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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

ii

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: _____________


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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.


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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

1.1 – History

1.2 – Cannabinoid receptors

1.3 – Endocannabinoid system

1.4 – Endocannabinoid chemistry

1.5 – In vivo effects of cannabinoids

1.6 – Cannabinoids in clinical use

7

7

8

10

12

19

21

Chapter 2 – Cannabinoid mediated diuresis in mice

2.1 - Introduction

2.1.1 – Cannabinoid and diuresis

2.1.2 – Cannabinoid receptors in the urinary system

2.1.3 – Standard diuretics

2.2 – Aim and rationale

2.3 – Material and methods

2.3.1 – Animals

2.3.2 – Diuresis

2.3.3 – Measurement of urine pH, Na+, K

+ and Cl

-

2.3.4 – Drugs

2.3.5 – Statistical analysis

2.4 – Results

2.4.1 – Validating diuresis

2.4.2 – Cannabinoid mediated diuresis

2.4.3 – Receptor mechanisms of cannabinoid mediated diuresis

2.4.4 – Urine analysis

2.5 – Discussion

2.5.1 – Validation of diuresis

2.5.2 – Cannabinoid mediated diuresis

23

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23

24

27

28

29

29

29

29

29

30

31

31

35

39

50

52

52

53

Chapter 3 - Cannabinoid mediated antinociception in mice

3.1 – Introduction

3.1.1 – Cannabinoid antinociception



3.3.1 – Animals

3.3.2 – Antinociception

3.3.3 – Drugs


3.4 – Results

59

59

59

61

62

62

62

63

63

64


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3.4.1 – Effects of cannabinoid agonists on antinociception

3.4.2Effects of antagonist pretreatment

3.5 – Discussion

Chapter 4 – Cannabinoid mediated tolerance

4.1 – Introduction

4.1.1 – Drug tolerance

4.1.2 – Cannabinoid and tolerance



4.3.1 – Animals

4.3.2 – Antinociception

4.3.3 – Diuresis

4.3.4 – Binding assay

4.3.5 – Drugs


4.4 – Results

4.4.1 – Tolerance to diuresis

4.4.2 – Tolerance to antinociception

4.4.3 – Changes in CB1 receptor levels

4.5 – Discussion

E. Conclusions

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85

86

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87

87

88

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93

97

99

103

F. Bibliography

107

G. Appendix: Laboratory Safety Training

115


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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.


2

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.


3

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


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,


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


4

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


5

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


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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


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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


8

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


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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,


10

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


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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


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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 Δ

8-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;


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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 Δ

9 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

http://www.drugs-forum.com/forum/showthread.php?t=117873


14

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.


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A) THC B) WIN55,212-2

C) AM7418 D) AM4054

Figure 2: Structures of cannabinoid agonists


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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.


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A) Rimonabant (SR141716A) B) AM6545

NN

NH

Cl

O

Cl

N

S

NC

OO

AM6545

C) AM630

Figure 3: Structures of cannabinoid receptor antagonists


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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


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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


20

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


21

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 ∆-9

THC 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.


22

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.


23

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


24

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


25

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


26

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.


27

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).


28

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.


29

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-


30

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.


31

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.


32

0 10 300

10

20

30

40

Saline injected (ml/kg)

Uri

ne (

g/k

g)

0 2 4 6 8 100

10

20

30

40

14

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).


33

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).


34

0

10

20

30

40

50

*

***

***

***

***

***

***

Furosemide

U-50,488

***

1.0 3.0 10.0 30.0 60.0V

Dose (mg/kg)

Uri

ne (

g/k

g)

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).


35

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.


36

0

1 0

2 0

3 0

4 0

V

***

***

0 .0 3 0 .1 0 .3 1 .0 0 .0 1

A M 7418

A M 4054

D o s e (m g /k g )

Urin

e (

g/k

g)

0

1 0

2 0

3 0

4 0

*****

V 1 .00 .3 3 .0 1 0 3 0 1 0 0

***

W IN 5 5 ,2 1 2 -2

9T H C

D o s e (m g /k g )

Urin

e (

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).


37

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.


38

0

1 0

2 0

3 0

4 0

V 0 .0 3 0 .1 0 .3 1 .0 0 .0 1

M a le

A M 4 0 5 4 (m g /k g )

Urin

e (

g/k

g)

F e m a le

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


39

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


40

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.


41

0

1 0

2 0

3 0

4 0

1 .00 .30 .10 .0 3 3 .0

A M 4 0 5 4 (m g /k g )

Urin

e (

g/k

g)

0

1 0

2 0

3 0

4 0

0 .0 m g /k g

1 .0 m g /k g

3 .0 m g /k g

1 0 .0 m g /k g

1 .0 3 .0 1 0 .0 3 0 .0 1 0 0 .0

T H C (m g /k g )

Urin

e (

g/k

g)

+ R im o n a b a n t

Figure 9: Dose response curves for THC and AM4054 on diuresis after 30 min pretreatment

with rimonabant or respective vehicle (n = 7-8).


42

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

response curves.

ED50 (mg/kg) a Potency Ratio

b

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


43

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

the AM4054 dose response curves.

ED50 ( mg/kg) a Potency Ratio

b

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





44

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.


45

0

1 0

2 0

3 0

4 0

5 0

F u ro s e m id e


U -5 0 ,4 8 8


1 .0 3 .0 1 0 .0 3 0 .0 1 0 0 .0

R im o n a b a n t

D o s e (m g /k g )

Urin

e (

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).


46

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.


47

0

10

20

30

40

V 0.03 0.1 0.3 1.0 3.0

3.0 mg/kg

0.0 mg/kg

+ AM6545

10.0 mg/kg

AM4054 (mg/kg)

Uri

ne

(g

/kg

)

Figure 11: AM4054 dose response curve 30 min after 0, 3 or 10 mg/kg AM6545, a peripherally

selective CB1 antagonist (n = 8).


48

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.


49

0.0

0.1

1.0

10.0

0

10

20

30

40

Uri

ne

(g

/kg

)

0.0

3.0

10.0 0.

010

.0

0.1mg/kg AM4054

10.0 mg/kg THC

100.0 mg/kg THC

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).


50

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.


51

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

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

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


52

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


53

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


54

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


55

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


56

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.


57

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


58

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.


59

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.


60

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.


61

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.


62

3.3 Materials and Methods:

3.3.1 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.

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.


63


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.


64

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).


65

0 1 2 3 4 5 6

0

20

40

60

80

100 0.1 mg/kg`

0.3 mg/kg

1.0 mg/kg

AM4054

Time (hr)

% M

PE

0 1 2 3 4 5 6

0

20

40

60

80

1000.3 mg/kg

1.0 mg/kg

AM7418

Time (hr)

% M

PE

Figure 13: Time course for AM4054 and AM7418 antinociception after single injections with

the respective doses as listed in legends (n = 6-8).


66

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.


67

0

20

40

60

80

100

0.1 0.3 1.0

Single dosing

Cumulative dosing

AM4054 (mg/kg)

% M

PE

Figure 14: Comparison of antinociception dose response curve after single dose or cumulative

dosing following AM4054 (n = 8).


68

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).


69

0

20

40

60

80

100

0.03

AM4054

THC

AM7418

WIN-55212

0.1 0.3 1.0 3.0 10.0 30.0 100.0

Saline

Dose (mg/kg)

%M

PE

Figure 15: Cumulative dose response curves for cannabinoid agonists in the mouse hot water

tail-withdrawal assay (n = 7-8).


70

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)

(95% CI)

0.3

(0.2, 0.4)

0.3

(0.2, 0.4)

2.7

(1.9, 3.7)

9.3

(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).


71

0

20

40

60

80

100Morphine

Pentobarbital

U 50,488

0.1 0.3 1.0 3.0 10.0 30.0 100.0

Dose (mg/kg)

%M

PE

Figure16: Cumulative dose response curves for non-cannabinoid compounds in the mouse hot

water tail-withdrawal assay (n = 7-8).


72

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.


73

0

25

50

75

100

0.1 0.3 1.0 3.0 10.0

3.0 mg/kg

0.0 mg/kg

1.0 mg/kg

10.0 mg/kg

+ Rimonabant

AM4054 (mg/kg)

% M

PE

0

25

50

75

100

10.0 30.0 100.0 300.0

0.0 mg/kg

3.0 mg/kg

10.0 mg/kg30.0 mg/kg

1.0 mg/kg

+ Rimonabant

THC (mg/kg)

% M

PE

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).


74

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.


75

Table 6

AM4054 THC

ED50 (mg/kg)a Potency Ratio

b 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




c 95% CI were not determined because ED50 value was calculated by interpolation of two points


76

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.


77

0

25

50

75

100

10.0 30.0 100.0 300.0

0.0 mg/kg

10.0 mg/kg

3.0

+ AM6545

THC (mg/kg)

% M

PE

0

25

50

75

100

0.0 mg/kg

3.0 mg/kg

0.3 1.0 3.0 10.00.1

10.0mg/kg

+ AM6545

AM4054 (mg/kg)

%M

PE

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).


78

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


79

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,


80

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


81

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.


82

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


83

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


84

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.


85

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.


86

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.


87

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 [125

I]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 [125

I]AM281 was measured to obtain the total radioactivity count and

determine the specific activity. All the samples were run in duplicates.


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.


88

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.


89

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.


90

1 3 5 7 9

0

1 0

2 0

3 0

4 0 V e h ic le

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

D a y s

Urin

e (

g/k

g)

1 3 5 7 9

0

1 0

2 0

3 0

4 0

D a y s

Wa

ter i

nta

ke

/ca

ge

(g

)

1 3 5 7 9

0

1

2

3

D a y s

We

igh

t lo

ss

(g

)

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).


91

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).


92

0

10

20

30

40

7 day 10 mg/kg THC

7 day vehicle

1.0 3.0 10.0 30.0 100.0

14 day post 7 day 10 mg/kg THC

THC (mg/kg)

Uri

ne (

g/k

g)

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).


93

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).


94

1 3 5 7 9

0

2 0

4 0

6 0

8 0

1 0 0

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

D a y s

% M

PE

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


95

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.


96

0

20

40

60

80

100

7 day vehicle

7 day 10 mg/kg THC

14 day post 7 day 10 mg/kg THC

1.0 3.0 10.0 30.0 100.0

THC (mg/kg)

% M

PE

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).


97

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).


98

0 30 60 90 120 1500.000

0.005

0.010 7 day vehicle

7 day 0.1 mg/kg AM2389

7 day 10.0 mg/kg THC

Bound [pmol/mg]

Bo

un

d/F

ree

0

500

1000

1500

2000

-12 -10 -8 -6 -40

AM281 [M]

CP

M (

tota

l b

ind

ing

)

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).


99

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


100

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).


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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


102

peripheral CB1 receptors are involved in producing the decreases in diuresis produced by

cannabinoid agonists in mice.


103

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-


104

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


105

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.


106

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.


107

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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.

cannabinoid mediated diuresis in mice - drsrx917h89d/fulltext.pdf · cannabinoid mediated diuresis...

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