1 SOFT DRUG APPROACH IN CANNABINOIDS Thesis presented By Jimit Girish Raghav To The Bouve’ Graduate School of Health Sciences In Partial Fulfilment of the Requirements for the Degree of Master of Science In Pharmaceutical Sciences with specialization in Pharmacology NORTHEASTERN UNIVERSITY BOSTON, MASSACHUSETTS 14 th , August, 2014 2 Northeastern University Bouve College of Health Sciences Thesis Approval Thesis Title: Soft drug approach in cannabinoids. Author: Jimit Girish Raghav. Program: Pharmacology. Approval for thesis requirements for the Master of Science degree in: Pharmacology Thesis Committee (Chairman): Dr. Torbjorn Jarbe Date: 08/ 14/2014. Other Committee members Dr. David Janero Date: 08/14 /2014. Dr. Rajeev Desai Date: 08/14 /2014. Dean of the Bouve College of Health Sciences: Dr. Tom Olson DATE: . 3 4 Table of Contents Page List of figures . .4 ACKNOWLEDGEMENTS. .5 ABSTARCT . 6 INTRODUCTION . 6 i. STATEMENT OF THE PROBLEM . 7 ii. BACKGROUND AND SIGNIFICANCE . 9 MATERIALS AND METHODS . 12 RESULTS . 14 SUMMARY AND DISCUSSIONS . 17 REFERENCES . 18 5 List of figures: Figure1: Chemical structures of all the drugs used in the project . 8 Figure 2: Classification of drugs used in this project . 8 Figure 3: Overview of metabolic pathway of drugs used in this project . 9 Figure 4: Overview of tail-flick latency analgesia assay . 13, 14 Figure 5 a& b: Tail-flick latency data for drug AM7410 and (-) - ∆8- THC DMH. 14, 15 Figure 6: Tail-flick latency data for drug AM7438 and AM7410 . 16 Figure 7: Dose response curve for drug AM7438 . 17 6 ACKNOWLEDGEMENTS I would like to take this opportunity and thanks Dr Torbjorn Jarbe, who is my PI and the advisor for the current thesis. Without your vital support and belief I would have never been able to complete this project. I also express my deepest gratitude to Dr. David Janero and Dr. Rajeev Desai for being on my committee, your crucial suggestions, corrections and comments on my project were invaluable. I would also like to offer special thanks to Dr. Alexandros Makriyannis for his indispensable support he gave me on all my projects here at CDD. I will also like to appreciate Dr. Spiros Nikas for providing me with all the test molecules without any hesitation for this project. I would also like to thanks Dr. Kiran Vemuri for guiding me on my research. Last but not least I will like to thanks Roger Gifford my colleague/supervisor in lab who trained me initially on all the assays and helped me acclimatized with the lab environment. My heartfelt to thanks my parents; it was their support and nurture which made me help accomplishing everything in life. A special appreciation to National Institute of Drug Abuse (NIDA) for providing all the monetary requirements via grants to support all the research done in this project. 7 Abstract: The only plant-derived cannabinoid (phytocannabinoid) agent currently used for medical purposes in the USA is (-) - ∆9- tetrahydrocannabinol (THC). Here, I report the analgesic effects of two novel synthetic cannabinergic agents, AM7410 and AM7438 which are designed to be “soft-drugs”. Both drugs have metabolically labile ester groups strategically placed in their chemical structure. This ester group makes AM7410 and AM7438 susceptible to degradation to inactive metabolites by plasma esterases. Both compounds profiled in this thesis are analogues of (-) - ∆8- THC DMH (AM 10808; DMH = dimethylheptyl). The in vivo data demonstrate that both AM7410 and AM7438 produce maximal analgesia (1 mg/kg) in a tail-flick withdrawal assay. Both AM 7410 and AM7438 (0.3 mg/kg and 1 mg/kg) showed quick onset and offset of action when compared to (-) - ∆8 - THC DMH (0.3mg/kg and 1mg.kg). Additionally, data also suggest that the effects induced by AM 7438 (0.3 mg/kg and 1 mg/kg) have a faster offset when compared to AM7410 (0.3 mg/kg and 1 mg/kg) in the tail-flick assay. Introduction: A: Statement of Problem : The aim of this thesis is to evaluate the concept of the “soft-drug” approach in the field of cannabinoid chemistry/synthesis. The “soft-drug” approach has not yet been extensively analysed in the cannabinoid field whereas it is a well-established concept in other medicinal chemistry fields such as opioids and anti-hypertensives.1 A compound is considered to be a “soft drug” if the compound is an analogue of an parent compound and the analogue has a more predictable and controlled metabolism compared to its parent compound. A compound can also be labelled as a “soft drug” if the given compound has minimal side effects when compared with its parent compound.2 In the former case, the analog is synthesised to be a soft drug by introducing a certain chemical group or certain chemical modification into the structure of the analog which will make the drug more susceptible to metabolic degradation by enzyme(s). 2 One of the most common chemical modifications employed to generate a soft drug is the introduction of an ester moiety into the parent compound in an attempt to make the parent compound susceptible to enzymatic inactivation by (plasma) esterases. The work carried out and presented in this thesis will focus on exploring the above concept for two cannabimimetic agents. In this current paper the concept of “depot effect” will be discussed along with the concept of “soft- drug”. The depot effect is typically observed with lipophilic drugs. Drugs with high lipophilicity tend to sequester into fat tissue before flowing into the systemic circulation and to produce their pharmacological effect. The 8 “depot effect” mainly depends on the log P and topological polar surface area (tPSA) values of the molecules. Both logP and tPSA are indices of a drug’s lipophilicity and its cell membrane permeability. 3 The higher the logP value of a compound, the higher the lipophilicity. The higher the lipophilicity, the higher the chances that the compound will get distributed into fat tissues and hence the more likely is the compound’s ability to produce the depot effect. 3 If one is to follow the Lipinski’s rule of five which is set of rules that determines the ability of a test compound to be used as orally available drug for future consumption by humans. 4 According to this rule, the logP of the compound should be 5 or less to avoid the distribution or sequestration into fat tissues and qualify as a lead compound for potential human consumption. 4 By introducing an ester group in the structures of AM7410 and AM7438 (Fig 1) the polarity (clogP values for AM7410 and AM7438 are 6.59 and 5.0, respectively) was markedly increased for these two chemical molecules as compared to their parent analogue i.e. (-) - ∆8 - THC DMH (clogP=9.1). This enhanced polarity would be expected to reduce the depot effect relative to the more lipophilic parent compound (Fig. 2). A more controlled deactivation of both AM7410 and AM7438 compared to the parent compound ((-) - ∆8 - THC DMH) will be achieved by plasma esterases which will lead to the production of inactive metabolites (Fig 3). 9 Fig 1: Chemical structures of the three compounds used in this study. Fig 2: (-) - ∆8 - THC DMH can be categorized here as type B drugs, which are highly lipophilic, and these types of drugs carry a longer depot effect and are very slowly degraded by plasma esterases. AM7410 and AM7438 would fall in the type A drug category, as these drugs are more polar and carry less depot effect because of increased polarity and are quickly hydrolysed by plasma esterases. (Reproduced from Sharma et.al.) 5 10 Fig 3: The ester introduced in the design of cannabinoids makes this class of novel cannabinoids susceptible to plasma esterases which convert these molecules into inactive acid metabolites. (Reproduced from Sharma et.al.) 5 B: Background and Significance : Research concerning medical uses of marijuana and extracts thereof has increased considerably over the last several decades. The legalization of marijuana for recreational use in two states of the USA (Colorado and Washington) has further intensified the need to examine cannabinoid agents both for their potential therapeutic as well as harmful properties.6 Cannabis sativa is the plant from which active components of marijuana are extracted. The plant has been used in traditional medicines for several centuries for conditions such as appetite stimulation, pain management and spasms. 7 The identification of cannabinoid receptors and endogenous cannabinoid-like ligands further helped our understanding of the pharmacological working(s) of THC as well as other cannabimimetic agents. Two principal cannabinoid receptors have been identified and named cannabinoid receptor 1 (CB1R), originally characterized by Devane et al. in 1988, 8 and cannabinoid receptor 2 (CB2R), originally described by Munro et.al. in 1991. 9 CB1R is primarily distributed in the CNS and likely is responsible for the major psychotropic activities of THC and other cannabimimetic agents. CB2R is primarily concentrated in the periphery and especially on immune cells like macrophages. 10 Following these discoveries, two principal endogenous cannabinoid ligands were identified, namely anandamide (AEA; arachidonoyl ethanolamide) and 2-arachidonoyl glycerol (2-AG). 10 2-AG is found in much higher concentrations in the brain as compared to anandamide, and thus it has been proposed that 2-AG is the major neurotransmitter molecule in the endocannabinoid signalling system. 10 Therapeutic areas: THC is mainly responsible for the psychotropic effects (“high”) of the cannabis plant. Some therapeutic effects of the cannabis plant are contributed by another cannabinoid constituent in the plant, cannabidiol (CBD). CBD may act as an anti-emetic, neuroprotective and anti-inflammatory agent. 11 There are 11 very few prescription-based cannabinoid preparations available for medical use.