Cannabinoid Mediated Diuresis in Mice Doctoral Dissertation Presented By

Cannabinoid mediated diuresis in mice Doctoral Dissertation presented by

Girish Rajmal Chopda on August 7th 2013

To The Bouve’ Graduate School of Health Sciences in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in Pharmaceutical Sciences with specialization in Pharmacology

Department of Pharmaceutical Sciences, Northeastern University, Boston, MA

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

Department of Pharmaceutical Sciences, Northeastern University, Doctoral Dissertation

Dissertation Title: Cannabinoid mediated diuresis in mice

Presented by: Girish Rajmal Chopda

Date to be presented: 7th August 2013

Thesis Committee:

Chair and Advisor Dr Carol A Paronis Approval date: ______

Member Dr David Janero Approval date: ______

Member Dr John Gatley Approval date: ______

Member Dr Torbjorn Jarbe Approval date: ______

Member Dr Jack Bergman Approval date: ______

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

I am dedicating my thesis to my father Rajmal I Chopda, my mother Sangeeta R Chopda, my wife Aditee, my brother Vishal and my advisor Carol A Paronis.

iii

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

Table of Contents: Page Number A. Abstract 1

B. Resources Available 2

C. Biographical Sketch 3

D. Specific Aims 5

Chapter 1 – Introduction and background to the cannabinoid system 7 1.1 – History 7 1.2 – Cannabinoid receptors 8 1.3 – Endocannabinoid system 10 1.4 – Endocannabinoid chemistry 12 1.5 – In vivo effects of cannabinoids 19 1.6 – Cannabinoids in clinical use 21

Chapter 2 – Cannabinoid mediated diuresis in mice 23 2.1 - Introduction 23 2.1.1 – Cannabinoid and diuresis 23 2.1.2 – Cannabinoid receptors in the urinary system 24 2.1.3 – Standard diuretics 27 2.2 – Aim and rationale 28 2.3 – Material and methods 29 2.3.1 – Animals 29 2.3.2 – Diuresis 29 2.3.3 – Measurement of urine pH, Na+, K+ and Cl- 29 2.3.4 – Drugs 29 2.3.5 – Statistical analysis 30 2.4 – Results 31 2.4.1 – Validating diuresis 31 2.4.2 – Cannabinoid mediated diuresis 35 2.4.3 – Receptor mechanisms of cannabinoid mediated diuresis 39 2.4.4 – Urine analysis 50 2.5 – Discussion 52 2.5.1 – Validation of diuresis 52 2.5.2 – Cannabinoid mediated diuresis 53

Chapter 3 - Cannabinoid mediated antinociception in mice 59 3.1 – Introduction 59 3.1.1 – Cannabinoid antinociception 59 3.2 – Aim and rationale 61 3.3 – Material and methods 62 3.3.1 – Animals 62 3.3.2 – Antinociception 62 3.3.3 – Drugs 63 3.3.4 – Statistical analysis 63 3.4 – Results 64

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

3.4.1 – Effects of cannabinoid agonists on antinociception 64 3.4.2Effects of antagonist pretreatment 72 3.5 – Discussion 78

Chapter 4 – Cannabinoid mediated tolerance 82 4.1 – Introduction 82 4.1.1 – Drug tolerance 82 4.1.2 – Cannabinoid and tolerance 82 4.2 – Aim and rationale 85 4.3 – Material and methods 86 4.3.1 – Animals 86 4.3.2 – Antinociception 86 4.3.3 – Diuresis 86 4.3.4 – Binding assay 87 4.3.5 – Drugs 87 4.3.6 – Statistical analysis 88 4.4 – Results 89 4.4.1 – Tolerance to diuresis 89 4.4.2 – Tolerance to antinociception 93 4.4.3 – Changes in CB1 receptor levels 97 4.5 – Discussion 99

E. Conclusions 103

F. Bibliography 107

G. Appendix: Laboratory Safety Training 115

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

A. Abstract: Cannabinoid receptor agonists increase urinary output in rats; however these effects have not been characterized in mice. This study investigates whether diuresis is a cannabinoid receptor mediated effect in mice, and further compares cannabinoid mediated diuresis with antinociception. Adult male CD1 mice were injected sc (10 ml/kg) with vehicle or novel and commercially available cannabinoid agonists [AM4054, AM7418, THC (∆9- tetrahydrocannabinol) and WIN55212-22]. Voided urine was measured over 6 hr using single dosing procedures. Antinociception was measured using cumulative dosing procedures and a warm water (52oC) tail-withdrawal assay. In antagonism studies, cannabinoid CB1 receptor selective antagonist rimonabant (0.1-10.0 mg/kg), peripherally restricted cannabinoid CB1 receptor antagonist AM6545 (1.0-10.0 mg/kg) or cannabinoid CB2 receptor selective antagonist

AM630 (0.1-10.0 mg/kg) were administered as a 30min pretreatment. All of the cannabinoid agonists increased diuresis, yielding biphasic dose response curves with maximum voided urine ranging from 28-35 g/kg; urine output after vehicle injection ranged from 7-15 g/kg. All cannabinoid agonists also increased analgesia dose-dependently with peak effects similar to morphine. Peak diuretic effects occurred at doses approximately ½ log unit lower than those that produced maximum antinociceptive effects. Rimonabant dose dependently shifted the diuretic and antinociceptive dose response curve of AM4054 to the right, and was marginally more potent in the diuresis assay. Repeated administration of THC resulted in tolerance to the diuretic and antinociceptive effects of cannabinoids, which was accompanied by CB1 receptor downregulation. Our results indicate that cannabinoid agonists produce increases in urine output by actions at central CB1 receptors and decreases in urine output by actions at both central and peripheral CB1 receptors.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

B. Resources available for the project:

Laboratory space: Our laboratory is located at 140 The Fenway, room 241, and has all the necessary space and apparatus for performing the experiments. THC is obtained from the National Institute of Drug

Abuse (NIDA), AM compounds are obtained from the Center for Drug Discovery (CDD) at Northeastern

University and other required chemicals are reagent grade and purchased from authorized sources.

Animals: Male CD-1 mice were used in the experiments. The mice were purchased from Charles River

Laboratories. The documentation for animal training is in the Appendix.

Laboratory Safety: All the work performed in the laboratory is in compliance with the safety and hygiene guidelines established in the university. I have completed all the necessary training provided by the

University.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

C. Biographical Sketch:

Girish R. Chopda Office Address: Email: [email protected] 140 The Fenway, Room 241 Phone: (228) 233-5799 Department of Pharmaceutical Sciences 360 Huntington Ave, Northeastern University Boston MA 02115

Education: 2006 - BS Pharmacy Maharashtra Institute of Pharmacy, University of Pune Pune, Maharashtra, India 2007 - Certificate Clinical Research and Data Management University of Pune, Pune, Maharashtra, India 2009 - MS Pharmaceutical Sciences Bouvé College of Health Sciences, Northeastern University Boston, MA 2013 - PhD Pharmacology Bouvé College of Health Sciences, Northeastern University Boston, MA

Positions held: 2009- Teaching Assistant, Pharmacology and Medicinal Chemistry (PHSC4501), School of Pharmacy, Bouvé College of Health Sciences, Northeastern University 2010- Teaching Assistant, Pharmacology for Health Professions (PHSC4340), School of Nursing, Bouvé College of Health Sciences, Northeastern University 2011- Teaching Assistant, Human Anatomy lab (PHSC2302), Human Physiology lab (PHSC2304), School of Pharmacy, Bouvé College of Health Sciences, Northeastern University 2012- Teaching Assistant, Pharmaceutics lab (PHSC3419), School of Pharmacy, Bouvé College of Health Sciences, Northeastern University 2012 - Intern – Scientist, Pharmacology formulations (Discovery Support), Novartis Institute for Biomedical Research, Cambridge, MA 2013- Biologist III – Preformulations/Pharmacology/DMPK, Abbvie Bioresearch Center, Worcester, MA

Awards and Honors: 2010 1st Place, Graduate Student Poster Competition, Physical and Life Sciences Division, Northeastern University, Boston, Massachusetts 2011 Travel award recipient at the 2011 ICRS meeting, St Charles, Illinois 2012 Travel award recipient at 2012 ASPET meeting, San Diego, California

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

Professional Societies: 2009- Society for Neuroscience, Student member 2010- American Association of Pharmaceutical Scientists, Student member, NU Chapter, Treasurer 2010- American Society for Pharmacology and Experimental Therapeutics (ASPET), Student member 2011- Behavioral Pharmacology Society, registered attendee 2011- International Cannabinoid Research Society (ICRS), Student member 2013- ASPET, Mentoring and Career Development Committee, member

Publications: 1. Chopda GR, Transdermal Drug Delivery System: A Review. Pharmainfo.net vol 4, issue 1, December 1, 2006 2. Chopda G.R, Thakur G, Vemuri K, Makriyannis A, Paronis C.A. “Diuretic effects of cannabinoids in mice” (Revision submitted to European Journal of Pharmacology - 7/2013) 3. Chopda G.R, Nikas S, Makriyannis A, Paronis C.A. “Cannabinoid agonists mediate lower lip retraction in rats by activation of CB1 receptors” (In preparation for Psychopharmacology)

Proceedings of Meetings: 1. Chopda GR, Deth RC. Differential inhibition of thioredoxin and thioredoxin reductase activity by thimerosal: A possible mechanism of mercury toxicity in neurodevelopmental diseases. 436.24. 2009 Neuroscience Meeting Planner. Chicago, IL: Society for Neuroscience, 2009. 2. Chopda GR, Sharma R, Makriyannis A, Paronis CA. Effects of CB1 cannabinoid agonists in rats. #1506, NEU research expo, 2010. 3. Chopda GR, Sharma R, Thakur G, Vemuri K, Makriyannis A, Paronis CA. Cannabinoid mediated diuresis in mice. FASEB J March 17, 2011 25:617.6. 4. Chopda GR, Anderson J, Thakur G, Makriyannis A, Paronis CA. Diuresis: A simple and efficient measure to screen cannabinoids (2011), 21st Annual Symposium on the Cannabinoids, International Cannabinoid Research Society, St. Charles IL, p3-28. 5. Chopda GR, Anderson J, Nikas SP, Makriyannis A, Paronis CA. Cannabinoid CB1 and serotonin 5-HT1A agonists mediate lower lip retraction by independent mechanisms. FASEB J March 29, 2012 26:661.8 6. Chopda G.R, Bergman J, Vemuri K, Makriyannis A, Paronis C.A. “Possible Efficacy Related Differences Among Cannabinoid Agonists” FASEB J 2013

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

Specific Aims:

The goal of this project is to evaluate whether cannabinoids mediate diuresis in mice and, if so, to identify the mechanism of cannabinoid induced diuresis. Some studies have reported that phytocannabinoids or endocannabinoids produce diuresis in rodents and humans, but the diuretic effects of cannabinoids have never been fully characterized in spite of the extensive research on the other cannabinoid effects. It is important to identify the mechanism of cannabinoid mediated diuresis, as it may provide valuable insight into other more complex issues associated with cannabinoids such as addiction and tolerance. The above aim will be addressed in an organized way by dividing it as follows:

Aim 1: Establish cannabinoid mediated diuresis as a quantitative and dose-dependent effect in mice

Aim 2: Characterize the mechanism of action of cannabinoid mediated diuresis in mice and compare it with a previously well established cannabinoid effect (antinociception)

a. Evaluate whether the cannabinoid-induced increase in diuresis is blocked by pretreatment with

rimonabant, which is a CB1 selective antagonist/inverse agonist, AM630 a CB2 selective

antagonist or AM6545 a peripherally restricted CB1 antagonist

b. Obtain dose response curves for cannabinoid antinociception in the mouse warm water tail-

withdrawal assay and determine the effects of cannabinoid antagonists on the antinociceptive

effects

c. Compare the antinociceptive and diuretic effects of cannabinoid agonists and the effects of

cannabinoid antagonists on the agonist-induced antinociception and diuresis

Aim 3: Evaluate whether cannabinoid mediated diuresis in mice is a free water diuresis or whether a loss of electrolytes accompanies the water loss by:

a. Comparing the concentrations of sodium (Na+), potassium (K+) and chloride (Cl-) in urine

samples from furosemide and cannabinoid treated mice

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

b. Comparing urinary pH between the above two groups

c. Evaluating whether urine composition varies according to dose of cannabinoid

Aim 4: Determine the effects of repeated administration of cannabinoids on diuresis and compare the diuretic effect with antinociception with respect to the magnitude and rate of tolerance/sensitization by:

d. Determining if tolerance or sensitization develops to the diuretic effects of cannabinoids after

repeated administration of THC

e. If tolerance develops to the diuretic effects, comparing the magnitude of this tolerance to that

which develops to the antinociceptive effects of cannabinoids using the same dosing regimen

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

Chapter 1 – Introduction and Background to the Cannabinoid System:

1.1 History: Marijuana has been used since ancient times for recreational, religious and medicinal purposes. Records of marijuana use in medicine date back more than 5000 years, when it was used to induce analgesia during primitive surgery. Its property to alter sensory perception and to produce euphoria were also noticed and, as a result, it has been used for recreational and religious purposes in various parts of the world also for millennia (Adams,

1940; Maickel, 1973). In ancient literature from various parts of the world there is mention of cannabis use for several conditions in addition to treating pain, these include cough suppression, improving appetite, venereal diseases, dysentery, sedation, nausea and urinary incontinence. In the western world, as recently as the early 1900’s, cannabis was indicated for treating most of the above mentioned conditions, especially pain and sleep disorders, and was available in the form of oils, tinctures and creams at pharmacy stores. After the advent of the hypodermic needle in the 1850’s, water soluble drugs like opioids, aspirin and barbiturates that could be injected were preferred over cannabis for treating pain and to induce sleep and the use of cannabis in medicine began to decline due to the lack of solubility, poor stability and variable pharmacokinetic profile

(Grinspoon and Bakalar, 1997). The steep increase in abuse of cannabis in the early 1900’s and a multitude of political and social reforms led to cannabis use being effectively criminalized in the United States in 1937 (NIDA info facts, 2010; (Grinspoon, 1969). Later, in 1942, it was removed from the United States pharmacopeia, although recreational use of marijuana along with research on the therapeutic properties of cannabinoids continued. In 1975, the first clinical trial of the major psychoactive ingredient of cannabis compared THC dissolved in sesame oil with placebo in 20 patients undergoing cancer chemotherapy. The results of this study showed statistically significant anti-emetic effects of cannabis as compared to placebo treatment (Sallan

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda et al., 1975). Another clinical study by Noyes et al., 1975 showed that 20mg THC was an equally efficacious analgesic compared to 60 or 100 mg codeine; a lower dose of 10 mg THC was better tolerated but was less efficacious as compared to higher dose of THC and the two doses of codeine (Noyes et al., 1975a; Noyes et al., 1975b). However, products of the cannabis plant currently are classified as schedule I controlled substances in the United States and are under strict regulation of Drug Enforcement Administration (DEA). Apart from their recreational use and abuse liability, cannabinoids retain their therapeutic efficacy as analgesics, antispasmodics, muscle relaxants, bronchodilators and appetite stimulants along with treating anorexia, overactive bladder, nausea and vomiting, which make them viable candidates to return to the pharmacopeia. Recently interest in cannabis has grown, and currently it is under phase III clinical trials for treating cancer pain (Anonymous, 2010). Sativex, an oromucosal spray consisting of Δ9-THC and cannabidiol, is approved in many European countries for the treatment of spasticity in multiple sclerosis (MS) patients. It is also approved in Canada for treating neuropathic pain associated with MS and has shown beneficial symptomatic relief in patients with urinary incontinence. Synthetic forms of Δ9-THC, dronabinol (Marinol) are available as third line treatment for chemotherapy-induced nausea and vomiting. Many of the noncannabinoid drugs that are approved by the USFDA and extensively used in clinics have a therapeutic index 1:10 – 1:20, however marijuana has a therapeutic index of 1:25,000, when smoked, making it one of the safest drugs known to mankind.

1.2 Cannabinoid receptors: Due to the high lipophilicity of all cannabinoids, it was initially hypothesized that they exert their pharmacological effects nonspecifically by altering membrane fluidity. However, in 1990, CB1 receptors were cloned from rat, mouse and human brain tissues and this was followed soon thereafter by the cloning and characterization of the

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

CB2 receptors, in 1993, from human immune cells (Matsuda et al., 1990; Munro et al., 1993;

Galiegue et al., 1995). There is 68% sequence homology between the CB1 and CB2 receptors in their transmembrane domain region (Galiegue et al., 1995). It is now known that cannabinoids exert their pharmacological effects by binding to CB1 and or CB2 receptors that are members of the seven transmembrane G-protein coupled receptor (GPCR) super family. The subjective effects of cannabinoids are mediated by the activation of CB1 receptors in the CNS and that the

CB1 receptors are present presynaptically (Tanda and Goldberg, 2003; Klumpers et al., 2013).

As a result, there is often a perception that all CB1 receptor effects are centrally mediated. This idea was reinforced by early evidence suggesting that CB2 receptors were exclusively found peripherally, on the cells of the immune system where they have a role during inflammation.

However, with growing research in the cannabinoid field, it has been shown that CB2 receptors are also expressed on the microglia cells in the CNS, where they might have some function during inflammation and CB1 receptors are extensive found in the periphery (Matsuda et al.,

1990; Galiegue et al., 1995; Onaivi, 2006; Walczak and Cervero, 2011). Studies have recently investigated the role of peripheral CB1 and CB2 receptors in alleviating pain and inflammation however, the role of CB2 receptors in producing analgesic like effects remains controversial

(Ibrahim et al., 2006; Yu et al., 2010). Another GPCR, designated “GPR55”, was identified and cloned in 1999 (Sawzdargo et al., 1999) and was proposed to be the third cannabinoid receptor, however it was classified as an orphan receptor (Amy E. Monaghan. Class A Orphans: GPR55.

Last modified on 06/11/2012. Accessed on 5/25/2013. IUPHAR database (IUPHAR-DB), http://www.iuphar-db.org/DATABASE/ObjectDisplayForward?objectId=109). Of importance here is the fact that neither the nonselective cannabinoid agonist WIN55,212-2 nor the CB1 antagonist rimonabant have any affinity for the GPR55 receptors (Ryberg et al., 2007). Hence,

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

GPR55 receptors may not be involved in cannabinoid mediated effects that are antagonized by rimonabant.

CB1 and CB2 receptors, upon activation, signal predominantly via the Gi/o pathway and have an inhibitory influence on cell firing by inhibiting adenylyl cyclase and decreasing c-AMP levels. Cannabinoid receptor activation also inactivates calcium channels and opens inwardly rectifying potassium channels, which ultimately hyperpolarizes the cells and prevent neurotransmitter release. Furthermore, prolonged cannabinoid receptor activation has been linked to downstream activation of mitogen activated protein kinase (MAPK) pathway, causing changes in gene transcription that result in changes in receptor localization or density (Pertwee,

1997; Piomelli, 2003). Taken together, these data suggest that activation of CB1 receptors triggers multiple downstream signaling pathways and selective activation of one downstream pathway over the other may vary depending on the receptor location, ligand bound or the frequency of receptor activation, similar to most drug receptor interactions.

1.3 Endocannabinoid system: Identification of CB1 and CB2 receptors was followed shortly thereafter by identification of endogenous compounds that bind to these receptors, called endocannabinoids; among the various endocannabinoids the two most commonly studied are N- arachidonyl ethanolamine (anandamide) and 2-arachidonylglycerol (2-AG). Unlike conventional neurotransmitters, endocannabinoids are not synthesized and stored in vesicles but are synthesized and released on demand. Anandamide and 2-AG are synthesized by cleavage of membrane lipid precursors N-arachidonoylphosphotidylethanolamine (NAPE) and diacylglycerol (DAG) respectively. The endocannabinoids signal in a retrograde manner, i.e., they are released from the postsynaptic ganglion and bind to the cannabinoid receptors on the presynaptic membrane. Cannabinoid receptors, being coupled to the inhibitory signaling

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda molecules, cause inhibition of neurotransmitter release from the presynaptic neurons that these receptors are localized on, and may interfere or regulate other neurotransmitter systems

(Rodriguez de Fonseca et al., 2005). Following their synthesis and release, the endocannabinoids are taken up by membrane-bound reuptake transporters, after which they are degraded by the lipid bound enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). FAAH and MAGL are responsible, respectively, for the breakdown of anandamide to arachidonic acid and 2-AG to ethanolamine/glycerol (Palmer et al., 2002;

Piomelli, 2003). Anandamide, in addition to being an endogenous cannabinoid ligand, is an endovanilloid that binds to the vanilloid receptor 1, also called the transient receptor potential cation channel subfamily V member 1 (TRPV1) and is involved in mediating TRPV1-dependent hypotension, analgesia and increased diuresis in mice (Pacher et al., 2004; Haller et al., 2006;

Xie and Wang, 2009). Given the overlap between the CB1 and TRPV1 effects, studies have looked at binding of the phytocannabinoids to the TRPV1 receptors and found that they lack affinity for these receptors (Lam et al., 2005; Li and Wang, 2006). Hence, most of the effects of the phytocannabinoids and other exogenous cannabinoids are attributed to actions at CB1 and/or

CB2 receptors and are independent of TRPV1 receptors. However, effects of endogenous cannabinoids anandamide and methanadamide occur by actions on both CB1 and TRPV1 receptors but are mutually exclusive according to the literature information available to date.

Several studies have specifically identified and distinguished differences between the effects of cannabinoids on the CB1 and TRPV1 receptors in vivo utilizing selective receptor antagonists as tools. Methanandamide produced disruption of operant responding in rats by TRPV1 receptor mechanisms, independent of cannabinoid receptors (Panlilio et al., 2009). In another study, anandamide produced decreases in mean arterial pressure in instrumented rats that was

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda antagonized by a CB1 receptor antagonist but not by a TRPV1 antagonist (Li and Wang, 2006).

There is a possibility that novel cannabinoid agonists might have affinity for TRPV1 receptors, however, as rimonabant lacks affinity for TRPV1 receptors, effects that can be antagonized by rimonabant would be independent of TRPV1 mechanisms.

1.4 Cannabinoid Chemistry: Cannabinol (CBN), cannabidiol (CBD) and ∆9- tetrahydrocannabinol (THC) are among the many compounds isolated from marijuana. Of these,

THC is the primary psychoactive component of crude marijuana; CBN has some marijuana-like effects, whereas CBD is inert. The structure of THC was identified by Gaoni, Mechoulam, et al.,

1964. THC exists in two isomeric forms Δ9-THC and Δ8-THC that structurally differ by the position of a double bond as seen in Figure 1. Δ8-THC is the more stable of the two isomeric forms of THC and has similar pharmacological effects as compared to Δ9-THC.

Figure 1: Structure of Δ8-THC and Δ9-THC

Δ9-THC has a tricyclic ring structure, as shown in Figure 1 and is synthesized in laboratories for research purposes. Synthetic THC helps minimize the variability in THC content that is obtained in different batches of crude marijuana and this facilitates comparison of pharmacological properties of the drug across various studies. After successfully synthesizing

THC in the laboratory, chemists have more recently synthesized other cannabinoid analogues;

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda based on their structure they are classified as classical (THC-like/tricyclic ring, Figure 2A) or non-classical cannabinoids such as bicyclic derivatives, aminoalkylindoles (Figure 2B), arylsulphonamides and eicosaonoids (Mechoulam, 1970; Maickel, 1973). This advance in cannabinoid chemistry has made available newer drugs which are more stable and potent as compared to THC, making a wide range of synthetic cannabinoid analogues available as tools for better understanding the cannabinoid system. One of the first synthetic analogues that has been widely studied is WIN 55,212 (Compton et al., 1992); WIN55212-2 is an aminoalkylindole, that binds CB1 and CB2 receptors nonselectively, and is equipotent to THC. More recently developed novel classical cannabinoid agonists that were used as part of this study are AM4054 and AM7418 (synthesized at the Center for Drug Discovery (CDD) at Northeastern University).

AM7418 is a Δ8-THC analogue with an ester group on the side chain (Figure 2C) of the pharmacophore which was anticipated to shorten its duration of action (Sharma, 2011). AM4054 has no double bond in the C ring (where the Δ8 or Δ9 double bond is present) and also has an adamantyl group on the side chain (Figure 2D) which makes it a shorter-acting analogue similar to AM7418 (Thakur et al., 2013). Table 1 lists the binding affinities for all cannabinoid agonists used in the present studies at CB1 and CB2 receptors, along with their relative selectivity for

CB1 receptors. All agonists have low nM affinities for the CB1 and CB2 receptors, the binding affinities for THC and WIN55,212-2 have been reported by several groups using various cell lines and tissue preparations [http://www.drugs-forum.com/forum/showthread.php?t=117873], and binding affinities for AM7418 and AM4054 are based on studies using expressed human, mouse, or rat CB1 or CB2 receptors (Thakur et al., 2013). None of the four cannabinoid agonists had more than a 10-fold difference in their Ki values for CB1 or CB2, hence none of the compounds may be considered selective for either CB1 or CB2 receptors. Although the Ki

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda values of THC and WIN 55,212 encompass a 40- to 50-fold range, AM4054 and AM7418 have higher affinities for CB1 than do WIN55,212-2 and THC, based on averages obtained from several values reported in literature (Thomas et al., 1998; Sharma, 2011; Thakur et al., 2013).

AM4054 and AM7418 are more selective for CB1 over CB2, they both have other, pharmacokinetic attributes that enhance their utility as tools to study cannabinoid effects. THC is highly lipophilic with unpredictable absorption, a high volume of distribution, and also very high plasma protein binding. In contrast, AM4054 and AM7418 are both less lipophilic than

THC (determined according to lower ClogP values) and as a result have a quick onset and offset of action. These pharmacokinetic features increase the potency of both AM4054 and AM7418, resulting in lower ED50 values in vivo, relative to THC, than would be predicted by their Ki values.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

A) THC B) WIN55,212-2

C) AM7418 D) AM4054

Figure 2: Structures of cannabinoid agonists

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

The first cannabinoid antagonist identified, SR141716A (rimonabant), is a diarylpyrazole derivative (Figure 3A), synthesized at Sanofi Recherche, Montpellier, France (Rinaldi-Carmona et al., 1994). Unlike the cannabinoid agonists, rimonabant is receptor selective, with a higher affinity for CB1 than CB2 receptors, it is also a CB1 inverse agonist as demonstrated by various in vitro assays. Other cannabinoid antagonists also are more selective for one or the other cannabinoid receptor. AM630 is a widely used CB2 receptor selective antagonist that has more than 150-fold selectivity for the CB2 receptors over the CB1 receptors making it a useful pharmacological tool for distinguishing CB1 and CB2 receptor effects of cannabinoid agonists

(Pertwee et al., 1995). AM6545, like rimonabant, is selective for CB1 receptors but, unlike rimonabant, has a sulfur in the unsaturated 6 membered ring at the para position to nitrogen

(Figure 3B). AM6545 is characterized as a neutral CB1 antagonist in vitro and is considered as a peripherally constrained CB1 selective antagonist in vivo. The mechanism by which AM6545 exerts effects primarily in the periphery is attributed to the presence of a glycoprotein efflux transporter in the blood brain barrier (BBB) which removes AM6545 rapidly from the CNS back into the periphery and results in potential peripheral localization of AM6545 (Tam et al., 2010).

The affinity of the antagonists for the CB1 and CB2 receptors is given in table 1 along with their relative selectivity for CB1 over CB2 receptors.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

A) Rimonabant (SR141716A) B) AM6545

O S O O N N H N N Cl

NC Cl

AM6545 C) AM630

Figure 3: Structures of cannabinoid receptor antagonists

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

Table 1: Binding affinities of cannabinoid compounds and their relative selectivity for CB1

Compounds CB1 (Ki) nM CB2 (Ki) nM CB1 selectivity

Agonists

THC 5 to 80 3 to 75 ~1

WIN55,212-2 2 to 123 0.3 to 16.2 0.1

AM4054 5 12 2.4

AM7418 0.6 1.2 2

Antagonists

Rimonabant 2 to 12 514 to 13200 > 500

AM630 5152 32 <0.01

AM6545 1.7 523 > 300

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

1.5 In vivo effects of cannabinoids: To better understand the mechanisms of THC and other cannabinoid compounds in vivo, animal models have been used for decades. Drug discrimination was a useful tool for screening cannabinoid like compounds before the identification of cannabinoid receptors, and to date remains a reliable tool for characterizing cannabinoid compounds in vivo. Rats, pigeons, and monkeys have been successfully trained in drug discrimination paradigms using standard cannabinoid agonists or antagonists. Once trained successfully to discriminate a standard cannabinoid, animals can be used repeatedly to screen novel cannabinoid compounds (Jarbe and McMillan, 1979; Jarbe et al., 2001; Jarbe et al., 2004;

McMahon, 2006; Ginsburg et al., 2012; Jarbe et al., 2012).

For many years, cannabinoid drugs also have been screened in vivo using four behavioral tests in mice: hypothermia, analgesia, catalepsy and locomotor activity with the requirement that novel ligands had to be active in all four assays to be identified as cannabinoids (Little et al.,

1988). This battery of four tests (tetrad) was developed prior to the availability of selective antagonists for characterizing cannabinoids, and is still used to screen cannabinoids as it provides a good in vivo measure of efficacy and potency. Since the discovery of rimonabant, it has been shown that the effects of cannabinoid agonists in all the four tests can be antagonized by pretreatment with the CB1 selective antagonist/inverse agonist (McMahon and Koek, 2007).

Other noncannabinoid drugs may be active in one or more of the tetrad tests, yet few other drugs produce all four effects, and presumably they are not antagonized by rimonabant (Martin et al.,

1991; Wiley and Martin, 2003). Even though the tetrad is used as a standard assay for testing cannabinoid compounds in mice, the complete pharmacological profile of cannabinoid class of compounds is unknown, and many studies now look beyond the tetrad to evaluate cannabinoid effects in vivo. For example, the elevated plus maze and other maze paradigms in mice and rats

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda have been used to identify anxiolytic-like effects of THC and other cannabinoid agonists (Onaivi et al., 1990), although others have reported that THC was found to be ineffective as an anxiolytic in punished responding procedures (Marco et al., 2004; Delatte and Paronis, 2008) . Still others have noted both dose-related anxiolytic and anxiogenic effects of cannabinoid agonists in plus maze paradigms, with some evidence that low doses of cannabinoid agonists produce anxiogenic effects by acting on the CB1 receptors, while higher doses acts via the 5-HT1A receptors to produce the anxiolytic-like effects (Marco et al., 2004). The antidepressant effects of cannabinoid agonists have also been evaluated using forced swim test in rodents (Hill and

Gorzalka, 2005), and other studies have identified effects of cannabinoids on CB1 receptors present in different regions of the brain on learning, memory, attention, stress and reinforcing effects using various animal models (Martin et al., 2002; Rubino et al., 2008). All or most of these effects produced by cannabinoids are thought to be mediated by actions at the central CB1 receptors.

In addition to their centrally mediated effects, cannabinoids also modulate the cardiovascular system (tachycardia and vasodilation), digestive (increase food intake), and respiratory (bronchitis) systems and have immunosuppressant effects (Pertwee, 1997). Effects of phytocannabinoids, synthetic cannabinoids, and the endocannabinoid anandamide on diuresis have been previously reported in rats (Li et al., 2006; Paronis et al., 2013), however this effect has not been previously reported in mice. Diuresis affords a cost effective and objective measure of drug action, as compared to the tests of the tetrad, and also is simple to assess in untrained animals, as compared to the long training needed for drug discrimination studies. In addition to these practical considerations, studying cannabinoid-mediated diuresis and further investigating

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda its mechanisms of action can provide valuable information towards identifying the full spectrum of cannabinoid mediated effects in intact behaving animals.

1.6 Cannabinoids in clinical use: Very few cannabinoid drugs are approved for use in the United States. Dronabinol, a synthetic Δ9-THC, was the first US FDA approved cannabinoid, in 1985, for treatment of nausea and vomiting in patients undergoing cancer chemotherapy and treating anorexia and weight loss in HIV/AIDS patients (Stott and Guy,

2004). Nabilone was the first synthetic cannabinoid to be approved by the US FDA, in 2006, as an anti-emetic in patients undergoing cancer chemotherapy and non-responsive to conventional anti-emetics. Currently, nabilone and dronabinol are used as fourth line treatment options, i.e., only when all the other available drugs are ineffective as anti-emetics (Beal et al., 1995; Haney et al., 2005; Berlach et al., 2006; Haney et al., 2007). In Canada and Europe, Sativex, an oral spray consisting of ∆-9THC and cannabidiol (CBD), is approved for treating neuropathic pain associated with multiple sclerosis and spasticity. Sativex is under phase III testing in the United

States for neuropathic pain associated with multiple sclerosis and cancer chemotherapy, in addition, it has been found useful in multiple sclerosis patients to control overactive bladder

(Brady et al., 2004; De Ridder et al., 2005; Barnes, 2006; Anonymous, 2010).

In contrast to the therapeutic uses of cannabinoid agonists in promoting food intake, the

CB1 antagonist/inverse agonist rimonabant is an appetite suppressant and was used to induce weight loss though increasing incidences of adverse effects like severe depression and anxiety causing suicidal thoughts led to its being withdrawn from the market. The mechanisms responsible for these severe adverse effects remain obscure, however, cannabinoid antagonists that are devoid of the inverse agonist like effects as well as antagonists that are peripherally restricted are being developed as weight loss drugs.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

Together, these studies indicate that in spite of the abuse potential of cannabinoids, novel cannabinoids are emerging as potential candidates for various indications including but not limited to bladder diseases, glaucoma and pain management, and as more new cannabinergic compounds are synthesized, there is a good possibility that in the future cannabinoids may emerge as the primary treatment for diverse medical conditions. Prior to this, however, it is imperative that we fully understand the full scope of the physiological effects of cannabinoids, including their impact on water homeostasis in vivo following either acute or chronic administration.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

Chapter 2: Cannabinoid mediated diuresis in mice

2.1 Introduction:

2.1.1 Cannabinoids and diuresis: Effects of marijuana on the urinary system were noted in ancient Indian and Chinese literature, and increase in urine output following marijuana ingestion also has been anecdotally reported in western medical literature before the discovery of the cannabinoid receptors. These effects may underlie its therapeutic effectiveness for treating kidney stones, glaucoma and edema (Stuart, 1911; Allentuck and Bowman, 1942; Chopra and

Chopra, 1957; Pryor et al., 1977). A study in the 1950’s by Frances Ames showed that oral cannabis ingestion increased urinary output in human subjects to an average of 420ml, as compared to 200ml after placebo treatment, over a 1-3 hr observation period; some individual subjects experienced 6-fold increases in urine output (Ames, 1958). Later, Barry et al., 1973 and

Sofia et al., 1977 showed that THC elicited dose dependent increases in diuresis in rats after i.p and oral administration, with effects that were quantitatively higher than those produced by thiazide diuretics. In these studies, increases in both urine output and corticosterone levels after

THC administration were observed in normal rats but not in adrenalectomized and hypophysectomized rats (Kubena et al., 1971; Barry et al., 1973). Together, these data indicate that effects of THC on corticosterone and diuresis were mediated via the pituitary adrenal axis with possible involvement of both central and peripheral sites (Barry et al., 1973; Sofia et al.,

1977). Because these studies predate the discovery of specific cannabinoid receptors, the involvement of receptor sites in mediating the diuretic effects of cannabinoids were not investigated. However, later studies on the role of THC and other cannabinergic compounds on the neuroendocrine system and the hypothalamic-pituitary axis demonstrated that cannabinoids increase corticosterone release by CB1 receptor mechanisms (Murphy et al., 1998). Despite this

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda clear evidence that increases in urine volume represent a quantitative, objective measure of THC effects in unrestrained rats, to the best of our knowledge there have been no reports of changes in urine output after cannabinoid treatment in mice, a species widely used in drug discovery.

2.1.2 Cannabinoid receptors in the urinary system: After the identification of specific cannabinoid receptors, many studies mapped the localization of the cannabinoid CB1 and CB2 receptors in different tissues across various species. In vitro and ex vivo studies report the presence of CB1 receptors on peripheral tissues including heart, fat cells (adipocytes), liver, intestine, kidney, and lungs, in addition to their extensive localization within the CNS (Gatley et al., 1996; Pertwee, 1997). In the lower urinary tract, CB1 receptor but not CB2 receptor mRNA, cDNA and protein are found in isolated bladder and kidney preparations across different species

(Pertwee and Fernando, 1996; Walczak et al., 2009; Larrinaga et al., 2010). In keeping with the presence of CB1 receptor protein, the effects of cannabinoid drugs in isolated bladder preparations confirm a role for CB1 receptors in modulating bladder activity. For example, structurally diverse cannabinoid agonists such as THC, WIN55,212-2, anandamide, dose- dependently inhibit electrically evoked contractions, and these effects are blocked by pretreatment with the CB1 antagonist rimonabant but not by the CB2 antagonist AM630, suggesting CB1 receptor involvement in producing bladder relaxation (Pertwee and Fernando,

1996; Martin et al., 2000). Another group of researchers have similarly shown that cannabinoid agonists inhibit mechanically-induced distensions in bladder preparations by acting on the CB1 receptors on the bladder, and further suggested that CB1 receptors may also modulate inflammatory pain mediated by TRPV1 receptors, which co-localize with CB1 receptors in the mouse bladder (Walczak et al., 2009; Walczak and Cervero, 2011). Studies have also reported a role for cannabinoid receptors upstream of the bladder as cannabinoid receptor binding has been

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda reported in rodent and human kidney tissues (Li and Wang, 2006; Larrinaga et al., 2010). While these ex-vivo studies indicate that CB1 receptors function in bladder and kidney preparations across various species, a specific role of cannabinoid receptors in normal urinary tract functioning remains to be determined. Moreover, these actions likely are not restricted to direct actions within the lower tract. Similar to the demonstration that cannabinoids increase corticosterone release within the hypothalamic-pituitary axis, activation of CB1 receptors also will inhibit the release of vasopressin and oxytocin from the posterior pituitary, possibly via inhibitory inputs from glutamatergic neurons projecting on the hypothalamus, which regulates release of the pituitary hormones (Tyrey and Murphy, 1984; Di et al., 2003; Tasker, 2004).

The involvement of CB1 receptors in modulating diuresis or micturition has been examined in isolated tissue preparations using not only synthetic cannabinoids and phytocannabinoids, but also the endogenous cannabinoid, anandamide. However, anandamide does not exclusively bind cannabinoid receptors; indeed, anandamide is also classified as an endovanilloid due to its high affinity for the TRPV1 receptors. Like CB1 receptors, TRPVI receptors are widely located on sensory cells and urothelial cells of the urinary tract (Avelino and

Cruz, 2006). Some studies have specifically evaluated the role of TRPV1 receptors in anandamide-mediated effects in rat bladder preparations and, in contrast to CB1 mediated effects, these studies found that activation of TRPV1 by anandamide increases reflexive bladder contractions (Dinis et al., 2004). Such results might raise questions regarding a role of TRPV1 receptors in the effects of exogenous cannabinoids (Li and Wang, 2006), although this is tempered by evidence that exogenous cannabinoids do not bind to TRPV1 (Ross et al., 2001).

This finding was confirmed in vivo by the demonstration that diuretic effects of a cannabinoid agonist was not blocked by the TRPV I antagonist capsazepine (Paronis et al., 2013). Although

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda evidence supports a role for CB1, but not TRPV1, mediation of the effects of cannabinoids on diuresis, other studies have suggested still other mechanisms might also be involved. In one study using anesthetized and catheterized rats, infusion of anandamide or its longer acting analogue methanandamide increased both glomerular filtration rate and urine output, and neither effect was attenuated by pretreatment with either a CB1 or TRPV1 antagonist, suggesting that activation of neuronal reflexes also may be involved in mediating the effects of endocannabinoids on the kidney (Li and Wang, 2006). In contrast, others have shown that anandamide increases renal blood flow, glomerular filtration rate, urine volume and decreases mean arterial blood pressure by dilatory effects on afferent and efferent arterioles via both cannabinoid receptor dependent as well as independent mechanisms (Koura et al., 2004). It was recently proposed that these changes result from actions of an intermediate metabolite of anandamide, prostamide E2 primarily in the renal medulla in catheterized rats (Ritter et al.,

2012). One interesting study in anesthetized rats demonstrated that WIN 55-212 reduced bladder motility and increased micturition threshold by peripheral CB1 receptor mechanisms, this presumably would lead to a decreased urine output and is in contrast to increased diuresis reported with cannabinoid agonists in awake rats (Sofia et al., 1977; Dmitrieva and Berkley,

2002; Paronis et al., 2013). The reasons for these differences are unknown, though they may reflect inherent differences in drug responses in awake and anesthetized animals. Nonetheless, there is strong evidence that exogenous cannabinoids administered to intact rats increase diuresis by actions at CB1 receptors, with no evidence supporting a role for CB2 or the TRPV1 receptors involvement in cannabinoid mediated diuresis in rats (Paronis et al., 2013). One goal of the present research was to extend and confirm these findings to another species, mice.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

2.1.3 Standard Diuretics: The diuretic effects of cannabinoid agonists were qualitatively and quantitatively compared to the effects of two other diuretics that work through different mechanisms; one was the standard loop diuretic, furosemide, and the other was a kappa opioid receptor (KOR) agonist, U50,488. Furosemide increases urine output by blocking the Na-

K-2Cl symporter in the thick ascending Loop of Henle, resulting in an increased loss of water along with Na+, K+ and Cl-, producing a true diuresis by increasing urine output without changing the electrolyte concentration in the excreted urine (Goodman et al., 2006). In contrast,

KOR agonists increase urine output that consists of increased water loss without accompanying electrolyte loss, as a result producing dilute urine, also referred to as free-water diuresis. KOR agonists produce water diuresis by inhibiting vasopressin secretion through activating KOR in the hypothalamus and in turn inhibiting the secretion of vasopressin (Slizgi and Ludens, 1982;

Brooks et al., 1993; Rossi and Brooks, 1996). When present, vasopressin acts on the collecting ducts of the kidney, increasing their permeability to water and, as a result, increasing water reabsorption and concentrating the urine. Hence, by inhibiting vasopressin release the KOR agonists prevent the water reabsorption and produce dilute urine. In addition to diuretic agents, water also acts as a diuretic that will increase urine output; more specifically, water loading in animals or humans will result in a volume-dependent increase in urine output by inhibiting vasopressin secretion (Slizgi and Ludens, 1982).

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

2.2 Aim and Rationale: The goals of this research were to:

1) Develop a simple, cost-effective assay for measuring diuresis in isolated, awake mice;

2) Using these methods, establish the diuretic effects of cannabinoids in mice by qualitatively and quantitatively comparing the effects of cannabinoid agonists to those of standard diuretics according to the volume and ion content of collected urine;

3) Determine the receptor mechanisms involved in producing cannabinoid diuresis by using appropriate pharmacological tools, including identifying potential roles for central or peripheral mechanisms involved in cannabinoid-mediated diuresis.

The findings of these studies may reveal new insights into the role of cannabinoid receptors in maintaining water homeostasis and further help better understand the full spectrum of the physiological effects of cannabinoids.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

2.3 Material and methods:

2.3.1 Animals: Male CD-1 mice, weighing 20-25 g at the start of the study (Charles

River Laboratories, Wilmington MA), were housed 4/cage in a climate controlled vivarium with food and water available ad libitum. Mice were acclimatized to the animal facility for 7 days, and to study procedures twice, prior to testing. Mice were re-used with a minimum of 7 days interval between drug testing. All experiments were performed during the light portion of the light/dark cycle. All studies were approved by the Northeastern University Animal Care and Use

Committee, in accordance with guidelines established by the National Research Council.

2.3.2 Diuresis: Urine output was measured over 6 hours during which mice did not have access to food and water. Mice were placed on an elevated grid floor and isolated under a plastic cup (10 cm diameter; 5 cm height); weigh boats were placed underneath each mouse to collect the voided urine. Voided urine was measured by determining the change in weight of the boats every 2 hours to minimize volume loss due to evaporation. Mice were used for 4-8 weeks; doses of drugs and vehicle were always randomized to minimize time dependent bias. For single drug studies the injection volumes were 1 ml/100g, when drugs were studied in combination, doses were delivered in half volumes, e.g., for antagonism studies 30 min pretreatment with 0.5 ml/100g vehicle or antagonist was followed by 0.5 ml/100g injection of the agonist.

2.3.3 Measurement of urine pH, Na+, K+ and Cl- : Urine samples were collected from individual mice and stored at – 4oC until analysis. Urine pH and concentrations of Na+, K+ and

Cl-, were quantified using ion selective microelectrodes according to manufacturer’s protocol

(Lazar Research Laboratory, Inc, Los Angeles, CA, USA).

2.3.4 Drugs: Δ9-THC and rimonabant were obtained from the National Institute on Drug

Abuse [(NIDA), Rockville, MD]; WIN-55-212 [((R)-(+)-[2,3-Dihydro-5-methyl-3-(4-

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate],

U50,488 [trans-(+/-)3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]-cyclohexyl)-benzeneacetamide methane sulfate] and furosemide were purchased from Sigma-Aldrich (St. Louis, MO). AM4054

[9β-(hydroxymethyl)-3-(1-adamantyl)-hexahydrocannabinol], AM6545 [5-(4-(4-cyanobut-1- ynyl)phenyl-1-(2,4-dichlorophenyl)-4-methyl-N-(1,1-ioxothiomorpholino)-1H-pyrazole-3- carboxamide] and AM630 [6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4- methoxyphenyl) methanone] were synthesized at the Center for Drug Discovery, Northeastern

University. U50,488 was dissolved in saline; furosemide was dissolved in 1% 1N NaOH and sterile water; all other compounds were prepared in 5% ethanol, 5% emulphor-620 (Rhodia,

Cranbury, NJ) and 90% saline, and further diluted with saline. Except where noted, injections were delivered s.c. in volumes of 1ml/100g body weight; drug doses are expressed in terms of the weight of free base.

2.5.5 Statistical analysis: To determine ED50 values for diuresis, 50% of the maximum effect was defined using the formula: [((maximum urine output with the drug – urine output with vehicle)/2) + urine output with vehicle]. ED50 values were calculated using linear regression when more than two data points were available, and otherwise were calculated by interpolation.

All ED50 values were calculated using data plotted on a log scale, to first obtain log ED50 and then converting to antilog. Data were analyzed using one way ANOVA followed by Dunnett’s or Bonferroni’s multiple comparison tests. Significance for all tests was set at p ≤ 0.05.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

2.4 Results:

2.4.1 Validating diuresis: Initial studies validated the procedure used for measuring and quantifying diuresis in mice. Mice that received sham injections voided an average of 4g/kg urine. After injection of 10 or 30 ml/kg saline (fluid-load), the amount of voided urine by weight was approximately 10 g/kg and 30 g/kg, respectively (Figure 4), indicating that diuresis can be measured and quantified accurately in mice using s.c. injections. 10 ml/kg was the standard injection volume for all subsequent experiments; as this volume of injection produced slightly increased urine output, the stability of this response over time was determined by injecting a group of mice with saline or vehicle for 14 weeks. There was no effect of repeated testing on urine output as seen in Figure 4, though it was noted that urine output was highest during week 1, possibly due to the stress of the novel test apparatus. This was taken into consideration for all studies measuring urine output by acclimatizing each group of mice to the test procedure and apparatus at least once before saline or drug testing.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

40 40

30 30

20 20

Urine (g/kg) Urine 10 10

0 0 0 10 30 0 2 4 6 8 10 14 Saline injected (ml/kg) Weeks

Figure 4: Urine output, after s.c., saline injections at different volumes, n = 8, (left) and after repeated exposure to 10ml/kg injection volume for 14 weeks, n = 7, (right).

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

The loop diuretic furosemide, 1.0 - 60.0 mg/kg, dose-dependently increased the amount of voided urine, maximum urine output of 50.8 ± 2.1 g/kg was obtained at the dose of 30.0 mg/kg, which plateaued on further increasing the dose to 60.0 mg/kg (Figure 5). These results suggest that ~50 g/kg is the average maximum urine a mouse can void over a 6hr period without access to water, as furosemide is not a ceiling diuretic. U-50,488, a selective κ-opioid receptor

(KOR) agonist, at doses of 1.0 - 60.0 mg/kg produced a dose-dependent increase in diuresis with maximum mean urine output of 33.7 ± 4.4 g/kg with a dose of 30.0 mg/kg; increasing the dose to

60.0 mg/kg did not further increase urine output. Figure 5 shows the full dose response curves for furosemide and U-50,488 in producing increases in diuresis in mice over a 6 hr test session.

Both furosemide and U-50,488, at doses as low as 3.0 mg/kg, produced significant increases in diuresis compared to diuresis after respective vehicle treatments. The ED50 (95%Cl) for furosemide and U-50,488 were 4.8 (3.6, 6.3) mg/kg and 3.8 (2.7, 4.9) mg/kg respectively, similar to values reported in literature (Sim and Hopcroft, 1976; Vonvoigtlander et al., 1983). The maximum urine output obtained with furosemide was statistically higher than the total maximum urine output obtained after U50,488 treatment. U50,488 increases diuresis by inhibiting vasopressin secretion, whereas furosemide acts locally in the ascending Loop of Henle to produce its diuretic effects and this difference in mechanisms may explain the difference in the maximum urine output produced by the two compounds. The amount of voided urine following vehicle administration was not significantly different between any of the groups tested (F4,34=

1.27; p > 0.05).

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

*** 50 *** *** 40 *** *** 30 *** *** * 20

Urine (g/kg) Urine 10 Furosemide U-50,488 0

V 1.0 3.0 10.0 30.0 60.0 Dose (mg/kg)

Figure 5: Dose-response curves for furosemide (n = 8) and U-50,488 (n =8) on diuresis in mice, measured over 6hr after drug administration. *** = p < 0.005 is statistically significant from vehicle treated controls (V).

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

2.4.2 Cannabinoid mediated diuresis: Δ9THC and WIN-55212-2 are well characterized cannabinoid receptor agonists that have been extensively studied; they are included here as standard compounds. AM4054 and AM7418 are novel cannabinoid agonists that have advantageous pharmacokinetic properties, including greater potency and, perhaps, shorter duration of action or half-lives. All four cannabinoid agonists were tested for their ability to produce increases in diuresis in mice and all four agonists dose-dependently increased voided urine with a maximum urine output ranging from 29 - 38 g/kg as shown in Figure 6. Peak effects of AM4054, AM7418, WIN55212-2 and THC occurred at doses of 0.1, 0.3, 3.0 and 10.0 mg/kg respectively, with maximum mean urine outputs of 38.0 ± 6.2, 29.7 ± 2.8, 29.3 ± 3.6 and 31.6 ±

3.4 g/kg respectively. An unexpected observation was the biphasic nature of the dose response curves for cannabinoid-induced diuresis. As seen in Figure 6, all cannabinoid agonists dose dependently increased urine output at lower doses, producing an ascending portion of the dose response curve. Further increasing the dose above that which produced maximum increases in diuresis, resulted in dose-dependent decreases in urine output such that at the highest doses tested, urine outputs were similar to those obtained with vehicle treatment; this constituted the descending limbs of the cannabinoid dose response curves. The biphasic nature of the cannabinoid dose-effect functions was dissimilar to the effects of furosemide and U-50,488, both of which produced increases in diuresis and doses above the peak diuretic doses did not further increase or decrease urine outputs, producing a plateau of their dose response curves.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

A M 7 4 1 8 4 0 A M 4 0 5 4 ***

*** )

g 3 0

g (

2 0

i r

U 1 0

V 0 .0 1 0 .0 3 0 .1 0 .3 1 .0 D o s e (m g /k g )

W IN 5 5 ,2 1 2 -2 9 4 0  T H C *** *** ) **

g 3 0

g (

2 0

i r

U 1 0

V 0 .3 1 .0 3 .0 1 0 3 0 1 0 0 D o s e (m g /k g )

Figure 6: Biphasic diuresis dose response curves for cannabinoid agonists AM4054 (n=7),

AM7418 (n=8), ∆9-THC (n=8) and WIN-55212-2 (n=8). All drugs were injected at a volume of

10 ml/kg and diuresis was measured over a 6 hr test session. V represents values after respective vehicle treatment. ** = p < 0.01 , *** = p < 0.005 is statistically significant from vehicle treated controls (V).

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

Biphasic dose response curves, while not unique, also are not commonly seen in physiological responses to drugs, therefore, a small series of studies further examined this phenomenon. Previous studies on the diuretic responses to cannabinoids in female rats did not produce biphasic dose response curves, hence, we questioned whether the biphasic dose response curve was either a gender or a species specific effect. To address this, female CD1 mice, injected with 0.01-1.0mg/kg AM4054 yielded similar results to our observations in male mice, that is AM4054 produced a biphasic dose response curve albeit with 0.5 log unit lower potency in females as seen in Figure 7. Maximum urine output in female mice was 36.6 ± 4.5 g/kg at a dose of 0.3mg/kg as compared to 38 ± 6.2 g/kg at dose of 0.1mg/kg in male mice. These results suggest that the biphasic dose effect function of cannabinoid agonists is seen in mice of either gender.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

4 0

) g

k 3 0

(

e 2 0

i r

U 1 0 F e m a le 0 M a le

V 0 .0 1 0 .0 3 0 .1 0 .3 1 .0 A M 4 0 5 4 (m g /k g )

Figure 7: Diuresis dose response curve for AM4054 in male (n=7) and female mice (n=8).

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

Based on the visual observations of gross behaviors over the course of the 6 hr test sessions, it seemed plausible that the decrease in urine output at higher doses may be due to the emergence of sedative effects of cannabinoids. To this end we tested whether high-dose sedative effects of AM4054 interfered with the voiding induced by a noncannabinoid. 3.0 mg/kg furosemide injected after 1.0 mg/kg AM4054 produced urine output of 15.7 ± 3.8 g/kg over a 6 hr test period, which was significantly different (p =0.0085) than produced 6 hr after 3.0 mg/kg furosemide alone (28.4 ± 1.7 g/kg), but not significantly different (p=0.118) as compared to 1.0 mg/kg AM4054 alone (7.4 ± 3.1 g/kg). This suggests that CB1 receptor mediated sedative effects at high doses interferes with voiding.

2.4.3 Receptor mechanisms of cannabinoid mediated diuresis: In order to evaluate the contributions of CB1 and CB2 receptors in mediating cannabinoid diuresis, the effects of THC and AM4054 were re-determined in the presence of one of the three cannabinoid antagonists, the

CB1 selective antagonist, rimonabant, the CB2 selective antagonist, AM630 or the peripherally restricted CB1-selective antagonist, AM6545. All three antagonists are competitive antagonists at the orthostatic site of the respective cannabinoid receptors. The CB1 selective antagonist rimonabant alone, at 1.0, 3.0 and 10.0 mg/kg produced urine output that was not significantly different from urine output obtained after vehicle treatment. However, 30 min pretreatment with rimonabant at these same doses elicited dose-dependent rightward shifts of both the ascending and descending limbs of AM4054 dose response curves, as seen in Figure 9 and as evident from the ED50 values reported in tables 2 and 3. Rimonabant at similar doses also shifted the ascending limb of the THC dose response curve to the right (Figure 9, Table 2), whereas its effects on the descending limb of the THC dose response curve could not be fully evaluated because of solubility issues with higher concentrations of THC. Nonetheless, it is worth noting

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda that even the lowest dose of rimonabant completely antagonized the decreases in diuresis produced by 100 mg/kg THC. The potency ratios for rimonabant were similar for antagonizing the ascending limbs of both THC and AM4054 suggesting involvement of the same CB1 receptors in mediating increased diuresis by AM4054 and THC.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

4 0

) 3 0

g (

2 0

i r

U 1 0

0 .0 3 0 .1 0 .3 1 .0 3 .0 A M 4 0 5 4 (m g /k g )

+ R im o n a b a n t 0 .0 m g /k g 1 0 .0 m g /k g 4 0 1 .0 m g /k g

3 .0 m g /k g

) 3 0

g (

2 0

i r

U 1 0

1 .0 3 .0 1 0 .0 3 0 .0 1 0 0 .0 T H C (m g /k g )

Figure 9: Dose response curves for THC and AM4054 on diuresis after 30 min pretreatment with rimonabant or respective vehicle (n = 7-8).

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

Table 2: ED50 values (with 95% CI) and potency ratios calculated from the ascending limb of dose response curves.

a b ED50 (mg/kg) Potency Ratio

AM4054 alone 0.05 (0.03, 0.07)

+ 1.0 mg/kg Rimonabant 0.20 (0.1, 0.9) 4

+ 3.0 mg/kg Rimonabant 0.67 (0.5, 1.3) 14

+ 10.0 mg/kg Rimonabant 0.96 (0.4, 4.0) 20

+ 3.0 mg/kg AM6545 0.04 (0.00, 0.07) 0

THC alone 2.5 (0.8, 5.1)

+ 1.0 mg/kg Rimonabant 12.6 (4.1, 27.5) 5

+ 3.0 mg/kg Rimonabant 17.4 (ND)c 7

+ 10.0 mg/kg Rimonabant 56.9 (40.5, 80.9) 23

a ED50 values were calculated from grouped data b Potency ratios were calculated by dividing the ED50 value of the agonist alone by the ED50 value obtained after antagonist pretreatment c 95% CI were not determined because ED50 value was calculated by interpolation of two points

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

Table 3: ED50 values (with 95% CI) and potency ratios calculated from the descending limb of the AM4054 dose response curves.

a b ED50 ( mg/kg) Potency Ratio

AM4054 alone 0.3 (0.2, 0.4)

+ 1.0 mg/kg Rimonabant 0.5 (ND) 2

+ 3.0 mg/kg Rimonabant 1.3 (0.7, 1.7) 4

+ 10.0 mg/kg Rimonabant ≥ 3.0 ≥ 10

+ 3.0 mg/kg AM6545 0.9 (0.6, 1.1) 3

a ED50 values were calculated from grouped data b Potency ratios were calculated by dividing the ED50 value of the agonist alone by the ED50 value obtained after antagonist pretreatment

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

The doses of rimonabant that shifted both the ascending and descending limbs of THC and AM4054 dose response curves to the right did not antagonize the increased diuresis produced by either 10.0 or 30.0 mg/kg furosemide or U50,488 (Figure 10). These results demonstrate that rimonabant selectively blocks changes in urine output produced by the cannabinoid agonists, without having any effects on diuresis produced by the loop diuretic furosemide or the KOR agonist U50,488. This provides clear evidence that rimonabant does not non specifically decrease drug induced increases in diuresis and further strengthens the supposition that effects of cannabinoids that are antagonized by rimonabant are CB1 receptor mediated.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

5 0

) g

k 4 0

g (

3 0 F u ro s e m id e e

n + R im o n a b a n t

i 2 0

r U -5 0 ,4 8 8 U 1 0 + R im o n a b a n t R im o n a b a n t 0

1 .0 3 .0 1 0 .0 3 0 .0 1 0 0 .0 D o s e (m g /k g )

Figure 10: Dose response curves for furosemide, U50,488 and rimonabant shown in solid symbols with solid lines. Furosemide and U50,488 in the presence of 10.0 mg/kg rimonabant shown ion open symbols and dotted lines (n = 6-8).

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

To determine whether the diuresis produced by cannabinoids is mediated by central or peripheral CB1 receptors, antagonism studies in the presence of AM6545 were performed. Mice treated with the peripherally selective CB1 antagonist AM6545 (3.0 mg/kg) alone had a mean urine output of 16.6 ± 4.2 g/kg, which was not significantly different than vehicle treatment. 30 min pretreatment with AM6545, at a dose of 3.0 mg/kg, had no effect on the ED50 for the ascending limb of AM4054 dose response curve, however, it produced a 3-fold rightward shift of the descending limb of the AM4054 dose response curve, according to the change in the ED50 values reported in table 2 and as seen in Figure 11. Increasing the dose of AM6545 to 10.0 mg/kg, did not further shift the descending limb but shifted the ascending limb of AM4054 to the right (Figure 11). These results, in accordance with other reported effects of AM6545 (10.0 mg/kg) on hypolocomotion and scratching in mice (Sherica Tai Thesis, 2012) suggests that high doses of AM6545 may saturate the glycoprotein efflux transporters in the BBB responsible for removing AM6545 from the CNS and as a result may attenuate central effects of cannabinoid agonists.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

40 + AM6545 0.0 mg/kg 30 3.0 mg/kg 10.0 mg/kg 20

Urine (g/kg) Urine 10

V 0.03 0.1 0.3 1.0 3.0 AM4054 (mg/kg)

Figure 11: AM4054 dose response curve 30 min after 0, 3 or 10 mg/kg AM6545, a peripherally selective CB1 antagonist (n = 8).

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

As with most currently available cannabinoid agonists, neither AM4054 nor THC is selective for CB1 or CB2 receptors. Therefore, to evaluate whether CB2 receptor mechanisms mediate any effects on changes in urine output by cannabinoid agonists, a CB2 selective antagonist, AM630, was used. Mice treated with 10.0 mg/kg AM630 yielded urine output of

12.4 ± 2.6 g/kg, a value which was not significantly different from vehicle treatment.

Pretreatment with AM630, 0.1 - 10.0 mg/kg, did not have any effect on the increase in urine output produced by the peak dose of 0.1 mg/kg AM4054 (Figure 12). Pretreatment with 3.0 or

10.0 mg/kg AM630 did not antagonize the increase in diuresis produced by 10.0 mg/kg THC, neither did 10.0 mg/kg AM630 antagonize the decrease in diuresis produced by 100.0 mg/kg

THC (Figure 12). Together, these data indicate that CB2 receptors are not primarily involved in the diuretic effects of cannabinoid agonists in mice.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

40 0.1mg/kg AM4054 10.0 mg/kg THC 30 100.0 mg/kg THC

Urine (g/kg) Urine 10

0.0 0.1 1.0 0.0 3.0 0.0 10.0 10.0 10.0 AM630 (mg/kg)

Figure 12: Effects of combinations of AM630 and AM4054 or THC. AM630 was injected as a

30 min pretreatment to the doses of AM4054 and THC that produced maximum increase in diuresis (see Figure 2), or a dose of THC that produced maximum decrease in diuresis (n = 6-8).

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

2.4.4 Urine analysis: To evaluate whether cannabinoid-induced diuresis is accompanied by proportional loss of electrolytes or is a free-water loss, the profile of electrolyte excretion after THC administration was compared with that after administration of furosemide or U50,488.

Urine samples were collected after saline, or a range of doses of furosemide, U50,488 and THC.

The total amount of excreted Na+ and Cl- increased dose dependently following furosemide as shown in table 4; these results concur with reports in the literature regarding furosemide, and indicate that the concentration of urine after furosemide administration is similar to that after saline administration (i.e., the increased amount of excreted Na+ and Cl- results from the increased volume of urine). Urine obtained from THC treated mice contained total amounts of

Na+ and Cl- similar to that in urine samples from saline treated mice, however, as THC increased urine volumes significantly relative to saline treatment, THC produced dilute urine which is indicative of a free water diuresis. The type of diuresis produced by THC is similar to that produced with KOR agonist U-50,488 reported in the literature and shown in Table 4 (Leander et al., 1985). Total K+ levels were unaltered after any of the three drugs. Table 4 also shows the urine pH following drug or vehicle treatment; pH for urine samples were weakly basic and there was no effect of drug or dose on the urine pH values.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

Table 4: Total amount of ions (in μEq; mean ± sem) excreted in urine over 6hrs

Na µEq K µEq Cl µEq pH

Saline 26.5 ± 4.7 16.6 ± 3.2 130.3 ± 12.8 7.5 ± 0.1

Furosemide

1.0 mg/kg 60.2 ± 11.9 5.4 ± 0.9 * 189.2 ± 31.0 7.8 ± 1.4

3.0 mg/kg 99.1 ± 12.8 *** 6.5 ± 1.2 249.9 ± 23.5 8.1 ± 0.1

10.0 mg/kg 149.3 ± 12.9 *** 12.6 ± 2.7 408.9 ± 32.1 *** 8.2 ± 0.3

30.0 mg/kg 232.2 ± 17.7 *** 30.4 ± 4.5 ** 586.3 ± 64.1 *** 7.0 ± 0.1

THC

1.0 mg/kg 17.2 ± 6.8 8.4 ± 3.8 95.9 ± 30.2 7.8 ± 0.3

3.0 mg/kg 41.8 ± 15.2 14.8 ± 4.4 175.1 ± 56.9 7.6 ± 0.2

10.0 mg/kg 73.2 ± 16.7 * 12.6 ± 3.0 202.1 ± 36.6 7.6 ± 1.1

30.0 mg/kg 71.4 ± 4.6 * 46.4 ± 34.6 206.6 ± 6.9 7.2 ± 1.0

U-50,488

1.0 mg/kg 43.8 ± 22.7 6.3 ± 2.4 125.5 ± 46.5 8.0 ± 0.2

3.0 mg/kg 27.9 ± 10.0 8.6 ± 2.4 117.0 ± 33.0 7.5 ± 0.2

10.0 mg/kg 27.2 ± 7.2 18.8 ± 4.3 127.3 ± 27.7 7.5 ± 0.2

30.0 mg/kg 20.1 ± 3.5 26.4 ± 7.8 103.8 ± 20.0 7.7 ± 0.3

* p = < 0.05; *** = p < 0.001

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

2.5 Discussion:

2.5.1 Validation of diuresis: These studies demonstrate that diuresis can be quantitatively and qualitatively measured and characterized in mice. The method used is distinct from the metabolic cages commonly used to study diuresis, as it allows the accurate measurement of diuresis in individual mice. In saline-loaded mice, injected volumes of 10-30 ml/kg were completely retrieved in a 6 hr study period confirming the validity of the method used. It has been reported that water loading with more than 10 ml/kg inhibits vasopressin levels significantly and as a result increases urine output (Slizgi and Ludens, 1982). With this in mind, all mice were injected with a constant volume of 10 ml/kg across various groups and treatments, including when multiple injections were given. The amounts of voided urine after vehicle or saline injections were not significantly different between any of the groups tested, and were stable in the group of mice tested repeatedly with saline for 14 weeks. The maximum urine output with furosemide was equivalent at 30.0 mg/kg and 60.0 mg/kg, suggesting that this is the average maximum urine that can be voided by a mouse without access to water over a 6 hr study period, as furosemide is a high ceiling diuretic. This was particularly important as a positive control, providing a maximum value (~50 g/kg, or 1.2-1.5 ml total volume) that could be expected in subsequent studies.

It is well established that furosemide exerts its diuretic actions by inhibiting the Na+-K+-

2Cl- symporter in the thick ascending Loop of Henle of the kidney (Goodman et al., 2006). To ascertain whether there is a difference, in mice, between drugs that produce diuresis via a direct or indirect action on the kidney, KOR agonist mediated diuresis also was measured, as it has been suggested that KOR agonists produce diuresis by inhibiting the secretion of vasopressin

(Rossi and Brooks, 1996; Craft et al., 2000). Vasopressin is known to act on the collecting ducts

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda of the kidney, hence, inhibiting vasopressin secretion results in increased production of dilute urine (Goodman et al., 2006). The KOR agonist U-50,488 dose-dependently produced increases in diuresis with a maximum urine output of ~ 34 g/kg, approximately 70% of that obtained after furosemide, suggesting differences in total voided urine volumes with the two compounds. If the difference in maximum urine output between furosemide and U-50,488 reflects the differences in site of action, then comparison of maximum urine output with cannabinoids with that of furosemide and U-50,488 may point to the possible site of action of cannabinoids in producing diuresis. As a final point, the ED50 values for furosemide and U-50,488 in the diuresis measurements were similar to values reported in the literature (Sim and Hopcroft, 1976;

Vonvoigtlander et al., 1983; Craft et al., 2000), a result that further validates the method of urine collection used herein, as compared to commonly used metabolism cages, which may yield confounded results in mouse studies due to the small volume voided and high surface area for evaporation. The methods used here are reliable, cost effective, less labor intensive and yield urine relatively free of dander and food contaminants, providing an efficient way to measure urine output in isolated mice.

2.5.2 Cannabinoid mediated diuresis: All cannabinoids tested produced dose- dependent increases in urine output, with an order of potency of AM4054 > AM7418 >

WIN55212-2 > ∆9THC. The maximum urine output with cannabinoid agonists was quantitatively similar to that produced by U50,488, suggesting cannabinoid-mediated diuresis may share common mechanisms with KOR mediated diuresis, thus, cannabinoids may directly or indirectly interfere with vasopressin secretion. In support of this hypothesis, other studies in rats have shown inhibitory influence of endocannabinoids and THC on the release of hormones from the anterior and posterior pituitary including vasopressin and oxytocin, suggesting that

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda cannabinoids decreases vasopressin secretion by effects on endocrine cells (Tyrey and Murphy,

1984; Tasker, 2004). Diuretics that act by decreasing vasopressin secretion or otherwise inhibiting the effects of vasopressin result in dilute urine, by contrast, loop diuretics such as furosemide do not change the electrolyte concentration of urine. The analysis of the electrolyte concentrations in urine collected after U50,488 or furosemide administration confirm that in mice furosemide does not alter the electrolyte composition of urine, whereas U50-488 yields dilute urine. Urine recovered after THC treatment was dilute urine, similar to that produced by

U50,488, further suggesting that THC and other cannabinoids may increase diuresis by inhibiting the effects or release of vasopressin.

The biphasic dose response curve for diuresis, with smaller effects observed at higher doses, was obtained with all cannabinoid agonists and in both male and female mice. This result was somewhat surprising, as an earlier study in rats did not find biphasic effects after same cannabinoid agonists were administered at identical dose ranges (Paronis et al., 2013) and suggests there might be multiple mechanisms involved in cannabinoid modulation of urine output in mice. Biphasic dose response curve for diuresis were not observed for either furosemide or U50,488 although biphasic effects have been previously reported for other compounds. In one study, the opioid ligand, BW942C produced biphasic dose response curve in diuresis measurements in rats, the biphasic dose response curve was attributed to independent mechanisms, as its partial KOR agonist actions lead to an increase in urine output, whereas the descending limb of the curve was attributed to antidiuretic effects mediated through MOR

(Vaupel et al., 1990). Similarly, it has been shown that dopamine has a biphasic effect for diuresis as a result of interactions with separate receptors; lower doses of dopamine will increase diuresis by acting on dopamine receptors whereas higher doses decrease diuresis by acting on

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda alpha adrenergic receptors (Olsen et al., 1997). In efforts to identify the mechanisms that underlie the complex nature of the biphasic dose response curves obtained with cannabinoids, the receptor mechanisms of cannabinoid diuresis were characterized using selective CB1 and CB2 antagonists. Initial studies determined that rimonabant, AM6545, and AM630 produced urine output similar to that produced after saline treatment, that is, the antagonists neither increased nor decreased the effects of the 10 ml/kg volume load that accompanied each drug injection.

Further, the highest dose of rimonabant did not antagonize the increase in diuresis produced by the noncannabinoid diuretics, furosemide and U50,488; together these results indicate rimonabant does not produce a physiological antagonism of increases in diuresis. Pretreatment with 1.0 – 10.0 mg/kg rimonabant, a CB1 selective antagonist/inverse agonist, dose-dependently shifted both the ascending and descending limb of the AM4054 dose response curve to the right suggesting a role for the CB1 receptors in cannabinoid mediated increases and decreases in diuresis. There were no significant differences in the slopes of the curves, suggesting parallel rightward shift as would be expected in the presence of a competitive antagonist. There seemed to be a slight decrease in the maximum diuretic effects of AM4054 in the presence of rimonabant, however the suppression of the magnitude of the maximum diuretic effect was not related to the dose of rimonabant and in no case was it statistically significant. Rimonabant also dose-dependently shifted the ascending limb of THC dose response curve to the right however, any rightward shift of the descending limb of THC dose response curve could only be implied, but not quantified as pretreatment with all doses of rimonabant produced maximum urine output at the highest dose of THC tested, 100 mg/kg. A further observation from these studies was that even the lowest dose of rimonabant, 1 mg/kg, completely blocked the decreases in urine output usually observed after 100 mg/kg THC while having lesser effects on the descending limb of the

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

AM4054 dose response curve. The difference in sensitivity of rimonabant towards the ascending and descending limbs of the two cannabinoid agonists might suggest involvement of different cannabinoid receptor subtypes in producing the two limbs of the diuresis dose response curves.

Like most cannabinoid agonists, THC and AM4054 have similar affinity for CB1 and CB2 receptors, therefore studies determined whether the CB2 receptors play a role in either the ascending or descending limbs of the cannabinoid dose response curves. AM630 is a widely used CB2 selective antagonist, and a dose-range was selected that included doses 10-fold higher than those shown to successfully antagonize a CB2 effect in mice (Maione et al., 2008). AM630 at the highest dose tested did not antagonize the increases in diuresis produced by AM4054 or

THC, neither did it antagonize the decreases in diuresis produced by high dose of THC. Thus these data do not support an involvement of the CB2 receptors in cannabinoid mediated increases or decreases in diuresis in mice. Another possibility is that separate central and peripheral mechanisms are responsible for the ascending or descending limbs of the cannabinoid dose response curves. To address this possibility, a set of studies used AM6545, a CB1 selective antagonist with limited CNS permeability (Cluny et al., 2010; Randall et al., 2010; Tam et al.,

2010). A dose of 3 mg/kg AM6545 did not antagonize the ascending limb of AM4054 dose response curve yet did shift the descending limb of the AM4054 dose response curve to the right.

These results may indicate some involvement of peripheral CB1 receptors, presumably those found in the urinary system, in modulating the diuretic responses to higher doses of cannabinoids, whereas the low dose increases in diuresis may be produced by activation of the central CB1 receptors.

Another possible explanation for the descending limb of the diuresis dose-response curve could be that sedation that results from high doses of cannabinoids may interfere with voiding.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

For WIN55212-2 and THC, the doses on the descending limb correspond to the doses at which decreases in locomotor activity and increases in catalepsy-like behavior have been reported in literature (Fan et al., 1994; Wiley et al., 2007). To address the hypothesis that decreased movement may have interfered with the expression of a diuretic effect of high doses of cannabinoid agonists, a high dose of AM4054 was given as a pretreatment to furosemide. Under these conditions, the maximum urine output after furosemide treatment was similar to that after saline treatment, suggesting interference of high dose AM4054 with voiding or micturition by virtue of its sedative effects or by producing relaxation of the bladder.

It is possible that the descending limb for cannabinoid diuresis is a function, both of peripheral CB1 receptor involvement as well as sedative-like effects. Studies using instrumented rodents have shown that cannabinoids produce relaxation of the bladder, increasing the micturition threshold at high doses and decreasing urinary frequency induced by nociceptive stimuli to the bladder by CB1 receptor mechanisms (Dmitrieva and Berkley, 2002; Hiragata et al., 2007). Although speculative, perhaps high doses of cannabinoids act peripherally to produce relaxation of the bladder and increase the micturition threshold, resulting in greater volume of urine stored in the bladder, and simultaneously act centrally to produce sedative-like effects that further prevent the voiding; as a result giving rise to the descending limb of cannabinoid dose response curve. A clinical study using placebo, THC and cannabis extract showed that both

THC and cannabis extract decreased urge incontinence episodes in patients suffering from multiple sclerosis further supporting the hypothesis (Freeman et al., 2006). If the increases and decreases in urine output produced by cannabinoid agonists are truly mediated by central and peripheral mechanisms respectively, as suggested by our studies, this may provide basis for

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda developing better cannabinoid compounds for treating overactive bladder and urinary incontinence in MS patients (Brady et al., 2004; Freeman et al., 2006; Capasso et al., 2011).

From all the above experimental findings we can conclude that structurally diverse cannabinoid agonists produce biphasic dose response curves for increasing diuresis in mice by acting on the CB1 receptors. Low dose increases and high dose decreases in urine output is most likely mediated by CB1 receptors located in the CNS and periphery, respectively. Identifying in further depth the mechanisms underlying opposing effects of cannabinoids on diuresis will help better understand the role of cannabinoids in the urinary system and as a result help provide better screening procedure for novel cannabinoids as well as developing better cannabinoid based medication for treating urinary tract conditions.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

Chapter 3: Cannabinoid mediated antinociception in mice

3.1 Introduction:

3.1.1 Cannabinoid antinociception: The discovery of CB1 and CB2 receptors and selective cannabinoid ligands has made the complete tetrad assay obsolete in terms of establishing novel compounds as cannabinoid-like in vivo. Nonetheless, individual tests from the tetrad continue to be useful in providing standard comparative measures of in vivo efficacy and potency for novel cannabinoid ligands or in characterizing novel cannabinoid effects (Lichtman and Martin, 1997; Paronis et al., 2012). Along these lines, antinociceptive effects were used in to compare the diuretic effects of cannabinoids with the measures of the tetrad assay in general and, in particular, a measure of cannabinoid effect that may have the most clinical relevance.

Antinociception has been commonly used in preclinical models to screen compounds that possess analgesic properties (Barrot, 2012). The tail-flick test is one of the oldest methods used to measure antinociceptive properties of compounds and has commonly been used to study pain relieving properties of drugs, including cannabinoid compounds, in rodents (D'Amour and

Smith, 1941; Compton et al., 1996; Welch et al., 1998). Radiant light or hot water has often been used as nociceptive stimuli for tail-flick measurements (Janssen et al., 1963; Raffa et al.,

1999). Hot water tail-withdrawal techniques have been commonly used in mice; it requires careful monitoring and control of water temperature, mouse handling and limited exposure of the mice to the test procedure to avoid effects of conditioning or learning on the tail-withdrawal response. The water temperature used as a noxious stimuli is set based on desired parameters for baseline latencies, cutoff latencies, and the design of the study, and normally ranges between 48-

55oC.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

One practical advantage of antinociception measurements is that they often can be obtained using cumulative dosing procedures. All cannabinoid agonists that produced diuresis were evaluated in an assay of warm water tail-withdrawal, and their efficacy and potency were compared across the two procedures. As similar ED50 values of cannabinoid agonists across all tetrad measurements are reported in the literature, identical order of potencies for multiple drugs of the same class across the two measures (antinociception and diuresis) would further suggest identical receptor involvement in producing cannabinoid mediated diuresis and antinociception

(Smith et al., 1994; Wiley and Martin, 2003). Antagonism studies of the antinociceptive effects of AM4054 and THC were also completed, in order to compare the potency ratios for antagonists across different measures, thus providing additional confirmation regarding the receptor sites involved in producing these two dissimilar effects. Furthermore, increases in tail-withdrawal latencies following cannabinoid administration has been associated with the actions of cannabinoids at the CB1 receptors in the spinal and supra-spinal sites, suggesting a primary CNS mechanism of antinociception (Martin et al., 1993; Martin et al., 1995; Welch et al., 1998). Most in vivo effects of cannabinoids, including but not limited to those of the tetrad, are thought to be produced by activation of CB1 receptors within the CNS. AM6545, a peripherally restricted antagonist that was used to differentiate the peripheral and central components of cannabinoid diuresis in chapter 2, was used in this study to determine whether peripheral CB1 receptors contribute to cannabinoid antinociception. The effects of AM6545 pretreatment on cannabinoid mediated antinociception were determined, using the same doses as in diuresis studies in order to allow direct comparisons across the two measures.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

3.2 Aim and rationale: Cannabinoid mediated antinociception is widely studied in laboratory animals and used as a robust assay for studying mechanisms of novel and existing cannabinoid ligands in mice. The rationale behind using antinociception was to provide a reliable pharmacological end point that could serve as a comparison for characterizing a novel cannabinoid effect (diuresis) with respect to:

1) Comparing rank order of potency of the different ligands across antinociception and diuresis

2) Determine and compare the potency ratios for CB1 antagonists across the two measure

This information will provide good validation for characterizing diuresis as a cannabinoid mediated effect in mice.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

3.3 Materials and Methods:

3.3.1 Animals: Male CD-1 mice, weighing 20-25 g at the start of the study (Charles

Committee, in accordance with guidelines established by the National Research Council.

3.3.2 Antinociception: Antinociceptive responses were determined using a warm water tail-withdrawal assay. A water bath maintained water temperature at 52.0 ± 0.5°C; temperature determined based on results of pilot studies. Each mouse was gently hand held and the distal 2-3 cm of its tail immersed in the water; latency to tail-withdrawal was measured using a stopwatch and a cut-off time of 8s was established to avoid tissue damage. Baseline latencies were determined twice with a 10 min interval; only mice with baseline latencies of 1-3s were used in drug studies. Complete dose response curves were generated in each mouse using cumulative dosing procedures similar to those described previously (Paronis and Woods, 1997). Briefly, 30 min (morphine, U50, 488, WIN 55,212-2 and pentobarbital) or 60 min (THC, AM4054 and

AM7418) after an injection, tail-withdrawal latencies were determined and mice were then injected with the next dose, such that the total cumulative dose was increased by 0.25 or 0.5 log units. This procedure was repeated until the tail-withdrawal latency reached the cut-off or no longer increased with subsequent increase in dose of the test drug. In studies utilizing pretreatment with antagonist or vehicle, the experimenter was blinded to the pretreatment conditions.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

3.3.3 Drugs: Δ9-THC and rimonabant were obtained from the National Institute on Drug

Abuse [(NIDA), Rockville, MD]; WIN-55-212 [((R)-(+)-[2,3-Dihydro-5-methyl-3-(4- morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate], morphine, sodium pentobarbital, and U50,488 [trans-(+/-)3,4-dichloro-N-methyl-N-(2-[1- pyrrolidinyl]-cyclohexyl)-benzeneacetamide methane sulfate] were purchased from Sigma-

Aldrich (St. Louis, MO). AM4054, AM7418, [9β-(hydroxymethyl)-3-(1-adamantyl)- hexahydrocannabinol] and AM6545 [5-(4-(4-cyanobut-1-ynyl)phenyl-1-(2,4-dichlorophenyl)-4- methyl-N-(1,1-ioxothiomorpholino)-1H-pyrazole-3-carboxamide] were synthesized at the Center for Drug Discovery, Northeastern University. Morphine, pentobarbital and U50,488 were dissolved in saline; all other compounds were prepared in 5% ethanol, 5% emulphor-620

(Rhodia, Cranbury, NJ) and 90% saline, and further diluted with saline. Injections were delivered s.c.in volumes of 1ml/100g body weight; drug doses are expressed in terms of the weight of free base.

3.3.4 Statistical analysis: Tail-withdrawal latencies are expressed as a percentage of maximum possible effect (%MPE), calculated using the formula: %MPE = [(test latency − baseline latency)/ (8 − baseline latency)] × 100. ED50 values were calculated using linear regression when more than two data points were available, and otherwise were calculated by interpolation. Data were analyzed using one way ANOVA followed by Dunnett’s or

Bonferroni’s multiple comparison tests. Significance for all tests was set at p ≤ 0.05.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

3.4 Results:

A warm water tail-withdrawal assay was used to quantify the antinociceptive effects of cannabinoids. First, to validate our procedures, a dose effect function for a standard analgesic compound was determined. Effects of the µ opioid receptor (MOR) agonist morphine, 0.3-30.0 mg/kg, were determined using cumulative dosing procedures [data are presented in Figure 16]; the ED50 value calculated from these data was 4.7 (2.8, 8.6) mg/kg. Repeated injection of saline or vehicle had no antincociceptive effects, with a maximum % MPE of 10.4 ± 4.6.

3.4.1 Effects of cannabinoid agonists on antinociception: Prior to full dose-effect determinations, time course studies were performed with two cannabinoid agonists to determine the time to reach peak effects of these compounds in producing antinociception. For AM4054, single injections were followed by determination of tail-withdrawal latency at 1, 2, 4 and 6 hr.

On average, peak effects were reached 1 to 2 hr after injection and >80% of the peak effect at any dose was always achieved at 1 hr (Figure 13). Time course studies with AM7418 at 30 min,

1, 3 and 6 hr showed that AM7418 had similar a onset of action as compared to AM4054, although the duration of action of AM7418 was a little shorter (Figure 13).

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

AM4054

100 0.1 mg/kg` 80 0.3 mg/kg 1.0 mg/kg 60

40 % MPE % 20

0 1 2 3 4 5 6 Time (hr)

AM7418

0.3 mg/kg 100 1.0 mg/kg 80

40 % MPE % 20

0 1 2 3 4 5 6 Time (hr)

Figure 13: Time course for AM4054 and AM7418 antinociception after single injections with the respective doses as listed in legends (n = 6-8).

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

As both the compounds had identical onsets of action, with peak effects between 1-3 hr, cumulative dosing procedures with these drugs used 1hr inter-injection intervals. A comparison of the cumulative dose-effect function for AM4054 and the dose-effect function obtained following single dose injections indicates that the two dosing procedures yielded equivalent results, shown in Figure 14.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

100

40 % MPE % 20 Single dosing 0 Cumulative dosing

0.1 0.3 1.0 AM4054 (mg/kg)

Figure 14: Comparison of antinociception dose response curve after single dose or cumulative dosing following AM4054 (n = 8).

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

A 30 min inter-injection interval was used for WIN55,212-2 whereas 1 hr was used as an interval for AM4054, AM7418, THC and to obtain cumulative dose response curves for antinociception. Like AM4054 and morphine, the cannabinoid agonists THC, WIN55212-2, and

AM7418 all produced dose-dependent increases in antinociception, and all were able to produce nearly 100% of the maximum possible effect (Figure 15).

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

100 AM7418 80 AM4054 THC 60 WIN-55212 Saline

40 %MPE

0 0.03 0.1 0.3 1.0 3.0 10.0 30.0 100.0 Dose (mg/kg)

Figure 15: Cumulative dose response curves for cannabinoid agonists in the mouse hot water tail-withdrawal assay (n = 7-8).

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

The ED50 values (with 95% CI) for the cannabinoid agonists AM4054, AM7418,

WIN55,212-2 and THC are shown in table 5; the rank order of potency of the drugs for increasing tail-withdrawal latency, determined from the ED50 values, was: AM4054 =AM7418 >

WIN 55,212-2 > THC, and this is similar to their rank order of potency for producing diuresis

(Ch 2).

Table 5: ED50 values (in mg/kg) for cannabinoid agonists, calculated using the linear portion of dose response curve.

Drugs AM4054 AM7418 WIN55,212-2 THC

ED50 (mg/kg) 0.3 0.3 2.7 9.3

(95% CI) (0.2, 0.4) (0.2, 0.4) (1.9, 3.7) (7, 12.3)

All cannabinoids at higher doses produce immobility and sedation in mice. To evaluate whether sedation in general impacts tail-withdrawal latency, leading to an overestimation of antinociceptive effects, the effects of the CNS depressant pentobarbital on tail-withdrawal latency were examined. Pentobarbital did not produce significant antinociceptive responses at doses up to 60.0 mg/kg, which represents an anesthetic dose in mice. Antinociceptive effects were also determined following injection of the KOR agonist, U50,488, which had diuretic effects as described in Chapter 2. U50,488, at doses 1.0-60.0 mg/kg, had some antinociceptive effects in the warm water tail-withdrawal assay; at the highest dose tested it produced approximately 60% of the maximum possible effect (Figure 16).

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

100 Morphine 80 Pentobarbital U 50,488 60

40 %MPE

0 0.1 0.3 1.0 3.0 10.0 30.0 100.0 Dose (mg/kg)

Figure16: Cumulative dose response curves for non-cannabinoid compounds in the mouse hot water tail-withdrawal assay (n = 7-8).

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

3.4.2 Effects of antagonist pretreatment: Full THC and AM4054 dose response curves were determined again in the presence of rimonabant. Mice were pretreated either with a single injection of rimonabant or vehicle and 30 min later, cumulative dose response curves with either

THC or AM4054 were completed. Rimonabant dose-dependently antagonized both AM4054 and THC, shifting the dose response curves to the right as shown in Figure 17. Effects of

AM4054 were attenuated to greater extent than the effects of THC after rimonabant pretreatment and this is most likely due to the difference in potency of the two agonists.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

100 + Rimonabant 0.0 mg/kg 75 1.0 mg/kg 3.0 mg/kg

50 10.0 mg/kg % MPE % 25

0.1 0.3 1.0 3.0 10.0 AM4054 (mg/kg)

100 + Rimonabant 0.0 mg/kg 75 1.0 mg/kg 3.0 mg/kg 50 10.0 mg/kg

% MPE % 30.0 mg/kg 25

10.0 30.0 100.0 300.0 THC (mg/kg)

Figure 17: Cumulative dose response curves for AM4054 (above) and THC (below) after 30 min pretreatment with vehicle or respective rimonabant doses as shown in legend (n = 7-8).

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

Potency ratios calculated for each dose of rimonabant are listed in table 6 and reveal that the rightward shifts of the AM4054 and THC dose response curves in the presence of rimonabant were similar. The slopes for AM4054 and THC dose response curves in the presence and absence of rimonabant were not statistically different, suggesting parallel rightward shifts indicative of competitive antagonism at CB1 receptors.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

Table 6

AM4054 THC

ED50 (mg/kg)a Potency Ratiob ED50 (mg/kg) Potency Ratio

Agonist Alone 0.28 (ND)c -- 21(3, 40) --

+ 1 mg/kg Rimonabant 0.44 (0.3, 0.6) 1.6 31.0 (ND) c 1.5

+ 3 mg/kg Rimonabant 0.59 (0.4, 0.8) 2.1 42.2 (18, 79) 2.0

+10 mg/kg Rimonabant 3.0 (ND) c 10.8 64.1 (29, 158) 3.0

+30 mg/kg Rimonabant 135 (83, 251) 6.4

+3 mg/kg AM6545 0.38 (ND)c 1.3

+ 10 mg/kg AM6545 0.38 (0.2, 0.6) 1.3 114.8 (ND) c 5.5

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

30-min pretreatment with the peripherally restricted CB1 antagonist, AM6545, at 3.0 mg/kg (the dose that antagonized descending limb of the AM4054 diuresis dose response curve) did not affect the AM4054 dose response curve (Figure 18) suggesting no role of peripheral CB1 receptors in AM4054 antinociception. Further, pretreatment with 10.0 mg/kg AM6545 (a dose that antagonized both the limbs of the AM4054 diuresis dose response curve), did not affect the

AM4054 dose response curve either. However, 10.0mg/kg AM6545 pretreatment shifted the

THC dose effect curve to the right. The ED50 values for THC in the presence of 10.0 mg/kg

AM6545 were similar to its ED50 values after 10.0-30.0 mg/kg rimonabant pretreatment (table

6). This differential antagonism of AM4054 and THC antinociception dose effect function by

AM6545 may be due to the fact that AM4054 primarily produces antinociceptive effects by actions at the central CB1 receptors, whereas, THC produces its antinociceptive effects by actions at both central and peripheral CB1 receptors.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

100 + AM6545 0.0 mg/kg 75 10.0 mg/kg

50 % MPE % 25

3.0 10.0 30.0 100.0 300.0 THC (mg/kg)

100

+ AM6545 %MPE 25 0.0 mg/kg 3.0 mg/kg 0 10.0mg/kg

0.1 0.3 1.0 3.0 10.0 AM4054 (mg/kg)

Figure 18: Dose response curves for THC (top) and AM4054 (bottom) following 30 min pretreatment with vehicle or the respective dose of AM6545 (n = 8).

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

3.5 Discussion:

Diuresis has not been previously identified as a cannabinoid receptor mediated effect in mice.

The antagonism of cannabinoid diuresis by rimonabant in chapter 2 established AM4054 and

THC-induced diuresis as a cannabinoid CB1 receptor-mediated effect, yet it is important to examine whether diuresis occurs at similar or different doses relative to other, previously well characterized, cannabinoid receptor effects. Antinociception was selected for use as a comparison to cannabinoid diuresis because of the four measures of the cannabinoid tetrad it has the greatest therapeutic potential. A warm water tail-withdrawal test was used and preliminary tests in mice using different water temperatures 48-55oC were performed to set optimum test parameters. As mice were used repeatedly in all test procedures, the temperature and cut-off latency were set in a way to minimize tissue damage while maintaining the integrity of the test.

In the warm water tail-withdrawal assay, morphine, THC and WIN55,212-2 produced linear dose response curves with ED50 values similar to those reported in literature (Paronis and

Holtzman, 1991; Zimmer et al., 1999; Wiley et al., 2007; Hull et al., 2010). Similarly, the KOR agonist U50,488 was less potent and efficacious as compared to other analgesic compounds in producing antinociception, the lower sensitivity of KOR agonists in mouse tail-flick test is in accordance with literature findings (Hayes et al., 1987). These results with standard compounds validate the procedure parameters used for measuring antinociception. Further validation was provided by using different and/or blinded experimenters to obtain dose response curves with

AM4054 and obtaining> 90% agreement among different experimenters; together, these observations attest to the robustness and reliability of the method in our hands. Finally, pentobarbital was used as a non-cannabinoid control to determine the extent to which sedation- induced immobility may impact antinociception measurements. Doses of pentobarbital higher

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda than those that produce sedation, and equal to those used for anesthesia (Vapaatalo and

Karppanen, 1969), did not produce significant antinociceptive effects, suggesting sedative effects alone of cannabinoids likely do not fully account for their antinociceptive effects.

THC, WIN 55212-2, AM4054 and AM7418 all dose-dependently increased antinociception. AM4054 and AM7418 were 10 to 30-fold more potent than THC and

WIN55212-2 in producing antinociception, evident from their ED50 values. The rank order of potency of the four cannabinoid agonists were identical for their diuretic and antinociceptive effects, further confirming a role of the same cannabinoid receptors in mediating both effects.

After comparing the rank order of potency of the cannabinoid agonists across diuresis and antinociception measurements, effects of antagonist pretreatment were compared between the two measures. Rimonabant a competitive CB1 selective antagonist, dose-dependently blocked

AM4054 and THC mediated antinociception, as expected of a CB1 receptor mediated effect and in accordance with published findings (Compton et al., 1996; Reche et al., 1996; Lichtman and

Martin, 1997). The potency ratio for rimonabant in antagonizing the AM4054 dose effect curve was slightly greater than that for antagonizing THC antinociceptive effects. Rimonabant produced parallel shifts of the agonist dose response curves suggesting competitive interactions at the CB1 receptors in producing both antinociception as well as diuresis.

In contrast to rimonabant, the peripherally restricted CB1 antagonist AM6545 did not antagonize the antinociceptive effects of AM4054, suggesting no involvement of peripheral cannabinoid receptors in antinociceptive effects. However, at a higher dose (10.0mg/kg),

AM6545 pretreatment antagonized THC antinociception without affecting AM4054 induced antinociception. Earlier diuresis studies (Ch 2) had shown that the dose of 3.0 mg/kg AM6545 antagonized the descending limb of cannabinoid diuresis without affecting the ascending limb,

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda whereas increasing the dose of AM6545 to 10.0 mg/kg antagonized both the ascending and descending limb of diuresis dose response curves. These data were interpreted as providing evidence that cannabinoid-mediated increases in urine output are produced exclusively by CB1 receptors in the CNS, while high-dose mediated decreases in diuresis are produced by central and peripheral CB1 receptors. In agreement with this interpretation, the antagonism by 10.0 mg/kg AM6545 of THC-mediated antinociception, similar to the antagonism observed with rimonabant, support CNS penetrability of this dose. The lack of antagonism of the antinociceptive effects of AM4054 by 10 mg/kg AM6545 is more difficult to explain, and may hint at differences in efficacy between AM4054 and THC.

Antinociception was used here primarily as a comparison measure for establishing diuresis as a cannabinoid CB1 receptor effect in mice. However, analgesic properties of cannabinoids were first mentioned in ancient literature and are being further evaluated in clinical trials for treating neuropathic and other forms of chronic pain. Secondary end points of some of these trials include alleviating symptoms of urinary incontinence and bladder over activity

(Anonymous, 2010). Findings of this research in whole animals suggest potential different roles of CB1 receptors in the CNS and periphery in mediating increases and decreases in urine output.

More detailed understanding of the mechanisms underlying cannabinoid effects on the urinary system will help better evaluate the effects observed in clinical trials and may aid in the development of better, more targeted, cannabinoid drugs for treating pain and or urinary tract disorders in the future.

To treat pain, current conventional approaches include the use of tricyclic antidepressants

(TCAs), anticonvulsants and opioid analgesics. All the above classes of compounds are associated with a risk of water retention, which hampers the quality of life of these patients and

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda requires the use of diuretics. Most of these drugs also produce nausea and vomiting warranting the use of concomitant medications. Cannabinoids are already approved for treating chemotherapy induced nausea and vomiting, they are also approved for treating neuropathic pain in many European countries. Carefully tailoring the effects of cannabinoid compounds on increasing and decreasing diuresis may provide an additional benefit for promoting cannabinoid compounds as therapeutics for treating pain and bladder disorders.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

Chapter 4: Cannabinoid mediated tolerance

4.1 Introduction:

4.1.1 Drug tolerance: Drug tolerance is defined as the adaptation to prolonged or continuous drug administration in a manner that requires higher doses of the same drug to produce the same magnitude of pharmacological effect. The rate at which tolerance develops depends on the drug and dosing regimen used. In most cases, drug tolerance is reversible and will disappear after cessation of drug taking, suggesting recovery from the adaptation. In terms of pharmacodynamics, drug tolerance is most commonly associated with changes in receptor numbers, receptor signaling, or both (Goodman et al., 2006); other mechanisms that underlie tolerance include increases in drug metabolism rates or decreases in receptor turnover.

4.1.2 Cannabinoids and tolerance: Marijuana is used frequently for recreational purposes, and hence its repeated use is very common. It has been reported that repeated exposure to cannabinoid agonists, either in vitro or in vivo, causes CB1 receptor down regulation

(decrease in receptor number) and receptor desensitization (decrease in downstream signaling)

(Breivogel et al., 1999; Sim-Selley et al., 2006). Behaviorally, tolerance has been reported to all the tetrad effects of cannabinoids, however, the rate and degree of tolerance development is different across the different effects (Wiley et al., 2007). The time required for tolerance development to the various effects in mice injected twice daily with 10 mg/kg THC varies between 0.5-6.5 days (Bass and Martin, 2000). Curiously, comparing 3, 6, and 13 day chronic cannabinoid treatment reveals no consistent differences in the magnitude of tolerance, although increasing the dose to twice daily injections of 80 mg/kg do further decrease the potency of THC for producing antinociception (Dalton et al., 2005). Another study comparing tolerance of 32 mg/kg THC administered once daily showed that complete tolerance to the hypothermic effects

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda of THC developed on day 2 and persisted until daily THC was administered (upto 56 days)

(Singh et al., 2011). Although THC tolerance may develop swiftly, recovery can be slow; the tolerance to the analgesic effect that developed after 6 day of repeated THC treatment took approximately 14 days to recover completely (Bass and Martin, 2000). Similarly, cross- tolerance to the effects of cannabinoids varies with the effect measured, the drug given repeatedly, and the drug acutely tested. In one study, mice injected twice daily with 10 mg/kg

THC showed tolerance to the effects of THC across all four tetrad measures but cross-tolerance to WIN 55,212-2 was observed only in hypolocomotion and antinociception and cross-tolerance to CP 55,940 was observed only in measures of hypothermia and antinociception. In contrast, twice daily treatment with 2.0 mg/kg CP-55,940 produced tolerance to CP-55,940 and cross- tolerance to THC and WIN 55,212-2 in all four measures, suggesting some difference between the naturally occurring and synthetic cannabinoids on receptor adaptation (Fan et al., 1994;

Wiley et al., 2005; Wiley et al., 2007). These disparate results make it difficult to define explicit effects that result from repeated exposure to cannabinoids, yet it has been demonstrated by others that tolerance does develop to the pharmacological effects of cannabinoids in rodents, nonhuman primates and humans, and this tolerance may be due to receptor adaptation (Lichtman and

Martin, 2005). Often related to drug tolerance are the phenomena of drug dependence and withdrawal. 10 mg/kg THC twice a day for 6 days is a dosing regimen commonly used to study the symptoms of precipitated cannabinoid withdrawal in mice. This dosing regimen has been shown to cause significant receptor down regulation associated with tolerance and drug dependence. Studies from our lab quantified withdrawal symptoms after THC administration at

10 mg/kg either once or twice a day for 6 days and found similar magnitude of withdrawal symptoms across both treatments (unpublished data). Similarly, a study has reported significant

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda increases in rimonabant-precipitated withdrawal symptoms in mice following a lower dose, 3 mg/kg THC, administered twice a day for 6 days (Cook et al., 1998), suggesting that, although not a common practice, administering THC at doses below 20 mg/kg/day for 6 days is adequate to produce some form of cannabinoid dependence in mice. The next series of studies evaluated the consequences of a 6 day dosing regimen with 10 mg/kg/day THC on the antinociceptive and diuretic effects of cannabinoids, as well as on cannabinoid CB1 binding parameters in mice.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

4.2 Aim and Rationale: It has been established that acute injection of cannabinoid agonists produce diuresis in mice, and these effects are mediated by their actions on CB1 receptors.

Other CB1 receptor mediated behavioral effects are subject to tolerance, thus a series of experiments examined whether, like other CB1 receptor effects, tolerance develops to the diuretic effects of cannabinoids.

The aims of this study were:

1. Determine if tolerance develops to the diuretic effects of THC.

2. Compare the degree of tolerance across measures of antinociception and diuresis.

3. Identify the time required for recovery from THC-induced tolerance to cannbinoid diuresis and antinociception.

4. Measure changes in CB1 receptor density associated with development of THC tolerance.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

4.3 Materials and Methods:

4.3.1 Animals: Male CD-1 mice weigh approximately 20-25 g at the beginning of the study (Charles River Laboratories, Wilmington MA). Mice were housed in groups of 4/cage in the Northeastern University animal facility in a climate controlled room with food and water available ad libitum. Mice were acclimatized to the animal facility for 1-2 weeks and to the study procedure 2 times before drug or vehicle test. All experiments were performed during the light portion of the light/dark cycle.

4.3.2 Antinociception: Antinociception was measured using a warm-water tail-water procedure and cumulative dosing techniques as described in chapter 3. Cumulative THC dose response curves were determined before (day 0) and after (days 8 and 15) being treated with vehicle or 10.0 mg/kg THC once a day for 7 days. In addition, antinociceptive effects of the daily injection of 10 mg/kg THC were determined on days 1, 3, and 5.

4.3.3 Diuresis: Diuresis was measured as described in chapter 2. Six groups of mice

(n=6/group) received vehicle or 1, 3, 10, 30, or 100 mg/kg THC and diuresis was measured over

6 hr. Mice that received 1 and 3 mg/kg were injected with 9 and 7 mg/kg THC respectively, after diuresis measurement and, along with the group that had received 10 mg/kg THC formed 3 groups of mice that were injected with 10 mg/kg THC every day for the next 6 days, such that each mouse received 10 mg/kg THC for 7 days; a fourth group of mice received vehicle for 7 days. Urine output was measured on day 1, 3, 5 and 7 after THC injection for 6 hr, and mice were weighed before and after every 6 hr diuresis session to determine weight loss. Water bottles were weighed every 24 hrs to determine amount of water intake per cage. On Day 8 and

14, the mice received 10, 30, or 100 mg/kg THC, and diuresis was again measured over 6hr.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

4.3.4 Binding assay: 3 groups of mice (n = 6) were injected daily with either 10 mg/kg

THC, 0.1 mg/kg AM2389 or vehicle for 7 days. 24 hr after the last injection mice were sacrificed using cervical dislocation and brain isolated and frozen at -80oC until further analysis.

On the day of the binding assay, cerebellum was isolated from the brain and weighed. The cerebellum from each animal was separately homogenized in TME (100 mM Tris, 5mM MgCl2,

1mM EDTA) buffer containing 3% BSA to obtain a 10 mg/ml homogenate. 3 ml homogenate was transferred to another tube, and 1.5 uCi radiolabeled [125I]AM281 was added. Cold AM251 was diluted in TME buffer, range - 1 pM – 10 uM and was used as the inhibitor for this assay. 100 ul of the inhibitor (non-radiolabeled AM251) or TME buffer was added to each eppendorf tube followed by

100ul of radiolabeled homogenate and 800ul homogenizing buffer. These samples were then incubated at room temperature for 90 min on a shaker followed by centrifugation at 14000 rpm (max) for 12 min at

4oC. The supernatant was aspirated and pellets were cut out using a sharp blade, dried using Kim wipes and placed into a glass tube for measurement of radioactivity using a gamma counter. Control with 100 ul of homogenate having [125I]AM281 was measured to obtain the total radioactivity count and determine the specific activity. All the samples were run in duplicates.

4.3.5 Drugs: Δ9-THC and rimonabant were obtained from the National Institute on Drug

Abuse [(NIDA), Rockville, MD]; AM2389 [9β-Hydroxy-3-(1-hexyl-cyclobut-1-yl)- hexahydrocannabinol] was synthesized at the Center for Drug Discovery, Northeastern

University. All compounds were prepared in 5% ethanol, 5% emulphor-620 (Rhodia, Cranbury,

NJ) and 90% saline, and further diluted with saline. Except where noted, injections were delivered s.c. in volumes of 1ml/100g body weight; drug doses are expressed in terms of the weight of free base.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

4.3.6 Statistical analysis: Tail withdrawal latencies are expressed as a percentage of maximum possible effect (%MPE), calculated using the formula: %MPE = [(test latency − baseline latency)/ (8 − baseline latency)] × 100. To determine ED50values for diuresis, 50% of the maximum effect was defined using the formula: [((maximum urine output with the drug – urine output with vehicle)/2) + urine output with vehicle]. ED50 values were calculated using linear regression when more than two data points were available, and otherwise were calculated by interpolation. For binding studies, data were normalized to the protein content of the brain homogenate and specific binding was determined by subtracting the non specific binding from the total binding. Scatchard plot was used for determining Bmax values by extrapolating the linear regression line on the x-axis.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

4.4 Results:

4.4.1 Tolerance to diuresis: Similar to effects reported in Chapter 2, THC increased urine output compared to vehicle treated animals, and doses higher than 10 mg/kg again formed a descending limb of a biphasic dose-effect function. Tolerance to the diuretic effects of 10.0 mg/kg THC developed gradually over the course of daily treatment, and total urine output following 10.0 mg/kg THC on day 7 was identical to urine output following saline treatment.

Changes in urine output following daily treatment with 10.0 mg/kg THC for 7 days correlated well with changes in weight loss in mice over the 6 hr test period and changes in water intake over 24 hr following testing (shown in Figure 19). This suggests that increases in urine output is accompanied by corresponding weight loss and an increase in water intake, and as tolerance develops to the diuretic effects of THC, effects on weight loss and water intake also dissipate.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

4 0 V e h ic le 1 0 .0 m g /k g T H C

) 3 0

g (

2 0

i r

U 1 0

0 1 3 5 7 9 D a y s

4 0

)

(

g 3 0

/ e

k 2 0

r 1 0

t a

W 0 1 3 5 7 9 D a y s

)

(

s 2

h g

i 1

e W

0 1 3 5 7 9 D a y s

Figure 19: Effects of 10 mg/kg THC or vehicle injections determined over time (days), on urine output (top), water intake over 24hr (middle) and weight loss over 6 hr (bottom) (n =6).

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

Tolerance that developed to the increases in urine output after 7 daily injections of 10.0 mg/kg THC extended to other doses of THC as the entire THC dose-response curve was shifted to the right after 7 days of 10.0 mg/kg THC administration as compared to vehicle treatment

(Figure 20). The ED50 value for the diuresis produced by THC in mice that received 10.0 mg/kg

THC for 7 days was 25.8 mg/kg as compared to an ED50 of 3.8 mg/kg in vehicle treated mice, corresponding to an approximate 7-fold increase in ED50 for the ascending limb of THC dose response curve. Tolerance also developed to the decrease in diuresis produced at higher dose

(30-100 mg/kg) of THC; doses higher than 100.0 mg/kg were not tested due to solubility issues, and so the magnitude of shift in the descending limb could not be determined.

The reversibility of tolerance to the diuretic effects of THC was evaluated by determination of THC dose response curve 14 days after stopping daily THC injections.

Increases in urine output after 10.0 mg/kg THC were intermediate to those obtained on days 1 and 8 and were not statistically different from either, suggesting partial recovery of the diuretic effects of THC. In contrast, recovery to the decrease in urine output produced by 100.0 mg/kg

THC was complete after the 14 day recovery period (Figure 20).

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

7 day vehicle 7 day 10 mg/kg THC 14 day post 7 day 10 mg/kg THC

10 Urine (g/kg) Urine

1.0 3.0 10.0 30.0 100.0 THC (mg/kg)

Figure 20: Urine output measured in mice treated with vehicle or 10.0 mg/kg once a day for 7 days and tested on day 8 with THC. THC dose response curve following 14 days after last injection of THC on day 7 (n = 6).

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

4.4.2 Tolerance to antinociception: Results describe in Chapter 3 indicate that

10.0mg/kg approximates an ED50 dose in increasing antinociception. The effects of daily 10.0 mg/kg THC on the development of tolerance to cannabinoid antinociceptive effects was evaluated as a comparison to the tolerance that was observed to the diuretic effects of this dose.

Tolerance to the antinociceptive effects of 10.0 mg/kg THC developed rapidly, within 3 days, and persisted for the duration of the daily dosing regimen (Figure 21).

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

1 0 0

1 0 .0 m g /k g T H C 8 0

E 6 0

P M

4 0 %

2 0

0 1 3 5 7 9 D a y s

Figure 21: Antinociception measured every other day 1 hr post 10.0 mg/kg THC (n = 6).

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

Similar to studies that were performed with diuresis, dose response curves for THC were determined before and after administration of 10.0 mg/kg THC once a day for 7 days. The THC dose response curve was shifted to the right (Figure 22) after 7 day exposure to THC, with the

ED50 changing from 9.5 mg/kg to 87.1 mg/kg, corresponding to an approximate 9-fold increase in ED50 values. Similar to diuresis studies, the mice that received 10.0 mg/kg THC for 7 days were allowed to recover for 14 days and then the antinociceptive effects of THC were re- determined. The dose response curve for THC after the recovery period was slightly to the left of THC dose response curve obtained immediately after the 7 day THC treatment period, as seen in Figure 22. The ED50 value for THC 14 days after stopping the daily injections was 60.5 mg/kg, indicating incomplete recovery of the antinociceptive effects of THC.

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

7 day vehicle 7 day 10 mg/kg THC 100 14 day post 7 day 10 mg/kg THC

40 % MPE % 20

1.0 3.0 10.0 30.0 100.0 THC (mg/kg)

Figure 22: Antinociception after cumulative THC injections, expressed as %MPE, in mice treated with vehicle or 10.0 mg/kg once a day for 7 days and tested on day 8 with THC, and THC testing 14 days after last THC injection on day 7 (n = 6).

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

4.4.3 Changes in CB1 receptor levels: Changes in CB1 receptor levels were determined in mice that received 7-day treatment with 10.0 mg/kg THC. The Bmax for CB1 receptors in the group of mice treated with vehicle was 170 ± 30 pmol/mg (n=6). Preliminary studies also determined effects of single injections of 1.0-30.0 mg/kg THC on CB1 receptor binding at 24 hr after injection and found no significant changes in CB1 receptor binding, with Bmax values that ranged from 116 to 245 pmol/mg (n=2-3). The Bmax value for mice that received THC for 7 days was 75 ± 9 pmol/mg (n = 6) and were significantly lower than Bmax values obtained from vehicle treated mice (p = 0.013). As a positive control, another group of mice was treated with the CB1 full agonist, AM2389, at a dose 0.1 mg/kg/day for 7 days; this dose is adequate to see signs of rimonabant-precipitated withdrawal symptoms in mice (unpublished data). Daily injection with

0.1 mg/kg AM2389 for 7 days resulted in a Bmax value for CB1 receptors of 34 ± 5 pmol/mg (n =

6) and was significantly different from vehicle treated mice (p = 0.001).

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

0.010 7 day vehicle 7 day 10.0 mg/kg THC 7 day 0.1 mg/kg AM2389

0.005 Bound/Free

0.000 0 30 60 90 120 150 Bound [pmol/mg]

2000

1500

1000

500 CPM (total binding) CPM(total 0 0 -12 -10 -8 -6 -4 AM281 [M]

Figure 23: Binding data for CB1 receptors, (bottom) total binding in the presence of increasing concentrations of cold AM281, dotted line represents non-specific binding. Top, Scatchard plot of the same data for determining Bmax by extrapolation (n = 6).

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

4.5 Discussion:

Cannabinoids produce diuresis in mice by activation of the CB1 receptors. Tolerance to many cannabinoid CB1-mediated effects, such as antinociception, hypothermia, rate of operant responding and hypolocomotion have been reported (Wiley et al., 2005; Wiley et al., 2007;

Nguyen et al., 2012; Desai et al., 2013). These studies sought to determine if the diuretic effects of cannabinoids are likewise subject to tolerance. The dose of 10 mg/kg THC is pharmacologically active and represents the peak dose for increasing diuresis (shown in chapter

2), however it is relatively low dose based on effects in other murine assays, for example, it is approximately the ED50 dose for antinociceptive effects. Often 20 mg/kg/day THC, or even higher doses, administered for 5-7 days are used to study cannabinoid physical dependence in mice and are considered necessary to produce tolerance to the pharmacological effects of THC in mice (Breivogel et al., 1999; Sim-Selley et al., 2006). Here, a dose of 10mg/kg/day for 7 days was selected to study tolerance, primarily based on unpublished work from our lab and evidence from the literature that indicate signs of precipitated withdrawal are obtained following this dosing regimen (Cook et al., 1998). Tolerance developed to both the diuretic and antinociceptive effects produced by 10 mg/kg THC, with diuretic tolerance perhaps emerging more gradually than tolerance to the antinociceptive effects of THC. Along with tolerance to the diuretic effects, the amount of water intake also proportionally decreased over 24 hr following diuresis testing and was accompanied by proportional decreases in loss of body weight over the 6 hr testing period. This suggests that loss in body weight was primarily due to fluid loss, which was recovered by fluid intake after the test session, although this was not directly assessed.

Complete dose response curve determinations with THC in mice after 7 days of 10 mg/kg

THC or vehicle demonstrated that tolerance developed to both the ascending and descending

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda limb of the diuresis dose response curves. The shift in the ascending limb of the diuresis dose response curve in THC-treated mice after 7 days of 10 mg/kg/day THC treatment was approximately 7-fold and was similar in magnitude to the shift in the dose response curve observed for antinociception (~9-fold). This suggests similar CB1 receptors might be involved in mediating the antinociceptive and diuretic effects of THC in mice and further supports the findings from chapter 2 that cannabinoid agonists produce increases in diuresis by actions at the

CB1 receptors in the CNS.

To investigate if the development of tolerance to the diuretic and antinociceptive effects of cannabinoids was accompanied by changes in CB1 receptor binding parameters in the brain, radioligand binding was performed on mouse cerebellum. Mice that were treated acutely with 1-

30 mg/kg THC showed no significant changes in CB1 receptor numbers in the mouse cerebellum as compared to vehicle treated animals. However, mice that received 10 mg/kg/day THC treatment for 7 days showed a statistically significant reduction in CB1 receptors when compared to vehicle treated animals. As the effects of only a single daily dose (10mg/kg) of THC were evaluated, one can only speculate that the Bmax for the CB1 receptors would decrease proportionally to an increase in dose. Others have also reported that CB1 receptors are down regulated significantly in the cerebellum following 6.5 day of 10 mg/kg THC twice daily dosing

(Nguyen et al., 2012).

100

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

The use of cerebellum tissue for determining CB1 receptor down regulation may not be ideal for understanding tolerance to cannabinoid-mediated diuresis; it is more likely that CB1 receptors in the hypothalamus are involved in endocrine functions responsible for maintaining fluid homeostasis (Goodman et al., 2006). However, binding studies in mouse hypothalamus using frozen brain tissues are difficult; hence the cerebellum was used as a proxy to indicate overall changes in brain CB1 receptors. One study comparing effects of sub-chronic THC dosing showed that although decreases in CB1 receptors in the hypothalamus were observed, they were not as significant compared to the decreases produced in the cerebellum (Nguyen et al., 2012). The regional differences in receptor downregulation following sub-chronic cannabinoid treatment could implicate possible role of CB1 receptors in specific regions of the brain in producing tolerance to the pharmacological effects of cannabinoids.

After demonstrating that tolerance developed to the diuretic and antinociceptive effects of

THC after 7 day 10mg/kg/day THC administration, and that this tolerance was accompanied by changes in CB1 receptors in the cerebellum, studies next tried to identify whether this tolerance was reversible after cessation of daily drug administration. 14 days after the last injection of

THC, dose response curves were re-determined for diuresis and antinociception and, surprisingly, complete recovery was not observed for the ascending limb of cannabinoid diuresis or for antinociceptive effects, suggesting the same (possibly CNS) CB1 receptors are involved in producing the two effects. However, the descending limb of cannabinoid diuresis recovered completely at day 14 indicative of the involvement of a distinct population of CB1 receptors

(possibly peripheral) in producing these effects. The above hypothesis supports the findings from chapter 2 that central CB1 receptor activation is associated with increasing diuresis while

101

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda peripheral CB1 receptors are involved in producing the decreases in diuresis produced by cannabinoid agonists in mice.

102

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

E. Conclusions:

This thesis research establishes diuresis as a robust cannabinoid-mediated effect in mice and, further, identifies the receptor mechanisms that underlie these effects. Initial parametric work involved developing and validating a simple, cost effective method of measuring urine output in individual mice. Once developed, these procedures were used to compare cannabinoid diuresis with diuresis produced by other drugs and, as well, to compare cannabinoid diuresis with another well characterized cannabinoid-mediated effect, antinociception. The major findings of this work, that THC and other synthetic cannabinergic compounds produce diuresis in mice, extend previous reports of the diuretic effects of cannabinoids in rats and humans (Ames, 1958;

Sofia et al., 1977; Paronis et al., 2013). The order of potency for the structurally distinct cannabinoid agonists - THC, WIN55,212-2, AM7418 and AM4054 – in producing diuresis was similar to the order of potency for antinociception, notably, however, peak diuretic effects occurred at doses lower than peak antinociceptive effects. The finding that all cannabinoids were more potent in terms of producing diuresis than they were antinociception suggests that diuresis may represent a more sensitive and objective measure of cannabinoid actions in vivo than other commonly used behavioral assays.

The cannabinoid agonists increased urine output in a manner qualitatively and quantitatively more similar to that produced by the κ-opioid agonist U50,488 than the loop diuretic, furosemide. Quantitatively, the four cannabinoids produced maximum urine outputs of

30-36 g/kg, equivalent to the outputs achieved with high doses of U50,488, and less than amounts voided after furosemide. Qualitatively, the relatively small Na+ loss following THC indicates weak naturetic effects that are more similar to the free water diuresis produced by U-

50,488 than the electrolyte loss that accompanies furosemide diuresis. However, unlike the κ-

103

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda opioid agonist and the loop diuretic, the cannabinoid agonists had biphasic dose-effect functions, and doses above those that yielded 30-36 g/kg urine led to dose-dependent decreases in urine output. Such biphasic functions were not noted in previous studies in rats and may represent a distinct difference between species.

The involvement of specific cannabinoid receptors in modulating urine output was investigated through pharmacological antagonism studies. To this end, receptor selective antagonists rimonabant or AM630, and the peripherally constrained antagonist AM6545, were used as pretreatment drugs (Rinaldi-Carmona et al., 1995; Ross et al., 1999; Tam et al., 2010).

The cannabinoid CB1 antagonist rimonabant had no intrinsic effects on diuresis yet did dose- dependently antagonize both the ascending and descending limbs of the AM4054 dose response curve. In contrast to rimonabant, the CB2 antagonist AM630 did not attenuate the effects of either moderate or high doses of AM4054 or THC. Together, these results suggest that, as in rats, cannabinoid agonists produce their diuretic effects in mice via actions at cannabinoid CB1 receptors with limited involvement of CB2 receptors. Moreover, since both limbs of the

AM4054 dose-response curve were antagonized by rimonabant, our data further indicate that both the increases and subsequent decreases in the magnitude of diuresis are CB1-mediated.

This was further confirmed by comparing the potency ratios for rimonabant across antinociception and diuresis, which revealed greater potency towards antagonizing increases in diuresis and identical potency ratios for antagonizing antinociception and decreases in diuresis.

Repeated administration of THC for 7 days resulted in development of tolerance to the diuretic as well as antinociceptive effects of THC. For diuresis, both the ascending and descending limbs of the THC dose response curve were shifted to the right, yet the recovery from tolerance was different for these two effects, suggesting that different sub-population of

104

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

CB1 receptor are responsible for the two limbs of cannabinoid diuresis dose response curve.

One hypothesis was that these effects occur by activation of CB1 receptors in two separate compartments, i.e., those found either centrally or peripherally. The quantitative and qualitative similarity between cannabinoid and κ-opioid diuresis suggested central mediation of the increase in urine output, as U50,488 is known to produce its diuretic effects through central actions

(Kapusta and Obih, 1993; Kapusta and Obih, 1995). To test this hypothesis, the peripherally constrained cannabinoid CB1 antagonist AM6545 (Cluny et al., 2010; Tam et al., 2010) was injected prior to determination of a full AM4054 dose-effect function. A moderate dose of

AM6545 did not affect the ascending limb of the AM4054 function, while shifting the descending limb of AM4054 diuresis to the right; a higher dose of AM6545 was able to shift both limbs of the AM4054 dose effect function. Although AM6545 does not readily cross the blood-brain barrier, higher doses will penetrate the CNS and have been associated with blockade of central antinociceptive effects of THC in the warm water tail-withdrawal measurement.

Though limited, these data suggest that diuresis produced by lower doses of agonists are central cannabinoid CB1 receptor effects, however, the decrease in the magnitude of diuresis produced at higher doses of agonists likely involves both central and peripheral cannabinoid CB1 receptors. If this is correct, than the results of the tolerance studies suggest that perhaps the peripheral cannabinoid receptors recovery more quickly during daily dosing regimens than do the central CB1 receptors. In concordance with this, there was very little recovery of the centrally-mediated antinociceptive effects of THC following daily dosing. Hence we can conclude that cannabinoids increase diuresis and produce antinociception by actions at the central CB1 receptors whereas they decrease diuresis possibly by actions at the peripheral CB1 receptors.

105

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

Clinical studies have reported beneficial effects of smoked or aerosolized cannabis on bladder dysfunction in patients with multiple sclerosis, primarily by decreasing urinary frequency in these subjects following marijuana use (Consroe et al., 1997; Brady et al., 2004).

These reports contrast with the earlier clinical reports demonstrating increase in urine output after cannabis administration (Ames, 1958). Our findings in mice demonstrate both dose related increases and decreases in urine output, providing a platform for understanding the mixed effects on urine output observed with marijuana in various clinical studies. As noted earlier in a study with rats (Sofia et al., 1977), the diuresis induced by THC in mice also is weakly naturetic compared to furosemide and further investigations in this area may yield a new, clinically beneficial diuretic. In contrast, our data suggest that development of peripherally selective cannabinoid CB1 agonists may be beneficial for patients suffering from bladder dysfunction.

106

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

F. Bibliography:

1. Adams R (1940) Marihuana. Science 92:115-119. 2. Allentuck S and Bowman KM (1942) The psychiatric aspects of marihuana intoxication. Amer J Psychiatry 99:248-251. 3. Ames F (1958) A clinical and metabolic study of acute intoxication with Cannabis sativa and its role in the model psychoses. J Ment Sci 104:972-999. 4. Anonymous (2010) GW Pharma initiates Sativex Phase III clinical trials, in, Pharma. Business Rev. 5. Avelino A and Cruz F (2006) TRPV1 (vanilloid receptor) in the urinary tract: expression, function and clinical applications. Naunyn Schmiedebergs Arch Pharmacol 373:287-299. 6. Barnes MP (2006) Sativex: clinical efficacy and tolerability in the treatment of symptoms of multiple sclerosis and neuropathic pain. Expert Opin Pharmacother 7:607-615. 7. Barrot M (2012) Tests and models of nociception and pain in rodents. Neuroscience 211:39-50. 8. Barry H, 3rd, Kubena RK and Perhach JL, Jr. (1973) Pituitary-adrenal activation and related responses to delta1-tetrahydrocannabinol. Prog Brain Res 39:323-330. 9. Bass CE and Martin BR (2000) Time course for the induction and maintenance of tolerance to Delta(9)-tetrahydrocannabinol in mice. Drug Alcohol Depend 60:113-119. 10. Beal JE, Olson R, Laubenstein L, Morales JO, Bellman P, Yangco B, Lefkowitz L, Plasse TF and Shepard KV (1995) Dronabinol as a treatment for anorexia associated with weight loss in patients with AIDS. J Pain Symptom Manage 10:89-97. 11. Berlach DM, Shir Y and Ware MA (2006) Experience with the synthetic cannabinoid nabilone in chronic noncancer pain. Pain Med 7:25-29. 12. Brady CM, DasGupta R, Dalton C, Wiseman OJ, Berkley KJ and Fowler CJ (2004) An open-label pilot study of cannabis-based extracts for bladder dysfunction in advanced multiple sclerosis. Mult Scler 10:425-433. 13. Breivogel CS, Childers SR, Deadwyler SA, Hampson RE, Vogt LJ and Sim-Selley LJ (1999) Chronic delta9-tetrahydrocannabinol treatment produces a time-dependent loss of cannabinoid receptors and cannabinoid receptor-activated G proteins in rat brain. J Neurochem 73:2447- 2459. 14. Brooks DP, Giardina G, Gellai M, Dondio G, Edwards RM, Petrone G, DePalma PD, Sbacchi M, Jugus M, Misiano P and et al. (1993) Opiate receptors within the blood-brain barrier mediate kappa agonist-induced water diuresis. J Pharmacol Exp Ther 266:164-171. 15. Capasso R, Aviello G, Borrelli F, Romano B, Ferro M, Castaldo L, Montanaro V, Altieri V and Izzo AA (2011) Inhibitory effect of standardized cannabis sativa extract and its ingredient cannabidiol on rat and human bladder contractility. Urology 77:18. 16. Chopra IC and Chopra RN (1957) The Use of the Cannabis Drugs in India. United Nations Office on Drugs and Crime:4-29. 17. Cluny NL, Vemuri VK, Chambers AP, Limebeer CL, Bedard H, Wood JT, Lutz B, Zimmer A, Parker LA, Makriyannis A and Sharkey KA (2010) A novel peripherally restricted cannabinoid receptor antagonist, AM6545, reduces food intake and body weight, but does not cause malaise, in rodents. Br J Pharmacol 161:629-642. 18. Compton DR, Aceto MD, Lowe J and Martin BR (1996) In vivo characterization of a specific cannabinoid receptor antagonist (SR141716A): inhibition of delta 9-tetrahydrocannabinol- induced responses and apparent agonist activity. J Pharmacol Exp Ther 277:586-594. 19. Compton DR, Gold LH, Ward SJ, Balster RL and Martin BR (1992) Aminoalkylindole analogs: cannabimimetic activity of a class of compounds structurally distinct from delta 9- tetrahydrocannabinol. J Pharmacol Exp Ther 263:1118-1126.

107

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

20. Consroe P, Musty R, Rein J, Tillery W and Pertwee R (1997) The perceived effects of smoked cannabis on patients with multiple sclerosis. Eur Neurol 38:44-48. 21. Cook SA, Lowe JA and Martin BR (1998) CB1 receptor antagonist precipitates withdrawal in mice exposed to Delta9-tetrahydrocannabinol. J Pharmacol Exp Ther 285:1150-1156. 22. Craft RM, Ulibarri CM and Raub DJ (2000) Kappa opioid-induced diuresis in female vs. male rats. Pharmacol Biochem Behav 65:53-59. 23. D'Amour FE and Smith DL (1941) A METHOD FOR DETERMINING LOSS OF PAIN SENSATION. Journal of Pharmacology and Experimental Therapeutics 72:74-79. 24. Dalton GD, Smith FL, Smith PA and Dewey WL (2005) Chronic Delta9-tetrahydrocannabinol treatment produces antinociceptive tolerance in mice without altering protein kinase A activity in mouse brain and spinal cord. Biochem Pharmacol 70:152-160. 25. De Ridder D, Ost D, Van der Aa F, Stagnaro M, Beneton C, Gross-Paju K, Eelen P, Limbourg H, Harper M, Segal JC, Fowler CJ and Nordenbo A (2005) Conservative bladder management in advanced multiple sclerosis. Mult Scler 11:694-699. 26. Delatte MS and Paronis CA (2008) Evaluation of cannabinoid agonists using punished responding and midazolam discrimination procedures in squirrel monkeys. Psychopharmacology (Berl) 198:521-528. 27. Desai RI, Thakur GA, Vemuri VK, Bajaj S, Makriyannis A and Bergman J (2013) Analysis of tolerance and behavioral/physical dependence during chronic CB1 agonist treatment: effects of CB1 agonists, antagonists, and noncannabinoid drugs, in J Pharmacol Exp Ther pp 319-328, United States. 28. Di S, Malcher-Lopes R, Halmos KC and Tasker JG (2003) Nongenomic glucocorticoid inhibition via endocannabinoid release in the hypothalamus: a fast feedback mechanism, in J Neurosci pp 4850-4857, United States. 29. Dinis P, Charrua A, Avelino A, Yaqoob M, Bevan S, Nagy I and Cruz F (2004) Anandamide-evoked activation of vanilloid receptor 1 contributes to the development of bladder hyperreflexia and nociceptive transmission to spinal dorsal horn neurons in cystitis. J Neurosci 24:11253-11263. 30. Dmitrieva N and Berkley KJ (2002) Contrasting effects of WIN 55212-2 on motility of the rat bladder and uterus, in J Neurosci pp 7147-7153, United States. 31. Fan F, Compton DR, Ward S, Melvin L and Martin BR (1994) Development of cross-tolerance between delta 9-tetrahydrocannabinol, CP 55,940 and WIN 55,212. J Pharmacol Exp Ther 271:1383-1390. 32. Freeman RM, Adekanmi O, Waterfield MR, Waterfield AE, Wright D and Zajicek J (2006) The effect of cannabis on urge incontinence in patients with multiple sclerosis: a multicentre, randomised placebo-controlled trial (CAMS-LUTS). International urogynecology journal and pelvic floor dysfunction 17:636-641. 33. Galiegue S, Mary S, Marchand J, Dussossoy D, Carriere D, Carayon P, Bouaboula M, Shire D, Le Fur G and Casellas P (1995) Expression of central and peripheral cannabinoid receptors in human immune tissues and leukocyte subpopulations. European journal of biochemistry / FEBS 232:54-61. 34. Gatley SJ, Gifford AN, Volkow ND, Lan R and Makriyannis A (1996) 123I-labeled AM251: a radioiodinated ligand which binds in vivo to mouse brain cannabinoid CB1 receptors. Eur J Pharmacol 307:331-338. 35. Ginsburg BC, Schulze DR, Hruba L and McMahon LR (2012) JWH-018 and JWH-073: Delta(9)- tetrahydrocannabinol-like discriminative stimulus effects in monkeys. J Pharmacol Exp Ther 340:37-45.

108

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

36. Goodman LS, Gilman A, Brunton LL, Lazo JS and Parker KL (2006) Goodman & Gilman's the pharmacological basis of therapeutics. New York : McGraw-Hill, c2006. 11th ed. / editor, Laurence L. Brunton ; associate editors, John S. Lazo, Keith L. Parker. 37. Grinspoon L (1969) Marihuana. Sci Am 221:17-25. 38. Grinspoon L and Bakalar JB (1997) Marihuana, the forbidden medicine. Yale University Press, New Haven. 39. Haller VL, Cichewicz DL and Welch SP (2006) Non-cannabinoid CB1, non-cannabinoid CB2 antinociceptive effects of several novel compounds in the PPQ stretch test in mice. Eur J Pharmacol 546:60-68. 40. Haney M, Gunderson EW, Rabkin J, Hart CL, Vosburg SK, Comer SD and Foltin RW (2007) Dronabinol and marijuana in HIV-positive marijuana smokers. Caloric intake, mood, and sleep. J Acquir Immune Defic Syndr 45:545-554. 41. Haney M, Rabkin J, Gunderson E and Foltin RW (2005) Dronabinol and marijuana in HIV(+) marijuana smokers: acute effects on caloric intake and mood. Psychopharmacology (Berl) 181:170-178. 42. Hayes AG, Sheehan MJ and Tyers MB (1987) Differential sensitivity of models of antinociception in the rat, mouse and guinea-pig to mu- and kappa-opioid receptor agonists. Br J Pharmacol 91:823-832. 43. Hill MN and Gorzalka BB (2005) Pharmacological enhancement of cannabinoid CB1 receptor activity elicits an antidepressant-like response in the rat forced swim test. Eur Neuropsychopharmacol 15:593-599. 44. Hiragata S, Ogawa T, Hayashi Y, Tyagi P, Seki S, Nishizawa O, de Miguel F, Chancellor MB and Yoshimura N (2007) Effects of IP-751, ajulemic acid, on bladder overactivity induced by bladder irritation in rats. Urology 70:202-208. 45. Hull LC, Rabender C, Gabra BH, Zhang F, Li PL and Dewey WL (2010) Role of CD38, a cyclic ADP- ribosylcyclase, in morphine antinociception and tolerance. J Pharmacol Exp Ther 334:1042-1050. 46. Ibrahim MM, Rude ML, Stagg NJ, Mata HP, Lai J, Vanderah TW, Porreca F, Buckley NE, Makriyannis A and Malan TP, Jr. (2006) CB2 cannabinoid receptor mediation of antinociception. Pain 122:36-42. 47. Janssen PA, Niemegeers CJ and Dony JG (1963) The inhibitory effect of fentanyl and other morphine-like analgesics on the warm water induced tail withdrawl reflex in rats. Arzneimittelforschung 13:502-507. 48. Jarbe TU, Harris MY, Li C, Liu Q and Makriyannis A (2004) Discriminative stimulus effects in rats of SR-141716 (rimonabant), a cannabinoid CB1 receptor antagonist. Psychopharmacology 177:35-45. 49. Jarbe TU, Lamb RJ, Lin S and Makriyannis A (2001) (R)-methanandamide and Delta 9-THC as discriminative stimuli in rats: tests with the cannabinoid antagonist SR-141716 and the endogenous ligand anandamide. Psychopharmacology 156:369-380. 50. Jarbe TU and McMillan DE (1979) Discriminative stimulus properties of tetrahydrocannabinols and related drugs in rats and pigeons. Neuropharmacology 18:1023-1024. 51. Jarbe TU, Tai S, LeMay BJ, Nikas SP, Shukla VG, Zvonok A and Makriyannis A (2012) AM2389, a high-affinity, in vivo potent CB1-receptor-selective cannabinergic ligand as evidenced by drug discrimination in rats and hypothermia testing in mice. Psychopharmacology 220:417-426. 52. Kapusta DR and Obih JC (1993) Central kappa opioid receptor-evoked changes in renal function in conscious rats: participation of renal nerves. J Pharm Exp Ther 267:197-204. 53. Kapusta DR and Obih JC (1995) Central kappa opioids blunt the renal excretory responses to volume expansion by a renal nerve-dependent mechanism. J Pharm Exp Ther 273:199-205.

109

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

54. Klumpers LE, Roy C, Ferron G, Turpault S, Poitiers F, Pinquier JL, van Hasselt JG, Zuurman L, Erwich FA and van Gerven JM (2013) Surinabant, a selective cannabinoid receptor type 1 antagonist, inhibits Delta(9) -tetrahydrocannabinol-induced central nervous system and heart rate effects in humans. Br J Clin Pharmacol 76:65-77. 55. Koura Y, Ichihara A, Tada Y, Kaneshiro Y, Okada H, Temm CJ, Hayashi M and Saruta T (2004) Anandamide decreases glomerular filtration rate through predominant vasodilation of efferent arterioles in rat kidneys. Journal of the American Society of Nephrology : JASN 15:1488-1494. 56. Kubena RK, Perhach JL, Jr. and Barry H, 3rd (1971) Corticosterone elevation mediated centrally by delta 1-tetrahydrocannabinol in rats. Eur J Pharmacol 14:89-92. 57. Lam PM, McDonald J and Lambert DG (2005) Characterization and comparison of recombinant human and rat TRPV1 receptors: effects of exo- and endocannabinoids. Br J Anaesth 94:649-656. 58. Larrinaga G, Varona A, Perez I, Sanz B, Ugalde A, Candenas ML, Pinto FM, Gil J and Lopez JI (2010) Expression of cannabinoid receptors in human kidney. Histol Histopathol 25:1133-1138. 59. Leander JD, Zerbe RL and Hart JC (1985) Diuresis and suppression of vasopressin by kappa opioids: comparison with mu and delta opioids and clonidine. J Pharmacol Exp Ther 234:463- 469. 60. Li J and Wang DH (2006) Differential mechanisms mediating depressor and diuretic effects of anandamide. J Hypertens 24:2271-2276. 61. Lichtman AH and Martin BR (1997) The selective cannabinoid antagonist SR 141716A blocks cannabinoid-induced antinociception in rats. Pharmacol Biochem Behav 57:7-12. 62. Lichtman AH and Martin BR (2005) Cannabinoid tolerance and dependence. Handb Exp Pharmacol:691-717. 63. Little PJ, Compton DR, Johnson MR, Melvin LS and Martin BR (1988) Pharmacology and stereoselectivity of structurally novel cannabinoids in mice. J Pharmacol Exp Ther 247:1046- 1051. 64. Maickel RP (1973) Pharmacology of Marihuana (Cannabis sativa). The American Biology Teacher 35:398-404. 65. Maione S, Morera E, Marabese I, Ligresti A, Luongo L, Ortar G and Di Marzo V (2008) Antinociceptive effects of tetrazole inhibitors of endocannabinoid inactivation: cannabinoid and non-cannabinoid receptor-mediated mechanisms, in Br J Pharmacol pp 775-782, England. 66. Marco EM, Perez-Alvarez L, Borcel E, Rubio M, Guaza C, Ambrosio E, File SE and Viveros MP (2004) Involvement of 5-HT1A receptors in behavioural effects of the cannabinoid receptor agonist CP 55,940 in male rats. Behav Pharmacol 15:21-27. 67. Martin BR, Compton DR, Thomas BF, Prescott WR, Little PJ, Razdan RK, Johnson MR, Melvin LS, Mechoulam R and Ward SJ (1991) Behavioral, biochemical, and molecular modeling evaluations of cannabinoid analogs. Pharmacol Biochem Behav 40:471-478. 68. Martin M, Ledent C, Parmentier M, Maldonado R and Valverde O (2002) Involvement of CB1 cannabinoid receptors in emotional behaviour. Psychopharmacology (Berl) 159:379-387. 69. Martin RS, Luong LA, Welsh NJ, Eglen RM, Martin GR and MacLennan SJ (2000) Effects of cannabinoid receptor agonists on neuronally-evoked contractions of urinary bladder tissues isolated from rat, mouse, pig, dog, monkey and human. Br J Pharmacol 129:1707-1715. 70. Martin WJ, Lai NK, Patrick SL, Tsou K and Walker JM (1993) Antinociceptive actions of cannabinoids following intraventricular administration in rats, in Brain Res pp 300-304, Netherlands. 71. Martin WJ, Patrick SL, Coffin PO, Tsou K and Walker JM (1995) An examination of the central sites of action of cannabinoid-induced antinociception in the rat. Life sciences 56:2103-2109. 72. Matsuda LA, Lolait SJ, Brownstein MJ, Young AC and Bonner TI (1990) Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 346:561-564.

110

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

73. McMahon LR (2006) Characterization of cannabinoid agonists and apparent pA2 analysis of cannabinoid antagonists in rhesus monkeys discriminating Delta9-tetrahydrocannabinol. J Pharmacol Exp Ther 319:1211-1218. 74. McMahon LR and Koek W (2007) Differences in the relative potency of SR 141716A and AM 251 as antagonists of various in vivo effects of cannabinoid agonists in C57BL/6J mice. Eur J Pharmacol 569:70-76. 75. Mechoulam R (1970) Marihuana chemistry. Science 168:1159-1166. 76. Munro S, Thomas KL and Abu-Shaar M (1993) Molecular characterization of a peripheral receptor for cannabinoids. Nature 365:61-65. 77. Murphy LL, Munoz RM, Adrian BA and Villanua MA (1998) Function of cannabinoid receptors in the neuroendocrine regulation of hormone secretion, in Neurobiol Dis pp 432-446, United States. 78. Nguyen PT, Schmid CL, Raehal KM, Selley DE, Bohn LM and Sim-Selley LJ (2012) beta-arrestin2 regulates cannabinoid CB1 receptor signaling and adaptation in a central nervous system region- dependent manner. Biol Psychiatry 71:714-724. 79. Noyes R, Jr., Brunk SF, Avery DA and Canter AC (1975a) The analgesic properties of delta-9- tetrahydrocannabinol and codeine. Clinical pharmacology and therapeutics 18:84-89. 80. Noyes R, Jr., Brunk SF, Baram DA and Canter A (1975b) Analgesic effect of delta-9- tetrahydrocannabinol. J Clin Pharmacol 15:139-143. 81. Olsen NV, Olsen MH, Bonde J, Kanstrup IL, Plum I, Strandgaard S and Leyssac PP (1997) Dopamine natriuresis in salt-repleted, water-loaded humans: a dose-response study. British journal of clinical pharmacology 43:509-520. 82. Onaivi ES (2006) Neuropsychobiological evidence for the functional presence and expression of cannabinoid CB2 receptors in the brain. Neuropsychobiology 54:231-246. 83. Onaivi ES, Green MR and Martin BR (1990) Pharmacological characterization of cannabinoids in the elevated plus maze. J Pharmacol Exp Ther 253:1002-1009. 84. Pacher P, Batkai S and Kunos G (2004) Haemodynamic profile and responsiveness to anandamide of TRPV1 receptor knock-out mice, in J Physiol pp 647-657, England. 85. Palmer SL, Thakur GA and Makriyannis A (2002) Cannabinergic ligands, in Chem Phys Lipids pp 3- 19, Ireland. 86. Panlilio LV, Mazzola C, Medalie J, Hahn B, Justinova Z, Drago F, Cadet JL, Yasar S and Goldberg SR (2009) Anandamide-induced behavioral disruption through a vanilloid-dependent mechanism in rats. Psychopharmacology (Berl) 203:529-538. 87. Paronis CA and Holtzman SG (1991) Increased analgesic potency of mu agonists after continuous naloxone infusion in rats. J Pharmacol Exp Ther 259:582-589. 88. Paronis CA, Nikas SP, Shukla VG and Makriyannis A (2012) Delta(9)-Tetrahydrocannabinol acts as a partial agonist/antagonist in mice. Behav Pharmacol 23:802-805. 89. Paronis CA, Thakur GA, Bajaj S, Nikas SP, Vemuri VK, Makriyannis A and Bergman J (2013) Diuretic effects of cannabinoids. J Pharmacol Exp Ther 344:8-14. 90. Paronis CA and Woods JH (1997) Clocinnamox dose-dependently antagonizes morphine- analgesia and [3H]DAMGO binding in rats. Eur J Pharmacol 337:27-34. 91. Pertwee R, Griffin G, Fernando S, Li X, Hill A and Makriyannis A (1995) AM630, a competitive cannabinoid receptor antagonist. Life Sci 56:1949-1955. 92. Pertwee RG (1997) Pharmacology of cannabinoid CB1 and CB2 receptors. Pharmacol Ther 74:129-180. 93. Pertwee RG and Fernando SR (1996) Evidence for the presence of cannabinoid CB1 receptors in mouse urinary bladder. Br J Pharmacol 118:2053-2058.

111

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

94. Piomelli D (2003) The molecular logic of endocannabinoid signalling. Nat Rev Neurosci 4:873- 884. 95. Pryor GT, Husain S, Larsen F, McKenzie CE, Carr JD and Braude MC (1977) Interactions between delta9-tetrahydrocannabinol and phencyclidine hydrochloride in rats. Pharmacol Biochem Behav 6:123-136. 96. Raffa RB, Stone DJ, Jr. and Hipp SJ (1999) Differential cholera-toxin sensitivity of supraspinal antinociception induced by the cannabinoid agonists delta9-THC, WIN 55,212-2 and anandamide in mice. Neurosci Lett 263:29-32. 97. Randall PA, Vemuri VK, Segovia KN, Torres EF, Hosmer S, Nunes EJ, Santerre JL, Makriyannis A and Salamone JD (2010) The novel cannabinoid CB1 antagonist AM6545 suppresses food intake and food-reinforced behavior. Pharmacol Biochem Behav 97:179-184. 98. Reche I, Fuentes JA and Ruiz-Gayo M (1996) A role for central cannabinoid and opioid systems in peripheral delta 9-tetrahydrocannabinol-induced analgesia in mice. Eur J Pharmacol 301:75-81. 99. Rinaldi-Carmona M, Barth F, Heaulme M, Shire D, Calandra B, Congy C, Martinez S, Maruani J, Neliat G, Caput D and et al. (1994) SR141716A, a potent and selective antagonist of the brain cannabinoid receptor. FEBS Lett 350:240-244. 100. Rinaldi-Carmona M, Barth F, Héaulme M, Alonso R, Shire D, Congy C, Soubrié P, Brelière JC and Le Fur G (1995) Biochemical and pharmacological characterisation of SR141716A, the first potent and selective brain cannabinoid receptor antagonist. Life Sci 56:1941-1947. 101. Ritter JK, Li C, Xia M, Poklis JL, Lichtman AH, Abdullah RA, Dewey WL and Li PL (2012) Production and actions of the anandamide metabolite prostamide E2 in the renal medulla. J Pharmacol Exp Ther 342:770-779. 102. Rodriguez de Fonseca F, Del Arco I, Bermudez-Silva FJ, Bilbao A, Cippitelli A and Navarro M (2005) The endocannabinoid system: physiology and pharmacology. Alcohol Alcohol 40:2-14. 103. Ross RA, Brockie HC, Stevenson LA, Murphy VL, Templeton F, Makriyannis A and Pertwee RG (1999) Agonist-inverse agonist characterization at CB1 and CB2 cannabinoid receptors of L759633, L759656 and AM630. Br J Pharmacol 126:665-672. 104. Ross RA, Gibson TM, Brockie HC, Leslie M, Pashmi G, Craib SJ, Di Marzo V and Pertwee RG (2001) Structure-activity relationship for the endogenous cannabinoid, anandamide, and certain of its analogues at vanilloid receptors in transfected cells and vas deferens. Br J Pharmacol 132:631- 640. 105. Rossi NF and Brooks DP (1996) kappa-Opioid agonist inhibition of osmotically induced AVP release: preferential action at hypothalamic sites. Am J Physiol 270:E367-372. 106. Rubino T, Guidali C, Vigano D, Realini N, Valenti M, Massi P and Parolaro D (2008) CB1 receptor stimulation in specific brain areas differently modulate anxiety-related behaviour. Neuropharmacology 54:151-160. 107. Ryberg E, Larsson N, Sjogren S, Hjorth S, Hermansson NO, Leonova J, Elebring T, Nilsson K, Drmota T and Greasley PJ (2007) The orphan receptor GPR55 is a novel cannabinoid receptor. Br J Pharmacol 152:1092-1101. 108. Sallan SE, Zinberg NE and Frei E, 3rd (1975) Antiemetic effect of delta-9-tetrahydrocannabinol in patients receiving cancer chemotherapy. The New England journal of medicine 293:795-797. 109. Sawzdargo M, Nguyen T, Lee DK, Lynch KR, Cheng R, Heng HH, George SR and O'Dowd BF (1999) Identification and cloning of three novel human G protein-coupled receptor genes GPR52, PsiGPR53 and GPR55: GPR55 is extensively expressed in human brain. Brain Res Mol Brain Res 64:193-198. 110. Sharma R, (2011). (2011) Design and synthesis of novel cannabinoids with controlled detoxification, in, Northeastern University.

112

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

111. Sim MF and Hopcroft RH (1976) Effects of various diuretic agents in the mouse. J Pharm Pharmacol 28:609-612. 112. Sim-Selley LJ, Schechter NS, Rorrer WK, Dalton GD, Hernandez J, Martin BR and Selley DE (2006) Prolonged recovery rate of CB1 receptor adaptation after cessation of long-term cannabinoid administration. Mol Pharmacol 70:986-996. 113. Singh H, Schulze DR and McMahon LR (2011) Tolerance and cross-tolerance to cannabinoids in mice: schedule-controlled responding and hypothermia. Psychopharmacology (Berl) 215:665- 675. 114. Slizgi GR and Ludens JH (1982) Studies on the nature and mechanism of the diuretic activity of the opioid analgesic ethylketocyclazocine. J Pharmacol Exp Ther 220:585-591. 115. Smith PB, Compton DR, Welch SP, Razdan RK, Mechoulam R and Martin BR (1994) The pharmacological activity of anandamide, a putative endogenous cannabinoid, in mice. J Pharmacol Exp Ther 270:219-227. 116. Sofia RD, Knobloch LC, Harakal JJ and Erikson DJ (1977) Comparative diuretic activity of delta9- tetrahydrocannabinol, cannabidiol, cannabinol and hydrochlorothiazide in the rat. Arch Int Pharmacodyn Ther 225:77-87. 117. Stott CG and Guy GW (2004) Cannabinoids for the pharmaceutical industry. Euphytica 140:83- 93. 118. Stuart GA (1911) Chinese Materia Medica: Vegetable Kingdom. 119. Tam J, Vemuri KV, Liu J, Bátkai S, Mukhopadhyay B, Godlewski G, Osei-Hyiaman D, Ohnuma S, Ambudkar SV, Pickel J, Makriyannis A and Kunos G (2010) Peripheral CB1 cannabinoid receptor blockade improves cardiometabolic risk in mouse models of obesity. J Clin Invest 120:2953- 2966. 120. Tanda G and Goldberg SR (2003) Cannabinoids: reward, dependence, and underlying neurochemical mechanisms--a review of recent preclinical data. Psychopharmacology (Berl) 169:115-134. 121. Tasker J (2004) Endogenous cannabinoids take the edge off neuroendocrine responses to stress, in Endocrinology pp 5429-5430, United States. 122. Thakur GA, Bajaj S, Paronis C, Peng Y, Bowman AL, Barak LS, Caron MG, Parrish D, Deschamps JR and Makriyannis A (2013) Novel Adamantyl Cannabinoids as CB1 Receptor Probes. J Med Chem 56:3904-3921. 123. Thomas BF, Gilliam AF, Burch DF, Roche MJ and Seltzman HH (1998) Comparative receptor binding analyses of cannabinoid agonists and antagonists. J Pharmacol Exp Ther 285:285-292. 124. Tyrey L and Murphy LL (1984) Effects of delta-9-tetrahydrocannabinol on reproductive neuroendocrine function in the female: animal studies. NIDA research monograph 55:42-51. 125. Vapaatalo H and Karppanen H (1969) Combined toxicity of ethanol with chlorpromazine, diazepam, chlormethiazole or pentobarbital in mice. Agents Actions 1:43-45. 126. Vaupel DB, Cone EJ, Johnson RE and Su TP (1990) Kappa opioid partial agonist activity of the enkephalin-like pentapeptide BW942C based on urination and in vitro studies in humans and animals. J Pharmacol Exp Ther 252:225-234. 127. Vonvoigtlander PF, Lahti RA and Ludens JH (1983) U-50,488: a selective and structurally novel non-Mu (kappa) opioid agonist. J Pharmacol Exp Ther 224:7-12. 128. Walczak JS and Cervero F (2011) Local activation of cannabinoid CB(1) receptors in the urinary bladder reduces the inflammation-induced sensitization of bladder afferents. Mol Pain 7:1744- 8069. 129. Walczak JS, Price TJ and Cervero F (2009) Cannabinoid CB1 receptors are expressed in the mouse urinary bladder and their activation modulates afferent bladder activity. Neuroscience 159:1154-1163.

113

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

130. Welch SP, Huffman JW and Lowe J (1998) Differential blockade of the antinociceptive effects of centrally administered cannabinoids by SR141716A. J Pharmacol Exp Ther 286:1301-1308. 131. Wiley JL and Martin BR (2003) Cannabinoid pharmacological properties common to other centrally acting drugs. Eur J Pharmacol 471:185-193. 132. Wiley JL, O'Connell M M, Tokarz ME and Wright MJ, Jr. (2007) Pharmacological effects of acute and repeated administration of Delta(9)-tetrahydrocannabinol in adolescent and adult rats. J Pharmacol Exp Ther 320:1097-1105. 133. Wiley JL, Smith FL, Razdan RK and Dewey WL (2005) Task specificity of cross-tolerance between Delta9-tetrahydrocannabinol and anandamide analogs in mice. Eur J Pharmacol 510:59-68. 134. Xie C and Wang DH (2009) Ablation of transient receptor potential vanilloid 1 abolishes endothelin-induced increases in afferent renal nerve activity: mechanisms and functional significance, in Hypertension pp 1298-1305, United States. 135. Yu XH, Cao CQ, Martino G, Puma C, Morinville A, St-Onge S, Lessard E, Perkins MN and Laird JM (2010) A peripherally restricted cannabinoid receptor agonist produces robust anti-nociceptive effects in rodent models of inflammatory and neuropathic pain. Pain 151:337-344. 136. Zimmer A, Zimmer AM, Hohmann AG, Herkenham M and Bonner TI (1999) Increased mortality, hypoactivity, and hypoalgesia in cannabinoid CB1 receptor knockout mice. Proc Natl Acad Sci U S A 96:5780-5785.

114

Doctoral Dissertation, Northeastern University Investigator: Girish Rajmal Chopda

G. Appendix:

Completion of Investigator assessment quiz for working with research animals

User: Girish Chopda

Submitted: 10/08

Name: Investigator Assessment Quiz

Status: Completed

Score: 100 out of 100 points

Instructions: This test consists of 20 multiple choice and/or True/False questions. You must answer all questions. You will be notified at the end of the test whether you passed (hopefully) or failed. 70% of the questions must be answered correctly to pass. If you fail you must read the training module and take the test again. If you pass, you will be given approval from the NU-IACUC and the DLAM to work with research animals at Northeastern University.

115