European Journal of Pharmaceutical Sciences 132 (2019) 118–120

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European Journal of Pharmaceutical Sciences

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Promoting cannabis products to pharmaceutical drugs T

ARTICLE INFO ABSTRACT

Keywords: Cannabis sativa is widely used for medical purposes. However, to date, aroma, popular strain name or the content of Cannabis sativa two phytocannabinoids—Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are mostly considered for ther- Cannabinoid apeutic activity. This is despite the hundreds of compounds in this plant and their potential synergistic interactions in Drug development mixtures. New, specific and effective cannabis-based drugs must be developed to achieve adequate medical standards First-in-human for the use of cannabis. To do this, the comprehensive molecular profile of cannabis-based drugs must be defined, and Terpene mixtures of compounds should be tested for superior therapeutic activity due to synergistic effects compared to Synergy individually isolated cannabis compounds. The biological pathways targeted by these new drugs should also be characterized more accurately. For drug development and design, absorption, distribution, metabolism and elim- ination versus toxicity (ADME/Tox) must be characterized, and therapeutic doses identified. Promoting the quality and therapeutic activity of herbal or synthetic cannabis products to pharma grade is a pressing need worldwide.

1. Introduction reproducibility, probably due to uncharacterized changes in the un- known compositions of active compounds. To overcome this difficulty, In recent years, medicines based on Cannabis sativa L., including C. sativa used for medical treatment should be better defined for their herbal cannabis (marijuana) products, have become increasingly precise content of phytocannabinoids, terpenoids, and perhaps addi- available to patients in many countries. However, scientific evidence of tional plant secondary metabolites. the medical activities of these herbal products and their targeted bio- logical pathways is scarce. Today, the specific cannabis type used ina 3. Promoting cannabis products: identification of synergistic given medical treatment is described by the strain's popular name, as- interactions sociated with its aroma, taste and/or content of the phytocannabinoids Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). These two Significant synergistic effects, popularly termed “entourage”, have phytocannabinoids are the decarboxylated forms of Δ9-tetra- been reported for whole cannabis extract versus the activity of its in- hydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA), re- dividual compounds in cells and animal models (Russo, 2011; Blasco- spectively, produced by the plant (Hanuš et al., 2016). Their ratio de- Benito et al., 2018; Nallathambi et al., 2018). Recently, synergistic com- fines most C. sativa strains, termed as either “high in THC”/sativa- binations of cannabinoids and terpenes have been found with increased originated or “high in CBD”/indica-originated (De Meijer, 2014). De- cytotoxic activity against colorectal cancer (CRC) cell lines (Nallathambi finitions of C. sativa strains and their herbal preparations based on et al., 2018). Synergistic interactions may occur between different can- aroma, taste, and/or CBD/THC content alone are less than satisfactory nabinoids (i.e., “intra-entourage”) (Berman et al., 2018), or between can- and far from meeting the requirements for drug development. nabinoids and terpenes (i.e., “inter-entourage”) (Nallathambi et al., 2018). Hence, cannabis preparations should be optimized to contain mixtures of 2. Promoting cannabis products: precise content of compounds these C. sativa-derived compounds and/or whole extract with composi- tions showing the greatest synergistic activity. Any individual C. sativa variety contains hundreds of different sec- ondary metabolites (Aizpurua-Olaizola et al., 2016), including a ple- 4. Promoting cannabis products: identification of mode of action thora of identified and categorized phytocannabinoids (Hanuš et al., 2016). Moreover, new studies show that the beneficial activity of 4.1. Cannabinoid receptors and signaling in cells induced by specific ligands cannabis cannot be attributed to a single compound (Russo, 2011; Blasco-Benito et al., 2018; Nallathambi et al., 2018). Besides cannabi- Another way to improve a given C. sativa preparation is by revealing noids, hundreds of other secondary metabolites, such as terpenoids, its mode of action. THC, synthetic compounds and endocannabinoids sterols and flavonoids, are produced by cannabis inflorescences (De have been suggested to activate G-protein-coupled cannabinoid re-

Meijer, 2014). Some of these volatile and semi-volatile compounds ceptor 1 (CB1). Another identified and isolated endocannabinoid re- account for the typical aroma of cannabis, and perhaps for part of its ceptor is cannabinoid receptor 2 (CB2)(Pertwee et al., 2010). These biological activity (Singh and Sharma, 2015). receptors mediate the synaptic and cellular effects of endocannabinoids As a result, patients are being treated with cannabis products that in various cells and tissues (Maccarrone et al., 2015). Recent evidence are not well defined, and suffer from a lack of efficacy or suggests that agonists that bind to the endocannabinoid receptors https://doi.org/10.1016/j.ejps.2019.02.027 Received 20 December 2018; Accepted 17 February 2019 Available online 07 March 2019 0928-0987/ © 2019 Elsevier B.V. All rights reserved. European Journal of Pharmaceutical Sciences 132 (2019) 118–120

demonstrate functional selectivity, activating or inhibiting CB1 and CB2 However, cannabis-based drug development will lead to new downstream signaling pathways via both canonical and non-canonical pharmaceutical products only if the new drug effectively targets a signaling pathways (Priestley et al., 2017). Therefore, there is a rela- specific receptor in a cell, without showing any relevant toxicity. In tively substantial amount of knowledge on the signal transduction fol- comparison to other drugs used for the treatment of some medical lowing CB receptor activation by specific ligands. conditions (e.g., epilepsy and psychotic disorders), CBD often has a better side-effect profile and comparable or superior efficacy (Russo, 4.2. Signaling in cells in response to C. sativa whole extract 2019). However, some important pharmacological and toxicological parameters have yet to be studied, particularly for cannabis compounds In contrast to the knowledge on ligand-activated signal transduction other than the most studied CBD and THC. In addition, more clinical via CB receptors, almost nothing is known about the induced or re- trials with a greater number of participants and longer chronic cannabis pressed signaling in cells, tissues and organs by cannabis whole extract. compound administration are needed for the individual cannabis This lack of knowledge on its mode of action may be due to the com- compounds and synergistic mixtures (Iffland and Grotenhermen, 2017). plexity of the whole extract as compared to a pure, single compound; This process involves specific preclinical development and testing, complexity that derived from the array of cannabinoids in C. sativa followed by trials in humans to determine the efficacy of the drug under whole extract being accompanied by an abundance of terpenoids. cost-effective conditions. Therefore, preclinical development strategies Numerous studies suggest that terpenoids have therapeutic prop- such as the crucial in vitro to in vivo extrapolation (IVIVE) of cell assays and erties and affect cytokine levels and enzymatic activity (de Santana animal in vivo to human in vivo extrapolations are needed to identify the Souza et al., 2014). Little is known about the mode of action of terpe- most promising individual cannabis compounds or mixtures of compounds noids in terms of upstream signaling pathways (e.g., receptor and in- for activity in humans. Accordingly, plasma exposure showing efficacy but teractor activation), and this should be further studied in relation to no toxicity in animals is routinely considered to calculate the first-in- drug development from plant material, C. sativa included. This com- human (FIH) dose projections that will give the same plasma exposure plexity, leading to the synergistic interactions reported between can- assuming similar mechanisms of action in each species. This should be nabis-derived compounds, may originate from the activation of more followed by clinical trials to determine whether the newly identified in- than one signaling pathway to affect a certain condition. dividual cannabis compounds or their combinations will show a sufficient To monitor intra- and inter-entourage effects, we recently made an initial safety margin at the expected therapeutic doses. effort to better understand the signaling pathways which are synergistically More importantly, we suggest that the goal of finding proper sy- induced by certain mixtures of phytocannabinoids and terpenoids that are nergistic mixtures of active cannabis compounds is key; hence, these active against CRC cell lines. CRC cells were treated with individual and studies should also be designed around this goal. In particular, drug–- combined fractions of C. sativa chemotype I extract. RNA profiling identified drug interactions (DDI), which are a common cause of adverse reactions more than 2000 differentially expressed genes that were affected by treat- and efficacy, must be investigated among the C. sativa molecules; sy- ment with the synergistic combination but not when treated with the in- nergistic efficacy responses may then result in a lower dosage inhu- dividual fractions (Nallathambi et al., 2018). Among the activated signaling mans. However, the large number of compound mixtures that can be pathway components related to the differentially expressed genes were the generated, even from small compound collections, all but negates the secreted Wnt glycoproteins that are involved in cell–cell communication, feasibility of exhaustive experimental testing in vivo or in humans. The and tumor suppressor P53 transcription factor. These pathways and others ability to predict the behavior of compound combinations in in vitro are involved in cell-cycle arrest and apoptosis; they are often dysregulated in biological systems, whittling down the number of combinations to be CRC patients and are a desired target for anticancer drugs (Cheng et al., tested, is therefore crucial. Moreover, to facilitate drug design, high- 2018). Their attenuation by certain synergistic mixtures of cannabis-derived throughput screening by in vitro facilities such as in vitro cell assays compounds suggests that certain compositions may target specific biological based on individual versus mixtures of cannabis compounds should be pathways in cells to affect cell death, and may prove to be a new modeof compared for efficacy and toxicity concerns. action for cannabis anticancer therapy compared to individual compounds Furthermore, the corresponding active concentration determined in (Nallathambi et al., 2018). However, more examples should be studied, and these in vitro assays could be challenged by using in silico molecular in particular proven in vivo in humans, to assess their mode of action. This is structure-based pharmacokinetics/pharmacodynamics (PKPD) modeling, discussed in the following. including the DDI effect, to reproduce the active concentration of each individual cannabis compound present in the mixtures under in vivo con- 5. Steps required to promote the quality of cannabis products ditions. These may serve as primary tools for early-stage drug discovery to facilitate the identification of promising candidates (synergistic mixtures) The steps required to promote the quality of herbal or synthetic can- based on their potential efficacy and plasma kinetics in vivo, to be further nabis products to that of a pharmaceutical drug are illustrated in Fig. 1. studied from more specific IVIVEs, animal studies and clinical trials. First, when a treatment with C. sativa extract is sought, a comprehensive Overall, the absorption, distribution, metabolism and elimination versus chemical profile of the given variety used should be generated andfully toxicity (ADME/Tox) should be characterized for each newly identified reported (De Meijer, 2014). Second, appropriate synergistic mixtures of active compound or mixture followed by an estimation of the expected cannabis compounds should be determined for use in medical treatments therapeutic doses (The Drug Development Process. US Food and Drug and as a basis for breeding and designation of new C. sativa strains. These Administration https://www.fda.gov/forpatients/approvals/drugs/; new strains will produce the proper combinations of compounds, be Bulusu et al., 2016; Poulin, 2016,), including integrative structure-phar- characterized by a full chemical profile and be labeled for a specific macokinetic-pharmacodynamic modeling (Vlot et al., 2018). medical use (i.e., a given activity or disease). Third, the compounds can be purified from cannabis plants or synthesized. The compounds may beused 6. Conclusions to mimic the designated activity of the compositions defined as biologi- cally active. Fourth, characterization of the biological pathways affected in Despite the high availability of C. sativa herbal products and their human cells and tissues and specific targeting of these biological pathways wide beneficial use in medicine, they remain mostly uncharacterized. by cannabis products may lead to drug innovation. This should be sup- With the worldwide progress in approval of cannabis for medicinal use ported by data resources such as the Therapeutic Targets Database, which the current approach of “botanical drug” needs to be changed. Medical covers the association of most target and drug entries to the corresponding cannabis product development should include safety and efficacy con- pathway and includes a pathway-interaction database supporting drug siderations as those used for a fully defined pharmaceutical product. discovery and design (Yang et al., 2015). Drug development of cannabis-based products should follow accepted

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Fig. 1. Redefining cannabis products as drugs for medical use. Extraction of cannabis inflorescence (1) and chemical analysis (2) of full molecularinventoryof cannabis compounds (3) should be performed. Active compounds and their synergistic mixtures (4) may then be identified, and the biological pathways affected by the cannabis-derived molecules defined (5). These studies should form the basis for drug discovery and design by characterizing the ADME/Tox foreachnewly identified individual and/or mixture of active molecules (7) purified from cannabis (1) or their synthesis (6), followed by estimation of the therapeutic dose(8).This information will also affect cannabis breeding and designation of cannabis strains for a specific activity or indication (9).IVIVEs, in vitro to in vivo extrapolations of cell assays; DDI, drug–drug interaction studies; FIH, first-in-human dose prediction. drug development and design schemes; however, the importance of the Maccarrone, M., Bab, I., Bíró, T., Cabral, G.A., Dey, S.K., Di Marzo, V., Konje, J.C., Kunos, G., synergistic interactions between the different cannabis compounds Mechoulam, R., Pacher, P., Sharkey, K.A., 2015. Endocannabinoid signaling at the per- iphery: 50 years after THC. Trends Pharmacol. Sci. 36, 277–296. must be taken into consideration. Cannabis medicalization has become Nallathambi, R., Mazuz, M., Namdar, D., Shik, M., Namintzer, D., Vinayaka, A.C., Ion, A., part of many governmental policies worldwide. Establishing common Faigenboim, A., Nasser, A., Laish, I., Konikoff, F.M., Koltai, H., 2018. Identification of synergistic interaction between cannabis-derived compounds for cytotoxic activity in col- and reasonable grounds for cannabis medical use by promoting the orectal cancer cell lines and colon polyps that induces apoptosis-related cell death and quality and therapeutic activity of herbal or synthetic cannabis pro- distinct gene expression. Cannabis Cannabinoid Res. 3, 120–135. ducts to a pharma grade is essential. Pertwee, R.G., Howlett, A.C., Abood, M.E., Alexander, S.P.H., Di Marzo, V., Elphick, M.R., Greasley, P.J., Hansen, H.S., Kunos, G., Mackie, K., Mechoulam, R., 2010. International Union of Basic and Clinical Pharmacology. LXXIX. Cannabinoid receptors and their ligands: Acknowledgments beyond CB1 and CB2. Pharmacol. Rev. 62, 588–631. Poulin, P., 2016. The need for human exposure projection in the interpretation of preclinical in vitro and in vivo ADME tox data. In: Will, Y., McDuffie, E., Jeffy, B. (Eds.), Drug Discovery We thank Prof. Yoram Kapulnik for his helpful comments on this Toxicology Book: From Target Assessment to Translational Biomarkers. John Wiley & Sons, manuscript. Inc., USA, pp. 584 The Pfizer Company. Priestley, R., Glass, M., Kendall, D., 2017. Functional selectivity at cannabinoid receptors. Adv. Pharmacol. 80, 207–221. References Russo, E.B., 2011. Taming THC: potential cannabis synergy and phytocannabinoid-terpenoid entourage effects. Br. J. Pharmacol. 163, 1344–1364. Russo, E.B, 2019. The case for the entourage effect and conventional breeding of clinical Aizpurua-Olaizola, O., Soydaner, U., Öztürk, E., Schibano, D., Simsir, Y., Navarro, P., cannabis: no “strain,” no gain. Front. Plant. Sci. 9, 1–8. Etxebarria, N., Usobiaga, A., 2016. Evolution of the cannabinoid and terpene content de Santana Souza, M.T., Almeida, J.R.G.D.S., de Souza Araujo, A.A., Duarte, M.C., Gelain, D.P., during the growth of Cannabis sativa plants from different chemotypes. J. Nat. Prod. 79, Moreira, J.C.F., dos Santos, M.R.V., Quintans-Júnior, L.J., 2014. Structure–activity re- 324–331. lationship of terpenes with anti-inflammatory profile–a systematic review. Basic Clin. Berman, P., Futoran, K., Lewitus, G.M., Mukha, D., Benami, M., Shlomi, T., Meiri, D., 2018. A Pharmacol. Toxicol. 115, 244–256. new ESI-LC/MS approach for comprehensive metabolic profiling of phytocannabinoids in Singh, B., Sharma, R.A., 2015. Plant terpenes: defense responses, phylogenetic analysis, reg- Cannabis. Sci. Rep. 8, 14280. ulation and clinical applications. 3 Biotech 5, 129–151. Blasco-Benito, S., Seijo-Vila, M., Caro-Villalobos, M., Tundidor, I., Andradas, C., García- Vlot, A.H.C., de Witte, W.E.A., Danhof, M., van der Graaf, P.H., van Westen, G.J.P., de Lange, Taboada, E., Wade, J., Smith, S., Guzmán, M., Pérez-Gómez, E., Gordon, M., 2018. E.C.M., 2018. Target and tissue selectivity prediction by integrated mechanistic pharma- Appraising the “entourage effect”: antitumor action of a pure cannabinoid versus abota- cokinetic-target binding and quantitative structure activity modeling. AAPS J. 20, 11. nical drug preparation in preclinical models of breast cancer. Biochem. Pharmacol. 157, Yang, H., Qin, C., Li, Y.H., Tao, L., Zhou, J., Yu, C.Y., Xu, F., Chen, Z., Zhu, F., Chen, Y.Z., 2015. 285–293. Therapeutic target database update 2016: enriched resource for bench to clinical drug Bulusu, K.C., Guha, R., Mason, D.J., Lewis, R.P., Muratov, E., Motamedi, Y.K., Cokol, M., target and targeted pathway information. Nucleic Acids Res. 44, D1069–D1074. Bender, A., 2016. Modelling of compound combination effects and applications to efficacy and toxicity: state-of-the-art, challenges and perspectives. Drug Discov. Today 21, ⁎ 225–238. Hinanit Koltaia, , Patrick Poulinb,c, Dvory Namdara Cheng, J., Dwyer, M., Okolotowicz, K.J., Mercola, M., Cashman, J.R., 2018. A novel inhibitor a Institute of Plant Science, Agriculture Research Organization, Volcani targets both Wnt signaling and ATM/p53 in colorectal cancer. Cancer Res. 78, 5072–5083. De Meijer, E.P., 2014. The Chemical Phenotypes (Chemotypes) of Cannabis. Handbook of Center, Rishon LeZion 7528809, Israel Cannabis. pp. 89–110. b Consultant Patrick Poulin Inc., Québec City, Québec, Canada Hanuš, L.O., Meyer, S.M., Muñoz, E., Taglialatela-Scafati, O., Appendino, G., 2016. c Phytocannabinoids: a unified critical inventory. Nat. Prod. Rep. 33, 1357–1392. School of Public Health, Université de Montréal, Montréal, Canada Iffland, K., Grotenhermen, F., 2017. An update on safety and side effects of cannabidiol:a E-mail address: [email protected] (H. Koltai). review of clinical data and relevant animal studies. Cannabis Cannabinoid Res. 2, 139–154.

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