Soft Drug Approach in Cannabinoids

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SOFT DRUG APPROACH IN CANNABINOIDS

Thesis presented

Jimit Girish Raghav

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

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: .

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

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

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.

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. Depending on the country, cannabinoid preparations available in pharmacies are: 1) oral THC marketed as Dronabinol (Marinol®); 2)

Nabilone (brand name: Cisamet), a hexahydrocannabinol structurally related to THC and 3) Nabiximols (Sativex®), which is marketed as a sublingual spray and contains THC and CBD in a 1:1 ratio. 12,13

In the 1970’s and 80’s, several clinical trials were performed to test the efficacy of dronabinol for inhibition of nausea and vomiting, side effects caused by chemotherapeutic agents. 14 The results of the clinical trials established that oral THC is effective as an anti-emetic to inhibit nausea and vomiting. 14 With further studies, it was found that twice-a-day dosing of 2.5 mg dronabinol results in an effective anti-emetic effect in cancer patients. 14 The orexigenic effect (increased appetite) induced by THC has been known for centuries, and this effect of THC has been utilized for treating loss of appetite in patients suffering from HIV/AIDS. 15 In recent decades, clinical trials have suggested efficiency of dronabinol for also treating anorexia. 16 Dronabinol has also been prescribed for the treatment of chronic neuropathic pain, eliciting analgesia in patients suffering from, e.g., multiple sclerosis. 17 THC can be beneficial to HIV-infected people in treating their neuropathic pain sensations. 18

Other small-scale studies have indicated that THC potentially can be used in various chronic pain-related diseases like rheumatism and fibromyalgia. 17

Pharmacokinetic issues with cannabinoids: THC is highly lipophilic and hence, in practical terms, not water soluble. Its partition coefficient in n-octanol/water is around 6000, which is an experimentally calculated ratio using a flask shake method. 18 THC is also thermo- and photolabile. The pKa value for THC is about 10.6, and the compound rapidly degrades in an acidic environment.18 For recreational purposes, the most common route of

THC administration is through smoking marijuana. For therapeutic purposes, THC is given by the oral route

(Dronabinol). When given orally, THC absorption is highly erratic and slow. 18 Peak plasma concentration may occur anytime between 60 min to 6 h after ingestion in different subjects. THC is rapidly degraded in the stomach’s acidic environment. In the stomach, THC is converted into various substituted cannabidiol-like- products as well as the minor ∆8-THC isomer. THC is also subject to an extensive first-pass metabolism, especially when given orally. A dronabinol capsule of 10 mg resulted in the bioavailability of only 6 to 7 % THC in the studied subjects, concomitant with high inter-subject variability. 18 12

THC distribution in the body is also one of the issues with oral THC and smoked cannabis. Studies with radiolabelled THC showed that after chronic THC administration, maximal concentration of THC is found in fat tissues, and the concentration ratio of THC in fat tissues to brain was 27:1 after 7 days of administration and 64:1 after 27 days of administration. 19 It has also been reported that only 1% of total administered THC is required for its psychoactive effects. 19

Given its high lipophilicity, THC rapidly partitions into tissues which are highly perfused, especially adipose tissues.

This sequestration of THC in fat tissue accounts for its appreciable volume of distribution along with a very slow elimination rate and, especially in the case of oral ingestion, delayed pharmacological effects resulting from the depot effect. 19 The depot effect seen with THC may be attributed to the direct deposition of THC in adipose tissues or, as several studies reported, THC’s active metabolite, namely 11-OH-THC. 11-OH THC forms conjugates with fatty acid in adipose tissues. It is still unclear whether the depot effect seen with THC is because of THC itself or because of the reactivity of11-0H THC with fatty acids in adipose tissues. Haggerty et.al 20 reported that 11-OH

THC forms a conjugate with palmitic acid, and the resulting conjugate, 11-palmitoyl –delta-9-THC, is a psychoactive compound in that it produces catalepsy (muscle rigidity) and hypothermia (lowered temperature) when injected to rats.

Effect of THC in laboratory animals: In parallel with humans, laboratory animals also experience behavioural and physiological effects of THC (e.g., catalepsy and hypothermia). Since the early 1980’s, the tetrad test has been employed to evaluate the effects of novel cannabinoid agents. 21 The tetrad test includes four characteristic behaviours induced by cannabimimetic agents in laboratory animals: 1- decrease in spontaneous activity, or hypo locomotion; 2- analgesia; 3- catalepsy; and 4- hypothermia. 22 This tetrad test was developed primarily for rodents. Mice display more profound hypothermia and analgesia compared to rats, which prompted me to use mice in the reported studies.22 The analgesia test component was selected over other tetrad tests based on my preliminary studies, which suggested that analgesia induced by cannabinoid agents and evaluated in the standard tail-flick test parallels the proposed “soft-drug” profile of the drugs. Tail-flick latency increases with an increase in drug level in the body, and latency decreases as the tested drug level deceases in body as it gets metabolized to its respective inactive metabolite. Tail-flick withdrawal in a hot water bath was selected over other analgesia assays because of its ease and efficiency. 13

The validity of the soft drug approach has already been demonstrated with drugs like esmolol (a class II anti- arrhythmic β-blocker) and remifentanil (a potent opioid used during pre and post-operative analgesic), both of which are now available as prescription medicines. Given the rise of interest of cannabinoids in the field of medicine (especially as potential pain medications), it will be extremely helpful to examine cannabinoid agonists designed to be potential soft drugs.

MATERIALS AND METHODS:

Animals: Young (aged 3-4 weeks) male CD-1 mice weighing between 25-35 grams were used. . Mice were housed in groups of 4 in a single cage. Mice were kept in a 12-hour day, 12-hour night light cycle routine. All the experiments were performed during the 12-hour light phase. All animals had free access to food and water. Mice habituated to the new environment of the animal facility for at least one week before any handling or experiments were performed. For each set of experiments, an experimentally naïve group of mice was used. All the animals were purchased from a listed Northeastern University approved vendor (Charles River Breeding

Laboratories, Wilmington, MA, USA). All experiments performed were in accordance to the protocol no: 13-1134

R approved by Northeastern University’s Institutional Animal Care and Use Committee (NU-IACUC).

Drugs: AM7410, AM7438 and (-) - ∆8 - THC DMH were provided by the chemistry section of the Center for Drug

Discovery, Northeastern University, Boston, MA. All drugs were stored at -20˚C. For preparing the drug suspensions, aliquots of thawed drug stock solutions were taken based upon dosing need. Total injection volume delivered to each mouse was 10 ml/kg. The organics used to prepare injectable forms of all three drugs were dimethyl sulfoxide (DMSO); Tween 80 and propylene glycol (PEG), in a final concentration of 2%, 4%, and 4%, respectively, in saline.

Analgesia: A standard mouse tail-flick assay was used to profile the in vivo analgesic effect of the test compounds. An effective analgesic agent increases the latency time before the animal withdraws its tail from a warm-water bath. A cartoon representation of the experimental set-up is shown below (Fig 4). Three compounds

(-) - ∆8 - THC DMH, AM7410 and AM7438 were tested in the tail-flick assay at different doses. For each dose, naïve mice (n=6) were used. Animals experienced 3 days of habituation followed by a testing session (day 4).

This assay was carried out at ambient room temperature (22-24°C). On any given day of testing or habituation 14 training, animals were acclimatized to the experimental room for 30 min prior to any handling. This acclimation training condition included dipping the terminal 2 -3 cm of tail in a water bath main tained at 38 ˚C for 10 sec. On the third day, each animal received a saline injection to acclimatize the animals to the injection procedure. On the test day, the water bath was in creased and maintained at 52°C, and the first reading was taken and noted at 0 min (the baseline reading). A cut-off period of 10 sec was used to prevent any injury to the tail because of the hot water. After 30 sec post-recording of the base line readings, each animal receive d the drug injection of a predetermined dose. S ubsequent readings were recorded at 20 min, 60 min, 180 min, and 360 min post -drug injection. Along with the drug injectio ns, one group (n=6) of mice was kept reserv ed for obtaining con trol data; this group only received the vehicle instead of drug. The raw data included tail flick withdrawal by each mouse , which was timed in seconds and then converted into a Maximum Possible Effect (MPE) score. The formula for converting raw analgesia data into a MPE score is:

% Maximum Possible Effect (MPE) = 100 * [(Test Response - Baseline Response)/(Maximum Response -Baseline

Response)]

The MPE data are used to generate a dose -response curve for each of the three test compounds, from which potencies, duration of action, onset and offset of action will be determined.

Fig 4: Brief overview the tail-flick analgesia experiment. Only 2 to 3 cm of the distal end of the tail will be dipped into the water bath.

Statistical Analysis: Statistical comparison of tail-flick latency data obtained from AM410 and (-) - ∆8 - THC DMH studies will use two-way repeated measures ANOVA along with Bonferroni’s post-hoc test to assess the time-and dose effect functions. The statistical analysis was performed using GraphPad Prism 5.03 (GraphPad Software, San

Diego, CA). To analyse and compare tail-flick latencies data from AM7438 and AM7410, a linear mixed model repeated measures ANOVA (IBM® software package, SPSS, v.21) was applied.

RESULTS:

Analgesic effect of AM7410 and (-) - ∆8 - THC DMH: Tail-flick latency data experiments conducted in mice demonstrated that AM7410 significantly differs from (-) - ∆-8 - THC DMH in terms of its duration of action. It is evident from Fig. 5 that AM7410 has a quicker onset and quicker offset of action when compared to (-) - ∆8 - THC

DMH. The ANOVA analysis of the tail-flick data showed significant effects for dose (D) [F ( 2,120 )=160.6; P<0.0001] and time (T) [F( 14,120 )=4.5; P<0.0001] along with the D × T interaction for three doses (0.1,0.3, 1.0 mg/kg) of each compound, i.e., AM7410 and (-) - ∆8 - THC DMH. .

100

80 AM7410

% MPE % 40

0 20 60 180 360 Time (min)

100 0.1 mg/kg 0.3 mg/kg 80 1.0 mg/kg Vehicle

60 ∆∆∆8-THC-DMH

% MPE % 40

0 20 60 180 360 Time (min) 5a 5b

Fig. 5 a&b: Tail-flick latencies of mice (n=6 for each dose) in a hot water bath (52 ˚C) post administration of

AM7410 , an ester analog of (-) - ∆8 - THC DMH are shown in Fig. 5a . Tail -flick latencies of mice (n=6) administered 16 a lower dose (0.1mg/kg) of AM7410, (-) - ∆8 - THC DMH respectively and vehicle are shown in Fig 5b. Latencies were converted into maximum possible effect (%MPE) displayed on the ordinate. MPE is expressed as the group mean ±SEM.

Analgesic effect of AM7438: The chemical difference between AM7438 and AM7410 is slight, i.e., AM7438 (log

P=5) contains a cyano group (Fig. 2), which makes the compound more polar vs. AM7410 (log P= 6.59). The tail- flick latency data is congruent with this difference (Fig. 6). AM 7438 has a shorter duration of action when compared to AM7410 at the two doses (0.3 mg/kg and 1 mg/kg) examined. A mixed repeated measures ANOVA applied to the tail-flick latency data with AM 7410 and AM7438 during 180-min and 360-min time points (offset phase) revealed significant effects for drug (D) [F1,44 =8.98; p<0.0004], dose level (L) [F 1,44 =40.95;p<0.001] and time

(T) [F 1,44 =28.46;p<0.001]. All the three parameters (D, L, and T) had significant differences when compared pair- wise using Sidak multiple comparison t-test (p=0.005). This pair-wise comparison of three parameters provides evidence of a faster off-set of AM7438 as compared to AM7410.

In both the experiments, tail-flick latency caused by vehicle is not compared using statistical analysis graphically because MPE did not exceed 20% in any of the examined four time-points.

100

%MPE 40

0.3 mg/kg AM7438 20 1.0 mg/kg AM7438 0.3 mg/kg AM7410 1.0 mg/kg AM7410 0 2060 180 360

Time (min)

Fig 6: Tail-flick latencies of mice (n=6 for each dose) in a hot water bath (52 ˚C) post administration of AM7438 and AM7410. Latencies are displayed as maximum possible effect (%MPE) and shown on the ordinate. %MPE is expressed as the group mean ±SEM. The data for AM7410 is reproduced from Fig. 5.

The results generated from testing two lower doses of AM7438 are displayed in Fig.7. A two-way repeated measures ANOVA indicated significance for Dose (D) [F3, 20 = 125.1], Time (T) [F3, 60 = 61.4] and the interaction

D x T [F9, 60 = 10.8]. 18

Fig 7: Tail-flick latencies of mice (n=6 for each dose) in a hot water bath (52 ˚C) post administration of four doses of AM7438. Latencies are displayed as maximum possible effect (%MPE) and shown on the ordinate. %MPE is expressed as the group mean ±SEM.

Summary and Discussion: The main aim of this thesis project was to evaluate the analgesic effect of two novel potential cannabimimetic agents using an in -vivo mouse model i.e. tail-flick latency assay. As a function of time, the analgesic effect in this mouse tail-flick model can be used as a surrogate indicator for the time course of the pharmacological effect of each tes t agent.

Results from the tail-flick analgesia studies suggest that both of the novel agent s AM7410 andAM7438 differs from their parent analogue (-) - ∆8 - THC DMH in terms of their analgesic time course profile . Thus, AM7410 and

AM7438 both have quicker onsets and offset s of action as compared to (-) - ∆8 - THC DMH . AM7438 is the mo st polar molecule of the three compounds evaluated in this study. The cyano group in the AM7438 (Fig. 3) makes the compound more polar in comparison with AM7410 and (-) - ∆8 - THC DMH, as observed from clogP values and as displayed in Fig6. T his increase in polarity is a likely determinant of the reduced duration of action of AM7438 when compared to AM7410. Future studies with these two molecules will involve screening them using other in- vivo and behavioural models to extend the characterization of these two compounds as “soft drugs”. As a specific example, hypothermia studies with AM7410 and AM7438 along with the presence of a CB1R antagonist will be 19 helpful for characterizing the involvement of CB1R in the analgesic effect of these agents. It will also be a significant step in the development of this project to evaluate these molecules in drug-discrimination models. If the cannabinoid esters were found substitute for THC in drug discrimination, the esters might represent potentially safer alternative analgesics with less risk of THC-induced psychobehavioral adverse events for potential use as pre and post-operative analgesics and pain management in e.g. multiple sclerosis.

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