientists and healthcare authorities [1][2][3][4]. Thus, new antibacterial strategies and antibacterial compounds are urgently needed to counteract the increasing occurrence of antibiotic-resistant and, especially, multidrug-resistant (MDR) pathogens, to keep the live-saving advantages toward bacterial pathogens [5,6]. In 2017, the World Health Organization (WHO) emphasized the crucial need for antimicrobial drug development against a group of 13 different genii, families, and specific species of pathogenic bacteria by categorizing them into critical, high, and medium priority groups, according to their prevalence of resistance, clinical relevance, contributions to global morbidity and mortality, and their economic burden, for global healthcare systems to guide antimicrobial research and development [7,8]. The vast majority of these prioritized bacterial pathogens are drug-resistant Gram-negative bacteria. The development of new antimicrobial drugs against Gram-negative bacteria is particularly challenging due to the general structure of the Gram-negative cell envelope, which leads to a very effective permeation barrier rendering a majority of drugs with antibacterial activity against Gram-positive pathogens inactive against Gram-negative ones.
However, antimicrobial resistances are also increasing in Gram-positive bacteria and, among the high priority pathogens, the Gram-positive pathogen Staphylococcus aureus is the leading cause of both healthcare and community-associated infections worldwide, and a major cause for morbidity as well as mortality, especially taking into account the emergence and rapid spread of Methicillin-resistant Staphylococcus aureus (MRSA). These bacteria can cause serious infections of the skin, surgical wounds, the bloodstream, the lungs, or the urinary tract. Furthermore, their distinct ability to form biofilms on tissue [9], in wounds [10,11], and on implants [12] can lead to severe chronic biofilm-associated infections [13,14].

Bioactivities and Medical Uses of Phytocannabinoids from Cannabis Sativa and Synthetic Analogues
Historically, different plant material, oils, and extracts of Cannabis sativa have been utilized for recreational, spiritual, and medical purposes since ancient times [61][62][63][64].
The strong psychotropic activity observed when marihuana (dried resinous flowers and proximal little leaves of Cannabis sativa) or hashish (dried and compressed resinous extracts of Cannabis sativa) are smoked, vaporized, or consumed orally; having been processed into pastries is related to the interaction of contained psychotropic cannabinoids as partial agonist with human endocannabinoid receptors CBR1 [65][66][67][68][69].
For several of the phytocannabinoids, especially the minor constituents of Cannabis sativa, the overall pharmacological profile is still incomplete or there are no pharmacological data available. In general, more investigations are necessary to understand their complex biophysiological activities.
Medical cannabis, as well as several Cannabis sativa derived products, which have been approved by the national drug approval authorities, contain mostly either ∆ 9 -THC (1), CBD (10), or a mixture of both cannabinoids. They are prescribed in several Western countries to allay symptoms, such as neuropathic pain, nausea, vomiting, muscle spastics, and weight loss within treatment of severe diseases, such as multiple sclerosis, epilepsy, AIDS, or cancer [33,62,83,87,88].
The broad variety of therapeutic effects and interesting bioactivities of phytocannabinoids has also stimulated their chemical total synthesis, bioengineered synthesis, as well as the synthesis of numerous analogues [75] with modified pharmacological profiles, which has recently been comprehensively reviewed [50,[89][90][91]. A particular highlight is the synthesis and in vitro evaluation of potent photo-switchable ∆ 9 -THC-analogues, which were reported by a consortium of the academic research groups of Trauner, Carreira, Frank, and their co-workers in 2017. These analogues (28-31, Figure 4) contained azobenzene moieties as lipophilic side chains, which allow optical control of CB1R signaling [92]. To date, fully synthetic, but naturally occurring ultra-pure cannabinoids, such as Dronabinol (synthetic ∆ 9 -THC (1)) and even the fully synthetic ∆ 9 -THC-analogue Nabilone (31) (Figure 4) bearing a modified lipophilic side chain and a ketone moiety in the upper ring are approved for different indications [87].
The synthetic exploration of the structure-activity relationships has also produced several cannabinoid mimetics with tremendously increased psychotropic activity, which entered the market of legally available designer drugs sold as herbal bath additives, leading to severe acute psychotropic and physical effects for consumers [93,94], and several cases of reported deaths upon acute organ failure [95]. Furthermore, in recent years cannabis or cannabinoids have become some sort of trend medication for several diseases strongly hyped by a growing cannabis lobby without significant medical data evidence. In 2019, US authorities for the Food and Drug Administration (FDA) published two consecutive warnings for misuse of cannabidiol products with unsubstantiated claims by producers [96,97].

Antimicrobial Activities of Phytocannabinoids from Cannabis Sativa
The application of plant material or seeds of Cannabis sativa for the preparation of early antibiotic treatments of different infections have already been reported from folk medicine at the end of the 19 th century, as described in early review articles by Kabelik [98,99].
First systematical evidence for the antimicrobial activity of constituents of Cannabis sativa was given by the Dissertation Krejci in 1950 [100]. His comprehensive bacteriological in vitro investigations on the antibacterial effects of extracts of Cannabis sativa on different bacteria were later published in 1958 [101]. Krejci reported a good activity of the cannabis extracts against the growth of Gram-positive bacteria, while Gram-negative bacteria were not affected. Furthermore, he prepared a salve containing 2% extracts and demonstrated its therapeutic efficacy in the treatment of skin infections with Staphylococci [101]. Independently, Drobotko et al. described the occurrence of antibacterial activity in extracts from hemp in 1951 [102], while Ferenczy et al. reported the finding of the occurrence of antibacterial activity in extracts obtained from seeds of hemp in 1956 [103,104]. Ferenczy et al. demonstrated the potency of the antimicrobial activity using dilutions of their extracts and observed that their activity was pH-dependent [104].
Since these early findings, numerous of reports on the antimicrobial activity of different extracts prepared from Cannabis sativa on Gram-positive, Gram-negative bacteria, and different fungi , which have also been partially reviewed elsewhere [98,130,131].
The additional scientific knowledge from these studies was rather limited, as in most cases, complex mixtures with undetermined composition or partially determined mixtures of over 60 compounds [112] have been utilized for the evaluation of the antimicrobial activity, thus, clear compound specific pharmacological conclusions are impossible. Considering the variety of natural products from different classes, which have been isolated from Cannabis sativa [132][133][134] and the complex pharmacology of phytocannabinoids mentioned above, studies on isolated single cannabinoids are required to fully understand their antimicrobial potential.
The first early study on the antimicrobial activity of specific, isolated cannabinoids from Cannabis sativa were reported by Schultz and Haffner in 1958 [27]. They isolated cannabidiolic acid (CBDA) (11) and compared its antibacterial activity against S. aureus, Bacillus subtilis, and Escherichia coli to cannabidiol (CBD) (10), the corresponding diacetate (31) as well as its sodium salt (32) ( Figure 5). While CBD (10) and CBDA (11) show a potent antibacterial activity against the Gram-positive bacteria S. aureus and B. subtilis, an activity against the Gram-negative E. coli was not observed. As the poor water solubility of the compounds was limiting their investigations, the diacetate 32 and its sodium salt 32a were generated, which showed significantly increased water solubility. Nonetheless, these derivatives were inactive against all tested bacteria [27].
In 1965, Melchoulam et al. mentioned the antibacterial activity of CBG (17) and CBGA (18) against Gram-positive bacteria in a short side note of their publication on the isolation and structure determination of natural cannabinoids. However, neither information on bacterial strains nor specific MIC (Minimal Inhibitory Concentration) data were given [135].
In 1976, Klingeren and Ham published a study on the antibacterial activity of ∆ 9 -THC (1) and CBD (10), in which both cannabinoids were screened for their antibacterial activity against the Gram-positive bacteria S. aureus, Streptococcus pyogenes, Streptococcus milleri and Enterococcus faecalis and the Gram-negative pathogens E. coli, Salmonella typhi and Proteus vulgaris [26]. Both compounds displayed significant antibacterial activity against all Gram-positive bacteria tested (Table 1, Entry 1 and 2), but were in accordance with the results of Krejci et al. [28] inactive against the Gram-negative ones. Furthermore, the authors proved on cultures of S. aureus that both compounds are bacteriostatic as well as bactericidal. Table 1. Overview on antibacterial activity of ∆ 9 -THC (1) and CBD (10) against different strains of Gram-positive and Gram-negative bacteria [26]. In 1981, Turner and ElSohly published a report on the in vitro evaluation of antimicrobial activity of CBC-type cannabinoids against different Gram-positive bacteria and fungi including an in vivo study in rats investigating the anti-inflammatory activities in an carrageenan-induced rat paw edema assay [25]. In 1982, the same research group published an expanded study on the antibacterial and antimycotic activities of further CBC-type and CBG-type cannabinoid derivatives [29]. The results of both studies are summarized in Table 2. In this study, CBC (19) showed the highest activity of all tested CBC-type cannabinoids against S. aureus and B. subtilis with a MIC of 1.56 µg/mL and 0.39 µg/mL, respectively, even superior compared to Streptomycin ( Table 2, Entry 1). Interestingly, isoCBC (33) ( Table 2, Entry 2), bearing the lipophilic side chain and one hydroxyl group in exchanged positions displayed similarly potent activity against B. subtilis with a MIC of 0.78 µg/mL. The truncation of the lipophilic side chain in both isoCBCand CBC-type cannabinoids led to a clear 2-8-fold drop in antibacterial activity (Table 2, Entries 3, 4 and 5), with the exception of isoCBC-C 1 (35) exhibiting a MIC of 0.78 µg/mL against B. subtilis (Table 2, Entry 6). Moreover, the double bonds in the GPP-derived side chain seem to be less important for high activity as the reduced derivative 38 (Table 2, Entry 7) showed comparable antibacterial activity as CBC (19). Interestingly, a moderate antimycobacterial activity Mycobacterium smegmatis was observed for some of the compounds with best activity for CBC-C1 (37) and 38 exhibiting MIC values of 3.12 µg/mL comparable to Streptomycin (Table 2, Entry 4). The structure-activity relationships for the antibacterial activity of CBC-type cannabinoids can be concluded as illustrated in Figure 6. The antimycotic activity of the compounds was less pronounced. Only CBC-C 1 (37) displayed a moderate antimycotic activity against Sachcheromyces cerevisiae with a MIC of 6.25 µg/mL, clearly less active compared to Amphotericin B. The activity against Trichophyton mentagrophytes, a pathogenic fungus, was comparable to Amphotericin B with 2 two-fold higher MIC of 6.25 µg/mL.  The authors also demonstrated that all tested CBC-type cannabinoids in this study reveal a higher anti-inflammatory activity compared to phenylbutazone or aspirin [25].
The CBG-type derivatives isoCBG-C 1 (39) and CBG-C 1 (40) displayed comparably strong antibacterial activity with MIC values in the range of 1.56-6.25 µg/mL [29]. According to the trends observed for the CBC-type derivatives, the reduction of the double bond in the GPP-derived side chain was well tolerated, as derivative 41 showed similar antibacterial activity against S. aureus and B. subtilis and good activity against M. smegmatis with a MIC of 3.12 µg/mL. Again, the antimycotic activities of the CBG-type compounds were less pronounced with only moderate activity. CBE (14) showed no noteworthy antibacterial or antimycotic activity [29].
In 2008, Appendino et al. [24] reported a more focused structure-activity relationship study on the anti-staphylococcal activity of different naturally occurring cannabinoids and synthetic derivatives against a broad range of various multi-drug resistant strains of S. aureus, including EMRSA-15, one of the main epidemic methicillin-resistant strains [141], SA-1199B, a multidrug-resistant strain that possesses a gyrase mutation, and in addition overexpresses the NorA efflux mechanism [142], RN-4220, a macrolide-resistant strain [143], XU212, and a tetracycline-resistant strain overexpressing the TetK efflux pump [144], as summarized in Table 4.

Entry Compounds
Antibacterial Activity IC 50    The carboxylic acid precursors CBGA (18) ( Table 4, Entry 6) and CBDA (11) ( Table 4, Entry 2) similar to ∆ 9 -THC (2) ( Table 4, Entry 11) retained their potent activity; however, esterification of the carboxylic acid moieties was detrimental for the activity. Thus, compounds 57, 58, 62, and 63 showed no noteworthy activity (Table 4, Entries 3, 8 and 9). Additionally, bigger substituents in the same position, such as in the prenylated CBD-derivative 67, were also not tolerated and led to complete loss of activity. While the biosynthetic cannabinoid precursor olivetol 68 showed very low activity ( Table 4, Entry 17), no activity at all was observed for resorcinol 69 missing the lipophilic side chain (Table 4, Entry 18). Thus, indicating that both the lipophilic C5 side chain as well as GPP-derived lipophilic side chain are crucial for high activity. In addition, the substantially decreased activity of camagerol (66), a dehydroxylated CBG-derivative revealed that the overall polarity in the GPP-derived lipophilic side chain is important for high activity as well. Interestingly, the synthetic isomers of CBD (Compound 64, Table 4, Entry 13) and CBG (Compound 65, Table 4, Entry 14), bearing the lipophilic side chain and one hydroxyl group in exchanged positions displayed potent activity comparable to their corresponding natural counterparts indicating that both moieties, although crucial for activity, can vary in their position at the phenolic core. A trend that has been observed for CBG-C 1 -type and CBC-type cannabinoid derivatives earlier [29], suggesting that they may rather serve as lipid affinity modulators influencing water solubility and cellular bioavailability [24].
Recently, a very comprehensive study on the antimicrobial, antibiofilm and anti-persister cell activities of phytocannabinoids from Cannabis sativa against MRSA was published by Brown and co-workers comprising in vitro and in vivo investigations of 18 commercially available cannabinoids, pre-cannabinoids and synthetic isomers [23]. The results of this study are summarized in Table 5.
Recently, antibiofilm activity of ethanolic extracts of Cannabis sativa, likely containing phytocannabinoids, has been reported by Frasinetti et al. [121]. However, the study of Brown and co-workers for the first time allowed a direct attribution of antibiofilm activity to the tested cannabinoid derivatives [23]. As summarized in Table 5, the antibiofilm activity of all tested cannabinoids against MRSA USA300 strain correlated well with their specific anti-staphylococcal activity. Thus, the major cannabinoids, such as ∆ 8 -THC (5), CBGA (18), CBG (17), and exo-THC (22) completely suppressed biofilm formation at concentration of 2 µg/mL (Table 5, Entries 5,11,12 and 16), CBG (17) displaying the most potent antibiofilm activity with an IC 50 value of 0.5 µg/mL [23]. Remarkably, in addition, CBG (17) was demonstrated to eradicate preformed biofilms of MRSA USA300 at concentrations of 4 µg/mL [23]. Brown and co-workers also screened for the ability of the cannabinoids to kill persister cells ( Table 5). The anti-persister activity correlated with the activity against actively dividing cells and CBG (17) showed the most active cannabinoid killing persisters in concentration-dependent manner starting at 5 µg/mL, again. Impressively, CBG (17) was able to completely eradicate a population of 108 CFU/mL MRSA persisters below detection thresholds within 30 min of treatment [23].
In order to elucidate the molecular target of CBG (17), Brown and co-workers repeatedly challenged MRSA USA300 with various lethal concentrations ranging from 2-16-fold MIC or sequential subcultures in solution with sublethal concentration of CBG (17) to select for spontaneous resistance. However, no spontaneous resistant mutants were obtained, suggesting a low frequency of resistance of below 10 −10 for MRSA [23]. A comprehensive chemical genomic analysis on the model bacterium B. subtilis at sublethal concentrations of CBG (17) than led to the identification of 41 transposon mutants, which revealed an enrichment of genes encoding for proteins that are localized at the cytoplasmic membrane, and genes encoding for proteins involved in processes taking place at the cytoplasmic membrane. Thus, indicating that CBG (17) acts via a disruption of the integrity of the cytoplasmic membrane, which was confirmed through treatment of MRSA cells with a membrane-potential sensitive fluorescent probe in the presence of CBG (17), leading to a dose-dependent increase of fluorescence occurring at MIC of CBG (17) [23]. Unfortunately, a hemolytic activity of CBG (17) on human erythrocytes was observed at 32 µg/mL, above its MIC of 2 µg/mL, but with a low therapeutic index of 16:1 [23].
However, a pharmacokinetic study of 120 mg/kg doses of CBG (17) in rats and mice have reported no signs of acute toxicity [153]. Brown and co-workers demonstrated the antibacterial efficacy of CBG (17) in vivo at 100 mg/kg doses in a murine systemic infection model, displaying a significant reduction of bacterial burden by a factor of 2.8 log 10 in CFU compared to the control, while being well tolerated in mice [23].
Finally, Brown and co-workers discovered that the antibacterial activity of several cannabinoids could be significantly increased against Gram-negative pathogens, which have been reported to be less susceptible towards cannabinoids [26,140] in the presence of sublethal concentrations of polymyxins perturbating the outer membrane of Gram-negative bacteria. The authors could show, that CBG (17), which was inactive against E. coli (>128 µg/mL), was active in the presence of a sublethal concentration of 0.062 µg/mL of polymyxin B with a MIC of 1 µg/mL. This observation uncovered the hidden broad-spectrum antibiotic activity within this synergistically acting polymyxin B adjuvant combination therapy approach, and allowed Brown and co-workers to demonstrate the potent efficacy of CBG (17) against clinical isolates of Gram-negative priority pathogens such as Acinetobacter baumannii, E. coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa.

Conclusions
Novel antimicrobial drugs are urgently needed to counteract the growing occurrence of bacterial resistance towards market antibiotics. Phytocannabinoids, including ∆ 9 -THC-type, ∆ 8 -THC-type, CBN-type, CBD-type, CBC-type, and CBG-type derivatives have been demonstrated to display potent antibacterial activity against Gram-positive pathogens, in particular against highly virulent and prevalent MRSA strains, which are the leading cause of both healthcare and community-associated infections [23][24][25][26]29]. Due to their non-psychotropic and non-sedative pharmacologic properties, CBD-type, CBC-type, and in particular CBG-type cannabinoids are the most promising candidates for further investigations. Although, the carboxylic acid bearing pre-cannabinoids, such as ∆ 9 -THCA (2) or ∆ 8 -THCA (6) do not show psychotropic effects either, their intrinsic instability through sun light or heat mediated decarboxylation or aromatization in the presence of oxygen leading to psychotropic ∆ 9 -THC-type, ∆ 8 -THC-type or CBND-type cannabinoids, makes them less attractive for further development. The recently demonstrated potent antibacterial in vivo efficacy [23], which previously published reports on the inactivation of CBG (17) by serum [135] proved to be wrong, the discovery of additional potent antibiofilm and anti-persister cell activities of CBG (17) against MRSA [23], as well as the synergistic activity of CBG (17) in the presence of sublethal concentrations of polymyxins against Gram-negative priority pathogens, such as A. baumannii, E. coli, K. pneumoniae, and P. aeruginosa, further support the role of CBG-type cannabinoids as lead candidates for the development of novel broad spectrum antibiotics.
However, considering the complex pharmacology [67,68,154] of CBG (17) acting within the endocannabinoids system as an inhibitor for the uptake of the endocannabinoid ligand anandamide (24) [155] and, furthermore, taking into account the crucial role of the endocannabinoid system within a multitude of essential physiological processes [66,70,73,156], such as embryo and child brain development [157,158], more studies on the pharmacological profile of CBG (17) are necessary to elucidate the poorly understood pharmacological relationships. For non-psychotropic natural cannabinoids, mostly mild, tolerable side-effects such as dizziness, dry mouth, gastrointestinal disorders, or tiredness have been reported; however, this might change drastically for derivatives and needs to be investigated [87,88]. For CBG (17), although hemolytic activity on human erythrocytes with a low therapeutic index have been an issue in vitro [23], preliminary pharmacokinetic results on in vivo show no acute toxicity and high tolerance in mice and rats [23,153,159].
Finally, it is important to emphasize that, owing to their ability to activate the reward system, cannabinoid consumption is potentially addictive, and long-term use might produce tolerance and dependence [87,88]. Nevertheless, considering relatively short administration periods of antimicrobial drugs, this is probably less of an issue.
While CBG (17) possess several desirable physiochemical properties as a medicinal chemistry lead in terms of molecular weight, number of hydrogen donors and acceptors, number of rings and rotatable bonds, it is suffering from its high lipophilicity with a calculated logP of 6.74 and an associated poor water solubility, [23] which need to be addressed in medicinal chemistry campaigns. Furthermore, the accumulation of therapeutic cannabinoids in adipocytes of fatty tissue [160,161] due to their highly lipophilic nature need to be investigated to exclude negative long-term effects. Thereby, the easy access through isolation from high content types of Cannabis sativa and easy synthesis of CBG (17) from readily available starting materials [162] are a huge benefit, which will boost the chemical exploration of CBG (17) as antibacterial lead candidate.
Cannabinoids can be administered through vaporization, intravenous injection, sublingual mouth spray, or oral application, for the latter, however, appropriate stomach-resistant galenic formulations have to be considered to avoid known gastric degradation [161], leading to lowered plasma levels [153].
The most promising and obvious application of CBG (17) based antibiotics might be in topical applications against MRSA caused skin infections as the usage of cannabinoids in medical salve has already been successfully demonstrated by Krejci [101] in 1959, and mupirocin, the standard-of-care antibiotic in this indication, suffers from resistances [163,164].
Beyond all irrational hype on cannabis products, cannabinoids are truly promising antimicrobial drugs and I am convinced that they will find their way into a novel medical application as antibacterial treatment of infections. With regard to that, more research to address structural and pharmacokinetic shortcomings is needed.
Funding: This work has been carried out within the framework of the SMART BIOTECS alliance between the Technische Universität Braunschweig and the Leibniz Universität Hannover. This initiative is supported by the Ministry of Science and Culture (MWK) of Lower Saxony, Germany. The content of this work is solely the responsibility of the author and does not necessarily represent the official views of the funding agencies.