CLOMIPRAMINE IN DOGS: PHARMACOKINETICS, NEUROCHEMICAL

EFFECTS, AND EFFICACY IN COMPULSIVE DISORDER

A Thesis

Presented to

The Faculty of Graduate Studies

of

The University of Guelph

by

CAROLINE J. HEWSON

ln partial fulfillment of requirements

for the degree of

Doctor of Philosophy

June, 1997

Q Caroline J. Hewson, 1997 Acquisitions and Acquisitions et Bibliographie SeMces services bibliographiques 395 Wellington Street 395, rue Wellington ûüawa ON K1A ON4 OttawaON K1AON4 Canada Canada

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The author retains ownership of the L'auteur conserve la propriété du copwght in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantid extracts from it Ni la thèse ni des extraits substantiels may be printed or otherwise de ceilen ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. ABSTRACT

Clomipramine in Dogs: Pharmacokinebics, Neurochemical Effects, and

EffÎcacy in Compulsive Disorder

Caroline Joan Hewson Co-advisors: University of Guelph. 1997 RO Bal& UA Luescher

Canine compulsive disorder is a syndrome of abnomal conflict behaviours,

possibly associated with central neurochemical dysfunction. There is no proven

treatment for the disorder, but the human anti-compulsive drug, clomipramine,

has been reported to be effective. Three expenments investigated biological and

behavioural effects of clomipramine in dogs. Experïrnent 1 was a

pharmacokinetic study. Six dogs received one oral dose (3 mgkg) of

clomipramine. After dosing, blood was taken at O, 15, 30,45min. 1, 2, 4. 8, 12h,

and then 12-hourly up to 120h. The dogs then received 28 daily doses (3mglkg

q 24 h); on d28, they were sampled as before. There was interdog variability in

the elimination half-life of clomipramine (1-9h) and its metabolite,

desmethylclomipramine (1-4h). Experiment 2 used six other, behaviourally

normal dogs to assess the effect of clomipramine (3 mglkg q 24 h PO) on turnover of serotonin, dopamine and norepinephrine, as indicated by concentrations of the respective metabolites in cerebrospinal fiuid (CSF). The experiment had a randomised, placebo-controlled, crossover design. CSF was taken after 1,2,4 and 6 weeks on each treatment: there was a 2-week washout period between treatments. No effect on neurotransrnitter turnover was detected. Experiment 3 was a randomised, placebo-controlled, double-blind, crossover clinical trial of clomipramine (3 rng/kg q 12h PO). Cases of canine compulsive disorder were diagnosed by a clinical ethologist. following telep hone interviews with owners. An independent rater also interviewed owners, using formal criteria to diagnose compulsive disorder. Diagnostic agreement between the ethologist and the criteria was low (~=0.02;n=60). Fifty-one cases were enrolled in the clinical trial. They received each treatment for 4 weeks, with a 2- week washout perÏod in-between. At the end of each treatment, owners rated the severity of the compulsive behaviour using hnro scales of acceptable construct validity (p4.01). Both scales indicated a treatment effect (odds ratio=4; pe0.015, n=50). These experimentç contribute new information about the pharmacokinetics, neurochemical effect and clinical use of clomipramine in dogs. They describe the development of a valid measure of behavioural change, and demonstrate that clomipramine is a useful treatrnent for canine compulsive disorder. ACKNOWLEDGEMENTS

My sincere thanks to all those who have made this research possible.

In particular, I thank my advisory cornmittee (DE. Ron Ball, Andrew Luescher,

Peter Conlon and Joane Parent) for their guidance and good humour. My

thanks also ta the Departrnents of Population Medicine, and Animal and Poultry

Science for their support.

I am deeply grateful to the sponsors of my research. Persona1 funding

was provided by the Canadian Commonwealth Scholarship Program and by

scholanhips from the University of Guelph and the Ontario Veterinary College.

The research funds were provided by Novartis Animal Health, Pet Trust and the

Animal Health Trust of Canada.

I acknowledge the contribution of al1 the participating dogs and their owners and handlers. Thanks also to the Central Animal Facility. and to Jodie

Karrow, Heather Cole, Jacky Adarnek. Craig Moseley and Cindy Leonard, al1 of whom provided excellent technical assistance. To VictorÏa Edge, Mohamed

Shoukri and Andrew Milan for statistical advice. To Muriel Burke and al1 in the

Clinical Research Building for their support. To al1 who have supported and encouraged me, including fellow students, family and fnends. Special thanks to

Christine Power, Tarcia Gennring, Ute Straube, Laura Taylor, Wiil Wistowsky,

Janet Schell, John Craven, Dee Britney, Lawrence Cahill. Nicola Pritchard,

Alison McDermott, Adrian Glasser, Patncia Harton-McCord, David and Caroline

Jordan, Anne Valliant, Don McKeown and Jack Halip.

CHAPTER 3 : THE PHARMACOKINETICS OFCLOMIPRAMINEAND DESMFTHYLCLOMIPRAMINE 1N DOGS: PARAMETER ESTIMATES FOLLOWING A SINGLE ORAL DOSE AND 28 CONSECUTIVE DAILY ORAL DOSES OF CLOMIPRAMINE

3.1 lntroduction ...... 32

3.2 Materials and Methods ...... 34 3.2.1 Single-dose study ...... 35 3.2.2 Multipledose study ...... 35 3.2.3 Handling of samples ...... 36 3.2.4 Detenination of clomipramine and de~rnethylclorniprarnine~~~~.~-~~~~~~--.36 3 .2.5 Pharmacakinetic analysis ...... **...... *...... *.....*~.... 38

3-3 Results ...... 39

3.4 Discussion ...... 52

CHAPTER 4: THE EFFECT OF CLOMIPRAMINE ON THE TURNOVER OF CENTRAL MONOAMINE METABOLITES IN BEHAVlOURALLY NORMAL DOGS

Introduction...... -59

4.2 Materials and Methods ...... 61 4.2.1 Sampling technique ...... 62 4.2.2 Sample handling ...... 63 4.2.2.1 CSF ...... 63 4.2.2.2 Blood ...... 64 4.2.3 Chrornatographic Analysis - monoamine metabolites ...... 65 4.2.3.2 5-HIAA and HVA ...... -66 4.2.3.2 MHPG...... 66 4.2.4 Determination of clomipramine and desmethylclomipramine ...... -67 4-25 Statistical Analysis ...... -67

4.3 Results ...... 69 4.4 Discussion ...... 6

CHAPTER 5: DIAGNOSIS OF CANINE COMPULSIVE DISORDER: EVALUATION OF THE DIAGNOSTIC ACCURACY OF FORMAL CRITERIA

Introduction ...... 83

5.2 Materials and Methods ...... 84 5.2.1 Recruitment of Cases ...... -84

iii 5.2.2 Diagnostic methods ...... 84 5.2.2.1 Interview #1 ...... -85 5.2.2.2 Interview #2 ...... 86 5 2.3 Measurement of diagnostic accuracy ...... 86

5.3 Results ...... 89 5.3.1 Description of the sample ...... 89 5.3.1 -1 Sarnple size ...... 89 5 .3.1.2 Gender ...... 89 5.3.1.3 Age ...... 89 5 -3-1 -4 Breed ...... 89 5.3.1 -5 Behaviour ..,...... -90 5.3.2 Diagnostic agreement ...... 92

5.4 Discussion ...... 96

CHAPTER 6: DEVELOPMENT OF TWO QUESTlONNAlRES TO MEASURE CHANGE IN BEHAVlOURAL SEVERITY IN DOGS WiTH COMPULSIVE DISORDER

6.1 Introduction ...... 1O0

6.2 Materials And Methods ...... ,...... -1 O1 6.2.1 Telephone questionnaire ...... 102 6.2.1.1 Item seledion and scaling ...... 102 6 .2.1 -2 Pre-Testing ...... -1 05 6.2.2 Owner-administered questionnaire...... *....1 05 6.2.2.1 Item selection and scaling ...... 105 6.2.2.2 Pre-testing ...... 106 6.2.3 Data collection ...... ,., ...... 1 06 6.2.4 Data analysis ...... *...... **...... *..*...... 1 07 6.2.4.1 Missing data ...... 1 10

6.3 Results ...... 1 10

6.4 Discussion ...... 111

CHAPTER 7 A RANDOMISED CONTROLLED CLlNlCAL TRIAL OF CLOMIPRAMINE AS A TREATMENT FOR CANINE COMPULSIVE DISORDER

7.1 Introduction ...... 1 19

7.2 Materials and Methods ...... 120 7.2.1 Recruitrnent of cases ...... -120 .. 7.2.2 Assessrnent of eligibility...... 121 7.2.3 Trial design ...... 123 7.2.4 Trial conduct ...... 127 7.2.4.1 Follow-up ...... 128 7.2.5 Data analysis ...... 128 7 .2.5. 1 Effectiveness of masking ...... 130

7-3 ResuIts ...... 130 7.3.1 Description of sarnple ...... 131 7.3.1 -1 Sex ...... ,...... 131 7.3.1 -2 Age ...... 131 7.3.1.3. Breed: ...... 131 7.3.1.4 Behaviour ...... ,...... 132 7.3.2 Clinical evaluation ...... 134 7.3.3 Statistical analysis ...... *...... *.....***.....*....*.....*-134 7.3.4 Cornpliance ...... -136 7.3.5 Adverse Effects ...... 1 37 7.3.6 Effectiveness of masking ...... 2 7.3.7 Fo~~ow-UP......

7.4 Discussion ...... 144

CHAPTER 8 SUMMARY AND FUTüRE RESEARCH

8.1 Description of affected subjectç ...... 152

8.2 Use of diagnostic criteria that exclude other disorders ...... 152

8.3 Laboratory studies ...... -153

8.4 Response to treatment and follow-up studies ...... 155 8.4.1 Pharmaco kinetics and response to treatment ...... 55 8.4.2 Measurement of response to treatment ...... 156 8.4.3 Clinical significance of response to treatrnent ...... 157

REFERENCES ...... 158

APPENDIX 1 Results of statistical model-building (general linear rnodel; backwards elimination) in a placebo-controlled crossover experiment to examine the effect of clomipramine on central turnover of serotonin. dopamine and norepinephrine ...... 170

APPENDIX 2 Consent fom and information about a randomised controlled double blind trial of clomiprarnine in canine compulsive disorder ...... -174 APPENDIX 3 Preliminary questionnaire sent to owners of dogs suspected of having canine compulsive disorder...... 7 78

APPENDIX 4 Telephone questionnaire used to obtain a behavioural case history of dogs suspected of having canine compulsive disorder...... 180

APPENDIX 5 Telephone questionnaire used at the behaviour service of the Ontario Veterinary College to obtain a behavioural history ...... 197

APPENDIX 6 Telephone questionnaire used to measure outcorne in a randomised controlled double-blind clinical trial of clomipramine as a treatrnent for canine compulsive disorder ...... 202

APPENDIX 7 Questionnaire used by owners to score the ffequency, duration and context of their dogs' compulsive behaviour during a randomised double-blind clinical trial of clornipramine...... 21O

APPENDIX 8 Checklist used by veterinarians to evaluate dogs with compulsive disorder, prior to en rollment in a clinical trial of clomipramine ...... 215

APPENDIX 9 Part of the randomisation Iist for a placebo-controlled clinical trÏal of clomipramine in dogs with compulsive disorder...... 217

APPENDlX 10. Information provided to owners of dogs with compulsive disorder, about how to unmask treatrnent allocation in a double-blind, randomised controlled clinical trial of clomipramine ...... 21 8

APPENDIX 11. Calendar used by owners of dogs with compulsive disorder, during a randomised, placebo - controlled, double-blind clinical trial of clomipramine (3rngkg q 12 h)...... 21 9

APPENDIX 12 Information supplied to the veterinarians attending dogs with compulsive disorder during a randomised, placebo-controlled, double-blind, clinical trial of clomipramine...... -.221

APPENDIX 13 The results of statistical model-building (ordinal regression, proportional odds model, stepwise selection) in a randomised placebo- controlled clinical trial of clomipramine in dogs with compulsive disorder...... 225

APPENDIX 14 Descriptive data on 75 dogs diagnosed with canine compulsive disorder by expert opinion...... 227 LIST OF TABLES

Table 2.1 Possible breed predispositions for canine compulsive disorder ...... 1 3 Table 2.2 Diagnoses proposed for dogs that show air-biting or tail-chasing behaviours ...... 17

Table 3.1 Pharmacokinetic parameter estimates (one-compartmental model) for plasma clomipramine in two dogs following a single oral dose of clomipramine (3 mgkg) ...... 47

Table 3.2 Pharmacokinetic parameter estknates (non-cornpartmental model) for plasma clomiprarnine in four dogs following a single oral dose of clomipramine (3 mgkg) ...... 48

Table 3.3 Pharmacokinetic parameter estimates (one-cornpartmental model, assuming equal rates of absorption and elimination) for plasma clornipramine in one dog following 28 consecutive daily oral doses of clomipramine (3 mgkg q 24 h)...... 49

Table 3.4 Pharmacokinetic parameter estimates (non-compartrnental model) for plasma clomiprarnine in five dogs following 28 consecutive daily oral doses of clomipramine (3 mgkg q 24 h) ...... 49

Table 3.5 Pharmacokinetic parameter estimates (one-compartmental model, assuming equal rates of absorption and elimination) for plasma desmethylclomipramine in three dogs following a single oral dose of clomipramine (3 mgkg)...... -50

Table 3 -6 Phamacokinetic parameter estimates (one-corn partmental model, assuming unequal rates of absorption and elimination) for plasma desmethylclomipramine in one dog following a single oral dose of clomipramine (3 mgkg)...... 50

Table 3.7 Pharmacokinetic parameter estirnates (one-compartmental model, assuming equal rates of absorption and elimination) for plasma desmethylclomipramine in five dogs following 28 consecutive daily doses of clomipramine (3 mgkg q 24 h PO)...... *...... *.....*...... 51

Table 3.8 Pharmacokinetic parameter estirnates (one-compartmental model, assuming unequal rates of absorption and elimination) for plasma desmethylclomiprarnine in one dog following 28 consecutive daily doses of clomipramine (3 mgkg q 24 h PO)...... 51

vii Table 4.1 Mean (SD) concentrations of 5-hydroxyindoleacetic acid (5-H IAA), homovanillic acid (HVA) and 3-methoxy 4-hydroxyphenylgtycol (MHPG) in the cerebrospinal fiuid of six dogs, following treatment with clomipramine (3mglkg q 24 h) and placebo ...... 71

Table 4.2. Mode1 parameters and estimates of the effect of clomipramine (3 mglkg q 24 h for 6 weeks) on the monoamine metabolites 5-HIAA, HVA and MHPG in canine cerebrospinal fluid, in a repeated measures AB-BA crossover experiment...... 74

Table 4.3 Mean (SD) concentration of clomipramine and desmethylclomiprarnine in plasma of 6 dogs during treatment with oral clomiprarnine (3 mglkg q 24 h) for six weeks...... 75

Table 4.4 Product moment correlation coefficients behveen the concentrations of clomipramine and desmethylclomiprarnine in plasma and the concentrations of 5-HIAA, HVA and MHPG in canine CSF during six weeks of treatrnent with oral clomipramine (3 rnglkg q 24 h)...... 75

Table 5.1 Criteria on which a formal diagnosis of canine compulsive disorder was made (after Hewson & Luescher (1 996).) ...... 87

Table 5.2 2 x 2 table to illustrate how measures of diagnostic agreement were calculated in a study comparing formal diagnostic criteria for canine CD with an expert's diagnosis ...... 88

Table 5.3 Behaviours and breeds of 82 dogs that were referred for diagnosis for possible canine CD ...... A

Table 5.4 Breakdown of the diagnoses of canine compulsive disorder made by an expert, and wÏth three diagnostic criteria...... 93

Table 5.5 2 x 2 table and indices of agreement between diagnoses of canine CD made by expert opinion and diagnoses made by formal criteria ...... 94

Table 6.1 Two global questions about behavioural severity, used as outcome measures in a clinical trial of clomipramine in canine CD...... 103

Table 6.2 Use of behavioural ratings from a 5-point Likert scale (Question 3) to describe change in behavioural severity...... 109

Table 6.3 Constnict validity of a five-point rating scale (Question 3) that measured behavioural change in a crossover clinical trial (n = 50)...... 11 3 Table 6.4 Constnict validity of a comparative 10-point rating scale (Question 15) that measured behavioural change in a crossover clinical trial (n = 50)...... 114

Table 7.1 Two global questions about behavioural severity, used as outcome measures in a clinical trial of clomipramine as a treatment for canine CD...... ,...... 126

Table 7.2 Behaviours and breeds of 51 dogs with canine compulsive disorder that participated in a clinical trial of clornipramine ...... 133

Table 7.3 Final results of analysing clinical trial data by ordinal regression (proportional odds model, stepwise selection)...... 1 37

Table 7.4 Data from dogs that showed identical occurrence of adverse effect on dnig and placebo during a randomised, placebo- controlled. double-blind, crossover clinical trial of clomipramine...... 138

Table 7.5 Data from dogs that showed different occurrence of adverse effects on dnig and placebo during a randomised, placebo- controlled, double-blind, crossover clinical trial of clomipramine ...... 139

Table 7.6 2 x 2 table and indices of agreement between owners' perceptions of treatment and true treatment allocation in Period 1...... 142

Table 7.7 2 x 2 table and indices of agreement between owners' perceptions of treatrnent and true treatrnent allocation in Period 2 ...... 143

Table 7.8 2 x 2 table and indices of agreement between owners' perceptions of treatrnent sequence and true treatment allocation over both periods ...... -......

Table 7.9 Results of follow-up on 51 dogs with compulsive disorder that participated in a randomised placebo-controlled clinical trial of clomipramine ...... -145

Table 7.1 0 Data from 13 cases of canine compulsive disorder that continued to receive clomipramine (CMI; 3mg/kg q 12h PO) following a randomised placebo-controlled clinical trial of the dnig ...... 146

Table Al. 1. Mode1 parameters and estimates of the effect of clomipramine (3 mgkg q 24 h for 6 weeks) on the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) in canine cerebrospinal fiuid, in a repeated measures AB-BA crossover experiment ...... -171 Table A1 -2. Model parameters and estimates of the effect of clornipramine (3 mgtkg q 24 h for 6 weeks) on the dopamine metabolite homovanillic acid (HVA) in canine cerebrospinal fiuid, in a repeated measures AB-BA crossover experiment ...... 172

Table A1 -3. Model parameters and estimates of the effect of clomipramine (3 mgkg q 24 h for 6 weeks) on the norepinephrine metabolite 3-methyl4-hydroxyphenylgiycol (MHPG) in canine cerebrospinal fluid. in a repeated measures AB-BA crossover experiment ...... 1 73 LIST OF FIGURES

Figure 3.1 Mean (+ SD) plasma concentrations of clomipramine (a) and desmethylclornipramine (O) in six dogs (0 following a single daily dose of clomipramine (3rngkg PO), and (ii)after 28 consecutive daily doses of clomiprarnine (3rngJkg q 24 h PO) ...... 41

Figure 3.2 Semilog concentration-time plots of plasma concentrations of clomipramine (a) and desmethylclomiprarnine (O)in six dogs following a single oral dose of cfornipramine (3 mgkg)...... 42

Figure 3.3 Sernilog concentration-tirne plots of plasma concentrations of clomipramine (a) and desmethylclornipramine (O)in six dogs after the last of 28 consecutive daily oral doses of clomipramine (3 mgkg q 24 h) ...... 44

Figure 3.4 Mean (+ SD) plasma concentrations of clornipramine (a) and desmethylclomipramine (O) in six dogs dosed daily with clomipramine (3mgkg q 24 h PO) for 26 days...... s3

Figure 4.1 Mean withindog concentrations of 5-HIAA and HVA in cerebrospinal fiuid during storage at 4" C-...... 70

Figure 4.2 Mean betweendog concentrations of 5-HIAA and HVA in cerebrospinal fluid during storage at 4°C (n = 6)...... 70

Figure 4.3-Figure 4.5. Mean treatrnent contrasts (+ 95% confidence inte~als) of 5-HIAA, HVA and MHPG in canine CSF during six weeks of treatment with clomipramine (3mglkg q 24 h) and placebo in a balanced AB-BA crossover experiment (n = 6) ...... 72

Figure 6.1 Median between-penod ratios of behavioural frequency, duration and context for three categories of between-period change in behavioural severity...... 112

Figure 7.1 Entry criteria for a dinical trial of clomipramine as a treatment for canine compulsive disorder...... 122

Figure 7.2 Bar charts showing the nuniber of dogs that were 'Better in the second period', 'The same in both pends' or 'Worse in the second period' in two treatrnent sequences, (clomipramine-placebo) and (placebo-clomipramine)...... *...... *...... ***..*.* 135 CHAPTER 1: INTRODUCTION

There is a behavioural syndrome in dogs that is comparable to spontaneously-

occumng stereotypies in farm. laboratory and zoo animals (Kennes et al 1988,

Carlstead et al 1991, Houpt 8 McDonnell1993, Lawrence 8 Rushen 1993). The

canine syndrome includes stereotypic and non-stereotypic behaviours (Fox 1964,

Luescher et al 1991, Overall 1994), and has been given various names induding

stereotypies (Luescher et al 1991), compulsive behaviour (Fox 1964), compulsive

disorder (Hewson & Luescher 1996), obsessive compulsive disorder (Luescher et

al 1991, Overall 1992). cornplex partial seizures (Colter 1989, Dodman et al 1996)

and psychomotor seizures (Sorjonen 1992). This variety of tems indicates that

systematic research on affected dogs is required. In this thesis, the canine

syndrome will be called canine compulsive disorder (canine CD).

Canine CD typically presents as one or more of the following behaviours:

air-biting, tail-chasing, walking or ninning in a circle or a figure-eight, self-licking,

licking objects, fixation with an object or light or shadow. "checkingnthe rear end, stereotypic barking, assurnption of a frozen body position and self-mutilation.

Canine CD is of concem for several reasons. First 1 is likely that affected dogs suffer in some way; that is, their subjective experience is unpleasant and is associated with aversive leveis of arousal (Duncan et al 1993). The author's experience with approximately 120 dogs suggests that severely affected dogs are in a state of anxiety when perfoning the behaviours, sometimes whining.

Similarly, Thompson et al (1956) reported that tail-chasing Scottish terriers would bark or yelp while chasing their tails. In other species, it has been difficult to

ascertain whether stereotypies indicate srnering. This difficulty stems from

rnethodological problems and a lack of research (Duncan et al 1993). However,

untii proven otherwise, it should be assumed that stereotypies indicate some

degree of suffering (Duncan et al 1993). The same may be said of canine CD.

Canine CD is also of concem because the behavioun can be maladaptive

in various ways. Some dogs injure themselves while performing their behaviour,

either because they self-mutilate (eg acral lick dematitis) or because they

inadvertently collide with objects in their vicinw (eg while tail-chasing). In extrerne

cases of locomotory compulsive behaviours, dogs may lose weig ht Other

severely affected dogs cease to eat. An additional concem is that canine CD

disrupts the ownerdog relationship: anecdotal evidence from the Ontario

Veterinary College suggests that affected dogs are commonly euthanased or given

to humane societies, because the owners cannot cope with the behaviour

(Luescher et al 1991). Furthemore, canine CD rnay be misdiagnosed and treated as a purely somatic problem (Hewson 8 Luescher 1996). This is likely to result in

non-resolution of the behaviour, as well as being a financial and emotional cost for owners,

There are few reports of research on canine CD. The cause and development of the disorder are unknown and there is no proven treatment for affected dogs. However, drugs that inhibit serotonin reuptake are reported to be effective treatments (Rapoport et al 1992. Overall 1994, Overa111997 pp238-239); these dmgs indude clornipramine (Anafranilm) and ff uoxeu'ne (Prozacm).

This thesis describes a series of investigations into the use of clomipramine as a treatment for canine CD. The research comprised a pharmacokinetic study, a laboratory experirnent to study the neurochemical effect of clomipramine in dogs, and a randomised, controlled. double bfind clinical trial of clomipramine in which issues of canine behavioural measurement were also studied . CHAPTER 2: LITERATURE REVIEW

This chapter provides a critical review of the literature on (Ï) ethological and clinical

aspects of canine CD, (ii) possible treatments for canine CD, including

clomipramine, and (iii) methodological issues in the clinical assessment of

treatments for affected dogs.

2.1 Ethological and clinical aspects of canine CD

2.1.1 Terminology

The behaviours seen in canine CD have been called stereotypies (Luescher

et al 1991). obsessive compulsive disorder (Luescher et al 1991. Overall 1992a),

compulsive disorder (Hewson & Luescher lW6), complex partial seizures (Colter

1989, Dodman et al 1996) and psychomotor seizures (Sorjonen 1992). These

tems are now reviewed.

2.7.1.1 Stereotypy

Mason (1991) has provided critical reviews of the terni stereotypy. The terni is most commonly defined as a repetitive and invariant pattern of behaviour that serves no obvious purpose (Odberg 1978) in the context in which it is

perfoned (Luescher et al 1991). The terni is typically used to mean behaviours that are considered abnormal because they are not known to occur in free-living animals (Manning 8 Dawkins 1992, p91; Mason 1993). Such a definition of abnorrnality may be criticised because it depends on the genetic distance between

captive and wild species. Furthemore, despite the popular understanding of

stereotypy as abnormal behaviour, the term includes normal behaviour,

pharmacologically-induced behaviour and abnomal behaviour. In dogs for

example, stereotypy includes the normal behaviour of circling before lying down to

sleep, the circling induced by high doses of apomorphine (Nymark 1972), and the

spontaneously-occurring, persistent circling that has been described by owners as

a source of concem (Blackshaw et al 1994, Overall.1994).

An additional limitation of the definition of stereotypy is that it has both

descriptive (operational) and causal (functional) elements. The descriptive

elements ("repetitive and invariant") are not adequate to distinguish normal

behaviour from abnonal behaviour, as discussed above. The causal element in

the definition is the behaviouts apparent lack of purpose. Lack of purpose is a

subjective assessment which could reflect ignorance and could also absolve the

animal's care-giver from any responsibility for the behaviour. Furthermore, the lack

of purpose is not consistent with the current literature in which stereotypy is

understood to be a response of confined animais to frustration induced by their environment (Lawrence & Rushen 1993). The purpose of stereotypic behaviour

could be said to be a signal to care-givers that the environment is inadequate. 2.1.7.2 Obsessive compulsive disorder

Obsessive compulsive disorder (OCD) is a human psychiatnc disorder that

is characterised by recurrent intrusive thoughts (obsessions) and ritualistic

behaviours (compulsions) (Arnerican Psychiatrie Association 1994). As with stereotypy, OCD has a descriptive definition and the symptoms may be common to a varÏety of psychiatnc illnesses (Hollander & Wong 1994). The term OCD has been recommended for use in companion animals, on the grounds of homology with human OCD, and the Iikelihood that animals have obsessive thoughts

(Overall 1997 pp219-220). That argument is anthropocentnc and takes no account of the development, ethology or psychology of either the human or the veterinary syndromes. In addition, there is no direct evidence that the animals' behaviours stem f'rom obsessive thoughts.

2.1.1.3 Compulsive disorder

In contrast to the definitions of stereotypy and OCD, the definition of compulsive disorder postulates causality. Compulsive disorder rnay be defined as a behavioural condition that is induced by past or continued confiict or frustration, and is indicative of neurochemiml dysfunction in the brah (Luescher et al 1991.

Hewson & Luescher 1996). The behaviours may be classified as reactive and abnormal (Luescher et ai 1991), and they appear to be associated with full consciousness and a low threshold of arousal (Hewson & Luescher 1996).

Amusal rnay be considered to occur when an animal detects discrepancies between observed and expected events, and attempts to cape with the situation

by means of behavioural responses (Cabib 1993, p 129).

In the definition of compulsive disorder, confiict behaviour incfudes

behaviours that are induced by fnistradion. Fnistraüon and conflict have been reviewed by Yates (1962) and Hinde (1970). and are now discussed. Fnrstration may be defined as the state of an organisrn when it is placed in an objectively defined fistrating situation. A fnistrating situation is defined as one in which a physical barrier prevents an animal hmobtaining a physical goal by behaviours which had fomerly obtained that goal (Yates 1962, p176). Frustration may therefore be understood to occur when the environment places constraints on the expression of a highly motivated behaviour. There is likely to be individual variation in the amount of environmental constraint which is required to induce frustration. Situations of frustration for dogs have been reviewed (Luescher et al

1991). Examples include (i) social isolation, which prevents the dog from expressing species-typical behaviours and has been associated with tailthasing

(Thompson et al 1956) and with destructive behaviour (ii) thunderstoms, because the dog is unable to escape fmm the fear-inducing stimulus. and (iii) unpredictable changes in social environment such as when the owner has an irregular work schedule - frustration anses because the dog's expectations of exercise and social contact are not met-

Conflict may be understood in a social or a psychological sense. In canine

CD, confiict is used in a psychological sense and connotes a motivational state in which "two incompatible tendencies are aroused equally...." (Immelmann & Beer

1989, p57) and "tendencies to perforrn more than one activity are sirnultaneously

expressedn(McFariand 1981, p98). Only one of these tendencies rnay be .

expressed, but in a modified way (Hinde 1970, p361). Cleariy, animais are almost

always motivated to perform two or more behaviours sirnultaneously; the important

issue in confiict is the incompataibility of highly motivated behaviours. In ethology,

three types of conflict are recognised: approach-avoidance, approach-approach

and avoidance-avoidance, and these are reviewed by Hinde (1970, pp360-365).

In dogs, an example of approach-avoidance conflict would be approaching a

stranger because of territorial aggression and running away from that person

because of fear. Normal responses seen in that situation may be selfdirected

aggression and running in circles (Luescher et al 1991). An example of approach-

approach conflict would be approaching the food bowl because of hunger and

approaching the owner because of social behaviour- An exampte of avoidance-

avoidance conflict would be running from a person because of fear, and avoiding

an invisible electric fence-line because of fear. Normal behavioural responses to situations of conflict have been reviewed by Hinde (1970, ~~396421)~and have

been described in dogs (Hewson & Luescher 1996).

The definition of compulsive disorder must be regarded as a 'working' one because neither the aetiology nor the pathophysiology of the disorder are known.

However, the definition is clinically relevant because it excludes normal and pharmacologically-induced behaviours, but includes conflict behaviours mat are stereotypic and those thaï are non-stereovpic. The definition also provides an

immediate framework for atternpting treatment and prevention. Use of the terni

'compulsive' in animals can be criticised because compulsions have been defined

subjectively in humans (Baron-Cohen 1989): the person usually feels driven ta

perfom compulsions in order to reduce the anxiety that accompanies obsessions

(Amencan Psychiatnc Association 1994). A terni like 'conflict disorder' might

therefore be more appropriate for animals. However, for the purposes of this

thesis, the term compulsive disorder will be retained.

2.I.l. 4 Cornplex partial sekures

Behaviours such as tailchasing, air-biüng and generalised agg ression have

been called complex partial seizures (Colter 1989. LeCouteur & Child 1989, Dodman

et al 1996) or psychomotor seizures (Sorjonen 1992). These diagnoses are

controversial (Parker 1991; Dr. Joane Parent, personal communication). A sekure

is a clinical sign of transient deœrebration and is associated with altered consciousness. A seizure has three stages: (Ï) an initial 'aura' of variable duration

(secondsdays) characterised by behavioural changes such as restlessness or hiding, (ii) a brief 'seizure stage' or ictus (30-120seconds) in which there is a sudden increase in tone of muscle groups, and (iii) a post-ictal phase of variable duration

(rninutesdays), in which the animal has decreased awareness (Kay 1989).

Cornplex partial sekums involve an acquired focal lesion in one cerebral hemisphere

(Colter 1989). Psychomotor seizures are wmplex partÏal seizures (Cotter 1989; So rjonen 1992) that are associated with stereotyped activity (Coiter 1989). A diagnosis of seizures is confirmeci by electroencephalography. However, methodological and other difficuities prevent electroencephalograms from being used to diagnose complex parfial seizures (Coiter 1989, Klemm 1989, Sorjonen

1992).

A discussion of the seizure hypothesis of canine CD is now presented.

Clinical presentafion: the clinical presentation of air-biting and tail-chasing is not consistent with seizures (Dr. Joane Parent, personal communication). Unlike seizures, tail-chasing and air-biting are corn plex. goaldirected behaviours that are often seen when the dog is excited. Dogs can usually be distracted while showing the behaviours, and are described by owners as being 'normal' as soon as the behaviour ceases. ln addition, examination of a video-recording of the behaviour does not demonstrate an aura or a post-ictal phase (personal experience; Dr. Joane

Parent, personal communication).

A history of confiict or fnistration is essential to a diagnosis of canine CD, as currently understood, but this information has not been presented in discussions of complex partial or psychomotor seizures (Colter 1989, Sojonen 1992. Dodman et al

1996).

SimiIa&y to stereotypies in other species: some behaviou rs shown in canine

CD are stereotypic and are similar to stereotypies described in other species.

Stereotypies in farm, laboratory and zoo animals are not regarded as seizures, but as abnomal behaviours that develop in response to environmental constraints on highly motivated behaviours (Lawrence & Rushen 1993). The findings of Thompson

et al (1956) are consistent with this view; in an inhumane study, 11 Scottish Teniers

were raised in total social isolation, and eight developed a syndrome of chasing their tails in response ta external stimuli. The authors noted that littemates that had been

raised in domestic settings did not show such behaviour. From an ethological standpoint, the tail-chasing was consistent wiVi canine CD, not seizures, and was likely to have been induced by the dogs' environment.

CIomipramine and seizures: a loose argument against the seizure hypothesis cornes from the reported efficacy of the human anti-compulsive dnig, clornipramine, in dogs with tail-chasing and other behaviours (Overall 1994, Dodman et al 1996, Hewson & Luescher 1996). Clomipramine is contra-indicated in humans who have seizures (Ciba Geigy 1991); if the canine behavioun were seizures, they could be expected to intensify with clomipramine. This has never been found at the

Ontario Veterinary College, nor has it been reported (Overall 1994, 1997c, Dodman et al 1996).

2.7.7.5 Summary

The above review of teminology indicates that spontaneously-occumng, abnomal canine behaviou~,such as tail-chasing and air-biting, are not consistent with seizure activity. In addition, the behaviours are not adequately described by the temis stereotypy or obsessive compulsive disorder. Until more is known about the syndrome, an alternative and clinically useful terni is canine compulsive disorder. Epidemiological studies of canine CD have not been reported. The

prevalence of stereotypies in thoroughbred horses in Europe has been estimated

at 2% (Vecchiotti 8 Galanti 1986). and the lifetime prevalence of OCD in humans

in North Arnerica is approxhately 2.5% (Kamo et al 1988). Based on these data,

an indirect estimate of the Iifetime prevalence of canine CD in North America has

been suggested to be in the order of 2% (Hewson & Luescher 1996).

2.1.3 Aetiology

Canine CD is hypothesised to result from exposure to situations of confiict

or frustration (Hewson & Luescher 1996). Dogs are commonly exposed to these situations, but only some dogs develop abnonal behaviour. This suggests that dogs Vary in their susceptibility to the effect of the situations. Such variation has been described in bank voles and mink (Odberg 1987. Mason 1993). Individual variation would suggest a possible genetic predisposlion to develop canine CD.

The heritability of a tendency to develop canine CD has not been reported, but there is anecdotal evidence that certain breeds are predisposed to develop certain behaviours (Voith 1984, Blackshaw et al 1994, Hewson & Luescher 1996).

Possible breed predisposition for certain types of compulsive behaviour are listed in Table 2.1. Table 2.1 Possible breed predispositions for canine compulsive disorder

Breed Behaviour

Doberman Flank-sucking (Voith 1984)

German Shepherd Tail-chasing Pacing or ninning in a circle

Miniature Schnauzer Tuming and "checking" rear end

English Bull Temer Tailchashg (Dodman et al 1996; Blackshaw 1994) Freezing in one position (Dodman et al 1996)

Large breeds Self mutilation by persistent licking, usually of carpus or tarsus (Acral lick dermatitis or 'lick granuloma') .

In canine CD, it is postulated that the dog initially shows a normal response to a specific situation of confiict or fnistration (Hewson 8 Luescher 1996). The response then becomes emancipated from the original context (Odberg 1978), and is generalised to situations where there is no apparent conff ict or f'stration. but which seem to induce a state of high arousal in the animal (Hewson &

Luescher 1996). Behavioural emancipation has also been described in stereotyping bank voles, developing as the anirnals mature (Cooper & Odberg

1991). The development of the canine behaviou~should be studied, to clarify the proposed genetic and environmental causes of canine CD.

The pathogenesis of canine CD is unknown but there is indirect evidence that the disorder involves serotonin, dopamine and Bendorphins. Much of this evidence cornes ftom studies of pharmacologically-induced and spontaneously- occumng stereotypies in other species (Odberg 1986, Cooper 8 Dourish IWO,

Cabib 1993). However, pharmacologically-induced behaviours may not be adequate models of the spontaneously occumng behaviours.

2.7.4-7 Serotonin and canine CD

Support for the role of a central serotonergic dyçfunction in canine CD comes from controlled, non-randomised trials of various antidepressants in dogs with acral Iick dematitis ('lick granuloma') (Goldberger & Rapoport 7991,Rapoport et al 1992). These authors reported that dogs only responded to serotmin- reuptake inhibiting drugs. In addition, in a pilot study of bank voles, animals that showed stereotypies (n=10) had lower concentrations of the serotonin metabolite,

5-hydroxyindoleacetic acid, in the caudate nucleus of the brain than did voles that did not have stereotypies (~6)(Vandebroek et al 1995). This finding could refiect the unequal group sizes.

2.1.4.2 Dopamine and canine CD

The role of dopamine in stereotypies has been reviewed (Cabib 1993).

Work in rats indicated that two dopaminergic systems are involved in stereotypies: stereotypic locomotory behaviour was associated wlh the nucleus accumbens, but stereotypic oral and head rnovements were associated with the caudate nucleus

(Cabib 1993). In dogs, stereotypic circling can be induced with the dopamine agonist,

apomorphine (Nymark 1972). This provides indirect evidence for the involvement

of dopamine in canine CD. because circling is a manifestation of the disorder

(Luescher et al 1991. Blackshaw et al 1994). In cats. studies with apomorphine-

induced stereotypies, suggested that dopamine in the caudate nucleus was

involved in stereotypic behaviour (Cook & van Rossum 1970). In pigs and bank voles. the dopamine antagoniçt, haloperidol, inhibited the performance of naturally- occurnng stereotypies (Kennes et al 1988, von Borel18 Hurnik 1991). However, in the pilot study of Vandebroek et al. (1995). no differences in dopamine and itç metabolites (homovanillic acid and 3,edihydroxyphenylacetic acid), were detected between bank voles with stereotypies @=IO) and voles without stereotypies (n=6).

Those findings may reffect the srnall sample size.

2.7.4.3 Bendorphins and canine CD

The involvement of Bendorphins in canine CD may be hypothesised following reports that dogs with lick granuloma and tail-chasing responded to narcotic antagonists (Brown et al 1987, Dodman et al 1988b. White 1990). There are similar reports in horses (Dodman et al 1987, 1988a) and pigs (Cronin et al

1986). However, work in bank voles (Kennes et al 1988) and pigs (Cronin et al

1986) has suggested that opioids may only facilitate the development of stereotypies, but are unlikely to have a dominant role in maintaining these behaviours. 2- 1.4.4 Other evidence

There is further indirect support for the hypothesis that canine CD involves

neurochernical dysfunction. In a study that examined the effect of environmental

improvernent on bank voles with established stereotypies, the performance of

stereotypies did not diminish when the environment was 'enriched' (Cooper et al

1996). There is also anecdotal evidence that when sorne stabled horses and zoo animals have developed stereotypies and are subsequently allowed to live in

naturalistic habitats, they continue to show stereotypies. This suggests that such

anirnals may have a neurochernical dysfunction which prevents them from responding to the irnproved environments.

2 1.5 Diagnostic validity

A group of clinical signs can occur together by chance, or because of an underlying disease (Szatrnari 1992). Diagnostic validity confirms whether the signs are caused by an underlying disease (Robins & Guze 1970, Szatmari 1992). The diagnostic validity of the ternis canine CD, stereotypy and OCD needs to be established. For example, two recognised clinical presentations in dogs are persistent tail-chasing with high excitement (Crowell-Davis 1992, Blackshaw et al

1994), and air-biting (Cash & Blauch 1979, Brown 1987). 60th presentations have been identifid under many difFerent names, as shown in Table 2.2

In psychiatry, a systematic approach to diagnostic validity includes studies of heritability, neurochemistry and aetiology (Robins & Guze 1970, Feighner 1982, Table 2.2. Diagnoses proposed for dogs that show tail-chasing or air-biting behaviours

Behaviour Diagnosis Reference

Tail-chasing Compulsive disorder Hewson and Luescher 1996

Obsessive compulsive Luescher et al 1991 ; Overall 1992; disorder Overall 1997

Stereotypy Luescher et al 1991; Overall 1994; Overall 7 997

Cornplex partial seizures Colter 1989; Dodrnan et al 1996

Endorphin-related Crowell-Davis 1992 neurochemical abnormality

Leamed behaviour Croweil-Davis 1992

Aberrant motor behaviour Jones 1987

Air biting Aberrant motor behaviour Jones 1987

Response to dietary Brown 1987 allergens

Ocular disorder Cash and Blauch 1979

Cornplex partial seizures Colter 1989

Kendler 1990). A similar approach could be taken in applied ethology and shoukl include developmental studies. Once the diagnostic validity of a condition has been established, definitive diagnostic tests cm be developed. 2.1.6 Diagnosis

Two diagnostic approaches to canine compulsive disorder have been

described. One is an algorithm in which compulsive disorder is diagnosed by default

(Overall l992a). This algonthrn is the first reported formai approach to the diagnosis of behavioural conditions in animals. Algorithms could be a useful tool for those offering services in canine behaviour to the public. Overall's algorithm commences with the vaccination status of the dog and rules out somatic diseases and behavioural disorders using clinical tests. This approach is systematic and somewhat useful, but it takes no account of ethological principles. In addition. most clinicians diagnose by the hypothetico-deductive methad, not by the exhaustive approach us4 in the algorithrn (Sackett et al 1991, pp14-17). Furthemore. the starting point (vaccination status) is not direcüy relevant to the most common differential diagnoses for canine CD (Hewson & Luescher 1996). and a nurnber of the clinical tests are expensive and their diagnostic properties are unknown. The algorithm therefore has limited clinical utility in its present fom, although it is systematic and thorough.

The second diagnostic approach to canine CD is based on the hypothesised development of the disorder. The diagnosis is made from the behavioural history

(Hewçon & Luescher 1996). This technique is inexpensive and non-invasive.

Moreover, the utility of behavioural measures has been demonstrated in human clinical research and practice. For example, behavioural inventories are used in obsessive compulsive disorder (Goodman 8 Price 1992), cerebral palsy (Russell et al 1989) and autism (Szatman 1992).

This review outlines an ethological approach to understanding the syndrome called canine CD- The review indicates the need for research into the development. diagnosis. prevention and treatment of the syndrome. There is an imrnediate need for treatrnent of affected dogs and one research approach is to evaluate treatrnents for canine CD under the assumption that, as currently understood, it forms a distinct clinical entity and has diagnostic validity. Such research would benefit dogs currently. and would generate hypotheses that could be tested in studies of diagnostic validity. The next section of this review examines . possible treatrnents for canine CD, with ernphasis on clomipramine.

2.2 Treatrnents for canine CD

There is no proven treatment for canine CD. but psychotropic drugs and behavioural modification have been recomrnended (Dodman & Shuster 1994,

Hewson & Luescher 1996, Overall 1997 p312). Blackshaw (1994) stated that euthanasia was the treatment of choice in some tailchasing dogs. 2-2.1 Behavioural therapy

In human OCD, behavioural therapy is being used more wideIy and is

reported to provide better long-terni recovery than drugs (McTavish & Benfield

1990, Baer 1993). In dogs with CD, recommended behavioural therapy is

designed to remove any conflict and to counterwndition the dog (Hewson &

Luescher 1996). However, dinical experience suggests that more severely affected

dogs are SU focused on their behaviour that counterconditioning is difficult to

implernent. ln such dogs, psychotropic drugs may rnake behavioural modification

more effective.

Various dnigs have been recommended for canine CD, including phenobarbital (Dodman et al 1996), narcotic antagonists (Brown et al 1987, Dodman et al 1988b, White 1990, Crowell-Davis 1992, Overall 1992b),and antidepressants

(clorniprarnine [Overall 1Wb, Rapoport et al 1992, Hewson & Luescher 19961, imipramine, amitnptyline and doxepin [OvefaII IgWb], and fiuoxetine [Overall 1992b,

Rapoport et al i 992, Hewson & Luescher 19961). There is scant scientific evidence for the efficacy of any of these dmgs.

2.2.2- 1 Narcotic antagonists

Naltrexone, nalmefene and naloxone are narcotic antagonists, but none of them are feasible treatments for canine CD. Naltrexone was evaluated in 11 dogs with self-licking, self-chewing and scratching (Dodman et al 198813). A single subcutaneous injection (1 mgkg) of the drug improved the behaviours of 711 1

dogs for 90 minutes after injection. In another case series. 9 dogs with acral Iick

dematitis (White 1990) received oral naltrexone (2.2 mgkg q 24 h), and some

dogs irnproved. That result is surprising because naltrexone has an elimination

haif-Ife of 47-85 minutes in dogs (Pace et al 1979, Garrett & el-Koussi 1985). The drug was associated with severe pruritus in one dog (Schwartz 1993). Nalmefene

(14 mgkg SC) was also reported to improve self-licking, self-chewing and scratching (Dodman et al 1988b). but 1 has a short elimination half-life

(approximately 3 h) (Dodman et al 1988b). and is not available for oral administration. Naloxone was reported to irnprove tailthasing in one dog (Brown et al 1987). but it has a short elirnination half-life in dogs (70 min) (Pace et al

1979)) and an oral preparation is not available. Work in pigs and bank voles suggested that narcotic antagonists may not be useful for well-established stereotypic behaviours (Cronin et al 1986. Kennes et al 1988).

2.2.2.2 CIomipramine

Clomipramine is the drug that has been reported most in the treatrnent of canine CD (Goldberger 8 Rapoport 1991, Rapoport et al 1992, Overall 1994,

Dodman et al 1996). Clornipramine, desipramine. fluoxetine, serbaline and fenfluramine were evaluated in a non-randomised clinical trial involving 42 dogs with acral lick dematitis (Rapoport et al 1992). Only clornipramine, fluoxetine and sertraline were said to be effective. They are al1 potent serotonin-reuptake inhibiton. In a case senes describing three dogs (Overall1994), all three responded to

clomipramine. Other dinical evidence for the eficacy of clomipramine cornes frorn

a tail-chasing Bull Terrier (Dodman et al 1996). In humans, clomipramine has

been shown to be helpful in treating OCD (Leonard et al 1989, McTavish &

Benfield 1990, Pato et al 1991). This information, together with clinical experience at the Ontario Veterinary College and the fact fhat serious adverse effects of clomipramine have not been reported in dogs (Ciba Geigy 1991). suggests that clomipramine may be useful for dogs with CD.

2.2.3 Phamacology of clomipremine

Clomipramine is the 3-chloro analogue of the tricyclic antidepressant, imiprarnine (Baldessarini 1996). Tricyclic antidepressants inhibit the presynaptic reuptake of monoamines (Baldessarini 1996). and this affects neurotransmission.

Clomiprarnine affects serotonin, dopamine and norepinephrine in the brain. but the net effect of this is cornplex and incompletely understood. For example, serotonin is released at synapses where it actç as a neurotransmitter; in addition, it is also released from non-synaptic sites and is believed to modify neuronal excitability

(Sanders-Bush & Mayer, 1996). To the author's knowfedge, the effect of clomipramine at non-synaptic sites has not been reported.

Clomipramine is the strongest inhibitor of serotonin re-uptake in its class

(Hall & Ogren 1981) and is more effective than the other tricyclics as a treatment for human OCD (McTavish & Benfield 1990). The dnig inhibits the synaptic reuptake of serotonin in vitro (HaII & Ogren 1981, Hofhan et al 1992) and in vÏvo

(Westerink et al 19n,Adell et al 1989, Adell & Artigas 1991, Fujita et al 1991).

Inhibition of serotonin reuptake potentiates the action of serotonin at the synapse, resulting in longer-term adaptive changes in neural circuits (Hyman & Nestler

1996). These changes include reduœd sensitivity (down-regulation) of post- synaptic and pre-synaptic serotonergic receptors (Hyman & Nestler 1996,

Sanders-Bush & Mayer 1996)- There are at least thirteen sub-types of serotonergic receptor in the brain; some are excitatory and others are inhibitory

(Sanders-Bush & Mayer 1996). Drug-induced changes in receptor sensitivity may be regarded as a markerfor therapeuüc response, not the cause of it (Hyman &

Nestler 1996). There is evidence that the clinical response to tricyclic antidepressants may be caused by molecular changes in gene expression or in protein phosphorylation (Barden et al 1995, Hyrnan & Nestler 1996).

The dopaminergic efFed of clomipramine is unclear, but the dmg increases dopamine turnover (Westerink et al 1977) and is thought to decrease dopamine release (Kapur & Remington 1996).

The noradrenergic effect of clomipramine is attributed to the active metabolite, desmethylclomipramine, which is primarily a norepinephrine re-uptake inhibitor (Thomas & Jones 1977, Benfield et al 1980). 2.2.4 Pharmacokinetics of clomipramirie in dogs

The pharmacokinetics of clomipramine in dogs have not been well

described. The only report used one dog, in which a single oral dose of

clomipramine (5 mgkg) was almost whoIly excreted after four days. In the same

study, an oral dose of 0.3 mgikg was given to two humans and took fourteen days

to be totally eliminated (Faigle & Dieterle 1973). The disposition of clomipramine in

humans varies widely between patients due to genetic polymorphisrn of the

hepatic microsomal enzyme, cytochrome P450 206 (Brosen & Gram 1989,

Preskorn 1993). In addition, clomipramine inhibits that enzyme in some patients

(Crewe et al 1992, Nemeroff et al 1996) although, in rats, the drug appears to

induce hepatic microsomal enzymes (Friedman & Cooper 1983). To the author's

knowledge, there are no reports about genetic polymorphism of cytochrome P450

in dogs.

60th dogs and humans metabolise clomipramine by demethylation to form

desmethylclomipramine (Faigle & Dieterle 1973). In humans, desmethyl-

clomipramine has an elimination half-life approximately 1.5 times longer than the

half-life of clomipramine (Nagy & Johansson 1977, Evans et al 1980, Perel et al

1986). This is clinically important because desmethylclomipramine is associated

with anti-muscarinic and anti-histaminic side-effects (Benfield et al 1980).

Clomipramine has a wide safety margin in dogs. In a oneyear toxicity study, 18 Welsh Corgis were divided into gmups of six and received clomipramine in daily oral doses of 12.5 mgkg, 50 mglkg and 100 mgikg respectively. One dog died on the highest dose and another produced fewer sperm. No other adverse

effects were reported (Ciba Geigy 1991).

2.2.5 Neurochemical effect of clomiptamine in the canine brain

The effect of dnigs on monoamines in the brain may be assessed by

measuring the concentrations of the respective monoamine metabolites in the

cerebrospinal fluid (CSF) (Moir et al 1970, Comrnissiong 1985). This in vivo

procedure is simple and relatively inexpensive. The respective metabolites of

serotonin, dopamine and norepinephrine in the brain and CSF are 5-

hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA) and 3-rnethyl4-

hydroxyphenylglycol (MHPG) (Moir et al IWO, Kopin 1985, Agren et al 1986).

These metabolites have been measured in the cisternal CSF of untreated behaviourally normal dogs, under general anaesthesia (Moir et al 1970, Vaughn et al 1988a. b). Anaesthetic agents may affect the turnover of monoamines

(Westerink et al 1977. Barkai 1979) and the rate at which monoamines are eliminated from the CSF (Bourgoin et al 1975). The efiect of clomipramine on monoamine turnover has not been described in dogs.

In humans, clomipramine has been reported to affect monoamine metabolites in the lumbar CSF of patients suffering from depression and OCD

(Asberg et al 1977, Traskman et al 1979, Thorén et al 1980. Altemus et al 1994).

These reports have been widely referenced in the psychiatrie literature and the results are discussed in more detail in Chapter 4. It is difficult to make inferences from these human data because of the following limitations: (i) non-randomised

design; (ii) treatment effect assessed by using pre- and post- measurements on

the same patient. but without comparing between different treatments; and (iii) lack

of discussion of the methodological assumptions. The chief assumptions are that

the metabolites measured in CSF originate only from the brain, and that their

concentrations in CSF refiect the concentrations in local areas of the brain (Moir et

al IWO).

The foregoing suggests that clomipramine may be a useful treatment for

canine CD, but that further information is required. The pharrnacokinetics of clomipramine and its neurochemicd and clinical effects should be described in dogs. The neurochemical effect of the drug might be assessed by using CSF, as described in Section 2.2.4. The clinical efFiwcy of clomipramine in canine CD can be assessed in randomised, controlled, double-blind clinical trials that use an appropriate dosing regimen and a valid measure of outcorne. Such trials have not been reported.

2.3 Methodological issues in clinical trials of clorniparnine as a treatrnent for canine CD

Pocock (1987) and Meinert (1986) provide detailed reviews of the design and conduct of trials to assess the clinical efficacy of a treatrnent (clinical trials). ln trials of treatrnents for canine behaviour, issues of special concem are the

accurate identification of the behaviour when dogs are admitteci to the trial. and the accurate measurement of response to treatment (Kirshner & Guyatt 1985).

2.3.1 Diagnosis of cases for admision to a clinicai trial

In clinical trials. it is important to ensure that dogs admitted to the trial have the condition of interest. Such assurance cornes from knowing the accuracy of the diagnostic method. Diagnostic accuracy can be quantified by comparing the diagnoses made using the said method with the diagnoses reached by a 'gold standard' diagnostic test (Sackett et al 1991 p53). A 'gold standard' test is a definitive diagnostic test (Sackett et al 1991, p33). There is no such test for canine

CD because the diagnostic valideRyof the disorder has not been proven (see

Section 2.1 -5);this creates a challenge in a clinical trial. This issue is also a problem in psychiatry and has been reviewed (Cicchetti & Feinstein 1990,

Feinstein & Cicchetti 1990, Zarin 8 Earls 1993, Faraone & Tsuang 1994).

In the absence of a 'gold standard', the accuracy of a diagnosis may be quantified by measuring chance-corrected agreement with another diagnostic approach (Sackett et al 1991 pp30-31). Reported methods for diagnosing canine

CD were reviewed in Section 2.1 -6.Additional rnethods could include measurement of the brain's blood flow, metabolism or electrical activity (impractical in dogs), response to treatment (unknown because there is no proven treatrnent for canine CD), expert opinion and the 'best estimate procedure' (Kosten & Rounsaville 1992). The 'best estimate procedure' requires experts not involved with collecting data, to evaluate those data and the subjectç' medical records.

However, any use of expert opinion suffers from lack of explicit guidelines for diagnosis. An alternative measure of diagnostic accuracy is based on latent class analysis (Walter 8 Irwig 1988, Faraone & Tsuang 1994). This approach requires a minimum of three raters; it estimates observer error rates and the probability of making an incorrect diagnosis with the (latent) 'gold standard' (Walter & lmig

1988).

2.3.2 Measurnent of response fo treahtent in a clinical trial

The outcome measure in a clinical trial should permit valid inferences to be made about the efficacy of the treatment. Objective outcornes would be the frequency and duration of episodes of the behaviour. Standard techniques for measuring behaviour have been reviewed (Marb'n & Bateson 1993), but they may not be easy to adapt for cornpanion animals in domestic setüngs. A subjective outcome that has considerable practical relevance, is the owner's perception of behavioural severity. This outcome can be measured with rating scales. The development and use of health rating scaies have been reviewed (Streiner 8

Norman 1995). In general, rating scales rneasure both the presence of a constnict and the degree to which 1 is present (eg 'never', 'sometimes', 'frequently' and

'always') (Merrell 1994. p67). The advantage of using a rating scale to measure change in canine behaviour is that the sale can be used by the owner, who knows the dog best and who cmobserve its behaviour over a period of time, in the various eliciting settings. The scale could therefore provide information on low frequency or context-specific behaviour that might be difficult to measure using standard quantitative approaches, and that might not be apparent during direct observation in a chic. Rating scales are inexpensive and require no technical eq uiprnent, nor do they require professional raters. The disadvantage of rating scales is that they rely on qualitative data and personal judgernent (Merrell 1994).

The only reported rating scale for canine CD has been a single global question which asked ownen to rate their dog's behaviour on a scale of zero ta ten: zero represented no compulsive behaviour and ten represented the worst compulsive behaviour ever (Rapoport et al 1992). The psychornetric properties of this scale were not reported.

The psychornetric properties of rating scales have been reviewed (Kirshner 8

Guyatt 1985, Guyatt et al 1987, 1989, Streiner 8 Norman 1995); they are validity, reliability and responsiveness. There are dÏfFerent aspects of validity. In a trial of a treatment for canine CD, content validity implies that al1 relevant aspects of the behaviour are represented. Content validity can be established by consulting with experts. Face validity implies that the questions make sense to the person using them; this can be assessed by pre-testing. Criterion validity is rneasured when an established scale exists and a new scale is compared to it in an assessrnent of relative cost and ease of use. Constnict validity is especially important, but can be dficult to measure. Constnict validity implies that the instrument measures what it purports to measure. Reliability is a measure of the instrument's precision,

indicating how well 1 can discriminate between subjects. Responsiveness is

another important property of rating scales mat are designed to measure change;

it quantifies the degree to which the scale is sensitive to the change (Guyatt et al

1987, 1993).

2.3.3 Summary

This section highlights important methodological issues in clinical trials of treatments for canine behaviours. The accuracy of the diagnosis used to include animals in a trial, should be known. The validity and reliability of the outcome measure should also be established.

2.4 Conclusion

This literature review highlights the need for research into canine CD and its treatment. Clomipramine appears to be a promising treatment, but studies are needed to assess its effÎcacy. The following project was designed to test the hypotheses that clomipramine affects central monoamine turnover and is an effective treatment for canine CD. The project involved three experiments: (i) a pharmacokinetic study of oral clomipramine in dogs, (ii) a laboratory experiment to assess the effect of clomipramine on the turnover of monoamine metabolites in the brain of behaviourally normal dogs, and (iii) a randomised, controlled. double blind cl inical trial of clomiprarnine in which diagnostic accuracy and the validity of the outcome rneasure were also assessed. CHAPTER 9 : THE PHARMACOIUNETES OF CLOMIPRAMINE AND

DESMETHYLCLOMIPRAMINE IN DOGS: PARAMETER ESTIMATES

FOLLOWlNG A SINGLE ORAL DOSE AND 28 CONSECUTIVE DAILY ORAL

DOSES OF CLOMIPRAMINE

3.1 Introduction

Clomipramine is the 3-chloro analogue of the tricyclic antidep ressant, imipramine.

Tricyclic antidepressants act through inhibition of the pre-synaptic re-uptake of serotonin and notepinephrine (Baldessarini 1996). In vitro, clomiprarnine is the strongest inhibitor of serotonin re-uptake in Ïts class (Hall & Ogren 1981).

Clomipramine is also more effective than the other tricyclics as a treatment for obsessive compulsive disorder (McTavish & Benfield 1990). This human psychiatrie disorder is characterised by recurrent intrusive thoughts and ritualistic behaviours (American Psychiatrie Association 1994). When clomiprarnine is prescribed for treatment of obsessive compulsive disorder, the maintenance dose is usually given once or twice daily (McTavish & Benfield 1990).

Dogs can suffer from compulsive disorder, a behavioural syndrome that is analogous to obsessive compulsive disorder. Affected dogs show persistent behavioun that are often repetitive; eg tail-chasing and self-mutilation (Hewson &

Luescher 1996, Overall 1992)- There is currently no proven treatrnent for canine compulsive disorder but clomipramine has been reported to be effective at dose rates of 3 mgkg q 12 h (Overall, 1994) and 1-3 mgkg q 24 h (Goldberger & Rapoport 1991, Rapoport et al. 1992)- The pharmacokinetics of clornipramine in dogs have not been reported, to the authors' knowledge.

Both dogs and humans metabolise clomiprarnine by demethyiation to fom desmethylclomipramine (Faigle & Dieterle 1973) which is pnmarily a norepinephrine re-uptake inhibitor (Benfield et al. 1980, Preskom 1993). In humans, the elimination half-life of desmethylclornipramine is approximately 1.5 times longer than the half-life of clomipramine (Nagy & Johansson 1977, Evans et al. 1980, Perel et al., 1986). This is clinically important because desmethylclomipramine is not anti-obsessional (Balant-Gorgia et al. 1991) and 1 is associated with anti-rnuscarinic and anti-histarninic side-effects (Benfield et al.

1980).

The pharmacokinetics of clomipramine and desmethylclornipramine have been reported in rats (Friedman & Cooper 1983) and humans (Nagy & Johansson

1977. Evans et al. 1980, Perel et al. 1986). In Divo humans, an oral dose of 0.3 rng/kg of clomipramine was 92% excreted after fourteen days (Faigle & Dieterle

1973). ln a study of one dog (Faigle & Dieterle 1973),a single oral dose of clomipramine (5 mg/kg) was 96% excreted after four days. These data indicate that the pharmacokinetics of clomipramine might differ between dogs and humans.

The objectives of this study were to describe the phamiacokinetics of clomiprarnine and desmethylclornipramine in dogs following both a single dose of clomipramine (3 mg/kg PO) and 28 consecutive days of oral dosing (3 mg/kg q 24 h PO). 3.2 Materials and Methods

The protocol was approved by the Animal Care Cornmittee of the University of

Guelph. A non-random sarnple of six healthy, conditioned, intact male dogs was

used. The dogs were al! hound-crosses, approximately 2 years old and in lean

body condition (average weight 28 kg, SD 4.5 kg); five had been purpose-bred by

a supplier and the sixth was frorn an animal shelter. The dogs were housed under

standard conditions in one room in which each dog had its own kennel. They were

walked outside for 25 min, three times per week. They received water ad libitum

and were fed once daily at approximately the same time every morning (between

8.00 am and 8.45 am). The average amount fed was 425 g, SD 409 (Maintenance

Diet. dry; Hill's, Topeka. Kansas, USA).

Clomipramine hydrochloride was supplied as 20 mg and 2.5 mg tablets

(Ciba Geigy, Base!, Switzerland). The dnig was given 20 min after feeding, at a dose of 3 mg/kg PO: doses were adjusted to the nearest 1.25 mg. To facilitate dosing, the tablets were administered in gelatin capsules (Capsugel, Wamer-

Lambert. Greenwood, South Carolina, USA; rupture tirne, 60 s; 90-95% dissolved in 7 min). Each capsule held four tablets. The dogs were weig hed weekly and if their weight changed by more than 0.5 kg frorn itç previous value, the total dose of clomiprarnine was adjusted accordingly. No dog changed weight by more than 1 kg during the experiment. 3.2.1 Single-dose stuw

Twenty min after feeding, an 18 G,2-inch catheter (Becton Dickinson,

Rutherford, New Jersey, USA) was inserted in the cephalic vein of each dog; no

chemical restraint was used. Four mlof blood were taken and, immediately

afterwards, each dog received a single dose of clomipramine. Four mL samples of

blood were withdrawn at the following times after dosing: 15, 30 and 45 min and

then at 1, 2.4, 8, 12, 24, 36.48.60, 72, 84,96, 1 O8 and 2 20 h- Ali samples were

collected in 4 mL evacuated lithium heparin tubes (Vacutainer, Becton Dickinson,

Rutherford, New Jersey, USA). The catheters were fiushed with 3 mL of heparinised saline after each sample was taken. Depending on the dogs' tolerance for them, the catheters were removed after 4 - 12 h. Subsequent samples were taken by cephalic or jugular venipuncture which the dogs tolerated weil.

3.2.2 Multiple-dose study

This study began on the day following the completion of the single-dose study, and involved the sarne dogs. The dogs received clomiprarnine once daily for 28 days; medication was given 20 min after their daiIy ration of dry food.

However, on day 1, hodogs vomited within 30 min of dosing; therefore, for the remainder of this study, each dog received additional food immediately after dosing (150 g canned food; Medical Convalescent Food; Veterinary Medical Diets,

Guelph, Ontario, Canada) Blood was taken by jugular venipuncture immediately before drug administration, and by cep halic venipuncture 1-5 h afier drug administration.

Samples were taken in this way on days 1.2, 3, 4, 5. 6.7, 9, 11, 13, 20.22. 24 and 26. The dogs received the final dose of cfornipramine on day 28 and samples were then taken in the same tirne series as in the singledose study.

3.2.3 Handling of samples

Within 20 min of collection. the blood samples were stored at 4°C. Samples were then centrifuged (1200 g for 20 min at room temperature) wlhin the next 90 min. The plasma was immediately transferred into 2 mlpolypropylene cryovials

(Nalgene, Rochester, New York. USA) and was then kept at 4°C for up to 90 min before being stored at 87°C for 18 months. The effect of storage at -87OC was not assessed, but it is considered that clomipramine and desmethylclomipramine are stable at this temperature (Jon King, Ciba Animal Health, CH4002. Basle,

Switzerland - personal communication).

3.2.4 Detemination of clomi~neand desmethylclomipramine

Chernical analysis of the samples was conducted by Ciba Geigy (CH4002,

Basle Switzerland). Clornipramine and desmethyiclornipramine were extracted from plasma using a three-stage procedure (Godbillon & Gauron 1981). Briefly, the compounds were extracted at pH 10 into heptane containing 1% isoamyl alcohol. They were then back-extracted into an acidic phase and were re- extracted at a basic pH into heptane. The concentrations of both compounds were then detemined by gas chromatography with mass-spectrometric detection (Sioufi

et al. 1988).

In brief, 5 mL n-heptane-isoamyl alcohol, 100 PL Ïntemal standard solution

(containing 60 ng clomipramine and 150 ng desmethylclomipramine) and 1 mL buffer (pH 10; 0.2 M borÏc acid, 0.2 M potassium chloride, 0.176 M sodium hydroxide) were added to 0.1 mL plasma. The mixture was shaken for 5 min at

3000 rpm and then centrifuged at 1400 g for 10 min. The organic phase was transferred to another tube. shaken with 1 mL 0.1 N sulfi~ricacid for 5 min at 3000 rprn and then centrifuged at 1400 g for 3 min. The organic phase was then discarded, and 300 pL of 1 M sodium hydroxide and 1.6 mL of n-heptane were added to the aqueous phase. This mixture was shaken for 10 min at 300 rpm and centrifuged at 2500 g for 2 min. The organic phase was transferred and 20 pL of dimethylformamide. 20 pL of pyndine and 100 pL of pentafluoropropionic anhydride were added to it The mixture was vortex-mixed for 15 s and was held for 1 h at 60 OC. Two mL of alkaline buffer were then added and this mixture was shaken for 5 min at 300 rprn and centrifuged. The organic phase was then transferred and evaporated to dryness under a stream of nitrogen; the residue was redissolved in 10 pL of pyridine and 2 PL of the solution were injected into the gas chromatograph at 250 OC, using splitless injection. The interface between the mass selective detector and the chrornatograph was kept at 280 OC and the capillary column was inserted direcüy into the ion source. Total (free and protein- bound) concentrations of clomipramine and desmethylclorniprarnine were

measured, and the Iimit of detedon was 2 ng/mL-

3.2.5 Pharmacokinetic analysis

Pharmacokinetic parameters for clornipramine and desmethylcIomipramine

were determined following ttie single dose of clomipramine and again after the

twenty-eig hth consecutive daily dose.

Following visual inspection of semiloganthmic plasma concentration versus

time plots, the following candidate models were applied to each dog's data using

standard software (PC-NONLIN Version 4.0, Statistical Consultants Inc,

Lexington, Kentucky, USA). The candidate models were one-comparûnental

(Baggot 1977, pp145-155) and non-compartrnental (Gibaldi 1984, ppl7-28). The

cornpartmental models were non-linear, with unweighted variance, and the associated parameters were estimated iteratively using least squares regression

and the Nelder-Mead simpIex algorithm (Metzler & Weiner 1992, pp3.3-3.7). Two separate one-compartmental models were applied to each dogrs data, One model assumed equal absorption and elimination rates; the other assumed unequal absorption and elimination rates. The assurnptions of the cornpartment models were first-order absorption and elimination (Gibaldi 1984, pp12-13) and a lag-time between administration and the start of absorption. The models selected were those that had prediction vs time plots with visually acceptable goodness of fit, and that showed homogeneous scatter about zero in plots of residuals vs. time (Myers 1990, pp209-248). Additional selection criteria were low values of Akaike's

information criterion (Yamaoka et al. 1978) and parameter estimates with narrow

confidence intervals; models were rejected if the confidence intervals included

zero. When one-cornpartmental models were rejected, using those criteria, a non-

cornpartmental analysis was performed, using the method of moments (Gibaldi

1984, ~~20-25).

The data rnodelled were the plasma concentrations from irnmediately pre-

dosing (O h) to the time when the plasma concentration reached the lime of

detection of the chrornatographic system (2 nglmL); plasma concentrations below

the limit of detection were not modeled. The following parameters were estimated.

as appropriate for the model: lag time (tlW),maximum plasma concentration (C,,),

time of maximum plasma concentration (t,,) , absorption rate constant (k&s) ,

absorption haif-life (t,n abSI. elirnination rate constant (k,). elirnination half-Ife

first-order elirnination rate constant 0,area under the plasma concentration vs.

time curve (AUC; calculated by trapezoidal estimation), area under the first

moment curve (AUMC, calculated trapezoidally) and mean residence time (MRT).

3.3 Results

The dogs remained clinically heaithy during the experiment. Dog 3 showed

extreme fear throughout. This dog crouched at the rear of the kennel and would leave; when handled, he had diiated pupils and rigid posture. This dog's data are descnbed separately.

Figure 3.1 shows the rnean (SD) plasma concentration of clomipramine and desmethylclomipramine, following single and multiple doses of clomipramine.

The large SD illustrates marked Riterdog variability. Figures 3.2 and 3.3 show the data for each dog following single and multiple doses of clomipramine; the data are presented in semilogarithmic plasma concentration vs. tirne curves of clomipramine and desmethylclomipramine. Figures 3.2 and 3.3 indicated monoexponential elimination. In addition, duhg the post-absorptive phase, there was a log-linear decline of drug and metabolite concentrations, indicating first- order elirnination (Figures 3.2 and 3.3).

Following a single dose of clornipramine. the drug was detected in the plasma of Dogs 1.2.4, 5 and 6, within 15 - 45 min of dosing; however, the drug was no longer detected after 8-12 h (Figure 3.2). In the same dogs, desmethylclomipramine was detected within 15 min-2 h of dosing, but was no longer detected after 4-12 h (Figure 3.2). ln Dog 3, neither dnig nor metabolite was detected at 15 min and a technical error with the three subsequent samples

(30 min, 45 min and 1 h) meant that neither compound was measured until2 h Figure 3.1 Mean (+ SD) plasma concentrations of clornipramine (a) and desmethylclomipramine (O)in six dogs (i) following a single daily dose of clomipramine (3mglkg PO), and (ii) after 28 consecutive daily doses of clomipramine (3mgkg q 24 h PO).

Single-dose study

O 4 8 12 16 20 24 28 32 36 Time (h)

Multiple-dose study

O 4 8 12 16 20 24 28 32 36 Time (h) Figure 3.2 Semikg concentration-time plots of plasma concentrations of clomipramine (0) and desrnethylclomipramine (O) in six dogs following a single oral dose of clomipramine (3 mgkg). Sarnpling commenceci immediately before dosing (O h). (Owing to a technical error, Dog 3's data were not available 30 min, 45 min and 1 h afier dosing). Tirne th)

Time (hl

O 2 4 6 8 tO 12 14 16 18 20 22 24 26 Tme (h) Figure 3.3 Semilog concentration-time plots of plasma concentrations of clomipramine (0) and desmethyldomipramine (a) in six dogs after the last of 28 consecutive daily oral doses of clomipramine (3 mgkg q 24 h). Sampling commenced irnmediately before the 28th dose (O h). (Owing to a technical error, Dog 2's data were not available at 4 h after dosing). Time (hl

Oog 3 Dog 4

Oog 5 Dog 6 1

O 4 8 12 16 20 24 28 32 36 40 44 48 Time (h) after dosing; both drug and metabolite were then detectable up to 24 h after the

drug was administered (Figure 3.2)-

On day 28 of the multiple dose study (Figure 3.3), dogs 1 and 4 did not

have detectable concentrations of drug or metabolite immediately predosing (O h).

The other four dogs had detectable concentrations of both clomipramine (range

4.8-1 5.6 ng/mL) and desmethylclomipramine (range 2.2-1 7.3 ng/mL) imrnediately

before dosing on day 28. Following dosing on day 28, these four dogs showed a

more gradua1 decline in drug and metabolite concentrations, both of which were

still detectable at 24-36 h (Figure 3.3).

The pharmacokinetic parameters of clomipramine were largely estirnated

from non-compartrnental models (Tables 3.1 to 3.4), because one-cornpartmental

analysis produced wide parameter estimates, and plots of the residuals and of

predicted values indicated poor goodness of fit The t,. of clomipramine affer a

single dose ranged from 1-2 h to 16 h. Following multiple doses, tln, of clomipramine ranged from 1-5 h to 9 h.

The pharmacokinetic parameters of desmethylclomipramine were estimated from a one-compartrnental model (Tables 3.5 to 3.8); however, parameters for

Dogs 1 and 5 following a single dose could not be estimated because the metabolite was only detectable twice during the sampling period (Figure 3.2). The t,,! ,, of desmethylclomipraminefollowing a single dose ranged from 1.2 h to 2 h

(Tables 3.5, 3.6); following multiple doses, it ranged from 1.4 h to 4.3 h (Tables

3.7, 3.8). Table 3.1 Pharmacokinetic parameter estimates (one-compartrnental model) for plasma clomipramine in two dogs fallowing a single oral dose of clomipramine (3 mgm

t m el (h) 1.2 3.7 k ,S wl) 0.58 0.78 TL? abs (h) 1.2 0.92 AUC ,(pg.h/mL) 121 657

lag time; Grna: maximum piasma concentration; t,,: time of maximum plasma concentration; k,, :elimination rate constant; t ,n ,, : elimination half-life; kas: absorption rate constant; t absorption half-Ife; AUC ,: area under the plasma concentration vs. time curve from O to infinity.

# h, includes 4,, Table 3.2 Phamiacokinetic parameter estirnates (noncornpartmental model) for plasma clomiprarnine in four dogs following a single oral dose of clomipramine (3 wks)

Parameter Dog 3 Dog4 Dos 5 Dog6 Mean ISD

t fael (hl AUC 1, (Iq-hfmU AUC O-, (pg-h/mL) AUMC ,,, (pg.h2/m~)

Cm : maximum plasma concentration; t,,: time of maximum plasma concentration; t,n, : elimination half-life, P: first-order elimination rate constant; AUC ,,: area under the plasma concentration vs. time cuwe from O to the last detectable concentration AUC ,-, : area under the plasma concentration vs. time cuive from O to infinity; AUMC ,a,: area under the first moment curve from O to the last detectable concentration AUMC ,., : area under the fint moment curve from O to infinity; MRT,ast,:mean residence time based on data from O to the last detectable concentration; MRT,: mean residence time based on data from O to infinity Table 3.3 Pharmacokinetic parameter estimates (one-cornpartmental model, assuming equal rates of absorption and elimination) for plasma clomipramine in one dog following 28 consecutive daily oral doses of clomipramine (3 mglkg q 24 h). (Abbreviations: see Table 3.1 )

** AUC measured to 12h postdosing, not to 24 h # t,, includes t,a,

Table 3.4 Pharmacokinetic parameter estimates (non-cornpartmental model) for plasma clomipramine in five dogs following 28 consecutive daily oral doses of clomiprarnine (3 mgkg q 24 h). (Abbreviations: see Table 3.2)

Parameter Dog 2 Dog 3 Dog 4 Dog 5 Dog 6 Mean kSD Table 3.5 Pharmacokinetic parameter estimates (one-compartrnental model, assuming equal rates of absorption and elimination) for plasma desmethy lclomipramine in three dogs following a single oral dose of domipramine (3 mgkg). (Abbreviations: see Table 3.1)

Dog 2 Dog 3 Dog 6 Mean t SD Mode1 ke=k, k,~=k, k,=k,

Table 3.6 Pharmacokinetic parameter estirnates (one-cornpartmental model, assuming unequal rates of absorption and elimination) for plasma desrnethylclomipramine in one dog following a single oral dose of clorniprarnine (3 mg/kg). (Abbreviations: see Table 3.1)

Doa 4

" Parameter estimate of k ,, included zero Table 3.7 Phamacokinetic parameter estimates (one-cornpartmental model, assuming equal rates of absorption and elimination) for plasma desmethyldomipramine in five dogs following 28 consecutive daily doses of clornipramine (3 mgkg q 24 h PO). (Abbreviations: see Table 3.1)

Parameter Dog 1 Dog 2 Dog 3 Dog4 Dog 5 Mean +SD

t 1, (h) 1.2 1.5 3.7 0.69 1-7 1.8 + 1.1 c ma WmL) 29 113 136 81 58 84 I43 [ma (h) 4.5 6.1 12 3.6 6.21 6.55 I3.5 k (h*') 0.49 0.33 0.19 0-46 0.36 0-37k 0-12 t m ‘d (hl 1-4 2.1 3.6 -1 -5 1-9 2.1 2 0-86 AUC (pg.hlmL) 163 924 1903 481 435 781 I684 A UC - 24 (trapezoidal) 168" 774" 1775 477" 471

** AUC measured to 12h postdosing, not to 24 h

Table 3.8 Phamacokinetic parameter estimates (one-cornpartmental model, assuming unequal rates of absorption and elimination) for plasma desrnethylclomipramine in one dog following 28 consecutive daily doses of clomipramine (3 mgkg q 24 h PO). (Abbreviations: see Table 3.1)

Parameter Dog 6 Throughout the experiment, desmethylclomipramine was present in lower

plasma concentrations than clomipramine (Figures 3.1 to 3-41. with the occasional

exception of Dog 3 (Figure 3.2). When peak concentrations of both compounds

were measured during the multiple-dose study, there was marked interdog

variability as indicated by the large SD (Figure 3.4). However, the ratio of

metabolite to parent drug for each dog remained less than 0.6. indicating that

desmethylclomipramine was present in lower concentrations than clomipramine.

On d 26, the peak concentrations of both compounds were lower than on the other

days. However, on d 28, plasma concentrations 2 h after dosing were in the range

of the peak concentrations detected throughout the multiple-dose period (Figure

3.4).

At trough, Dogs 1,4 and 5 did not have detectable concentrations of either clomipramine or desmethylclomipramine. In the other dogs, the trough concentrations of both compounds were low (clomipramine, range 2-17 ng/mL; desmethylclomipramine, range 2-1 5 ng/mL), and the trough ratios of metabolite to parent dnig ranged from 0.4 to 1.1, Dog 3's ratio being consistently highest.

3.4 Discussion

To the knowledge of the authon. this is the first reported study of the pharmacokinetics of clomipramine and desmethylclomipramine in dogs. The study had a non-randomised design and a small sample size; these factors, coupled with Figure 3.4 Mean (+ SD) plasma concentrations of clomipramine (O) and desmethylclomipramine (O) in six dogs dos& daily with cfomiprarnine (3mgkg q 24 h PO) for 26 days. Samples taken 1.5 h after dosing on days 1-7, 9, 11. 13.20, 22.24.26 and 28. (On day 28, samples were taken 2 h after dosing).

large interdog variability, preclude statistical cornparison between the results of the single-dose and multipledose studies.

The one-compartmental analyses were unweighted because the datasets were small and the use of inappropriate weights can Iead to errors in parameter estimates (Myers1990, p280; Powers 1990). There were insuficient data tu

calculate weights fiom the standard curve of the assay (Myers 1990. p280).

Altematively, weights could have been derived hmthe dogs' data. However,

Peck et al. (1984. p146) state that "the use of weighting algonthms which are

functions of the observed data is inherently approximate and risky". Furthermore,

any influence of heteroscedasticity on parameter estimates for individual dogs was

likely to be insignificant, given the high betweendog varîability and the relatively

sparse data.

The data reported here support earlier evidence that the pharmacokinetics of clomiprarnine differ between dogs and humans (Faigle & Dieterle 1973). The estimated elimination half-lives in the present dogs were much shorter than those reported in humans (clomipramine, approximately 20 h; desmethylclomipramine. approximately 36 h; Nagy 8 Johansson 1977, Evans et al. 1980, Perel et al. 1986), but were similar to those in rats (clomipramine. 6 h; desmethylclomipramine. 2.4 h;

Friedman & Cooper 1983). The short t,ne, of clomipramine in dogs suggests that frequent dosing rnight be necessary to maintain steady state plasma concentrations and achieve an optimal clinical effect. However. in humans, the relationship between clinical response and the plasma concentration of clomipramine is unclear (Balant-Gorgia et al. 1991), and the dnig's efficacy might not depend on the maintenance of steady state plasma concentrations. Further pharmacokinetic and clinical work are required to ascertain an appropriate dosing regirnen for dogs. Our data showed interdog variability in plasma concentrations of

clomipramine and desmethylclomipramine. In humans, the main sources of

vanabiiity in plasma clomipramine following oral dosage are (i) first-pass

metabolism and enterohepatic recirculation (Nagy & Johansson 1977. Evans et al.

1980, Preskorn 1993); (ii) genetic differences in metabolisrn and clearance

(Brosen & Gram 1989, Preskom 1993); and (iii) inhibition of hepatic microsomal enzymes (Crewe et al. 1992, Nemeroff et al. 1996). These sources of variabiliity may also exist in dogs. First-pass rnetabolism may affect the pharrnacokinetics of clomipramine in dogs, but cornparison between oral and parenteral administration would be needed to detennine this.

The role of genetics in canine pharrnacokinetics has not been described, to the authors' knowledge. In humans. 7-10% of Caucasians exhibit genetic polymorphism of the hepatic isoenzyme P450 206 which catalyses the hydroxylation of clomiprarnine and desrnethylclomipramine (Brosen & Gram 1989).

Genetically polymorphic patients can produce sub-toxic concentrations of desrnethylclomipramine (Balant-Gorgia et ai. 1991). This is clinically important because desrnethylclomipramine is associated with side effects (Benfield et al..

1980); also, high concentrations of this metabolite can cause treatment failure in obsessive compulsive patients treated with clomipramine (Balant-Gorgia et al.

1991, Preskom 1993). The present study showed plasma concentrations of desmethylclomipramine that were consistently less than those of clomipramine

(Figures 3.1 to 3.4). This is the opposite of what has been reported in humans, in whom oral clomipramine produced plasma concentrations of desmethyl-

clomipramine that were markedly higher than those of clomipramine (Nagy &

Johansson 1977). The present data indicate that dogs rnay differ fkorn humans,

and rnay not develop high concentrations of desrnethylclomipramine; however,

given the short tm, of this metabolite, more frequent sampling would have helped

to confinn this.

The third source of pharmacokinetic variability of clomiprarnine in humans is

enzyme inhibition. To the authors' knowledge, this has not been reported in

humans in vivo; however, in vitro work with human hepatocytes has indicated that

clomipramine inhibits the activity of P450 2D6 (Crewe et al. 1992, Nemeroff et al.

1996). Conversely, when rats reœived 28 consecutive daily injections of clomipramine (15 mgkg q 24 h i.-p.), the t, ,of clomipramine decreased by 66%

(from 6 h to 2 h) and the t, ,, of desrnethylclornipramine decreased by 80% (frorn

2.4 h to 0.5 h; Friedman & Cooper 1983). These data suggested autoinduction of hepatic microsomal enzymes (Friedman & Cooper 1983). In our study, the t,ne, of both drug and metabolite following 28 consecutive daily doses of clomiprarnine were numerically greater than the t,De, following a single dose (Tables 3.1 to 3.8).

In addition. the AUC,, following multiple doses was numencally greater than the

AUC- after a single dose, indicating that clomipramine and desrnethyl- clomipramine accurnulated during the 28day dosing period. However, these data do not allow us to detemine whether enzyme inhibition occurred, because the study design did not control for period and order effects. Another possible source of variability in the plasma concentrations of

clomipramine and desmethylclomipramine was the amount of clomipramine

absorbed, and the rate of absorption. Clomipramine is basic (pKa = 9.5, Faigle &

Dieterie 1973) and Iipophilic (Balant-Gorgia et al. 1991); when given orally to

humans (Faigle & Dieterle 1973, Preskom 1993) and dogs (n = 1; Faigle & Dieterle

1973), it was also totally absorbed. In the single- and multiple-dose studies

reported here, clomipramine was administered shortly after meals. During the

multipledose study, dogs received more food after having been dosed, but this

should not have had a significant effect on the elimination of clomiprarnine

because the dogs had already eaten a full meal. Food was given because

experience at the Ontario Vetennary College Behaviour Clinic indicated that some

dogs might vomit if they received clomipramine on an empty stomach. Moreover,

it is often convenient for ownen to give medication to their anirnals when feeding

them. To the authors' knowledge, the effect of food on the absorption of

clomipramine has not been reported. Food could have delayed the absorption of the drug, reducing or increasing the amount absorbed (Gibaldi 1984, pp38-40;

Spilker 1986, pp145-157; Welling 1989). Clornipramine was administered in gelatin capsules and food coold have delayed or hindered their rupture. However, the capsules themselves should not have affected absorption sig nificantly.

In addition to the possible effect of food, the absorption of clomipramine might also have been affected by the dogs' ernotional state. Dog 3 showed chronic generalised fear and also had the highest AUC and AUMC. Following the final dose of clomipramine, the t,, of the drug in Dog 3 was long (8 h) and, unlike

the other dogs, Cm was less than 1 was after the single dose of clomipramine.

To the author's knowledge. there are no published experiments on the effect of

chronic fear on the absorption of dmgs from the gastro-intestinal tract Dog 3's

fear may have delayed gastric emptying and caused gut stasis, slowing the rate of

drug absorption but increasing the extent of it.

In summary, our data indicate that the t,ae, of clomipramine and

desmethylclomipramine in dogs are very short, and that dogs are not subject to

high concentrations of desmethylclornipramine with its attendant potential for side-

effects. These findings are in contrast to human data. If a clinical trial provides evidence for the efficacy of clomiprarnine as a treatrnent for canine compulsive disorder, further phamacokinetic studies will be required. In future studies the

period of blood sampling should be shorter than in our study and sampling should be more frequent in the first 12 h after dosing. In particular. the bioavailability of clornipramine in dogs should be quantified. The pharmacokinetic parameters foilowing single and multiple doses rnight also be compared in a randomked design.

This study provides preliminary information about the phamacokinetics of clomipramine in dogs. Clomiprarnine and other human psychotropic drugs are being used increasingly tu treat behavioural disorders in cornpanion animals. The data suggest that. before such use, the pharmacokinetics of these dnigs in cornpanion animals should be detenined. CHAPTER 4: THE EFFECT OF CLOMIPRAMINE ON THE TURNOVER OF

CENTRAL MONOAMINE METABOLITES IN BEHAVIOURALLY NORMAL

DOGS

4.1 Introduction

Clomiprarnine is a tricyclic antidepressant that has shown promise as a treatment for canine compulsive disorder (canine CD) (Rapoport et al 1992, Overall 1994).

Canine CD is syndrome of abnormal confiict behaviours, some of which are stereotypic (Hewson & Luescher 1996). The pathophysiology of the condition is not well understood. In other species, the neurochemistiy of stereotypic behaviours has been reviewed and is known to involve serotonin and dopamine

(Cooper 8 Dourish 7990; Cabiib 1993, pp119-145). Clomipramine affects both these neurotransrnitters, and also norepinephrine (Baldessarini 1996). If the drug proves to be an effective treatment for canine CD. information about its neurochemical effect could provide indirect evidence for the pathophysiology of this disorder.

Clomipramine inhibits the pre-synaptic reuptake of serotonin (Hall & Ogren

1981, Hoffman et al 1992). This results in decreased breakdown of serotonin in the pre-synaptic cell, and decreased production of the serotonin metabolite 5- hydroxyindoleacetic acid (54IAA) (Baldessafini 1996). Clomipramine has a similar effect on norepinephrine because the dnig's metabolite, desmethylclomipramine, is a norepinephn'ne re-uptake inhibitor (Thomas & Jones 1977, Benfield et al 1980). Treabnent of depressed patients with clorniprarnine

has been associated with reduced concentrations of both 5-HIAA and the

norepinephrine metabolite, brnethy 14-hydroxyp henylgIycoI (MHPG) in

cerebrospinal fluid (Asberg et al 1977, Traskman et al 1979, Altemus et al 1994).

The dopaminergic effed of clomiprarnine is unclear. The dnig has been

associated with an increase in dopamine metabolism, reflected by increased

concentrations of the dopamine metabolite. homovanillic acid (HVA) (Westerink et al 19n,Kapur 8 Remington 1996).

The effect of drugs on brain neurochemistry may be assessed in vivo by measuring the concentrations of the neurotransmitter metabolites in cerebrospinal fiuid (CSF) (Moir et al 1970. Commissiong 1 985). The respective metabolites of serotonin, dopamine and norepinephrine in the brain and CSF are 5-HIAA, HVA and MHPG (Moir et al 1970, Kopin 1985). These metabolites have been measured in the CSF of untreated, behaviourally normal dogs (Moir et al 1970,

Faull et al 1982, Vaughn et al 1988a,b).

The objective of the foilowing experknent was to compare the effects of clomipramine and placebo on the turnover of serotonin, dopamine and norepinephrine in the brains of behaviourally normal dogs, as indicated by the concentrations of 5-HIAA, MHPG and HVA in CSF. Secondary objectives were (i) to examine the relationship between these metabolites and the concentrations of clomipramine and desmethylclomipramine in CSF and plasma, and (ii) to describe the duration of treatrnent required before an effect. if any, could be identified. 4.2 Materials and Methods

This protocol was approved by the Animal Care Cornmittee of the University of

Guelph. Six behaviourally normal, male dogs was obtained from an animal shelter.

Following a Wo-week period of quarantine, the dogs were allowed to acdimatise

to the laboratory facility for 10 d. Five dogs were cross-bred and intact; one was a

Husky and had been castrated. The dogs were approximately 4 years old. They

were housed under standard conditions in one room in which each dog had its

own kennel. They were walked outside for 25 min every day. The dogs received

water ad libitum and were fed once daily (400 g, SD 35 g; Maintenance Diet dry;

Hill's, Topeka, Kansas, USA) between 15.30 and 16.30 h. Approximately 20 min

after feeding, medication was given by hand with 150 g of canned food (Medical

Convalescent Food; Veterinary Medical Diets. Guelph, Ontario, Canada). No

other food was given during the study.

Clomipramine hydrochloride and placebo were supplied as 20 mg and 2.5

mg tablets (Ciba Geigy, Basle, Switzerland) and were given orally (3 mg/kg q 24 h)

in gelatine capsules (Capsugel, Warner-Lambert. Greenwood, South Carolina.

USA. Rupture time. 60 S. 90-95% dissolved in 7 min). The dose was derived from the available evidence (Goidberger & Rapoport 1991. Rapoport et al 1992) and from clinical experience at the Ontario Veterinary College. The dogs were in lean body condition when acquired (bodyweight 22.2 kg, SD 2.8) and they were weighed weekly. Their weight increased during the experiment but no dog gained more than 3.5 kg. When a dog's weight changed by more than 0.5 kg from its

previous value, the total dose of clomipramine was adjusted accordingly.

The study had a balanced, AB-BA crossover design. Dogs were randomly

allocated to treatment sequence, using cornputer-generated random numbers

(Statistical Analysis System, Version 6.1, SAS Institute, Cary, NC). The dogs

received each treatment for six weeks. SampIes of blood and cerebrospinal fi uid

(CSF) were taken at the end of the first, second, fourth and sixth weeks of each treatment period, under general anaesthesia. On each occasion, samples were taken approximately 18 h after medication had been given. The sampling technique is described in Section 4.2.1 -

Between the treatment periods there was a two-week washout period during which the dogs did not receive treatment but continued to receive canned food as above. Dogs were not sampled during the washout period.

4.2.1 Sampling technique

The dogs were anaesthetised using 1% thiopental (Intraval, Rhone

Merieux, Victoriaville, Quebec) without premedication; aceprornazine (0.1 mglkg

IM ; Atravet, Ayerst Laboratories, Guelph, Ontario) was given after sampling, to aid anaesthetic recovery- Following tracheal intubation, anaesthesia was maintained with isoflurane (Aerrane, Ohmeda, Mississuaga, Ontario) and nitrous oxide (BOC

Gases, Guelph, Ontario). The dose of thiopental and the interval that elapsed between anaesthetic induction and CSF-sampling, were recorded as possible covariates (Bourgoin et al 1975, Westerink et al 19n,Barkai 1979). Each dog

was placed in lateral recumbency and CSF was collected tom the cerebello-

rnedullary cistem following percutaneous puncture with a 20 gauge, 1.5 inch spinal

needle (Becton Dickinson. Franklin Lakes, New Jersey, USA). On each occasion,

a total of 4 mL of CSF was collected by fkee-fiow, in aliquots of 1 rnL. In addition,

blood was collected from each dog, by jugular venipuncture, into 4 mL evacuated

lithium heparin tubes (Vacutainer, Becton Dickinson. Rutherford, New Jersey,

USA), for detection of clomipramine and desrnethylclomiprarnine. At the end of

each six-week treatment perÏod, additional blood was taken from each dog for

cornplete blood counts and biochernical profiles.

At the end of the experimental period, three dogs were euthanased while

under anaesthesia. The other three dogs were adopted by rnembers of the public.

4.2.2 Sample handling

4.2-2.7 CSF

One aliquot of CSF From each sampfe was anaiysed for total protein, routine cell counts and cytology. within 20 min of collection. Erythrocyte counts were later used as a covariate to control for any contamination of CSF by blood during sampling (Kopin 1985).

The remaining aliquots of CSF were held at room temperature for up to 15 min after being obtained. They were then held at 4OCfor 0.5-6 h, before being stored finally at -80°C.There was a possibility that initial storage at 4°C could affect the stability of the monoamnie metabolites, reducing their concentrations in

the CSF samples. This issue was examined as follows: at the final sampling of the

experirnent, additional aliquots of CSF were taken from each dog. These aliquots

were held at 4°C and were then serially transferred to -80°C at the following

intervals after sampling: 5, 10, 15. 17, 30. 62, 122,246.270 and 360 min. Fieen

months later, these aliquots were analysed for dHIAA and HVA; there was not

enough CSF to quantitate MHPG.

All the other CSF samples Rom the study were also held at -80% for fifteen

months before being analysed for monoamine metabolites and for clornipramine

and desmethylclomipramine. The effect of storage at -80% was not assessed.

However. when human lumbar CSF is stored at ôO°C, concentrations of 5-HIAA,

HVA and MHPG are not affected (Linnuila et al 1983, Scheinin et al !983). In

addition, clomipramine and desmethylclomipramine are considered to be stable at

-80°C (Jon King, Ciba Animal Health, CH-4002. Basle, Switzerland - personal communication)

4.2.2.2 Blood

Aliquots of blood were centrifuged at 1200 g for 20 min at room temperature; the supematant was immediately removed, transferred into 2 mL polypropylene cryovials (Nalgene, Rochester, New York, USA) and stored at -

80°C. FÏfteen months later, the concentrations of clomipramine and desmethylclomipramine in the samples were measured.

64 4.2.3 ChromafogtaphicAnalysis - monoamine metabolites

The concentrations of 5-HIAA, HVA and MHPG in CSF were rneasured

using high performance liquid chrornatography with coulometric detection

(Scheinin et al 1983). In brief, the mobile phase was a mixture of 55 mM sodium

phosphate, 77.5 mg/L disodium edetate (Na, EDTA; antioxidant) and 3%

rnethanol, adjusted to pH 3.5 with phosphoric acid; the mobile phase was

delivered at a constant flow of 1.3 rnUrnin (Waters, Mississuaga. Ontario).

Prepared samples were injected onto a C-18 reversed phase column (5 Pm. 150

mm x 3.9 mm i.d.) (Nova Pak; Waters, Mississuaga, Ontario) using a Waters WlSP

71OB autosampler with a 50 PL injection loop. The samples were quantitated

using a Coulochem II electrochemical detector (SPE Ltd. Concord. Ontario) and

chromatograms were recorded using Millennium 20 10 software (Waters,

Mississuaga, Ontario).

The intemal standard was 3.4 dihydroxybenzylamine (DHBA; Sigma

Chernical Company. St. Louis, MO). The standard mixture for HVA and 5-HIAA was 100 nM HVA, 100 nM 5-HIAA and 100 nM DHBA in 0.25 N acetic acid containing 180 mgIl Na, EDTA. Similarly, the standard mixture for MHPG contained 100 nM MHPG and 100 nM DHBA in 0.25 N acetic acid with 180 mg/L

Na, EDTA. The stability at room temperature of 5-HIAA, HVA and MHPG in the standard mixture was noted by holding aliquots of standard mixture for periods up to 24 h and then measuring the concentrations of 5-HIAA, HVA and MHPG that

remained in the mixture.

Samples were analysed in random order, in lots of three, with one standard

mixture per lot Technicians were blind to the treatrnent allocation in the experiment.

4.2.3.7 5-HM and HVA

After thawing, 100 pL CSF were vortex-mixed in a 1.5 mL Eppendorf tube with 100 pl of a solution of 180 mgIl Na, EDTA and 40 mg/mL DHBA in 0.26 N acetic acid. Prepared samples were kept on ice and were injected onto the column within 2 h of preparation.

4.2-3.2 MHPG

After thawing, 0.5 mL CSF was mixed with 0.1 mL of an enzyme mixture of

5.8 IUfrnL beta glucuronidase and 3.7 IUfmL aryl sulphatase ('Glucolase',

Calbiochem, Boehringer Diagnostics, LaJolla, Califomia); 0.5 rnL of 1 N sodium acetate was added and the mixture was incubated ovemight at 37°C. MHPG was then extracted by agitating the mixture with 2 mL ethyl acetate, and separating the organic solvent from the aqueous phase; this extraction process was repeated once. The two ethyl acetate extracts were combined, dried with sodium sulphate and the solvent was evaporated. The residue was reconstituted with 2.5 mL of 0.25N acetic acid wntaining 180 mglL Na2 EDTA. Prepared samples were kept

on ice until injected.

Standard curves were developed over the range of 25 nM to 250 nM for al1

metabolites. The Iimit of detection for al1 metabolites was 25 nM).

4.2.4 Detemination of clomipramine and desmethyIcIomipamine

The method for determining clomipramine and desrnethylclomipramine is

described in Chapter 3. Briefly, clomipramine and desrnethylclomiprarnine were

extracted from plasma using a three-step procedure (Godbillon & Gauron 198 1).

Concentrations of total clomipramine and total desmethylclomipramine were then determined by gas chromatography with mass-spectrometric detection (Sioufi et al

1988). The Iimit of detection was 2 ng/mL.

4.2.5 Staüstical Analysis

Four dogs had missing data on one occasion each because CSF could not be obtained due to the development of fibrosis at the site of sampling. Missing observations were estimated as the within-period means of the respective dog's metabolite levels. Treatrnent contrasts were then calculated for each dog at each week of sampling (treatment contrast = [concentration of metabolite on placebo] -

[concentration of metabolite on clomipramine]).

A repeated measures analysis was performed using a general Iinear mode1 to test whether the treatment contrasts for each metabolite were significantly dRerent frorn zero (Senn 1993, pp43, p67; Wallenstein & Fisher 1977). The

assumption of normality was not tested because the sample size was srnall

(Kleinbaum et al 1988, pl08). The assumption of hornoscedasticity was tested

using BartletYs test (Shoukn 8 Edge 1995. p9) and was upheld (x2 c 11,5 df).

The experiment had a nested factorial design. Dogs were a random effect

and were nested within treatment order. The hypothesis of no treatment effect

was tested using a rnixed model with the error term specified as dog(treatrnent

order) (Montgomery 1991, pp440450). The variables modelled were treatrnent

order. dog(treatrnent order), week, order*week, erythrocyteç (lm1 CSF), thiopental

dose (mgkg) and the interval between the induction of anaesthesia and the time of

CSF sampling (min). Week 6 was the reference variable for 'Lveekn. Canyover

effects were not included (Senn 1993, ppl4-15,226-238). Modelling was by

backward elimination; parameten were excluded if their associated p-value

exceeded 0.2. The variables 'treatrnent order' and 'week' were required in the

model and were retained independent of their associated p-value. Because the

same outcome was measured by hoquestions, the probability of erroneousiy

declanng a treatment effect (Type I error) was increased (Kleinbaum et al 1988.

pl3; Streiner 1993). Consequently, the Bonferroni approach was used: when each question was modelled, the level of significance was 0.01 5 (0.0513 = 0.01 5) so that the overall probability of a Type I error was maintained at a = 0.05 (Pocock 1987,

pp230-231; Kleinbaum et al 1988, Streiner 1993). The correlation of plasma concentrations of clomipramine and

desmethylclomipramine with the concentrations of metabolites in the CSF was

estimated using the product moment correlation coefficient (Steel 8 Torrie 1980,

~~272-281).

4.3 Results

The dogs remained clinically healthy throughout the experiment. None showed

abnormalities on haematological and biochemical tests. All CSF samples were

normal on routine analysis. After CSF was thawed for analysis, al1 metabolites

were stable at room temperature for at least 24 h. However, the exogenous 5-

HlAA used in the standard mixture showed 10% loss after 2.5 h at room

temperature.

Figures 4.1 and 4.2 demonstrate no marked change in concentrations of 5-

HIAA and HVA in CSF following 6 h of storage at 4°C. Figure 4.1 shows the mean

(SD) concentration of 5-HIAA and HVA within each dog throughout the increasing periods of storage at 4°C. The small SD indicates that there was little variation within dogs during storage. Figure 4.2 shows the mean (SD) concentrations of metabolites, at each of the successively longer periods of storage at 4°C. Only one dog (Dog 4) had enough CSF for analysis at 6 h; his concentrations of HVA

(100.6 pmollml) and 5-HIAA (60.8 pmoUrnL) were similar to his concentrations throughout the 6 h period (cf Figure 4.1). Figure 4.2 shows a marked peak in Figure 4.1 Mean withindog concentrations of 5-HIAA and HVA in cerebrospinal fluid during storage at 4' C. Samples were held at 4OC for increasing periods, to a maximum of 6 II.

Figure 4.2 Mean behveen-dog concentrations of 5-HIAA (O) and HVA (a) in cerebrospinal fluid during storage at 4°C (n = 6). Samples were held at 4°C for increasing periods, up to a maximum of 6 h

D uration of storage at 4C (min) mean HVA after 10 min at 4°C- The peak was due to a transient, 100% increase in Dog 6's HVA.

Table 4.1 shows the dogs' mean (SD) concentrations of monoamine metabolites after 1, 2 .4 and 6 weeks of treatment with dnig and placebo. Figs

4.3 to 4.5 show the mean (wlhin-âog) treatment contrasts * 95% confidence intervals (CI), at weeks 1,2,4 and 6. These figures suggest a treatment effect on al1 three metabolites at week 6.

Table 4.1 Mean (SD) concentrations of 5-hydroxyindoleacetic acid (5-HIAA), hornovanillic acid (HVA) and 3-methoxy 4-hydroxyphenylglycoI (MHPG)in the cerebrospinal fiuid of six dogs, following treatment with clomipramine (3mgkg q 24 h) and placebo

Treatrnent Week of 5-HIAA t SD HVA ,t SD MHPG k SD Treatment (pmoVml) (p rno VrnL) (p mollm L)

Placebo

Clomipramine Mean [concn of MHPG Mean (concn of HVA on Mean [concn of 5-HIAA on placebo - concn on placebo - concn on on placebo - concn on clomiprarnine] (pmollmL) clomipramine] clomipramine] (prnollrnL) II CL 4 A -* (prnoI/rnL) uio~ouioui The final results of statistical modelling are summarised in Table 4.2. The

model for HVA was significant (p = 0.01 1) and showed a rnarked dog effect (p =

0.007), but there was no treatment effect (p = 0.16). The models for 5-HIAA (p =

0.093) and MHPG (p = 0.061) were not significant, nor were their respective

treatment effects (5-HIAA p = 0.47; MHPG p = 0.045). The covariate RBC, which

refiected blood contamination of the CSF dun'ng sampling, approached

signifieance in the case of 5-HIAA (p = 0.03). However, the anaesthetic cuvariates

were not significant for any of the metabolites and were the first parameten to be

removed from the respective models. The results of the rnodelling procedure for

each metabolite are shown in Appendix 1.

Neither clomipramine nor desrnethylclomiprarnine were detected in CSF.

However, in plasma, the concentration of clomipramine and desmethyl-

clomipramine was above the lima of detection in al1 samples except one. The weekly concentrations of both compounds in plasma are summarised in Table 4.3: the large SDs suggest large interdog variability and, in relation to this, week is

unlikely to have had a significant effect on the concentrations of dmg and metabolite. The correlations between the levels of dmg and metabolite in plasma and the concentrations of 5-HIAA. HVA and MHPG in CSF are shown in Table 4.4.

There were small negative correlations, but none were significant. Table 4.2. Model parameters and estimates of the effect of clomiprarnine (3 mglkg q 24 h for 6 weeks) on the monoamine metabolites 5-HIAA, HVA and MHPG in canine cerebrospinal fluid, in a repeated measures AB-BA crossover experiment. Statistical analysis was by multivariate linear regression (backward elimination); CC = 0.015.

Metabolite F Value Pr > F Parameters in final F Value Pr > F Estimated treatment T Pr > 7 model effect (SE) (Ho:effect=O) (95% CI)

5-HIAA 2.38 0,093 Dog(order) 332 0.04 24.8 (33.2) 0.75 0,47 Order 0.16 0.71 (-40.3 -89.9) RBC 5.91 O,03 Week 1.93 0.17

HVA 4.16 0.071 Dog(order) 5.92 0.007 37 (24.3) 1.52 0.76 Order O. 39 O, 566 (1 0.6-84.6) Order*week 3.43 O. 052 Week 3,15 0.065

MHPG 2.49 0,061 Dog(order) 2,27 0,11 7.1 (3.3) 2.18 0.045 Order 2.1 0,22 (0.6-13.6) Week 2.02 O,15

HVA: homovanillic acid 5-HIAA: 5-hydroxyindoleacetic acid MHPG: 3-methoxy 4-hydroxyphenylglycol RBC: erythrocyte count in cerebrospinal fluid Order: treatment sequence SE: standard error 95% CI: 95% confidence interval Table 4.3 Mean (SD) concentration of clornipramine and desmethylclornipramine in plasma of 6 dogs during treatment with oral clomipramine (3 mgkg q 24 h) for six weeks. Plasma was obtained approxirnately 18 h after dosing.

Table 4.4 Product moment correlation coefficients between the concentrations of clomipramine and desrnethylclomipramine in plasma and the concentrations of 5- HIAA, HVA and MHPG in canine CSF during six weeks of treatment with oral clomipramine (3 mg/kg q 24 h). n = 6.

Metabolite HIAA HVA MHPG

Clomipramine -0.1 1 -0.29 -0.01 Desmethylclornipramine -0.1 3 -0.22 -0.71

- 5-HIAA: Ihydroxyindoleacetic acid - HVA: homovaniiiic acid - MH PG: 3-methoxy 4hydroxyindoleacetic acid 4.4 Discussion

This study investigated whether the turnover of serotonin, norepinephtine and dopamine in the brains of behaviourally nomal dogs, measured by CSF concentrations of 5-HIAA, HVA and MHPG, was affected more by clomiprarnine than by placebo. The unadjusted data (Table 4.1) show concentrations of MHPG of the order reported in other normal dogs (Faull et al 1982). However, the concentrations of 5-HlAA and HVA in the present study were higher than those reported previously (Faull et al 1982, Vaughn et al l988a); these differences rnay reflect inter- dog variability and differences in chrornatographic techniques.

The treatrnent contrasts at week 6 suggested an effect on al1 three metabolites (Figs 4.343, but statistical analysis dernonstrated little evidence against the nuIl hypothesis (a = 0.01 5) (Table 4.2). Only HVA had a significant model (p = 0.01 1) but the treatrnent effect was not significant (p = 0.16). With a less conservative approach (a= 0.05). the model for 5-HIAA was not significant, but that of MHPG approached significance (p = 0.061), and the treatrnent effect on

MHPG was significant (p = 0.045). At the present tirne, the biological significance of a treatment effect on MHPG in dogs is not obvious.

Overall, our results suggest that clorniprarnine does not affect the turnover of serotonin, dopamine and norepinephnne in the brains of behaviourally normal dogs, as indicated by the concentrations of 5-HIAA, HVA and MHPG in CSF.

However, in a randomised, controlled, double-blind clinical trial (Chapter 7) clorniprarnine was given twice daily (3 mgkg q 12 h) to dogs with canine CD, and a significant behavioural effect was observed. matfinding is consistent with the

hypotheses that clorniprarnine affects central monoamine turnover andior neuronal

activity. The present results are discussed in light of the cfinical efficacy of

clomipramine, with ernphasis on the methodology of the present experïment, the pharmacokinetics of clomipramine and the reports from other species.

Methodological sources of variation in the measurement of monoamine metabolites in CSF have been reviewed (Berb'lsson 8 Asberg 1984), but were minimised in Our experknent. The dogs' diet and exercise. the site of rneningeal puncture and the time of sampling were consistent throughout. The effectç of anaesthesia and of any contamination of CSF by plasma were also taken into account. The chromatograp hic rnethod was very reproducible and, unlike earlier methods reported in dogs (Vaughn et al 1988a), it permitted measurernent of

MHPG with minor modifications. The effect of storage at 4% on the concentrations of 5-HIAA and HVA in CSF was also considered. During storage at

4" C, there was littie wiaiin-dog variability over time, but Dog 4 showed a transient peak in HVA after 10 min. The cause of the peak is not clear; it is unlikely to be significant because, throughout the experhent al1 the dogs' samples were held at

4°C for at Ieast 30 min before being fiozen. Following fifteen months' storage at -

80" C,the 5-HIAA, HVA and MHPG were stable in CSF for at least 24 h both at room temperature and at 4" C. These findings are consistent with human data

(Langlais et al 1982) and confin that when canine CSF is taken for the rneasurement of monoamine metabolites, it need not be fiozen as soon as it is obtained.

Other methodological issues which coukl have affected our data, are the rnethodological assumptions, the small sample size, the dosing and sampling regimens, and the fact that the dogs were behaviourally normal. The study protocol was simple and inexpensive and has been widely reported in humans

(Asberg et al 1977, Thoren et al 1980, Altemus et al 1994). However, the rnethodological assumptions (Moir et al 1970, Kopin 1985) have been less widely discussed, nor are they easy to test One assumption is that the metabolites in

CSF originate only from the brain: this holds for 5-HIAA and HVA in dogs (Moir et al IWO), but not for MHPG, which can diffuse across the blood-brain bamer from plasma (Kopin 1985). If the concentration of MHPG in plasma is known, the concentration in CSF can be corrected accordingly (Kopin 1985). However, the concentration of MHPG in plasma was not measured in the present study. The assurnption can also be violated if there is blood contamination of CSF during sampiing; Our data indicated that such contamination couid have inffuenced 5-

HIAA. Another assumption is that concentrations of monoamine metabolites in cistemal CSF reflect the concentrations in local areas of the brain (Moir et al 1970).

This assumption may not hold for 5-HIAA, HVA or MHPG, because their concentrations Vary amongst brain regions (Eccleston et al 1968). For example, in

Dobermans, the highest concentrations of 5-HIAA and HVA were in the forebrain

(Mefford et al 1982), but Eccleston et al. (1968) and Moir et al. (1970) showed that 5-HlAA in cistemai CSF may not reflect 5-HIAA in the forebrain. Also, only a small

proportion of 5-HlAA and HVA passes directly from the brain into CSF. Most 5-

HlAA and HVA are acüvely transported out of the brain (Guldberg et al 1966,

Moir et al 1970, Kopin 1985); therefore, regional changes in the metabolites may not be detected in cistemal CSF. Another limitation of the methodology in this experirnent is that the concentrations of monoamine metabolites in CSF refiects neurotransmitter turnover, but not neuronal acüvity (Comrnissiong 1985). This

Iimits the inferences which can be made about the gross effect of the dnig on brain activity .

An anaesthetic effect was not detected. This could reflect an adequate study design, or the smali sarnple size. Anaesthetic effects on monoamines have been reported in other species (Bourgoin et al 1975, Westennk et al 1977, Barkai

1979).

In this experiment, a sample size could not be calculated a priori. The variances of the treatment contrasts were unknown because no similar data existed; however, the crossover design had the potential for high power.

Behaviourally normal dogs were used because they were readily available. Data from dogs with CD are also needed, but data from normal subjects would confin whether any differential neurochemical response to clomiprarnine is a direct effect of the dnig rather than being secondary to syrnptomatic improvernent (Altemus et al 1994). The dosing regimen in the experiment was the same as that used clinically at the Ontario Veterinary College; also, Rapoport et al (1 992) reported that it was clinically effective. However, the results of Our phamiacokinetic study

(Chapter 3) indicate that the dosing frequency may not have been sufficient to produce an effect on the monoamine metabolites.

Neither clomipramine nor desmethylclornipramine were detected in the CSF and both may have been cleared rapidly from the brain: in rats, the elimination half-lives of both dnrg and metabolite in the brain were 0.5 - 2 h (Friedman &

Cooper 1983). Both drug and metabolite were detected in plasma, but there was no signifiant correlation between their concentrations in plasma and the concentrations of monoamine metabolites in CSF. This could reflect the small sample size or Ït could be due to an inadequate dose of drug and/or a low density of drug-binding sites in the brain (Adell & Artigas 1991, Fujita et al 1991).

An additional factor that may have contributed to the results of this study is the sampling regimen. Samples of CSF were taken 18 h after dosing, so that the dogs would not be anaesthetised with food in their gastrointestinal tract. By contrast, when clornipramine was given chronically to patients with depression

(Traskman et al 1979) and obsessive compulsive disorder (Thorén et al 1980,

Altemus et al 1994), samples of lumbar CSF were taken approximately 8 h after medication (maximum of 250 mg divided TID in adolescents; maximum 150 mg divided TID in adults), and the concentrations of 5-HIAA in CSF had decreased.

The effect of clomipramine on HVA and MHPG was not consistent across these studies. HVA either decreased (Altemus et al 1994). or increased (Thorén et al l98O), or did not change (Asberg et al 1977, Trâskman et al 1979). MHPG either was not affeded (Thorén et al 1980) or was reduced (~sberget al 1977, Traskrnan et al 1979, Altemus et al 1994). Similar results were reported in rats, depending on the region of the bain that was examined WesterÏnk et al 19n. Mousseau &

Greenshaw 1989, AdelIl? Arügas 1991, Fujita et al IWl). Our results cannot be directly compared to those in humans or rats because of differences in study design. The human research papers did not describe healthy subjects (Altemus et al 1994, Thorén et al 1980, Traskman et al 1979, Asberg et al 1977). lacked a control treatrnent (Altemus et al 1994, Traskman et al 1979, Asberg et al 1977). did not address the rnethodological assumptions (Altemus et al 1994. Thorén et al

1980, Traskman et al 1979, Asberg et al 1977) and lacked appropriate statistical analyses (Altemus et al 1994, Thorén et al 1980, Traskman et al 1979. Asberg et al 1977). In rats, presumptively normal animals were used, but the brain was examined, not CSF.

In conclusion, this experirnent did not demonstrate an effect of clomipramine (3 mgkg q 24 h) on monoamine turnover in the brains of behaviourally normal dogs, as indicated by the concentration of the respective metabolites in CSF. However, further studies of central neurotransmitters are warranted in compulsive and behaviouwlly nomal dogs. The present experiment provides a basis for such studies: it allows a sample size to be estirnated, it demonstrates that CSF need not be frozen as soon as it is obtained. and 1 also describes a pracücal chromatographie method for detecting 5-HIAA, MHPG and

HVA. A close review of the Iiterature suggests that cistemal CSF may not be adequate to detect an effect of clorniprarnine on monoamine metabolites. More localiseci techniques such as microdialysis (Adel18 Artigas 1991. Rutter 8

Auerbach 1993) may be more appropriate for canine experiments. CHAPTER 5: OlAGNOSlS OF CANINE COMPULSNE DISORDER:

EVALUATION OF THE DIAGNOSTIC ACCURACY OF FORMAL CRfTERlA

5.1 Introduction

There is ernpirical evidence that canine compulsive disorder (canine CD) is a distinct clinical condition that is associated with a central neurochernical dysfunction (Hewson & Luescher 1996)- However. the pathophysiology of canine

CD has not ben systematically investigated and there is no 'gold standard' test which indicates tnie disease status. A 'gold standard' test is a definitive diagnostic test (Sackett et al 1991, p33). When no 'gold standard' is available, the accuracy of a diagnosis of canine CD can be quantified as the agreement between two diagnostic methods (Feinstein & Cicchetti 1990). Cohen's kappa, an index of chance-corrected agreement, may be used for this purpose.

Two diagnostic approaches to canine CD have been describeci. One is an algorithm in which CD is diagnosed by defauit, alter ciinical tests have mIed out other conditions (Overall1992). This approach has limited because the tests are expensive and their diagnostic properties (eg sensitinhty and specificity) are largely unknown (Sackett et al 1991, pp69-152). The second diagnostic approach uses formal diagnostic criteria that are based on the hypothesised development of canine CD; the diagnosis is made from the direct assessrnent of the behavioural history (Hewson & Luescher 1996). This technique is inexpensive and non- invasive, but its diagnostic accuracy has not been described. The following study used data from a clinical trial that assessed the effiwcy

of clomipramine in the treatment of canine CD. The objectives of the study were (i)

to quantify the agreement between an expert's diagnoses of canine CD and

diagnoses made using formal criteria derived fiorn Hewson & Luescher (1996). and

(ii) to review the formai criteria in light of any diagnostic disagreement.

5.2 Materials and Methods

5.2.1 Recruifment of Cases

During a 12-month period (March 1995-March 1996)' dogs with possible

CD were recniited through the veterinary press and public media. Following

referral by their veterinarian, interested owners received written information about

the clinicat trial, with a consent form that had to be signed and retumed before

owners couid participate further (Appendix 2). A total of 82 owners consented to

participate in the study.

5.2.2 Diagnostic methods

Participating owners received a list of questions about the history of their

dog's behaviour (Appendix 3). They were instructed to write down answers to these questions and to use them as a memory-aid during subsequent diagnostic

interviews. Owners then received two telephone interviews, approximately 3 h

apart. The same two interviewers were used consistently throughout the study; they were 'blind' to each other's diagnosis. In any household, both interviews

were conducted with the same household mernber- The order of interviews was

not randomised; approximately 85% of the sample received the expert's interview

second.

5.2-2.7 Interview #7

Interview #1 was conducted by the principal investigator (CH), using a questionnaire (Appendix 4) that was based on the hypothesised development of canine CD (Hewson 8 Luescher 1996). The questionnaire was developed from (i) a review of cases of canine CD that had been seen at the chic during the previous five years, and (ii)the case hiçtory forms used at the Ontano Veterinary

College Behaviour Service. Most of the questions had continuous response options and the remainder had dichotomous response options. The questionnaire was reviewed by three experts in clinical ethology and it was then pre-tested once.

The pre-test sarnple comprised ten owners whose dogs either were not known to have a behavioural disorder or had been referred to the Ontario Veterinary College

Behaviour Service with possible CD. These owners did not participate in the clinical trial.

Minor changes in wording resulted from the pre-testing and review procedures. The final questionnaire comprised 133 questions: 83 questions were about the problern behaviour, 13 questions were about housing, diet, training and exercise and the rernaining seven questions were about the dog's general behaviour and interactions in the household (Appendix 4). The interview lasted

approximately 35 minutes.

Canine CD was diagnosed by evaluating the behavioural history with

respect to formal diagnostic criteria (Table 5.1 ). The criteria were designed to

differentiate the disorder from normal conflict behaviour, which is context-specific

(Question B4d, Appendix 4), leamed behaviour (Question B5, Appendk 4) and

seizures (Question B6, Appendix 4) (Hewson 8 Luescher 1996). A dog was

considered to have CD if all the criteria were met.

52-2.2 Interview #2

The interviewer (AL) was an expert on canine CD and used the case history form of the Ontario Veterinary College Behaviour Service (Appendix 5). This form permits a less structured interview than the first interview. The expert's interview lasted approximately 20 min.

5.2.3 Measurement of diagnostic accuacy

Diagnoses were compared in a 2x2 table (Table 5.2) and the expert's opinion was aie referenœ standard. Cohen's kappa was calculated and the appropriate confidence interval was estirnated after testing for bias with

McNemar's chi-square (Shoukri & Edge 1995, pp83-90). Table 5.1 Critena on which a formal diagnosis of canine compulsive disorder was made (after Hewson & Luescher (1996)).

A dog has CD if al1 of the following critena are tnie

(i) physical examination indicates that the dog is 'clinically normal'

(ii) the dog shows one or more of the following behaviours: - tail-chasing with or without tail-mutilation - air-biting - pacing or running in circles - Iicking andlor nibbling of one area of the body sufkient to cause a sore ('lick - granuloma'); persistent licking of the body without causing mutilation - licking of objects or the air - flank-sucking - self-directeci aggression - 'checking' the rear end. - stereotypic barking - fixation with eg lights, a toy - othef?

(iii) there was an identifiable conflict or fnistration when the behaviour first started, andfor currently. Conflr'cf connotes a motivational state in which two incompatible behavioural tendencies are aroused equally (Immelmann & Beer 1989, p57). Frustration may be understood to occur when the environment places constraints on the expression of a highly motivated behaviour (Yates 1962, p176). Situations of conflict or frustration include - confiicting motivation (fear and other) - separation anxiety - absence of releasing stimuli - physical restraint - changes in social environment

(iv) the number of contexts in which the behaviour was show increased from the time the behaviour started is now shown in a number of different contexts

(v) in the intewal since the behaviour started, the daily frequency of the behaviour has increased

(vi) the behaviour is not purely conditioned. It is shown when the owners are not in the immediate vicinity of the dog or when the owners are absent

(vii) the behaviour is not a seizure: there is no history of pre-ictal or post-ictal seizure actïvrty. Also, the dog can be distracteci while showing the behaviour Table 5.2: 2 x 2 table to illustrate how rneasures of diagnostic agreement were calculated in a study cornparhg forma1 diagnostic criteria for canine CD with an expert's diagnosis

Diagnosis of Format Criteria CD+ CD- TOTAL CD + a b G1 Expert Diagnosis CD - c d G2 TOTAL F-l F2 N

- CD+: dog has compulsive disorder - CD-: dog does not have compulsive disorder - Observed agreement, Po = a + dM - Agreement expected due to chance, Pe = /FlxGl )IN + (F2xG21fN N - Cohen's kappa = Po - Pe 1 -Pe

Cases in which there was diagnostic disagreement were reviewed, to identify

sources of the disagreement.

The consequences of misdiagnosis (Szatrnari 1992). for the dogs and their owners, were not addressed. All dogs that did not go on to participate in the clinical trial were referred to the Ontario Veterinary College Behaviour Service. 5.3.1 Description of the sample

5.3.1.7 Sample size

A total of 84 dogs underwent diagnosis for canine CD. The dogs came from 82 households; two household each provided two dogs in the study. The total number of interviews was 86 because. for reasons unrelated to the study, the owners of two dogs that were eligible for the clinical trial could not participate following the standard interviews. These owners were therefore re-interviewed six months later and parh'cipated in the clinical trial.

5.3.7.2 Gender

The sample was 35% neutered male. 37% neutered female, 21 % intact male, and 7% intact female.

5.3.7.3 Age

The dogs ranged in age from 0.5-1 1 y (mean 4 y. SD 2.4; 25th percentile 2 y; median 3.25 y; 75th percentile 6 y).

5.3.7-4 Breed

The three most comrnon breeds were Doberman (12%. n=10). German

Shepherd (13%. n=l 1) and cross-bred (12%, n = 10). The remaining breeds were

English Bull Terrier (7%, n=6), Miniature Schnauzer (7%, n=6), Rottweiler (3.5%.

89 n=3), Labrador (5%. n=4), English Springer Spaniel(3.5%, n=3), Amencan Cocker

Spaniel(3.5%, n=3), Flatcoated Retriever (2.5%. n=2), Jack Russell Terrier (2.5%.

n=2), Dalrnatian (2.5%, n=2), and Standard Poodle (2.5%. n=2). The rernaining

24% of the breeds (n=20) were Amencan Bull Terrier, Bearded Collie, Border

Collie, Bouvier de Flandres, Cairn Terrier, Cavalier King Charles Spaniel, Chinese

Crested Dag , Miniature Dachsund (long-haired), lrish Setter. Keeshund,

Neapolitan Bull Mastiff, Old English Sheepdog, Pomeranian (miniature),

Pomeranian (standard), Pug, Shetland Sheepdog. ShiTzu, Staffordshire Bull

Terrier. Miniature Poodle and Toy Poodle (al1 n=l).

5.3.1.5 Behaviour

The behaviours for which the dogs were referred are shown in Table 5.3.

The three cornmonest behaviours were spinning in tight circles (27%, n=22), chronic licking with self mutilation ('lick granulorna') (20%, n=16). and licking the body without injury or Iicking objects (12%, n=10). The dogs had been showing their behaviours for an average of 2.2 y, (SD 1.8 ; range 1 mo-8.5 y ,25th percentile 10 mos, median 1.5 y; 75th percentile 3 y). The mean age of onset of the behaviour was 2 y (SD 1.8 ; range 2 mos-10 y ,25th percentile 6 mos, median

1-5 y; 75th percentile 2.5 y) Table 5.3 Behaviours and breeds of 82 dogs that were referred for diagnosis for possible canine CD

Behaviour Breed and Number of Dogs

Chronic licking with self-mutilation 5 cross-bred, 4 Doberman, 2 German ('lick granuloma') (n=16) Shepherd, other

Licking body with out self-injury, Miscellaneous or licking objects (n=1 O)

Spinning on the spot in a tight circle 5 German Shepherd, 4 English Bull as if tail-chasing (n=22) Terrier, 1 Amencan Staffordshire Bull Terrier, 3 Doberman, 9 Miscellaneous

Running or pacing in a large circle 3 German Shepherd, 2 Standard (n=6) Poodle, 1 Flat-Coated Retriever

Checking (n=7) 6 Miniature Schnauzer, 1 Miniature Poodle

Fixation with Iight and shadows (n=5) 2 Rottweiler, 1 Cairn, 1 Labrador 1 Staffordshire Bull Terrier

3 Doberman, 1 Dachshund, 1 English Bull Terrier

2 cross-bred, I Miniature Pomeranian

"Pacing back-and-forth (n=2);toy fixation (n=2); chewing the fur on the ears (n=1); assuming a Frozen 'sawhorse position' (n=l); polydipsia, (FI); barking (FI) 5.3.2 Diagnostic agreement

Cases of canine CD are denoted as CD+; dogs that were judged not to

have CD are denoted CD-. The expert diagnosed 77 CD+ (go%), but the criteria

only diagnosed four dogs as CD+ (45%)-

The formal diagnostic criteria (Interview #1) could not be applied to 26 dogs

(30%) because the behavioural histoty was not fully known (Table 5.4). Sixteen of

these dogs already had the behaviour at the time when the owners acquired them.

In the other 10 cases, the owners were unable tu judge whether the behavioural frequency and the number of eliciting contexts had increased from the time when the behaviour began.

The 16 dogs that showed the behaviour when the owner acquired them, were diagnosed with CD by the expet These dogs were known to have had a history of physical abuse andlor prolonged close confinement and lack of exercise. ln one case, the dog had been flown to Canada from a breeder in the US and showed severe tail-chasing from the time the owner colfected the dog at the airport. The breeder reported that the dog had never shown that behaviour before and it is possible that the plane joumey may have initiated the behaviour. Of the other ten dogs, the expert diagnosed nine of these cases as CD+ and one as CD-. Table 5.4: Breakdown of the diagnoses of canine compulsive disorder made by an expert, and with three diagnostic criteria. The criteria were (i) identifiable conflict at the time when the behaviour began, (ii) increased frequency of the behaviour from the time the ownerç first noticed it, and (iii) increase in the number of situations in which the behaviour was performed.

Dog has compulsive Dog does not have disorder (expert compulsive disorder diagnosis) (expert diagnosis)

Criteria could not be used (n=26)

Dog showed behaviour before acquired, therefore insufficient history for use of criten'a (n=16)

Owners couldn't recall information 9 therefore criteria could not be used (n=i O)

TOTAL

Criteria could be used (n=60)

None of the 3 criteria met (n=l3)

All three criteria met (n=4)

Initial confiict and increased context (~4)

Initial conflict and increased Frequency (n=7)

1 ncreased context and increased frequency (n=6) lncreased context (n=l ) increased frequency (n=9)

Initial conflict (n=16; includes 1 case of conditioned behaviour)

TOTAL Chance-corrected diagnostic agreement was calculated from the remaining

60 interviews. The 2x2 table of the data dernonstrated very discordant marginal distrÏbMons (Table 5.5). The criteria and the expert agreed that 4 dogs had CD and 8 did not. The remaining 48 dogs were al1 diagnosed as CD+ by the expert, but were diagnosed as CD- by the criteria. The observed and chance agreements were approximately equal and the kappa statiçtic was correçpondingly low (0.02).

Table 5.5 2 x 2 table and indices of agreement between diagnoses of canine CD made by expert opinion and diagnoses made by formal criteria. (Abbreviations - see Table 5.2)

Diagnosis of Formal Criteria CD + CD - TOTAL CD + 4 48 52 Expert Diagnosis CD- O 8 8 TOTAL 4 56 60

Estirnated indices of inter-method agreement:

- Observed agreement: 0.2 - Agreement expected due to chance: 0.1 8 - Chance-corrected agreement. kappa (95% confidence interval): 0.02 (- 0.004 - 0.05) - McNemar's chi-square, I d-f.: 48 (p = 0)

Prevalence of canine CD in the study sample (expert diagnosis): 52/60 = 86% The dmerence between the cells of disagreement (Table 5.2,48/60 vs. 0160) iç

reffected in McNemar's teçt for bias tL2,1 d.f. = 48; p =O). The prevalence of

canine CD in the 60 dogs from which K was calculated, was 86%, according to

expert opinion.

When the behavioural histories of the 48 cases of diagnostic disagreement

were reviewed, all the dogs met the criterion of not having seizures (Question 86,

Appendk 4). Only one dog was diagnosed with conditioned behaviour (Question

B5, Appendix 4). The other 47 dogs failed to meet one or more of the following

criteria :(i)identifiable conflict at the time when the behaviour began; (ii)increased

frequency of the behaviour frorn the time the owners firçt noticed it (iii) increase in

the number of situations in which the behaviour was perfomed. Twelve dogs did

not meet any of these criteria.

The remaining thirty five dogs met one of the criteria, or different

combinations of two of the criteria. These data are shown in Table 5.4. In the dogs that only met one of the criteria, the most commun criterion was an identifiable conflict when the behaviour was first shown (n = 13). The dog with conditioned behaviour also met this criterion. Approximately equal numbers of dogs met the dflerent combinations of two of the criteria. 5.4 Discussion

The results demonstrated rnarked diagnostic disagreement between Mo

diagnostic tests for canine CD. This finding must be interpreted in the conïext of

prevalence (Walter 1984. Streiner 1995). The prevalence of CD in the present

study was high (86%). perhaps because many dogs were referred by veterinan'ans who were aware of the clinical trial. When prevalence is much greater than 50%, the maximum value of K is 0.1-0.2 (Streiner 1995). However, even with those

maximum values, the diagnostic agreement in the present study was very low.

This result is surprising because the diagnostic methods were similar conceptually and methodologically. Both conceived canine CD as a syndrome of abnomal confiict behaviours that have become emancipated from their original context, and both made a diagnosis by interviewing the owner on the telephone. The chief difference between the two diagnostic methods was that expert opinion was more subjective than the fomal criteria and would have involved more inference.

Under the assumption that the expert's diagnoses were correct, the likely causes of diagnostic disagreement were (i) the order of the interviews, (ii) the potentially low reliability and validity of the information supplied by the respondents

(Sudman & Bradbum 1982 pp36-53; Streiner 8 Norman 1995. pp69-84), and (iii) the setting of the interviews (Sackett et al 1991 pp40-41). The order of interviews was not randomised. The first interview could have led sorne owners to think that the behaviour was wone than they had imagined. They could then have given a more extreme assessment during the expert's interview, causing diagnostic

disagreement-

The test-retest reliability of the interviews was not assessed owing to lack of

resources. Also, when the first questionnaire was pre-tested, owners indicated that repeat interviews mig ht discourage hem fiom participating further in the clinical trial. The validity of the information provided by the owners could not be assessed because much of the information was historical (Sudman & Bradbum

1982, pp36-53). The three criteria which caused disagreement involved historical information. Although each owner received a preliminary questionnaire in advance of the interviews, to aid recall, it is possible that they recalled historical infomation inconsistently. This could have caused disagreement between the diagnostic methods. Other response biases that could have reduced the validity of the infomation are social desirability bias (unconscious tendency to create a positive impression) and 'faking good' (intentionally creating a false poslive impression).

Either of these biases could have caused some owners not to admit to adverse events in the dogts history (Streiner 8 Noman 1995, pp72-75). There was no suitable scale to measure these biases in the present study.

Additional sources of diagnostic disagreement relate to the interviewers and to the setting of the interviews (Sackett et al 1991 pp3542). In the fomal interview, al1 ownen were asked the same questions in a consistent fashion and this would have minimised inference. In contrast, the expert's interview was less stnictured and could have led to the recording of inference rather than evidence. Another source of variabiiii in the two diagnoses may have been the settings in

which they were conducted. Owners were requested to provide ample

undistracted time for both interviews, but this was not always possible and may

have reduced the respondents' concentration and thus the quality of the

infonation provideci-

Expert opinion was chosen as the reference standard because it was the

only one available. A limitation of expert opinion is that the diagnosis is based on

personal clinical experience, therefore diagnostic guidelines cannot be stated

explicitly (Sackett et al 1991 p5). Another limitation of expert opinion, and of the

formai criteria. is that their respective sensitivity and specificity are unknown, and K

is infiuenced by the sensitivity and specificity (Walter 1984, Byrt et al 1993). To

overcome this problem, Walter (1984) recommended the use of three independent

observers, but that was not logistically possible in the present study.

The three criteria which caused diagnostic disagreement were based on the

hypothesised development of canine CD. Their poor agreement with expert

diagnosis could mean that canine CD does not develop as hypothesised. This

demonstrates the need for developmental studies of the disorder. Studies of

diagnostic validity are also needed. They would confirm whether the behaviours are caused by a comrnon disease (Robins & Guze 1970, Szatmari 1992). In

psychiatry, there is a systernatic approach to diagnostic validity. The approach

includes studies of heritabitity, neurochemistry and aetiology (Robins & Guze 1970,

Feighner 1982, Kendler 1990). A similar approach would be appropriate for the syndrome that is currently known as canine CD. If diagnostic validity were esta blished, definitive diagnostic tests could be developed.

The critena which caused disagreement could be explored in developmental studies of canine CD. Among the cases of diagnostic disagreement, 13 dogs met the single criterion of initial conflict. Conflict andlor frustration are necessary criteria for diagnosing canine CD, as currentiy understood. However, neither is not a sufkient criterion because normal confiict or frustration-related behaviours can occur in dogs (Hewson 8 Luescher 1996). In the present study, the criterion of initial confiict or fnistration required the interviewer's inference and may not have been accurately assessed in every case.

The criteria of increased behavioural frequency or increase in the number of eliciting conte- are not sufficient for a diagnosis of canine CD, because either criterion could be met by conditioning alone. Compulsive behaviours may have a conditioned component, and developmental studies could help to describe the role of conditioning in the disorder.

in summary, these results confimi that before diagnostic tests for canine CD can be assessed, additional research is needed on the development and diagnostic validity of canine CD. Following such work, the present questionnaire and diagnostic cnteria might be adapted and evaluated for use in clinical practice. CHAPTER 6: DEVELOPMENT OF TWO QUESTIONNAIRES TO MEASURE

CHANGE IN BEHAVIOURAL SEVERITY IN DOGS WlTH COMPULSIVE

DISORDER

6.1 Introduction

The outcome measure in a clinical trial should permit valid and clinically relevant

inferences to be made about the efficacy of the treatments. In the only reported clinical trial of clamipramine as a treatrnent for canine compulsive disorder (canine

CD) (Rapport et al 1992)' the outcome was measured by the owners who had been taught to score the behavioural severity using a ten-point rating scale. The dogs' behaviour was persistent Iicking with self mutilation (acral lick dematitis) and. on the rating scale, zero represented no licking and ten represented the worst licking ever. The limitations of the sale are that it is comparative ('worst') and its psychornetric properties are unknown (Streiner & Norman 1995. p164).

An important psychornetric property is wnstnict validity. which descnbes how well a scale measures what it purports to measure. One index of constmct validity is the relationship between measurements made with the scale of interest, and measurements of other aspects of the 'constnict that the scale is measuring

(Streiner & Norman 1995, pp150-151). Canine CD is manifested by specific behaviours (Hewson & Luescher 1996), the seventy of which cannot be measured directly. However, there are indicaton of behavioural severity that can be measured. These indicaton include the hourly frequency of the compulsive behaviour, the duration of a typical behavioural episode (Martin 8 Bateson 1993.

pp62-68) and the number of eliciang contexts in which the behaviour is shown

(Hewson 8 Luescher 1996). If a global question, such as that of Rapoport et al.

(1992). were really measuring changes in behavioural severity, the following

predictions might be made: changes in score on the global scale (eg a change from ten to zero) would be accompanied by changes in behavioural frequency and/or duration andlor number of cuntexts. The changes in those three parameters would be in the same direction as the change in score on the global scale and would be statistically significant.

When a rating scale is used to measure change, the scale should be able to detect the minimal clinically important dfierence (MCID). The MClD is the smallest change which subjectç perceive as beneficial (Jaeschke et al 1989).

This chapter describes the development of a questionnaire that contained two global measures of behavioural severity, and questions about MCID. The chapter also describes the assessrnent of the global measures' construct validity.

The data were colIected in the course of a randomised controlled clinicat triai of clomipramine in dogs with CD. The trial is described fully in Chapter 7.

6.2 Materials And Methods

Two questionnaires were developed and used to measure the severity of canine compulsive behaviour. One questionnaire (Appendix 6) included two global questions about the behavioural severity (Questions 3 and 15) and was adrninistered to owners by telephone. The other questionnaire (Appendix 7) was used by the owners, to record behavioural frequency, duration and context. These data were used to validate the global questions. Both global questionnaires were based on dinical experience and on a current hypothesis about canine CD (Hewson

& Luescher 1996). Questions were devised after reviewing standard ethological methods (Martin & Bateson 1993) and after consulting with three experts (veten'nary behaviourist, clinical epidemiologist and social scientist). Questions were

'individualised' for each dog, and ternis used in the questions were cleady defined.

6.2.1.7 Item seiecfion and scaling

The telephone questionnaire is found in Appendix 6. A global question that was not explicitly comparative, was developed to measure behavioural severity

(Question 3). In that question, severïty was defined as "how many times a day

'Dog's Name' shows the 'Behaviour' and how long he shows it for". Owners were read fnre response options, on a Likert scale, and were asked to choose one response to describe the dog's behavioural seventy during the past five days. This question was asked at the beginning of the interview. The second global question about behavioural severity was the ten-point global scale of Rapoport et al, (1992); this question was asked at the end of the interview (Question 15). Both questions are shown in Table 6.1. In addition, owners of dogs with acral lick dermatitis were asked to rate the overali appearanœ of the dogs' lesions. using both aie Likert sale

(Question 1),and the ten-point scak (Question 16).

Table 6.1 Two global questions about behavioural severity, used as outcome measures in a clinical trial of clornipramine as a treatment for canine CD. Questions were 'indkidualised' for each dog. Question 15 was modelled on the rating scale of Rapoport et al (1992)

Question 3

Next, I would like to get an overall idea of how bad is the 'Behaviour' that vou see at present By 'bad' I rnean how often 'Dogs show the Behaviouf and how long he 'Behaves' for. Taking everything together dufina the past 5 davs, would you Say that the 'Behaviouf has been:

1 extremely bad 2 very bad 3 quite bad 4 somewhat bad 5 not at al1 bad

Question 15

Similarly, we can score 'Dog's' 'Behaviouf durina the past 5 davs on a scale of zero to ten. A score of 'O' rneans no 'Behaviour'. A score of '10' means that the 'Behaviour' is the worst it has ever ken. Overall how would you rate 'Dog's' 'Behaviour' during the past 5 davs (mat is, since Monday)?

No The worst 'Behaviour' 'Behaviour' ever Additional questions were designed for Mure use as measures of the MClD

(Questions Za, 2b, 4a, 4b, 5-7 ). Ownen were also asked about any other changes in the dog's behaviour (Questions 8 and 9) and about side effects (Questions 10 and 11). The questions on side effeds were 'funnelled' (Sudman & Bradbum 1982, pp219-221): firçt, owners were given the opportunity to describe any signs of il1 health that they had noticeci. They were then asked whether there had been an increase, a decrease or no change in each of the following: the dog's appetite, water consumption, frequency of bowel movernents, fkquency of urïnation, amount of tirne spent sleeping, level of alertness when awake, and bodyweight These çpecific questions were based on experience with clomiprarnine at the Ontario Veterinary

College. and on side effects descrïbed in humans (Ciba Geigy 1991).

Ownen were also questioned about any changes in the dog's routine. including additional treatments (Questions 12a, b). At the end of the second treatrnent, al1 owners were asked to describe anything that might have occurred during the trial and that might have affected the dog's behaviour (Question 17).

Other questions concemed cornpliance (Question 13a. 13b). Following the approach of Sackett et al (1991, pp258-261), owners were asked whether the dog had missed any treatrnents. The efficacy of masking was also assessed, by asking owners which treatment they thought their dog had been receiving cunently

(Question 14). 6.2.1.2 Pre-Testing

The questionnaire was pre-tested twice by telephone. The first pre-test

involved five owners whose dogs had CD (based on an expert's diagnosis) and were

being treated for 1 through the Ontario Veterinary College. This pre-test resulted in

grammatical changes and re-ordering of some questions. The questionnaire was

then reviewed by 20 graduate students as a class assignment in epidemiology.

Further minor changes in wording were made and the questionnaire was again pre-

tested by telephone on two more owners of compulsive dogs that were being treated

with medication. No changes resulted from this pre-test. The final questionnaire

contained 16 questions and took 12-1 5 minutes to administer.

6.2.2-1 Item selecfion and scaling

The owner-administered questionnaire was designed to be used daily.

Questions were asked about the nurnber of episodes of the behaviour that the owner had seen in the past 24 h (behavioural fkquency), the typical duration of episodes during the past 24 h (behavioural durabion) and the number of eliciüng contexts that had occurred in the past 24 h and to which the dog had responded with compulsive behaviour (behavioural context). Because each dog showed the behaviour in different contexts, Question 4 was "individualised'. The questionnaire was pre-tested twice; the same owners described for the

telephone interview took part in these pre-tests (Section 6.2.1.2).The

questionnaires were mailed to the owners who were instnicted in their use and were

asked to use them daily for fiedays. This pre-test resulted in some changes in format and the addition of questions about the owners' feelings about the dogs' behaviour. The latter asked owners to rate their level of wony, fnistration and anger about Be dog's compulsive behaviour, and also to rate the degree to which the behaviour had prevented them fiom enjoying the dog's Company.

The questionnaire was then reviewed by 20 graduate students as before

(Section 6.2.1 -2)and the question on behavioural duration was reworded and expanded to account for the shortest, longest and typical episodes. The final form of the questionnaire is shown in Appendix 7. In that questionnaire, the behaviour was clearly defined at the beginning, as was the terni 'behavioural episode'. The questions al1 referred to events that had occurred during the past 24 hours, and covered behavioural frequency (Questions 2a-c), duration (Questions 3a-f) and context (Questions 4-6). There were also questions about the owners' feelings

(Questions 7ad). The questionnaire took 5-8 minutes to complete.

6.2.3 Data collection

Data for validation of the outcome measures were obtained fiom dogs that participateci in the clinical trial of ckmipramine (n=51). The trial was double-blind, randomised and placebo-controlled, with a balanced AB-BA crossover design.

Dogs received each treatment for four weeks, with a two-week, treatment -Free

'washout' period between treatments. The owner-adrninistered questionnaires

were sent to the owners a few days before cornmencing the trial. Every owner

was then telephoned, and was taught how to use the questionnaires by completing

one under the guidance of the der(CH). The owners used the questionnaires

daily for the next five days, to obtain pradce.

On the last five days of each treatrnent period, owners used their questionnaires daily. The sarne household member was required to score their dog's behaviour throughout the trial, at approxirnately the same time each day. On the final day of each treatment period, the owner who had been scoring the dog's behaviour had a telephone interview with a trained 'blind' interviewer who used the telephone questionnaire. In 88% of cases, the owner had both interviews with the same interviewer.

6.2.4 Data analysis

AI1 analyses were conducted 'blind' to treatment sequence. The data were analysed following the approach of Senn (1 993, pp109-112). For each global question (Table 6.2)' the data from the two treatment periods were used to categorise each dog as 'Better in the second period', 'The sarne in both periods' or 'Worse in the second period'. For Question 3, any change in rating between

Period 1 and Period 2 was categorised as 'Better' or 'Worse', as appropriate. For example, a change from "Somewhat badn in Period 1 to "Quite bad" in PerÏod 2

was categorised as 'Worse in the second period'. This is illustrated in Table 6.2.

For Question 15, a change in score of 2 or more was classified as 'Better' or

'Worse' as appropriate.

The construct validÏty of the three categories of outcome ('Better', 'Same'

and 'Worse') was assessed by using the data on behavioural frequency. typical

duration of a behavioural episode, and behavioural context These behavioural

data were summarised to provide 5-day means of frequency. duration and context for each dog in each treatrnent period. Then. the ratio of each dog's mean in

Period 1 to its mean in Period 2 was calculated; rneans of zero were recorded as

0.01 so that a ratio could be calculated. Construct validity was then assessed according to the following predictions:

- Dogs that were 'The same in both periods', would have the same

behavioural frequency (and/or duration andfor context) in Period 2 as in

Period 1. Consequently, the median ratios of the behavioural parameters

would not be signifcantly different from 1.

- Dogs that were 'Better in the second period' would have a lower

behavioural frequency (and/or duration andlor context) in Period 2

compared to Period 1. Consequently, the median ratios of the behavioural

parameters would be significantly greater than 1. Table 6.2 Use of behavioural ratings from a 5-point Likert scale (Question 3) to describe change in behavioural severity, Behaviour was rated at the end of each of two treatment periods; change was classified as 'Better in Period 2', 'The same in both periods' or 'Worse in Period 2'

eriod 2

Not at al1 Bad Somewhat Bad Quite Bad Very Bad Extrernely Bad

Not at al1 Bad Same Worse Worse Warse Worse

Somewhat Bad Better Same Worse Worse Worse

Quite Bad Better Better Same Worse Worse

Very Bad Better Better Better Same Worse

Extremely Bad Better Better 8etter Better Same - Dogs that were 'Worse in the second period', would have a higher

behavioural frequency (andfor duration andfor context) in Period 2

compared to Period 1 Consequently, the rnedian ratios of the behaviourai

parameters would be signifkantfy less than 1.

Each of these predictions was assessed using the ordinary sign test

(Dickinson Gibbons 1971, pp 100-202;Freund 1992, pp570-575). Owing to the

use of three validators for each level of outcorne, the significance level was set at

u = 0.01 7, in accordance with the Bonferroni approach (Streiner 1993).

6.2.4.7 Missing data

Four dogs had missing data from Period 2 because the owners failed to

return their questionnaires. A further two dogs had missing data on behavioural duration from both treatrnent periods- Missing data were estimated as the relevant

median ratio for that treatrnent sequence. Another dog improved so much after the first period that the owner withdrew the dog from the trial; this dog was not included in the analysis.

6.3 Results

The characteristics of the dogs are described in detail in Chapter 7. Briefly, 35 breeds were represented and the compulsive behaviours fell into four categories: locomotion (n=22), self mutilation by Iicking (n=12), licking of self or of objects without causing damage (n=12; this group included Rank-sucking) and

'hallucination' (n=5).

Figure 6.1 shows the median ratios of behavioural frequency, duration and context within each outcome category for Question 3 and Question 15. The plots suggest that al1 outcome categories might be valid, because the median ratios for each outcome were numerically consistent with the predictions. The results of the ordinary sign test (Tables 6.3 and 6.4) demonstrate that the outcornes were valid.

The only exceptions were the outcome 'Worse' in Questions 3 and 15, as validated by behavioural duration (p=O.l509, Table 6.3; p=0.1661, Table 6.4), and the outcome 'Bettef in Question 15, as validated by behavioural context

(p=0.0835. Table 6.4).

6.4 Discussion

This study assessed the constnict validity of three categories of change in behavioural severity. Vaiidity is not a general property of a rating scale; it is qualifieci by the population and the circumstances in which the scale is used

(Bergner & Rothman 1987). In the present case, the results suggest that Question

3 and Question 15 pemlvalid inferences to be made about treatment efficacy, in othenivise unbiased clinical trials of treatment for canine CD. This is interesting because Question 15 is a comparative scale (Table 6.1) and it was not expected to be valid owing to recall bias in use of the anchor points (Streiner & Noman 1995 Figure 6.1 Median bebveen-period ratios of behavioural frequency, duration and context for three categories of between-period change in behavioural severity. Categories of change were measured wiUi hrvo rating scales (Question 3 and Question 15). Data were obtained during an AB-BA crossover clinical trial.

Question 3

t 1 iFrequency

Better Same Worse (n=l8) (n=I 7) (n=15) Category of change

Question 15

Better Sarne Worse (n= 19) (n= 14) (n=17) Category of change Table 6.3 Construct validity of a five-point rating sale (Question 3) that rneasured behavioural change in a crossover clinical trial (n = 50). Validfty was assessed by the sign test (a= 0.017)

Measure of Validator p Prediction ns x p Conçtruct be havioural validity change

Better Frequency 2.6 ~'-1 18 16 0.0007 Yes (n=18) Duration 1.51 ~'1 18 15 0.0038 Yes Context 1.57 p > 1 15 13 0.0037 Yes

Same Frequency 1.08 p=1 17 11 0.3322* Yes (n=17) Duration 1.23 p=l 16 10 0.4544' Yes Context 0.95 p=l 16 5 0.2100* Yes

Worse Frequency 0.37 p < 1 14 13 0.001 Yes (n=15) Duration 0.38 p < 1 15 10 0.1509 No Context 0.53 ~<114 14 0.0001 Yes

- Validator: behavioural frequency, duration and context. For each dog, each validator was calculated as the ratio of [its mean value over five days in treatrnent period 1: its mean value over five days in treatment period 21 - CL: median ratio of each validator - Prediction: predicted value of p, if the measure of behavioural change is valid - n, :number of observations used in the sign test (observations quaito 1 were excluded) (Dickinson Gibbons 1971, pl02). - x: nurnber of observations that were greater than 1 or less than 1, as appropriate to the prediction (Dickinson Gibbons 1971; pp101-102). - p: binomial probability that X r x *: two-tailed Table 6.4 Construct validity of a comparative 1O-point rating scale (Question 15) that measured behavioural change in a crossover clinical trial (n = 50). Vafidity was assessed by the sign test (a=0.017). Table headings explained in Table 6.3.

Measure of Validatar p Predîction ns x p Constnict behavioural validity change

Better Frequency 2.67 ~~1 19 19 0.0000 Yes (n = 'l9) Duration 1.67 ~'1 19 15 0-0095 Yes Context 1.70 ~'1 19 13 0.0835 No

Same Frequency 0.84 p=l 14 7 1 * Yes (n = 14) Duration 1.23 1=1 13 9 0.2668* Yes Context 0.98 ~=l 10 3 0.3438* Yes

Worse Frequency 0.30 vc1 16 14 0.0020 Yes (n = 17) Duration 0.63 pC1 17 11 0.1661 No Context 0.63 pC1 16 14 0.0020 Yes

pp164-166). When this scale was used previously (Rapoport et al 1992)' anchor points were discussed with the owners at the beginning of the study, but they were not discussed with owners in the present study. An explanation for the scale's validity in this study is that owners had been observing their dogs closely during the last five days of both treatment periods and therefore could remember the behavioural severity from one period to the next.

The predictions about the construct validity of 'Worse' and 'Bettet were not fully supported (Tables 6.3 and 6.4). Behavioural duration did not validate the category 'Worse' (Questions 3 and 15) and context did not validate the category 'Better' (Question 15). Bergner & Rothman (1987) review reasons for poor construct validity. In the present study, poor validity could mean (i) 'Worse' and

'Better' were not valid outcorne categories, or (ii) the validators were not rneasured precisely, or (iii) the validators were inappropriate. These points are now discussed.

Overall, the categories 'Worçe' and 'Better' are likely to have been valid because, in most instances, predictions about their validity were supported (Table

6.3 and 6.4). An alternative to using these categories, would have been to assign numbers to the response options and then to measure the change in score.

Question 15 was a numeric rating scale and Question 3 could have been made numeric, by assigning sequential numbers to the responses. However, the use of nurneric data to represent qualitative outcomes can be misleading. In Question 15 for example, the two-point change from a score of 8 to 6 may not be exactly twice the magnitude of the one-point change from a score of 10 to 9. Similarly, in

Question 3, the three-level change from "Quite bad" to "Not at al1 badn rnay not be three times greater than the one-level change from "Extremely badnto "Very bad".

However, both examples describe behavioural improvements which may reasonably be classified as 'Better'.

The precision and accuracy with which the validators were measured may be questioned. The measurements were summary estimates, made every 24 h, and they were dependent on the owners' recollection. Owners were not asked to record data as the behavioural episodes occurred because pre-testing indicated

that this would have been too burdensorne. More refined rneasuring techniques

are available (Martin & Bateson 1993. pp62-113), but would have been difficult to

implement in a dornestic setting. Behavioural duration is a standard measure of

behaviour (Martin & Bateson 1993, pp63-65) and validated al1 the outcome

categories except for 'Worse' in Question 3 and Question 15. The poor validity of

'Worçe' may have arisen because dogs that were worse in the second period were

interrupted more by their owners, therefore the duration of a typical episode was

the same as in the first perÏod.

The outcome 'Better' in Question 15 had poor validity when assessed against context. This is difficult to explain because al1 the other predictions about context were supported. Context was chosen as a validator in accordance with the hypothesis that the behaviours of canine CD may becorne emancipated from the origina( context, generalising to several different conte& (Hewson 8 Luescher

1996). The present study provides indirect support for this hypothesis because, with the exception of 'Better' in Question 15, changes in context were consistent with changes in behavioural seventy. However, developmental studies of canine CD are required to test the hypothesis. An additional finding about context in the present study was that approximately 50% of the dogs licked themselves or licked objects, but most of these dogs did not Iick in readily defined conte&. Instead, they licked when they appeared relaxed and nothing was happening in the household. Context rnay not have been a sensitive validator for behavioural change in #ose

dogs. Their behaviours may represent a different type of canine CD, or may be

symptornatic of another condition. Developmental studies would help to clarify

this. The use of 'individualised' questions on context could be criticised for not

being generalisable. However, these questions al1 addressed the same issue, and

were appropriate to measure change within individuals (Guyatt et al 1993).

The outcome categories 'Better', 'Same' and 'Worse' are intuitively

appealing, and their validity justifies further studies of their utility. The accuracy of

these outcornes might be increased if, at the tirne of re-evaluation, owners were

reminded of the rating that they gave at the first evaluation (Guyatt et al 1985,

1993). Measurement of construct validity should be ongoing as better validators

are found. The responsiveness and reliabiiity of the rating scales should also be

establis hed. Responsiveness describes the sensitivity of a measure to clinically

important change, even if such change is small (Guyatt et al 1987). In the present study, the clinical importance of the changes was not established and would have required studies of the MClD (Jaeschke et al 1989, Guyatt et al 1993). Such studies might indicate additional levels of outcome ('Much Worse' and 'Much

Better') (Senn 1993, ppl09-112). Question 4 of the telephone questionnaire

(Appendix 6) could be used in studies of the MCID. The reliability of Questions 3 and 15 would indicate the reproducibility of the ratings, and should be estimated.

The reliability of owners' estimates of behavioural frequency, duration and context should also be established. The estimation of reliability requires at least two administrations of a questionnaire when the subjeds condition is stable (Streiner

& Norman 1995, pp104-127; Chinn 8 Burney 1987).

In conclusion. this study used an existing rating scale and a new scale to provide new and valid measures of change in the behavioural severity of canine

CD. The rating scales could be adapted and evaluated for use in other studies. CHAPTER 7 A RANDOMISED CONTROLLED CLlNlCAL TRIAL OF

CLOMlPRAMlNE AS A TREATMENT FOR CANINE COMPULSIVE DISORDER

7.1 Introduction

Clomipramine has been widely evaluated as a treatrnent for obsessive compulsive disorder in humans (McTavish 8 Benfield IWO, Pato et al 1991). The drug has also been recomrnended as a treatment for canine compulsive disorder (canine

CD) (Dodman & Shuster 1994, Hewson & Luescher 1996, Overall 1997). Canine

CD is syndrome of abnomal confiict behaviours for which there may be breed predispositions (Blackshaw et al 1994, Hewson & Luescher 1996). To the knowledge of the author, there are no reports of rigorous clinical trials of clomipramine in a variety of canine compulsive behaviours.

Anecdotal evidence for the efficacy of clomiprarnine in canine CD is found in case reports (Overall 1994, Dadman et al 1996),and in clinical experience at the

Ontario Veterinary College Behaviour Service (Hewson & Luescher 1996). In addition, in two controlled. non-randomised, double-blind AB-BA crossover trials

(Goldberger & Rapoport 1991. Rapoport et al 1992) involving only dogs with acral lick dermatitis, clomipramine was found to be effective. However, these results are difficult to interpret because the trials were not randomised and the validity of the outcome rneasure was not reported. Clornipramine appears to be safe in dogs

(Ciba Geigy 1991), but it has been associated with sedation, anorexia and

'nausea' (Goldberger 8 Rapoport 1991. Rapoport et al 1992). The objectives of the present study were (i) to assess the clinical efficacy of

clomipramine as a treatment for canine CD, given the hypothesis that

clomipramine reduces behavioural severity more than placebo does. and (ii)to

describe any adverse effects of clomipramine.

7.2 Materials and Methods

The following protocol was approved by the Animal Care Cornmittee of the

University of Guelph.

7.2.1 Recmitment of cases

Over a period of 12 months (March 1995-March 1996). cases of canine CD were solicited from veterinarians and owners. Recruitment from veterinary clinics was by (i)articles in magazines of provincial veterinary associations (Onta rio,

British Columbia and Alberta), (ii) articles in newsletters of the Calgary. Toronto and Edmonton Academies of Veterinary Medicine, and in the annual report of the research charity "Pet Trust", (iii) advertisernent at the annual meeting of the

Canadian Veterinary Medical Association. and (iv) addresses to veterinary meetings in London, Mississuaga, Ottawa and Winnipeg. Dogs were also referred from the Behaviour Service of the Ontario Veterinary College.

Media interest was generated through the University of Guelph's publicity office. This resulted in interviews on national radio and television, and with regional newspapers in Ontario, Alberta, British Columbia, Saskatchewan and New Brunswick. Owners were also contacted through letters in breed society

magazines (Miniature Schnauzer, Geman Shepherd and English Bull Terrier) and

lay joumals ("Dogs in Canada", "Pets Magazine", "The Pet Gazette" and 'The Dog

Dispatch"),

The incentive for participating in the trial was to assist one's dog and to

advance understanding of canine CD. In addition, cornplimentary follow-up from

the Ontario Veterinary College Behaviour Service was available to participants.

Owners were required to meet the cost of providing a video-recording of the dog's

behaviour and of submitting their dog to one clinical evaluation that induded

urinalysis.

7.2.2 Assessment of eligibiiity

Cases of CD were diagnosed by the method descnbed in Chapter 5. Briefly,

after giving inforrned consent (Appendix 2). owners received two telephone

interviews about the history of the dog's behaviour. One interview was very structured (Appendix 4), and a diagnosis was made according to formal cnteria that were based on the hypothesised development of canine CD (Hewson &

Luescher 1996). The second interview was held with an expert (AL) who used the behavioural case history forms used at the Ontario Veterinary College (Appendix

5). Dogs that the expert diagnosed with CD were candidates for further evaluation according to the entry criteria Iisted in Figure 7.1. The owners of these dogs were asked to supply a video-recording of the dog's behaviour showing (i) the dHerent Figure 7.1 Entry criteria for a clinical trial of clomipramine as a treatment for canine compulsive disorder. lnclude if al1 of the following apply

i) The dog has one or more of these CDS (as diagnosed by expert opinion) - tail-chasing - whirling - tuming and checking rear, or staring at abdomen (Schnauzers) - fly-snapping - selfdireded aggression - fiank-sucking - freezing - barking ii) The dog has shown the behaviour on daily basis for at least 2 months iii) The owner is capable of understanding the purposes and risks of the trial iv) The owner can provide a video-recording of the behaviour v) The dog has normal haematology and biochernistry profiles vi) The dog has not received psychotropic dmgs during the previous 4 weeks

Exclude if any of the followhg apply

i) The dog is undergoing treatrnent wiVi clornipramine currently or had received it in the past ii) The present environment is a major contributor to the behaviour (eg dog is tied up al1 day) iii) The dog has a history of these medical conditions: - glaucoma - diabetes meIlitus - epilepsy - liver disease - heart disease iv) The dog has a gastro-intestinal disorder which would interfere with drug absorption e.g. recunent diarrhoea of any aetiology '4 The dog is a breeding animal vi) Major events are expected during Vie 1l -week study penod e.g. - moving house - birth of baby - holidays - training classes - routine surgery on dog e.g. neuter, dentistry vii) The dog shows concurrent aggression of any kind - daily andlor - puts the owners at risk situations in which the behaviour was performed, (ii)for how long the dog would

perform the behaviour if not intempted, and (iii) how readily the behaviour could

be intempted (e-g. by a loud noise). The recordings were reviewed by the

principal researcher (CH) and, if a behaviour was considered atypical of CD,

confirmation of the original diagnosis was sought from the expert (AL).

The dogs were also taken to their veterinarians for a physical examination, weighing and measurement of urinary glucose and specific gravity (Appendix 8).

In addition, the veterinarians took blaod fram the dogs, for a complete blood count and limited biochemical profile (urea, creatinine, total protein, albumin, glucose, alkaline phosphatase and alanine transferase). AIl blood samples were sent to one analytical laboratory within each province. and the cost of the blood analyses was met by the study budget These clinical data and the results of the physical exam were used to exclude dogs with diabetes mellitus, Iiver disease, glaucoma, and cardiac disease-

Once all this information was received about a dog, the entry criteria were reviewed and the dog's eligibility was decided upon.

7.2.3 Tris1 design

The trial had a randomised, placebo-controlled, double-blind, AB-BA crossover design. There is anecdotal evidence that some behaviours seen in canine CD may be breed-specific (Hewson & Luescher 1996); therefore, the present trial included six behavioural categories: (i)tail-chasing Gerrnan S hep herds, (ii) spinning English Bull Terriers, (iii) fian k-sucking Dobermans, (iv)

'checking' Miniature Schnauzers, (v) acral Iick dermatitis ('lick granulorna') and (vi)

othen. Simple randomisation was perfomed by third parties who were not

involved in any aspect of the trial. Wrthin each behavioural category, cases were

allocated to treatment sequence by drawing a piece of paper beanng the case

number from a bag, and the treatment sequence from another bag. There was

balanced treatment allocation within each behavioural category: case numbers were considered in successive pairs and when treatment sequence was randomly

allocated to one of a pair, the remaining member of that pair received the other treatment sequence. Part of the randomisation list is shown in Appendix 9.

Randomisation was camed out before enrolling cases in the trial: following enrollment, dogs were assigned successive case numbers and the associated treatment sequence.

Clomipramine hydrochloride and lactose placebo were supplied as 20 mg and 2.5 mg tablets (Ciba Geigy, Basel, Switzerland). The doses of both treatments were based on the dog's weight at the beginning of the study. The dose was 3 mgkg q 12h; the dosing interval was dependent on the owner's daily schedule, but dogs received the doses 8-12 h apat Doses were adjusted to the nearest 1.25 mg. Tablets were administered in opaque gelatin capsules

(Capsugel, Wamer-Lambert, Greenwood, South Carolina, USA; rupture tirne, 60 s;

90-95% dissolved in 7 min). Treatment was given five minutes prior to feeding, butif dogs were fed ad libitum or only once daily, treatment was given with approximately 15-309 of food. Owners were instructed not to feed the dogs

anything in addition to the regular diet.

Oogs received clomiprarnine and placebo for 4 weeks each, with a 2 week,

treatrnent-free wash-out period in between. On the final day of each treatment

period, owners participated in a telephone interview (Appendix 6) in which they

assessed their dogs' behavioural severity using two valid measures, as described

in Chapter 6. Briefly, one measure (Question 3) rated behavioural severity on a

five-point Likert scale; the other measure (Question 15) was adapted from

Rapoport et a1.(1992) and used a numeric scale where zero represented no

behaviour and ten represented the worst behaviour ever. Both questions are

shown in Table 7.1.

ûther questions asked in the interview concemeci compliance, adverse

effects and the efFectiveness of 'blinding' (Appendix 6). In the question about

adverse effects, owners were first given the opportunrty to describe any signs of il1

heaith that they had noticed. They were then asked whether there had been an

increase, a decrease or no change in each of the following: the dog's appetite, water consumption, frequency of bowel movements, frequency of unnation, amount of tirne spent sleeping, level of alertness when awake and bodyweight. These specific questions were based on experience with clomipramine at the Ontario Vetennary

College, and on side effects described in humans (Ciba Geigy 1991). The Table 7.1 Two global questions about behavioural severity, used as outwme measures in a clinical trial of clomiprarnine as a treatrnent for canine CD. Questions were 'individualised' for each dog. Question 15 was rnodelled on the rating scale of Rapoport et al (1992)

Question 3

Next, I would like to get an overall idea of how bad is the 'Behaviouf that vou see at present By 'bad' I mean how often 'Dog' shows the Behaviour' and how long he 'Behaves' for. Taking evecything together durino the past 5 davs, would you Say that the 'Behaviour' has ken:

1 extremely bad 2 very bad 3 quite bad 4 somewhat bad 5 not at al1 bad

Question 15

Similarly, we can score 'Dogss' 'Behaviour' during the past 5 davs on a scale of zero to ten. A score of 'O' means no 'Behaviour'. A score of '1means that the 'Behaviour' is the worst it has ever been. Overall how would you rate 'Dog's' 'Behaviouf during the past 5 davs (that is, since Monday)?

No The worst 'Behaviour' 'Behaviou r' ever

development of the telephone questionnaire, and the validation of the global ratings are described in Chapter 6.

In addition, owners were supplied with questionnaires with which to record the daily frequency, duration and context of the behaviour during the last five days of each treatment period (Appendix 7). These data were used ta validate the outcome rneasure, as described in Chapter 6. Owners also used the questionnaires to describe their feelings about their dog's behaviour (Appendix 7;

Questions 5ad). for possible use as a secondary outcome rneasure in the clinical trial.

7.2.4 Trial conduct

The trial was home-based and dogs were not required to vis& the Ontario

Vetennary College at any the. Following a dog's enrollment, a package was sent by couder to the owner's veterinarian, for the owner to collect. The package contained (i)a covering letter, (ii)the first treatment (the second treatment was sent by courier immediately pnor to the second treatrnent period), (iii)information about how to 'unmask' the treatrnent in the event of an emergency (Appendix 10).

(iv) a calendar detailing the daily requirements for each day of the trial (Appendix

11). and (v) a supply of questionnaires (Appendix 7). Every owner was telephoned

2448 h before commencing the trial, and the contents of the package were explained. The dog's veterinarian also received information about how to

'unmask' the treatment, and suggestions about how to proceed in the event of side effects (Appendix 12). During the third week of each treatrnent period, CH telephoned the owner to remind them to commence daily recording of behavioural frequency, duration and context. and also to arrange the telephone interview on the last day of the treatment period. As each dog completed the trial, CH mlled the owner to discuss options for

further treatment Support and behavioural advice was provided from the Ontario

Veterinary College if desired. Details of the dog's treatment sequence were then

given to the owner's veterinarian. Owners and veterinarians were instructed not to

infonn CH of their dog's treatment sequence. Owners were asked to avoid

changing the dog's routine (eg food, exercise and training) and environment dunng

the study.

All cases were followed-up 1-2 years after they had completed the trial.

Owners were telephoned and asked (i) if the dog still showed the behaviour, (ii)

whether the dog had continued to receive clorniprarnine affer completing the trÏal,

and (iii) the reasons for continuing, or not continuing, to give the dog clomipramine.

Owners who continued to give the drug were asked what the long-term effect of

the drug was on the dog's behaviour, and if any adverse effects had occurred.

7.2.5 Data analysis

All statistical analyses were conducted 'blind' to treatrnent assignment. The trial outcome was the change in behavioural severity frorn Period 1 to Period 2, as measured by Question 3 and Question 15. The validation of the outcome measure is described in Chapter 6. The data from each question were used to categorise each dog's behaviour as 'Better in the second period', 'The same in both periods', or 'Worse in the second period' (Senn 1993, pp109-113). These three levels of outcome were analysecl for each question in turn, using ordinal

regression (proportional odds model; McCulIagh 1980, Scott et al 1997).

Carryover effects were not included in the model (Senn 1993, pp14-15,226-238).

Data were modelled using SAS (Sbtistical Analysis Systern, Version 6.1, SAS

Instiiute, Cary, NC) and gave the odds of being 'Better in the second period' for

dogs receiving the treatrnent order 'placebodrug', compared to dogs receiving the

treatment order 'drug-placebo'. Exptanatory variables were treatrnent order, type

of behaviour, breed and duration of the behaviour with the present owner. Models were built by stepwise selection, and parameters were excluded if their associated

p-value exceeded 0.1. Because the same outcome was measured by two questions, the probability of erroneously declaring a treatment effect (Type I error) was increased ( Kleinbaum et al 1988, p13; Streiner 1993). Consequently, the

Bonferroni approach was used; when each question was rnodelled, the level of significance was 0.025, so that the overall probability of a Type I error was rnaintained at az0.05 (Pocock 1987 pp230-231, Streiner 1993).

The questions about the owners' feelings (owner-administered questionnaire, Questions 5a-d, Appendix 7) were not used because inspection of the raw data indicated that owner ratings of wony, frustration, anger and enjoyment did not Vary between treatrnents. 7.2.5 1 Efectiveness of masking

The effecüveness of masking was assessed by calculating chance-corrected

agreement between each owner's perception of the treatment received in a gîven

period. and the treatment allocated. Agreement was estimated by the kappa

coefficient; the appropriate 95% confidence interval was estimated after testing for

bias with McNemar's chi-square (Shoukri & Edge 1995. pp83-90).

7.3 Results

The most successful methods of recruitrnent were public addresses at veterinary meetings and media interviews. Eighty-four dogs were recruited; 75 of them were diagnosed with canine CD and 51 of these participated in the clinical trial. Twenty four cases did not participate for the following reasons: (i)the expert changed his diagnosis to one of normal conflict behaviour following examination of the video recordings (n=2). (ii) owner nontompliance: although expressing interest in the trial, owners did not provide a video-recording and/or take their dog to the veterinarian for a check-up (n=6), (iii) decision to pursue a medical cause of the behaviour with their veterïnarian (n=2), (N) withdrawal due to changes in the owner's personal circumstanœs (n=2), (v) remission of the behaviour following changes in the environment (n=2) (vi) euthanasia because of the behaviour (n=2),

(vii) coincident development of unrelated disease (osteosarcorna, Cushing's syndrome; n=2), (viii) history of liver disease when received amitriptyline 12 mos previously (n=l), (ix) the behaviour was not shown frequently enough for any

change to be measured (n=3), (x) the behaviour was logistically dÏfficult to monitor

(polydipsia; n= l),and (xi) the owner wished to breed the (fernale) dog during the

study perîod (n=?).

7.3.1 Description of sample

The 51 dogs that participated in the clinical trial were located in Ontario

(n=42), British Columbia (n=3). Alberta (n=3), Manitoba (n=2) and Saskatchewan

(n=l).

7-3.7.7 Sex

The sample was 37% neutered male, 37% neutered fernale, 20 Oh intact

male, and 6% intact female.

7.3.1.2 Age

The dogs ranged in age from 1.25 y to 11 y (mean 4.2 y, SD 2.4; 25th percentile 2 y; median 4y; 75th percentile 5.75 y).

7.3.7.3. Breed:

The three most common breeds in the clinical trial were Doberman (16%, n=8), cross-bred (14%, n=7) and German Shepherd (10%. n=5). The remaining breeds were English Bull Terrier (8%, n=4), Miniature Schnauzer (6%. n=3),

English Springer Spaniel(4%, n=2),American Cocker Spaniel(4%, n=2), Jack

Russell Terrier (4%, n=2), Standard Poodle (4%, n=2) and Pomeranian (4%, n=2); also, Arnerican Bull Temer, Bearded Collie, Bouvier de Flandres, Cairn Terrier,

Cavalier King Charles Spaniel, Chinese Crested Dog, Miniature Dachsund (long- haired), Irish Setter, Keeshund, Pug, Rotîweiler, Shetland Sheepdog, ShiTzu, and

Miniature Poodle (n=l). The sample size was too small (n=51) to accommodate al1 the breeds in the statistical analysis; consequently, the breeds were dichotornised as pure-bred working dogs (n=31) and others, for analytical purposes.

7.3.1.4 Behavbur

The behaviours of the 51 dogs are shown in Table 7.2. With the exception of acral lick dennatitis, there were insufficient cases in the proposed categories of behaviour, for analytical purposes, the behaviours were divided into three categories: locornotory (n=21), acral lick dermatitis (n=12) and others (n=17)

The dogs had been showing their behaviours for 2 mos to 8.5 y before entering the trial (mean 2.25 y, SD 1.75; 25th percentile 1 y; median 1.5 y; 75th percentile 3 y). Three of the dogs had been treated with amitryptiline, but none of the other dogs had been treated with psychotropic dmgs before entering the trial,

Al1 the dogs with acral lick dermatitis had had at least two treatments before.

These treatments included antihistamines, topical corticosteroid creams, oral corticosteroids, bandaging, use of an Elizabethan collar. Table 7.2 Behaviours and breeds of 51 dogs with canine compulsive disorder that participated in a clinical mal of clomipramine

Behaviour Breed

Acral lick dermatiüs ('lick granuloma') (n=12) 5 cross-bred, 3 Doberman Geman Shepherd Dog. English Springer Spaniel, Irish Setter, Keeshund

Licking body wlhout selfinjury, or licking Bearded Collie, Chinese Crested objects Dog, Cavalier King Charles Spaniel, (n=7) English SprÏnger Spaniel. Miniature Poodle, Pug, Rothnreiler

Tail-chasing (spinning on the spot in a tight 2 German Shepherd Dog circle and attempting to catch tail in mouth) 3 English Bull Terrier (n=7) American Bull Terrier, Pomeranian

Spinning on the spot in a tight circle without 3 Doberman attempting to catch tail (n=10) 2 cross-bred Bouvier de Flandres, Jack Russell Terrier, ShiTzu Shetland Sheepdog Amencan Cocker

Running or pacing in a large circle (n=4) 2 Geman Shepherd Dog 2 Standard Poodle

2 Doberman Dachshund. English Bull Terrier

Checking (n3) 3 Miniature Schnauzer

Fixation with light and shadows (n= 1) Cairn Temer,

Fixation with a stable wall Jack Russell Temer (n=1)

Air-biting (n=l ) Miniature Pomeranian

Chewing fur without causing self-injury Arnerican Cocker Spaniel (n=l ) 7.3.2 CIinical evaluation

No abnomialities were detected in any of the dogs at the tirne of clinical examination. Most of them were afso normal on biochemical and haematological tests; however, three dogs had either biochemical or haernatological values outside the laboratories' normal range. These dogs were re-tested 10-14 days later, at which tirne al1 were found to be normal. Another dog had long-established

Addison's disease but was stable on medication with mineralocorticoid and g lucocorticoids.

7.3.3 Statisocal analysis

There were 24 dogs in the sequence placebo-dnig, and 27 in the sequence drug-placebo. One dog that had been showing circling behaviour daily for the past

7 yearç ceased to show the behaviour after receiving the first treatrnent. The dog relapsed during the washout period and the owner wïthdrew the dog from the trial without comrnencing the second treatrnent That dog's data were excluded from

.the analysis. When the trial was unmasked, foilowing analysis, it was revealed that the dog's first treatment was clornipramine.

Figure 7.2 shows the number of dogs in each treatrnent sequence that were

'Better in the second period ' 'The same in both periods ' or 'Worse in the second period'. Figure 7.2 illustrates that, when both treatment sequences are considered, 50% of dogs were better while receiving drug; this was found with either Question 3 (24150) or Question 15 (25/50). Figure 7.2 Bar charts showing the number of dogs that were 'Better in the second period', 'The same in both perïods' or 'Worse in the second period' in two treatment sequences, (clornipramine-placebo) and (plaœbo-clornipramine). The outcornes were measured with hoseparate questions (Question 3 and Question 15).

Question 3 1 placebo - dnig 1 adnig - placebo

Better Same Worse Outcorne category

Question 15

placebo - drug O dnig - placebo

Better Same Worse Outcome category The results of stepwise model-fitang are shown in Appendix 13. The final

models for both Question 3 and Question 15 contained treatrnent order; in

Question 15, duration of the behaviour with the present owner approached

significance. These statistics are summarised in Table 7.3. For Question 3, the

results indicate that the dogs were approximately four times more likely to be

'Better' when receiving clomipramine than when receiving placebo (model

p=0.007; parameter p=0.009). For Question 15, dogs were approximately 3.5

times more likely to be 'Better' when receiving clomipramine than when receiving

placebo (model p=0.0134; parameter p=0.024). In addition. there waç a trend

towards being less likely to have improved if the behaviour had been shown for a

long tirne (duration p=0.038).

7.3.4 Cornpliance

Reported cornpliance was high. In sequence 'placebodrug', 58% dogs

(n=14/24) did not miss any treatrnents in either period, and the remaining dogs missed a maximum of two treatrnents in either period. In sequence 'drug-placebo',

54% dogs (n=14/26) did not miss any treatrnents in either period, 35% (n=9/12) rnissed up to 4 treatrnents in one or both treatment periods. One dog missed

15/56 treatments while receiving the drug in the first period owing to sideeffects of anorexia and lethargy. Table 7.3 Final results of anaiysing clinical trial data by ordinal regression (proportional odds model, stepwise seledion). The outcorne was measured by two rating scales. In each case, treatment order was significant; in Question 15, the duration for which the dog had been showing the behaviour before entering the clinical trial approached signficance (u=0.025).

Rating Scale Score test for - 2 Log L Variable Cumulative Odds Wald f p proportional (P) Ratio" odds (95 % confidence assurn~tion interval)

Question 3 p=0.648 102-23 Order 4.36 (1-45-1 3.09) 6.88 0.009 (p=O. 007)

Question 15 p=0.257 100-47 Order 3.60 (1.19-1 0.89) 5.1 3 0.024 (p=O.OI 3) Duration 0.97 (0.94499) 4.32 0.038

*Estimate of the odds of canine compulsive disorder being 'Better' wlh clomipramine (3mgkg q 12h for 4 weeks) cornpared to placebo

7.3.5 Adverse Effects

These data were assessed descriptively as the withindog differences

between drug and placebo. The data are shown in Tables 7.4 and 7.5. Data on

differences were not available from sorne dogs because the owners were unsure of whether the parameter of interest had increased, decreased or remained the same on a given treatment. Owners were unsure about appetite (n=3/51), water consumption (5/51), and frequency of urination (9151) and the amount of thethat the dog spent sleeping (1151) (see Table 7.4). In addition, 4% of the data on side F b Cr) Table 7.5 Data from dogs that showed different occurrence of adverse effects on drug and placebo during a randomised, placebo-controlled, double-blind, crossover clinical trial of clomiprarnine (n=51). Occurrence of rnissing data is also shown.. (** : numbers are already given, in "Decreased on placebo, increased on drug" *: nurnbers are already given, in "Decreased on drug, increased on placebo") - Appetlte Water Frequency of Frequency of Time spent Alertness when Bodywelght consumption defaecation urination aslee~ awake

Decreased on unchanged on drug placebo

increased on placebo

Decreased on unchanged on drug placebo

increased on drug

Increased on unchanged on drug placebo

decreased on placebo

lncreased on unchanged on drug placebo decreased on drug effects were missing because of the wiütdrawal, as descrïbed, and an occasional

error in data collection frorn other owners (Table 7.4).

Seven adverse effects were rnonitored in this trial. When al1 the effects

were considered, 61% of the dogs showed no difference between drug and

placebo (Table 7.4). In the remaining dogs, the occurrence of adverse effects

when the dog received drug was different from when the dog received placebo.

Table 7.5 shows that several effects occurred more when dogs received drug than when they received placebo. The most notable of these effects was an increase in the time spent sleeping. Many dogs (41%; 21/51) slept more on drug, but did not sleep more on placebo. Six were markedly lethargic. causing the owners to contact the trial office or their veterinarian. Conversely, only 6% (n=3/51) slept more on placebo, but not on drug. Several dogs were less alert while receiving clomipramine; in 14% (7/51), the alertness decreased on drug, but not on placebo, whereas 4% (Z51)were less alert on placebo but not on drug. Appetite was reduced more with clomipramine than with placebo; 20% (10151) had a reduced appetite on drug, but not on placebo; 6% (3151) had a reduced appetite on placebo but not on drug. Water consumption increased with the drug; 14% (7151) dogs drank more on drug, but not on placebo. whereas 6% (3/51) drank more on placebo, but not on drug.

Other notable effects reported while dogs were receiving clomipramine, but not placebo are as follows. Two dogs (Keeshund and English Springer Spaniel) occasionally vomited within one hour of treatrnent with clornipramine. A Miniature Schnauzer became subdued and markedly anorexic after two days of treatment-

The owners then withdrew treatment for two days but the effects returned when they resumed treatrnent A veterinary examination revealed no clinical abnonality and, after a fumer five days without treatment, a lower dose was prescribed (2 mgkg q 12 hours) without further il1 effects. Another Miniature Schnauzer was lethargic and anorexic during the first three days of treatrnent, but recovered spontaneously. During the first week on clomipramine, a Standard Poodle was lethargic, ataxic and uncharactenstically aggressive to neigh bouring dogs, but showed no further side effects thereafter. The owner had just moved house and the aggression is likely to have been territorial. This dog was removed from the trial after receiving the drug, because his compulsive behaviour had improved so much. A Cairn Terrier that was fixated on refiections of moving light and on shadows, and that would lick the floor where the light was, replaced those behaviours wlh daily house-soiling (urine) and extensive licking of the carpet. A

Pug, Chow-cross, English Bull TeMer and English Springer Spaniel were al1 noticeably lethargic while on the dmg. One dog (Rottweiler) showed adverse effects while receiving placebo; it vomited six times in the first week of treatment, growled at the owner for no apparent reason and was lethargic. Haematology and clinical biochemistry were repeated in this dog and were normal. The dose was then decreased and the undesirable behaviour disappeared. 7.3.6 EWécfikeness of masking

When asked which treatrnent their dog had just been receiving, 25%

owners were unsure in both perÏods (Period 1. n=l 1; Period 2, n=10). The

remaining owners expressed an opinion and the resulting 2 x 2 tables and

estimates of kappa are shown in Figures 7.6-7.8. Chance-corrected agreement

was moderate in Period 1 (~=0.4)and higher in Period 2 (~=0.6)(Sackett et al.

1991, p30). Of the 50 owners who completed the trial, 28 (56%) expressed an

opinion in both periods and 24 of these owners identified the dog's treatment

sequence correctly (~=0.7;Table 7.8).

Table 7.6 2 x 2 table and indices of agreement between owners' perceptions of treatment and the true treatment aIfocation in Period 1.

Treatment Drug* Placebo" TOTAL Drug 13 3 16 Owner's Perception Placebo 8 16 24 TOTAL 21 19 40

"'Didn't know': n = 6 "'Didn't know': n = 5

- Observed agreement 0.725 - Expected agreement: 0.495 - Chance-corrected agreement, kappa (95% confidence interval): 0.46 (0.16-0.71 ) - McNernar's chi-square, 1 d-f.: 2.273 (p=0.132) Table 7.7 2 x 2 table and indices of agreement between owners' perceptions of treatment and the tnie treatrnent allocation in Period 2

Treatment Dnig* Placebo* TOTAL DW 17 5 22 Owner's Perception Placebo 3 16 19 TOTAL 20 21 41

"'Didn't know': n = 5 "'Didn't know': n = 4

- Observed agreement: 0.81 - Expected agreement: 0.5 - Chance-corrected agreement. kappa (95% confidence interval): 0.61 (0.34-0.82) - McNemar's chi-square, 1 d-f.: 0.5 (p=0.478)

Table 7.8 2 x 2 table and indices of agreement between owners' perceptions of treatment sequence and the true treatment allocation

Treatrnent Sequence Drug- Placebo- TOTAL Placebo DW Drug- 10 1 II Placebo Ownei's Perception Placebo- 3 14 17 Dw TOTAL 73 15 28

Observed agreement: 0.86 Expected agreement: 0.5 1 Chance-corrected agreement. kappa (95% confidence interval): 0.71 (0.4-0.92) McNemar's chi-square, 1 d-f.: 1 (p=0.317) 7.3.7 FONOW-up

The results of follow-up are summarised in Tables 7.9 and 7.1 0. Six dogs

(12%) were fost to follow-up. The rernaining dogs (n=45) had continued ta show

their behaviour. Of these, 32 did not continue to receive clomipramine after the

trial (Table 7.9), but 13 dogs continued to receive the drug (Table 7.1 0). The owners of the latter dogs found the drug was ineffective (n=4), somewhat effective

(n=3), or effective (n=6) (Table 7.1 0).

7.4 Discussion

To the knowledge of the author, this paper describes the first randomised, controlled, double-blind dinical trial of clomipramine in dogs with various behavioural manifestations of canine CD. The results suggest that clomipramine

(3 mgfkg q 12h) is a safe and effective treatrnent, as rneasured by the descrïbed questionnaires. This finding is supported by eariier case reports (Overall 1994,

Dodman et al 1996) and non-randomised clinical trials (Goldberger & Rapoport

1991, Rapoport et al 1992).

Treatment was the only significant variable in the statistical models. An effect of duration of the behaviour approached significance in Question 15; wïth a less conservative level of significance (a=0.05),duration had a slight effect on the outcome (odds ratio=0.9?; 95% confidence interval, 0.94-0.99). The low odds ratio indicates that the effect of duration in this trial was probably not of practical Table 7.9 Results of follow-up on 51 dogs with compulsive disorder that participated in a randornised placebocontrolled clinical trial of clomipramine

Result of follow-up Reasons Details of individual cases

Lost to follow-up (n=6) Owner moved (n=l) Owner did not respond to contact (n=5)

Dogs did not continue Dmg was ineffectnle or - Rottweiler that licked forepaws stopped clornipramine (n=32) not sufficiently effective showing the behaviour &ter being (n=24) treated for yeast infection. - Doberman that showed clockwise spinning was euthanased after developing neurological deficits that the attending veterinarians attn'buted to the behaviour

Adverse effects during - House-soiling, the tnal (n=3) - "Reduced spontaneity" - Reduced appetite. lethargy

Other reasons (n=5) - 'Lick granuloma' associated with radial paralysis; lim b am putated - Family member against the use of psychotro pic drugs in animals - The dog's behaviour was not severe enough - Owner lived in remote area and did not wish to run out of medication - Not specified

Continued clomipramine See Table 7.1 0 (n=13) importance. However, it could be investigated in future research. Neither breed nor behaviour were significant in this experiment The trial design provided breed- categories of behaviour, but few dogs were recruited into them and there was insufficient power to detect any differential treatment effect in these dogs, if such an effect existed. Observational studies of the behaviour woutd help to establish the existence of any breed predisposition.

The present trial has fie following strengths. lt was randomised and double-blind, cornpliance appeared to be high, and only one dog withdrew before completing the trial. In addition, the outcome measure had been validated. 60th the outcome measure and the validators were measured subjectively; therefore, the efficacy of btinding was assessed. The chance-corrected agreement between owners' identification of treatment and true treatment allocation was moderate in the first period and high in the second period, regardless of treatment sequence

(Tables 7.6 to 7.8). Owners were not asked how they decided which treatment their dog had been receiving, but adverse effects could have helped owners to recognise the drug. Such recognition could have biased their assessrnent of their dogs' behaviour. Appetite reduction, increased time asleep and decreased alertness were reported more when dogs were on dnig than on placebo (Table

7.4). Goldberger & Rapoport (1991) and Rapoport et al (1 992) used desipramine as a control because it has a similar side effect profile to domipramine. They reported a significant treatrnent effect, using the rating scale of Question 15 (Table

7.1). This suggests that the treatrnent effect in Our trial was real; ownerç may have identified treatments correctly because the dnig had a pronounced effect. This

effect could have been more apparent after the dog had received both treatrnents,

which could explain the higher K in the second period.

In this trial, ciorniprarnine appeared to be associated with sedation, reduced

a p petite, decreased frequency of defaecation, increased water consumption and

frequency of urination (Table 7.5). The latter two effects may result from the anti-

rnuscarinic effect of the drug, which causes a dry mouth in people (Preskarn

7 993). In studies of clomipramine (3 rngkg q 24h) in canine acral lick dematitis

(Goldberger & Rapoport 1991, Rapoport et al 1992). anorexia and lethargy

occurred in some dogs. Rapoport et al (1992) also reported 'nausea' in the dogs,

but did not state whether those dogs received the medication with food. Clinical

experience at the Ontario Veterinary College (Dr. Andrew Luescher, personal

communication) suggests that clomipramine may cause vomiting. In our multiple- dose pharmacokinetic study (Chapter 4). two dogs vomited within 30 min of their first treatment; thereafter, food was given immediately after treatment and no further vorniting occurred. In the dinical trial, treatment was given with food; owners were not asked specifically about vomiting, but did not report it as a problem (Appendix 5, Question 11).

Adverse effects of clomipramine may be seen in some dogs but not others, because of interdog variability in pharmacokinetics (see Chapter 4). This variability is a characteristic of clomiprarnine in humans (Brosen & Gram 1989), in whom therapeutic drug monitoring is recommended (Preskom 1993). Such monitoring is irnpractical in dogs, but the dosing regirnen for individuals could be

established empirically. Overail (1997, p313) recornmends commencing

clomiprarnine at 1 mgkg q 12 h. and increasing the dose by 1 mgkg every two

weeks to a maintenance dose of 3 mglkg.

Forty nine percent of dogs in our trial improved with clomipramine. The

remaining 51% were the same or were wone. One reason for non-response rnay have been an insufficient dose (Chapter 3). Further work is needed to ascertain if there is an optimal dose and dose ffequency in dogs. Another explanation for lack of response is that some of the dogs did not have the same disorder as those that did respond. In humans, obsessive compulsive symptoms are seen in different psychiatrie disorders (Hollander and Wong, 1994); the sarne may be true of the behaviours observed in this trial. However, the fact that some dogs improved significantly in our trial, supports the hypothesis that there is a distinct canine behavioural condition that responds to monoamine reuptake-inhibiting drugs.

The dogs and ownen who participated in this trial were selected purposively because canine CD is not weii recognised, and it would have been difficult to obtain a suficiently large random sample of affected dogs. The owners in the trial were highly motivated; they provided data using a valid measure and a significant effect of clomipramine was found- If these ownen represent most owners of dogs with canine CD. the results of the trial are generalisable. The generalisability of clinical trial results to those who would not participate in a clinical trial has been reviewed (Llewellyn-Thomas et al 1991). This randomised, controlled, double-blind clinical trial showed that

clornipramine diminished the severity of behaviours that are shown in canine CD.

However. none of the dogs in the trial were cured by clornipramine. Follow-up

indicated that most owners did not continue to give the drug to their dogs because

of adverse efieds. cost and insufficient efficacy. The information provided at follow-up is likely to refiect some recall bias, but is interesting nonetheless. On completing the trial. behavioural advice and support were offered from the

Behaviour Service of the Ontario Veterinary College. Owners who availed thernselves of the Service showed poor cornpliance. This may have been because they had already spent 11 weeks in the trial. Behavioural modification is likely to be an important aspect of the treatment of canine CD because of the role of the environment in inducing conflict or frustration (Chapter 2). In humans wiai obsessive compulsive disorder. behaviour therapy is an important part of case management (McTavish 8 Benfield IWO. Greist 1992). This information. and the fact that clomipramine was not effective in dogs in the long-tem, supports the hypothesis that dogs with CD woufd respond to a behavioural programme in conjunction with clornipramine. In dogs with separation anxiety, there is preliminary evidence that such a combination is more effective than behavioural modification alone (Simpson 1997). In canine CD, behavioural modification should address any sources of conflict or fnistration, and eliminate any learned component of the behaviour (Hewson & Luescher 1996). More work is needed to ascertain the optimal duration of treatment with clornipramine, and the most appropriate foms of behavioural therapy. CHAPTER 8 SUMMARY AND FUTURE RESEARCH

This thesis describes the first rigorous evaluation of a treatment for canine CD.

The resuIts indicate that clornipramine is a useful treatment for canine CD. The

research also provides some evidence for the diagnostic validity of the tem

'canine CD'. Robins and Guze (1970) recornmended a systematic approach to

diagnostic validity. Their approach comprised dinical descrÎption, laboratory

studies, specification of exclusionary criteria, studies of response to treatment. and studies of familial tendency. The present findings are discussed in light of those

recomrnendations. with emphasis on methodological issues.

8.1 Description of affected subjects

The clinical trial provided descriptive data on the history and presentation of 84 dogs suspected of having canine CD (Chapter 5). An expert diagnosed the disorder in 75 of these dogs, and their data are described in Appendix 14. This information will be of interest following future studies of the development of canine

CD. Breed-specific categories of behaviour were not identified in this thesis, but could be investigated in developmental studies, and in population-based, observational studies.

8.2 Use of diagnostic criteria that exclude other disordem

The forrnal diagnostic criteria for canine CD (Chapter 5) were based on the hypothesised development of the condition. The criteria excluded leamed behaviour, normal behavioural responses to conflict or fistration, and çeizures. In

addition, a history of conflict or fnistration, with behavioural ernancipation (increase

in the number of eliciting contexts), was required for a diagnosis of canine CD.

Many dogs did not meet the latter criteria and there was poor diagnostic agreement with expert opinion. One explanation for the poor agreement is recall bias. because the criteria describe histoflcai events. The clinical trial provided indirect support for the hypothesis of behavioural emancipation; a reduction in behavioural seventy was associated with a reduction in the number of contexts that elicited the behaviour (Chapter 6).

The diagnostic criteria would be clarified by studies of the development of canine CD. If diagnostic criteria for canine CD could be established. this would facilitate studies of treatment; it would also help clinicians to diagnose and treat cases.

8.3 Laboratory studies

The laboratory experiment (Chapter 4) examined the effect of clomipramine on central monoamine turnover in behaviourally normal dogs. The drug waç given once daily (3rngkg q 24h), and no effect was detected. However, the phannacokinetic study (Chapter 3) indicated that the dose may have been too low.

In the clinical trial, clomipramine was given twice daily (3mgikg q 12 h) and it reduced behavioural seventy. This suggests that clomipramine has a central neurochernicai effect in dogs with CD. However, a role of serotonin in the

pathogenesis of canine CD should not be inferred from a behavioural response to

clomipramine (Insel and Winslow 1992). Prelhinary evidence in rats suggests

that the therapeutic effect of antidepressants results from molecular changes in

gene expression or protein phosphorylation (Barden et al 1995, Hyman & Nestler

1996). Monoamines rnay therefore be markers for therapeutic response. rather

than the root of a given disorder.

Future neurochemical research could compare dogs that have CD, with

dogs that show conditioned behaviours and dogs that are behaviourally normal. If

dogs with CD were neurochemically different f'orn the others, this cauld suggest that canine CD has diagnostic validity. However, differences in the

neurotransmitter turnover of the above groups would not prove the pathology of canine CD, because neurotransmitter turnover does not indicate neuronal activity

(Commissiong 1985).

The methodology of Mure neurochemical studies should be carefully chosen. Serotonergic and dopaminergic synapses are concentrated in the forebrain, but a close review of the literature indicates that cistemal CSF rnay not reflect monoaminergic changes in the canine forebrain (Eccleston et al 1968, Moir et al 1970, Kopin 1985). Microdialysis may be a more useful technique, and has been used to demonstrate differences in 5-HIAA in the caudate nucleus, behrveen stereotyping and non-stereotyping bank voles (Vandebr~eket al 1995). The analyses in Chapter 4 indicate that the concentration of erythrocytes in CSF

should be included as covariates, in future work.

8.4 Response to treatment, and follow-up studies

The clinical trial indicates that there is a syndrome of canine behaviours that

responds to clomipramine (Chapter 7). Some confidence can be placed in this

result because the trial was carefully designed, with attention to pharmacokinetics

and outcome measurement. Follow-up indicated that clomipramine was not an

effective treatment in the long-tem. Follow-up also indicated that one dog thaï

licked its paws was unlikely to have had canine CD, but to have had a skin

infection. This suggests that bacterial and yeast cultures should be used to exclude dermatological causes of persistent Iicking behaviour.

8.4.1 Phannacokinetics and response to treatment

The dose of clomipramine in the clinical trial was estimated following a phannacokinetic study. That study indicated that dogs and humans metabolise clomipramine differently, which illustrates the importance of studying pharmacokinetics in the species of interest before conducting efficacy trials.

Clomiprarnine might have been more effective in the clinical trial and laboratory study if larger or more frequent doses had been used. However, increased dosing might have caused adverse effects. In the clinical trial, clomipramine was associated with reduced appetiie and sedation in some dogs. This interdog variability is consistent with the pharmacokinetic study which

demonstrated marked interdog variability in plasma concentrations of

desmethylclornipramine (Chapter 3, Tables 3.5-3.8), although its elimination haf-

life was short (approximately 2h). These results indicate that the dose of

clornipramine may need to be adjusted on an individual basis.

Chapter 3 provides a framework for future pharmacokinetic studies of

clomipramine in dogs. In particular, an intravenous study is required and sampling

should be more frequent during the first 24h after dosing. The minimal effective plasma concentration should be described. Cornparisons might also be made between different breeds of dog, given the effect of genetic polymorphism in hurnans and also the finding that the pharmacokinetics of clomipramine differ between strains of mice (Marty et al 1992).

8.4.2 Measurement of response to treatment

Special attention was paid to outcome measurement in the clinicaI trial

(Chapter 6). Two rating scales were used to produce outcome measures, and the construct'validity of these measures was demonstrated under the conditions of the triai. The reliability of these outcome measures should be assessed. and construct validÏty should continue to be evaluated when the scales are used in other trials.

The possibility of recall bias in the use of the comparative scale (Question 15,

Appendix 7) should be considered. Furthemore, owners may need to record behavioural frequency, duration and context, in order to monitor the behaviour sufficiently closely to provide a valid rating with the global scales. The minimal

clinically important change in behaviour should be detemined for both scales, and

their responsiveness should be estimated.

8.4.3 Clinical significance of response to freafment

Chapter 1 outiines why canine CD is a source of concern. The clinical trial showed that with the exception of minor side efiects, clomipramine was a safe and effective treatment that offers some hope for affected dogs and their owners.

Follow-up indicates that clornipramine is unlikely to be an adequate treatment and further research shouid evaluate a combination of behavioural modification and clomiprarnine. Other drugs might also be evaluated, and the work described in this thesis provides a frarnework for other studies. Until more information on cambined behavioural and pharmacological treatment is available, veterinarians and owners should not rely on clomipramine or other drugs to help affected dogs. Sources of conflict or frustration in the dog's environment should also be addressed (Hewson

& Luescher 1996).

In conclusion, this thesis contributes to the understanding of canine CD, and demonstrates that oral clomiprarnine (3 mgkg q 12h) is a usefùl treatment.

The thesis also addresses important methodoiogical issues, and describes the development of valid measures of change in the behavioural severity of canine

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Ab breviations - Order: treatrnent sequence. - Week: week of treatrnent - Thio: dose of thiopental used to anaesthetise the dog. - Ival: interval that elapsed between anaesthetic induction and CSF- sampling. - RBC: number of erythrocytes per mL of cerebrospinal fluid Table Al.1. Model parameters and estimates of the effect of clomipramine (3 mgkg q 24 h for 6 weeks) on the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) in canine cerebrospinal fiuid, in a repeated measures AB-BA crossover experiment

Model Overall F R Variable OF F Pr >F

1 1 .13 0.638 Order 1 O- f 0-765 (0-4374) Week 3 1.32 0-327 Dog(order) 4 2.16 0-155 O rdefweek 3 0.42 0,744 Th io 1 0.01 0,942 lval 1 0-06 0-81 6 RBC 1 3.41 0.098

2 1.35 0.638 Order 1 0.19 0.686 (0.319) Week 3 1-47 0.282 Dog(order) 4 2.69 0.093 Order*week 3 0.47 0.71 1 lval 1 0.06 0.81 RBC 1 3.81 0.079

3 1.6 0.636 Order 1 0.16 0.72 (0.223) Week 3 1.7 O. 224 Dog (order) 4 3.08 0.063 Order'week 3 0.53 0.67 RBC 1 4.29 0.063

4 2.1 8 0.583 Order 1 0-16 0.72 (0.093) Week 3 1-93 0-172 Dog(order) 4 3-52 0-035 RBC 1 5.91 0,029 Table A1.2. Model parameters and estimates of the effect of clomipramine (3 mgkg q 24 h for 6 weeks) on the dopamine metabolite homovanillic acid (HVA) in canine cerebrospinal fluid, in a repeated measures AB-BA crossover expenment

Model Overall F RL Variable DF F Pr >F (P)

1 2-78 0.81 2 Order 1 0.1 1 0,752 (0 -064) Week 3 1-74 0.228 Dog(order) 4 2.89 0,086 Ordefweek 3 2.68 0-11 Thio 1 0.01 0.91 9 lval 1 O. 1 0-76 RBC 1 0.9 0-366

2 3.32 0.81 2 Order 1 0.41 OS8 (0.032) Week 3 1.93 0.188 Dog(order) 4 3.2 0.062 Ordefweek 3 2.98 0-083 lval 1 0.12 0.741 RBC 1 0.99 0.343

3.9 0.809 Order 1 0-36 0.579 (0.016) Week 3 2.5 0.1 14 Dog(order) 4 3.67 0.039 Ordefweek 3 3.27 0.063 RBC 1 1.02 0.333

4 4.16 0.792 Order 1 0.39 0.566 (0.01 1) Week 3 3.15 0-065 Dog(order) 4 5-92 0.007 Ordertweek 3 3-43 0,052 Table A1.3. Model parameters and estimates of the effect of clomiprarnine (3 mgkg q 24 h for 6 weeks) on the norepinephrine metabolite bmethyl4- hydroxyphenylglycol (MHPG) in canine cerebrosphal fiuid, in a repeated measures AB-BA crossover expenment

Model Overall F (pl R Va riabLe DF F Pr =+

1 1 -46 0.694 Order 1 0.63 0.473 (0.290) Week 3 1 -77 0.222 Dog(order) 4 1.38 0.31 5 Ordefweek 3 0-67 0-594 Thio 1 1.19 0.673 [val 1 0.21 0.657 RBC 1 0-32 0-585

2 1.69 0.687 Order 1 2-18 0-214 (0.205) Week 3 1-99 O. 179 Dog(order) 4 2-22 0.1 39 Ordefweek 3 1-09 0.398 lval 1 0.26 0.61 9 RBC 1 O -42 0.53

Order 1 2.2 0.21 2 Week 3 2-14 0.153 Dog (order) 4 2-36 0.1 17 Ordefweek 3 7-09 0-396 RBC 1 0.52 0.485

4 2.15 O. 664 Order 1 2-1 0.221 (O. 102) Week 3 2.07 0.1 58 Dog(order) 4 2-32 0.1 16 Order*wee k 3 1-11 0.386

5 2.49 0.571 Order 1 2-1 0.221 (0.061) Week 3 2-02 0.1 54 Dog(O rder) 4 2.27 0.1 i APPENDIX 2 Consent fom and information about a randomised cantrolled double blind trial of clomipramine in canine compulsive disorder.

THE CANINE CD PROJECT A study of ttie treatment of compulsive behaviours in dogs.

Dr. Caroline Hewson Ontario Veterinary College

Why are we doing this study?

Compulsive behaviours rnay affect up to 2% of al1 dogs. Compulsive dogs are thought to have a chernical imbalance in their brain. They become stressed easily and that makes them perform unusual behavioun (for example: snapping at imaginary Ries). At present there is no proven treatment to restore the balance in the brain chernistry of affected dogs.

The drug cium@ramine is used to treat obsessive-cornpulsive disorden in hurnans. Preliminary studies indicate that clomipramine is not hanful to dogs and that it rnay be a useful treatment for compulsive behaviours. We are doing this study to confirm whether or not dogs with compulsive behaviours can be helped by clom@ramine.

This kind of study is known as a clinical trial. It has been approved by the Animal Care Cornmittee of the University of Guelph.

Who is taking part?

Sixty dcgs will be studied. They are al1 pets and they will live at home throughout the study.

Deciding if your dog is a suitable candidate.

Dr. Luescher and Dr. Hewson will discuss your dog's behaviour with you in two separate telephone interviews. We will then decide if your dog's behaviour is suitable for the study. If the behaviour is suitable. we will ask you to send us a video-recording of the behaviour and to take your dog to your veterinarian for a check-up and routine blood and urine tests.

Once we are sure that your dog is suited to the study, we can begin. The trial itself

The trial compares clomipramine with a control treatment called a placebo. The placebo is an inadive substance which has no effect on brain chemistry.

The trial takes eleven weeks altogether- There is a one-week "nin-in" penod at the beginning. ARer that your dog will receive bath treatments: four weeks on each, with a two-week 'sniashout period" in between.

Jreatments are in the fom of capsules. They are given twice daily, with food, at approximately the same times every day.

Monitoring your dog's behaviour

You will score your dog's behaviour daily for five days on three separate occasions: five days in the first week, five days at the end of the first treatment period and five days at the end of the second treatment period. ScorÏng is done using a short questionnaire (ten minutes). Dr. Hewson will go over the questions with you on the telephone before you start to score your dog.

At the end of each treatment penod Dr. Hewson will ask some general questions in a short telephone interview (ten minutes).

OTHER INFORMATION

Side Effects Clomipramine: We are not aware of any major side-effech of clornipramine in dogs. Occasionally we see constipation, dianhoea. vomiting or slight sedation (sleepiness). Placebo: The placebo should not cause any side-effects.

if your dog became ill during this trial, you would infom Dr. Hewson and contact your vetennarian for treatment. costs You are asked to meet the following costs: i) the video-recording of your dog's behaviour at the beginning ii) the vetennary check-up at the beginning

The study budget will meet the cost of treatments. sconng sheets. 'phone calls made to you, mailing and the laboratory fees for the samples taken at the beginning. Also, if your dog's behaviour continues after the trial ends we will advise you and your veterinarian about how to deal with 1, at no charge. Data will be stored in a cornputer at aie Ontario Veterinary College. All personal information is confidential and available only to study personnel. No reports will be presented or published which reveal your identity.

Randomisation The order in which your dog receives the treatments is determined at random. This means uçing a chance procedure, Iike rolling a dice.

Blinding The treatments look identical. At any given time, neither you nor your veterinarian nor Dr. Hewson will know which treatment your dog is on. This deliberate ignorance is known as blinding; it ensures that no-one is biased when assessing the dag's behaviour.

When aie trial is over, we will tell you the order of your dog's treatments.

Please note that, dnecessary, it wiil be possible to find out at once what treatment your dog is receiving during the triai.

Follow-up We will keep in regular contact with you and your vet for the duration of the trial. We would like to know how your dog is doing 6 months after he/she completes the trial: we will telephone you then to ask you. We will infom you of our overall findings when the trial has been completed and al1 the data have been analysed.

7) Liability Neither the University nor Dr. Hewson nor Dr. Luescher nor the sponsors of this trial (Ciba Geigy and Pet Trust) will be held financially Iiable in the event that the treaurients fail. reactions to the treatments occur or complications result from the implementation of the protocol.

8) Your obligations if you enroll Entry into the clinical trial is voluntary. You rnay withdraw at any time if you so wish. Withdrawal will not affect any care or treatment you may receive in the Behaviour Clinic or other clinics within the Ontario Veterinary College.

Please note that withdrawing your dog aRer enrollment will reduce the scientifc ment of this clinical trial. You should only enroll if you are certain about the value of the tnal and Eyou feel that you can fulfill the requirements listed above. If you have any questions please phone Dr. Hewson at 519 824-4120, extension 4472. CONSENT TO PARTICIPATE IN THE CANINE CD PROJECT

If you are satisfied with the explanation of The Canine CD Project and wish to take part in if, please complete the form below and then retum it to Dr. Hewson in the envelo pe provided.

Please do not sian until vou have had al1 vour auestio ns concemina the trial and your ~artici~ationin it. answered to your satisfaction.

DOG'S NAME (please print):

OWNER:

NAME (please print):

SIGNATURE:

Date:

WITNESS:

NAME (please print):

SIGNATURE:

Date:

For Office Use only: APPENOlX 3 Prelirninary questionnaire sent to owners of dogs suspected of having canine compulsive disorder.

The questionnaire was customised for male and female dogs, and was filled out by owners prior to a fomal diagnostic interview about their dog's behavioural history. The diagnostic procedure is described in Chapter 5.

THE CANINE CD PROJECT : Case Histoty

A. BACKGROUND INFORMATION

How old was your dog when you got her?

Where did you get her?

If neutered, how old was she when she was neutered?

THE BEHAVIOUR ITSELF

When the behaviour first started

Approximately how old was your dog when you first noticed the behaviour?

Did she show this behaviour before you got hep

If Yes -> Do you know in what situations she showed it?

Please think about the situation or situations when you first noticed the behaviour:

* what exactly was happening in the roorn or area where she was?

" how exactly did she behave? - what was her body language? - confident or stinking low to the ground - were her ears pricked up? - was her taii up or between her legs? * how did you respond ta her behaviour?

Did she show the behaviour in only one situation or did she show it in a number of differenf situations? Please specify 5. Can you think of anything that occurred in her Ife amund the time when she first started to show the behaviour?

The way the behaviour is now:

1. At present, does she show the behaviour evew day?

If 'Yesr-> For how long would you Say she has been showing this behaviour on a daily basis?

2. How many tirnes on an average day does she show the behaviour?

3. When she is showing the behaviour, how long does she do it for if you don? intempt hef?

4. PIease think about the ty~icalsituations in which your dog cunently shows the behaviour: * what exactly is going on in the room or area where she is when she shows the behaviouf?

- what exactly does she do? - what is her body language? - confident or slinking low to the ground? - are her ears pricked up? - is her tail up or between her legs?

- how do you respond to the behaviour?

5. Does your dog show the behaviour when you are not around? For example, does she show the behaviour when she is - out in the vard on her own? - in a kennel at the vers or elsewhere? - left with a pet-sitter?

6. Can she be distracted while she is showing the behaviour?

If 'Yesf -> Does she quickly start the behaviour again (that is within the next 2-3 minutes?)

7. 1s it possible to get her attention imrnediatelv after the behaviour has ended?

8. Daes your dog show any other behaviours that concern you? Please specv. APPENDIX 4 Telephone questionnaire used to obtain a behavioural case history of dogs suspected of having canine compulsive disorder.

ltalicised phrases were not read to the owner. A diagnosis was made by evaluating the history wïth respect to formal diagnostic criteria. These criteria are listed in Table 5.1.

REFERRING VETERINARIAN

Name: Add

Phone

Home Phone: Best time to call: Work Phone: Info' re trial protocol: Y N

DOG'S NAME: P i) Sex: M' MT P iv) How old was " " when you gc P ii) Age Neutered: himlher:

P iii) Breed: P v) Age:

A. 8efore we discuss " " 's behaviour I would first like tu have a few background details about hislher social group.

Al. Do you have any other pets:

a- Other dogs (#) Does " " get dong with the dog(s)? For example, do they ever fight with each other (PROBE: besides playing with each otherj

b- Cats (#) Does " " get dong with the cat(s)? For example, do they ever fight each other How many other people Iive at home with you at present: a- # of adults (49 yn) b- # of children (name, age):

Now let's talk about " ".

I would like to know if helshe shows any of the following behaviours at present Please answer 'Yes"or "No" or "1 don? know" as I list each behaviour. Does helshe (Yès = 2; No = 1; Don? know = 9) a- lick any parts of hisher body consistently, causing a sore? b- chase hislher tail for minutes at a time?

C- spin round and round on the spot d- when sith'ng down, does he/she have episodes when helshe tums around as if 'checking' hislher rear or where helshe has been sÏtting e- snap at the air as if catching flies f- growl at or tiy to attack any part of hisjher body g- suck hislher Rank, that is curl up, catch his/her flank in hislher mouth and remain in that position h- freeze in one position and stare fixedly at an object or freeze and gaze fixedly into space

1- bark rhythrnically and monotonously without any change in volume or pattern, like this: wruff wMwruff wruff j- pace about, for example in a circle or in a figure of eight k- show any other repetitive or unusual behaviour. Mat? 1- If the answer to a1of the above is "No",ask: 'What behaviour does " " show that concems you?"

rn - Besides STATE, does " " have anv other behavioun or habits that concem you or are a problem for you?

B2a At present, does heishe STATE eve~day ? If "No" - NOT ELIGIBLE

B2b For how long would you Say " " has been showing this behaviour on a dailv basis? 1 1 - 2 weeics ) 2 3 - 4 weeks )If c 2 müts - NOT ELlGIBLE 3 1 - 2 months ) 4 more than two months

How many times a day does he show the behaviour? 1 once or Nice a day - NOT ELIGIBLE 2 three tirnes a day 3 more than three times a day

When " " is actually STATE, how long would you Say that a typical episode lasts if you don? intempt him? 1 less than one minute - NOT EUGIBLE 2 between one and three minutes - NOT ELIGIBLÇ 3 more than three minutes

63 HISTORY OF CONFUCT

B3a yrs Approximately how old was " " when you first noticed the STATE CD? B3b - Do you know if helshe ever showed this behaviour before you got him? (Ves = 2; No = 7; Don? how = 9) If "Yes": Do you know in what situations helshe showed it?

B3c I would like to know the situation or situations when you fi=# noticed " " STATE. I am interested in what was happening in the room or area where helshe was, how helshe behaved and how you responded to that Can you please think back and tell me as much as you can about the situation when you fim# noticed " " STATE CD.

PROBES: Who was in the area with " "?

PEOPLE

ANIMALS

What exactly were the peoplelanimals doing?

What exactiy did " " do?

ma#did the people do in response to this?

Wha# did " " then do?

- what was hisher body language? - confident or slinking low to the ground - were the ears pricked up? - what was hisher tail camage - between the legs?

B3d - Can you think of anything that occurred in hislher life around the time that helshe finit started to STATE. For exampte: had there been builders in the house- B3e I would Iike to know if any of the follom'ng events in particular occurred around the the that " " Crst started to STATE CD? Please answer 'Yes" or "No" or "1 donY know" as I Iist eacb event (Yes = 2; No = f; Don%know = 9)

1- was there a new baby in the house

ii - had you just moved into a new house/aparbnent

iii - had a new pet been brought into the house

iv- had a member of the household (human or animal) gone away or moved out; FOR EXAMPLE on business?

v - had someone just moved in to your house (human or animal)

vi - had there been a change in your daily routine. For example, had you started shift work or a new job

vii- was there a change in hisher daily routine (walks / feeding / etcetera)

viii - had you had the house or yard altered in any way; FOR EXAMPLE new fumiture in the house, new turf laid in the yard

ix - had " " had a fnghtening experience? For example: had he/she been hit by a car or attacked by another dog?

X- had " " just had an operation or major treatrnent at the vetls?

xi - Had " " just started or just finished a course at obedience school?

xii - Had you changed hisher diet? From which food to what food? xiii - Can you think of any other event or events that occurred around the tirne that " " first started to show the behaviour? DIAGNOSTIC INFORMATlON

B3a - Initial conffict identifable?

836 Type of conflict?

0- conflicting motivations (FEAR + other) ii)... - separafion anxiety 111) - absence of releasing sfiinuli (e-g. social contacf, objecfs to chew, prey to catch) iv) - physicaf msfraint (chained up/confned) v) - change in social envimnmenf v0 - unstable sochl orûer (sibling rivalry) vil-)- compefition viil-)- lack of predictability of the envimnmenl (inconsistent treatment by the owner)

Yes = 2; No = 1; Don? know = 9 B4 EMANCIPATION FROM ORMUAL CONTDCT

&la - You Say that " " first began to STATE CD when STATE...... Does helshe show the behaviour in more situations now compared to n weekslmonths ago when helshe first started to show the behaviour? (Yes = 2; No = 1; DonY know = 9)

B4b I would also like to know the situations in which helshe currently shows the behaviour. Could you please describe four instances during the past week when " " has STATE CD. B4c CouM you please tell me if" " currentlyshows the behaviour in any of the following situations.

Does " " STATE CD when helshe is being: (Yes = 2; No = f; Don't know = 9; N.A.= O)

i) - petted ii) - groomed iii) - scolded or disciplined

iv) - when a command is given.

v) - when helshe is playing

va) - on her own vb) -wiai your other dogs

vc) -with you vd) - with the children

'4 - when you're getting hisher food ready vii) - when his food is put down for him

N.B. Yes = 2; No = 1; Don? know = 9

viii) -does helshe STATE CD when you take him/her out for walks:

**.b via- before the leash is put on ? VIII -once the Ieash on?

ix) - does helshe STATE CD when you are not paying attention to himfher . For example if you are watching television

x) - does he/she STATE CD when you are going out, leaving him/her on hisher own

N.B. Yes = 2; No = 1; Don? know = 9; MA. = O

xi) - when you retum home.

xii) -does helshe do it when you take hirnher in the car?

xiii) -does helshe do it at the vet's? " xiv) does helshe do it when you have visitors to your house and " knows the people: xv) - if the visitors are strangers and " " does not know them: xvi) -when the children are playing or running around xvii) -when he/she is with other people's children:

xviia) -If " knows the children xviib) - " " does not know the children xviii) -when " " is with other dogs:

xviiia) -" knows the dogs wiiib) -" " does not know the dogs

xvii?) -the dogs are on " "'s territory xviiid) they are on neutral%nitory xix) -does helshe do it when there is a loud noise outside or inside. FOR EXAMPLE: when the garbage is being collected or if you drop a saucepan xvii) -does helshe do it when someone leaves the room Who? Owners lStrangersl Friends. Which room? xxi) -Cari you think of any other situation in which " " shows the behaviour(s)

PROBES: - situations of excitement; - when relaxed; - when everyone quiet in the house; - when there is certain music playing or a certain radio or TV programme B4a - lncreased from single confext initially to several $HfereM confexts 846 - # difintconferts m- IdentifiabIe conflict cumntly? (Yes = 2; No = l;)

0- confliciing motivations (FEAR + other) ii) - separation anxr'ety iii) ,absence of releashg stimuli (eg. social contact, objects to chew, prey to catch) physicaf restraint (chained upiconfned) change in social environment unstable social order (siblhg nvalty) cornpetition lack of predictability of the envimnment (inconsistent treatment by the owner)

Yes = 2; No = 7; Don? know = 9; N.A, = O

85. ROLE OF CONDlTIONING

- Do you know if" " STATE CD when no one is around and helshe is by himself. For example: when you are inside and helshe is out in the yard. N.B. Yes = 2; No = f; Don't know = 9

Describe

86 SEIZURE RULE-OUT

B6a. i) - Is it possible to get " "'s attention while helshe is showing the behaviour?

ii) - Can you then get himlher to da something else? III)... - Ooes " " quickly go back to STATE CD (mat is within the next 2- 3- mins?) B6b. i)- 1s it possible to get " "'s attention immediatelv after an episode has ended naturally without you intemipüng it)?

ii) - Can you then get himlher to do something else?

III)-0. - Does " " quickly go back to STATE CD (that is within the next 2- 3- mins?)

. .. - DIA OSTIClAiFûRMA TION

B6- Epiepsy (ictal or post-ictal unconsciousness)

87 OTHER TREATMENTS TRIED

I would like to know if you have tried to treat " "'s behaviour in any way. For EACH of the following treaûnents that 1 am going to mention please indicate whether or not you have tned them for " "'s behaviour by saying "Yes" or "No" Surgery What

Have you tried any kind of behavioual modification as a treatment for the STATE? Mat

Medication f Drugs Mat?

Have you tried any other treatment for " "'s ..STATE? For example, homeopathy or herbal remedies. mat

Is " " on ggy kind of medication for anvthing at present? For example heartwonn treatment, skin dreseings, vitamins, oaier.

190 88- PUNISHMENT

Undentandably, a lot of peopk find it frustratirtg when their dog perfonns a compulsive behaviour and they sometimes discipline the dog by shouting at himlher or swottïng himlher.

a) Was " " ever disciplined for STATE? What was done? How did " " respond to that

b) Is " " ever disciplined now for showing the behaviour? What do you do? How does " " respond to that

B9a Do you think that " " has developed any oaier changes in hislher general behaviour since helshe started to STATE.... Describe 69b Does " " ever show any of the following behaviours? Please answer 'Yes" or "No" in each case: (If "yes" ask how owner nesponds and what dog is response to mat)

EUMINA TNE TEMPERAMENT RELATED

i) Housesoiling that is wetting or dirtying xiv) Depressedilnappetant in the house xv) Hyperexcitablefhyperactive xvi) Afra id of noisefthunder xvii) Affaid of people SOCIAL xviii) Afiaid of situations / objects lanimals ii) Barkingfhowlinglwhining xix) Does hefshe follow you around iii) Rolling in unsavoury items alot?

AGGRESSIVE BEHAVIOUR

iv) Eating things such as his stool or Does heishe garbage xx) Growi/nip/bite or stones? dirt? xxi) Jump up v) Catching prey xxii) Steal food vi) Stealing food xxiii) Demand touch vii) Wooi eatingfsucking xxiv) Want his own way

Dues helshe show aggression,(for example by growling) when xxv) hisfher nails are trirnrned viii) Groorning excessively xxvi) he/she is groomed xrmii) you stare into hislher eyes xxviii) you stand over or put your REPRODUCTION hand out over himlher xxix) you pick himfher up ix) Faise pregnancy agitation xxx) 1s " " PROTECTIVE of x) Mounting peoplelanimals food/bones/toys/bed/car 33 anv other house/yard/farniIy xxxi) Is hefshe AGGRESSIVE TO a. family LOCOMOTORY b. strangers c. other animaldpets xi) Digging xii) Roaming? Running away on walks C. OTHER BACKGROUND

. To finish up, I would like to ask some more general questions so as to get an îdea of a typical day in " " 's life.

Ci HOUSING

Cla First I'd like to ask you about hisher housing. Has helshe been crate- trained 1 no 2 yes

Clb During the day is " " kept inside the house or outside?

IF INSIDE 1 in crate or one room 2 confined to part of the house 3 run of house IF OUTSIDE 4 loose in yard 5 tied up outside 6 in nin outside

Clc Where is " " kept at night when you have gone to bed?

1 sleeps on bed. Whose? 2 sleeps in bedroom. Whose? 3 cagedfconfined in house 4 loose in house 5 outside

C2 FOOD

C2a. What do you feed C2b. How many tirnes a day do you himlher? feed himlher? 1 canned 1 free choice 2 moist 2 sid 3 dry 3 bid 4 table food 4 tid 5 combination 5 > tid C3a Does " " get out for a walk NERY day?

2 Yes_..-..., Go to 2c 1 No ...,,.... Go to 26 C2b Would you Say that O- " " is never taken for walks Ski, to 2e 1. he/she is only walked at weekends 2, hels he is walked several tirnesiweek 3, it varies

C2c - How many tintes p. day is hefshe taken out?

C2d min How long is each outing?

C2e What sort of collar or restraint is helshe on you have to take " " out?

1 Flat collar 2 A pinch (prong) collar 3 Choke collar 4 Head halter (ProrniseRlalti) 5 Body hamess 6 Off leash 7 Other

C4 TRAINING C4a Has " " ever had any obedience training? 1 No..*.-SKIP TO 3f 2 Yes

C4b Was the training 1 at obedience school? 2 On your own? 3 With private lessons at home?

C4c What method of training did you use? corrections? food rewards? choke chain

C4d Do you still train hidher? O Never 1 It varies 2 Every week 3 Daily c& - Has everyone in your family been involved in training " "?

C4f I am interested to know what level of training " " has reached? Would you Say that hisher training is

1 Basic (corne, sit, heel) 2 lntemediate (stay, down-stay) 3 Advanced. What level? (Corn petitive Dog ; C.D- Excellent; Utility Dog; Obedience Training Champion

C4g How obedient is " "? Would you Say that heishe 1 Is completely disobedient 2 Is variable 3 Obeys cornrnands most of the tirne

Thank you very much for answering al1 these questions. I am now going to read through everything you have told me about " " as well as having a look at your vers report. When would be a good time to cal1 you back and discuss things further? In order to be diagnosed as having a CD the dog must fuffill al1 of the following criteria, as indicated in italics:

1. Behaviour itself: one or mon?of the following behaviours i) lick granuloma vi) self-directed aggression ii) tail-chasing (Shepherds) vii) flank-sucking (Dobermans) iii) whirling (Bull Terriers) viii) freezing iv) tuming and checking rear ix) barking (Schnauzers) x) pacing v) fly-snapping xi) other ?

2. Contexts :behaviour must fulfil this criterion Occurred initially in a single context but now occurs in more than one contexts. "Context" = triggenng event If that is not specifically known, context = place where animal shows the behaviour e-g. in back yard, front yard. and in house)

3. Past or present confiict or frustration: one or more of the following eïther at present or when behaviour started i) conflicting motivations (FEAR + other) ii) separation anxiety iii) absence of releasing stimuli (e-g. social contact, objects to chew, prey to catch) iv) physical restraint (chained uplconfined) v) change in social environment vi) unsta ble social order (sibling rivalry) vii) cornpetition viii) lack of predictability of the environment (inconsistent treatment by the owner)

4. The frequency of the behaviour has increased since the behaviour first started

5. The behaviour occurs when owner is not present Le. is not a purely conditioned behaviour

6. The dog is conscious while performing the behaviour: helshecan be distracted both during and immediately after the behaviour Le. no evidence of ictal or post-ictal phases APPENDM 5 Telephone questionnaire used at the Behaviour Service of the Ontario Veterinary College to obtain a behavioumf history

BEHAVIOR CASE REPORT SPECIES CASE # Dog 1 DATE Cat 2

OWNER COMPLAINTS Name Add City Postal Code REFERRING VI3 Y N Home Phone: Name Chic Work Phone: Phone Best tirne to calt Informed of charge: Y N

PET 1 PET 2 - conflict cases Name Name Age: (if dyr) mos Age: (if lyr)vrs (if 4 yr) yrs

Breed: colour Breed: colour

Sex: M MC F FS Sex: M MC F FS

Age Neutered: Age Neutered:

STUDENT'S NAME DATES CALLED f IMEISTATUS FACULTY SIGNATURE PET 1 Pm2 OBTAINED FROM: OBTAINED FROM: 1 private home 1 private home 2 breeder 2 breeder 3 own breeding 3 own breeding 4 hurnane society 4 humane society 5 stray 5 stray 6 pet shop 6 pet shop

AGE OBTAINED: (if cl yr)-mas AGE OBTAINED: (if 4 yr)-mos (if >lyr)jrs (if>l yr)_vrs

DECLAWED? DECLAWED? O no O no 1 yes 1 yes 2 N/A (dogs) 2 NIA (dogs)

OTHER PETS: WHICH ANIMA - IS DOMINA # other dogs Pet 1 1 # other cats Pet 2 2

ENVIRONMENT DAlLY SCHEDULE # of adults (49 yrs): someone home all day 1 # of children: someone home haif days 2 Children's Ages: owner works full time 3 varies 4 HOUSING OF PET DAY NlGHT 1 caged or confined 1 sleeps on bed 2 nin of house 2 sleeps in bedroom 3 loose in yard 3 cagedconfined in house 4 tied upfin nin outside 4 loose in house 5 outside WALKS (WERCISE) 1 NIA (cats) CRATE TRAINED? 2 never O no 3 several timesheek 1 yes 4 daily 5 > once a day How long?

TRAINING HOW WELL OBEDIENCE TRAINED? I NIA (most cas) 1 NIA (no training ,cats) 2 None 2 poor 3 Basic (corne, sit, heel) 3 good 4 Average (stay, down-stay) 4 excellent 5 Advanced WHAT IS PET FED? HOW OFTEN FED? 1 canned 1 fke choice 2 moist 2 sid 3 dry 3 bid 4 table food 4 tid 5 combination 5 >tid

MEDICAL HISTORY LAST MEDICAL EXAM? RELATED MEDICAL PROBLEM? 1 less than 1 month ago O no 2 within the last 6 months 1 yes 3 more than 6 months ago what? HAVE DRUGS BEEN USED TO TREAT THIS BEHAWOR PROBLEM? O no 1 yes

DRUGS USED: DRUG 1 DRUG 2 DRUG 3 Narne

Code -

Dose (mglkglday)

# timeslday

Effect: very effective some effect no effect made worse unknown

Side effects:

O no 1 yes (what) 1 vomiting 1 vorniting 1 vomiting 2 diarrhea 2 diarrtiea 2 diarrhea 3 sedation 3 sedation 3 sedation 4 excitation 4 excitation 4 excitation 5- 5 5 CASE HISTORY - What exactiy happens? - What happens before & after? - Were there any changes when the proMern started? - What was done to correct pet? - When and where did it happen initially? - When & where does it happen now? - Did the behavior change overürne? - Owner's description of pet

AGE THE PROBLEM STARTED? HOW LONG DOES IT LAST? (ifclyr) mos (if ~1 yr)__yrs PRELlMINARY DlAGNOSIS:

FREQUENCY? 1 the same 2 increasing 3 decreasing PROGNOSIS HOW OfTEN DOES IT OCCUR? 1 Excellent 1 >IO times a day 2 Good 2 daily 3 Fair 3 2-3 times a week 4 Poor 4 less than once a week 5 Hopeless PROBLEM LIST ELlMlNATlVE LOCOMOTORY 1 Bed wetting 35Circlingfigure 8'sftail 2 DiarrheaNomiting chasinglpacinghnmiriing 3 Housesoiling 36 Digging 4 Urine marking 37 Lameness (conditioned) 5 UrÎnating on phemrnones 38 Roaming/Running away 6 Urine retention/anuna 39 Scratching objech 7 40

SOCIAL TEMPERAMENT RELATED 8 Barkinghowlinghvhining 41 Depressed/lnappetant 9 Feline vocalkation 42 Hyperexcitable/hypemctive 10 Rolling in unsavoury items 43 Fear of noise/thunder 11 44 Fear of people 45 Fear of situations 1objects 1 animals INGESTIVE 12 Anorexia AGGRESSIVE BEHAVIOR 13 Chewing 46 Growlslnips/bites 14 Coprophagia 47 Jumps up 15 Compulsive eatingldrinking 48 Steals food 16 Eating grasslplants/garbage 49 Demands touch 17 Pica SO Wants own way 18 Prey Catching 19 Stealing food 51 AGGRESSIVE RESISTANCE 20 Wool eathg/sucking punishment 21 training nail trîmlgrooming GROOMING eye stare 22 Licking/sucking/chewing standinglhand over 23 Excess grooming being picked up 24 Lack of grooming 25 Scratching self 56 Self mutilation 53 PROTECTIVE BEHAVIOR 26 foocübonesltoysibedfcar house/yard/family REPRODUCTION 27 Cannibalism 28 False pregnancy agitation 55 AGGRESSIVE TO 29 Masturbation a. family 30 Mounting peoplelanimak b. strangen 31 Self nursing c. other animaldpets 32

HALLUCINATORY 33 APPENDiX 6 Telephone questionnaire used to rneasure outcome in a randomised controlled double-blind clinical trial of clomipramine as a treatrnent for canine compulsive disorder. Italicised phrases were not read to the respondent

DATE: CASE NUMBER: OWNER:

"Hello - this is 'John SmÏth' calling from the Ontario Veterinary College. I work with Dr. Caroline Hewson and l'm calling to interview you about how Brandy has been doing during the past four weeks. 1s this a good tirne to talk? - the intewiew will take about 10 minutes.

I would like to ask you some questions about how Brandy has been doing? First of all, does she still lick her leg?

2 Yes 1 No

Q - 1 a) Next I would Iike to ask about hno sepaate things. One is the appearance of Brandy's leg; the other is the licking behaviour that you see. Fiist I'd like to ask about the appearance of Brandy's leg - I'd Iike to get an idea of how bad it looks at present By "bad" I mean the oveall swelling, redness and rawness. Taking everything together during the past 5 davs would you Say that the leg looks

1 extremely bad 2 very bad 3 quite bad 4 somewhat bad 5 not ai al1 bad

Q -2 a) I would like to know if there has been any change in the appearance of the leg, since he started the present treatrnent Com~aredto how it was four weeks aao, would you Say that overall her leg now looks . - - worse O about the same Go to Q-3 + - bette?

IF WORSE: Would you say $IF B~ER:~ould you say -1 a little worse 1 a little better -2 moderately worse 2 moderately better -3 quite a bit worse 3 quite a bit better 4 much worse 4 much better -5 very much worse 5 very much better

Go fo Q - 26 below Q - 2 b) How significant is this change in the appearance of her leg? Would you say the change is

1 too small to be of significance 2 a little significant 3 quite significant 4 very significant 5 extremely signifiant

Next, I would like to get an ovenll idea of how bad is the Iicking behaviour that vou see at present By "bad" I mean how often she licks and how long she licks for. Taking everything together during e past 5 davs, would you Say that the licking you have seen has been:

1 extrernely bad 2 very bad 3 quite bad 4 somewhat bad 5 not at al1 bad

Q - 4 a) I would Iike to know if there has been any change in the intensity of the licking behaviour, since he started the present treatment Com~aredto how it was four weeks ago, would you Say that overall her licking behaviour now is . - - worse O about the same Go to (2-5 + - bette?

'IF WORSE $IF BE~R -1 a IMle worse 1 a little better -2 moderately worse 2 rnoderately better -3 quite a bit worse 3 quite a bit better 4 much worse 4 rnuch better -5 very much worse 5 very rnuch better

Go to Q - 46 below Q - 4 b) How significant is this change in the licking behaviour? Would you say the change is

1 too small to be of signficance 2 a tiile significant 3 quite significant 4 very signficant 5 extremely significant

I am now going to ask you about diffbrent aspects of Brandy's Iicking behaviour and Iwould like to know ifthey have changed

Q-5 Cornoared to how she was four weeks amwould you say that average dav now. you see Brandy licking

1 much more 2 somewhat more 3 the same as four weeks ago 4 somewhat less 5 rnuch less 8 (N.A.) 9 (Dan 't know)

Qdi Compared to how it was four weeks ago, woutd you say that naw a typical episode of licking that you see is

1 much longer 2 somewhat longer 3 about the sarne length 4 somewhat shorter 5 rnuch shorter 8 (N.A.) 9 (Don't know)

Q-7 Iwould like to know if Brandy shows her licking behaviour in 3s manv situations now. Compared to how she was four weeks ago, would you Say that Brandy now shows the behaviour in

1 many more situations 2 a few more situations 3 approximately the same number of situations 4 fewer situations 5 many fewer situations 8 (MA) 9 (Don? know) Q-8 We have now talked about the fkquency of Brandy's Iicking episodes, their average duration and the situations in which Brandy has them. Has anvthina else about the licking changed since he started taking the last treatrnent (For example: has she started to develop other sorer because of Iicking?)

2 Yes. Please specify what has changed 1 No. 8 (MA) 9 (Don't know)

Apart from her Iicking, I am interested to know if Brandy's general behaviour has changed since he started aie last treatment For example: she might seern more excitable p~ more relaxed. Compared to how she was 4 weeks aao would you sry her general behaviour has changed in any way

2 Yes Please descnbe bnefy the changes you have observed 1 No 9 Not sure Please describe briefly the changes you have observed

Have you noüced any changes in Brandy's oveall health durina the last four weeks.

i No 9 Not sure 2 Yes. .... Please descnbe them briefly. Did you katthem in any way - with a home remedy or by going to your veteniiarian I am going to ask you about some specific aspects of Brandy's health. First, during the last four weeks do you think that her APPETlTE has increased, decreased or stayed the same?

lncreased No Decreased Don Y change know i) appetite .,..2 ...... O -.--. .-..1 ....- ...9 ... ii) has her water consumption?...... iii) the frequency of her bowel ....2 ...... ---O ..... --.-1 .-.-- ... 9 ... movernents? iv) her frequency of passing -2.....,.. O .-.-. --1 ..,.. 9 ... urine? v) amount of time she has spent ....2 ...... O ...... 1 ...... 9 ... sleeping? vi) her overall level of alertness .... 2 ...... -.O -.--- ....1 ...... 9 ... when she's awake vii) her body weight

OTHER FACTORS

So that we can interpret the information mat you have given us about Brandy's behaviour, we are interested to know if there have been any changes in Brandy's routine during the last four weeks. Do you think there have been any changes in Brandy's mutine dun'ng the last four weeks? (For example: going away and leaving her in a kennel; change in diet)

2 Yes. Please specify what has changed 1 No. 9 (Don't know)

b) Has Brandy been given d~lyother treatment for hef leg during the last four weeks?

2 Yes. Please specw 1 No. 9 (Don Y kno w) COMPUANCE

-i3 a) I am interesteci to know if there were any problems with the dosing schedule that Brandy has been on. During the last four weeks has she ever missed a treatment?

1 No 2 Yes.

b) - Approximately how many treabnents would you say she missed?

c) Overall, has it been d'icult to get her to take the medication? Would you Say

1 Very dificuit 2 Somewhat diicult 3 Fairly easy 4 Very easy

BUNDNESS

Q - 14 What treaûnent do you think Brandy has been receiving during the iast four weeks?

1 The drug 2 The placebo

Q-15 FinaIl% we can score Brandy's licking durina the past 5 davs on a scale of zero to ten. A score of 'O' means NO Iicking. A score of '10' means that the licking was the worst it has ever been. Overall how would you rate Brandy's Iicking behaviour during the gast 5 davs (that is, since Monday)?

No The worst licking licking ever 0-16 Similady, we can score the appearame of Brandy's leg durina the past 5 da= on a scale of zero to ten. A score of 'û' means the leg has healed and the hair started to grow back. A sore of 'la' means that the leg look the wont it has ever looked. -[ how would you rate the appearance of Brandy's leg during the past 5 davs (that is, since Monday)?

Enor! 1 2 3 4567 8 9 10 Bookma rk not defined. O Healed Looks the worst ever ADDITJONAL QUESTIONS TO ASK AT THE LAST GLOBAL RATING

Q - 14 So that I can interpret the results of this project, I would like to know if you are aware of anvthing that rnay have occurred during the Icut 11 weeks that could have changed Brandy's compulsive behaviour. Can you think of anything that may have kendone during the to try and change Brandy's behaviour. For example - have you got a new dog since you started ta take part in this study?

2 Yes. Ptease specw 1 No- 9 (Don't know)

Yes No Don't know

a) Hae Brandy been given any

extra training? ...... CC.-...... 2 ...... *1 *. .-.*9.** please specm

b) exercise? ...... 2 ...... please specm

c) Have (any of) you ...... 2 ...... consciously tried to change please specrfy the ways in which you interact with Brandy (For example: giving him more, or less, attention).

d) Have (any of) you consciously changed any aspects of her environment ...... 2 ...... please specw

Q - 15 Which treatrnent do you think Brandy has done better on during this trial?

1 the first 2 the second (O no prefemce) APPENDIX 7 Questionnaire used by owners to score the frequency, duration and context of their dogs' compulsive behaviour during a randomised double-blind clinical trial of clomipramine

THE CANINE CD PROJECT

A study of the treatment of compulsive disorders in dogs

The following questions are about events that have occurred durina the last 24 hou^. Please read the questions carefully and answer them as accurately as you can.

IMPORTANT: (i) The terni "circling" refen to the times when 'Dog' goes in a circle. The terni does NOT refer to the shoulder-licking behaviour.

(ii) Some questions refer to "episodes of circling". Please note that an "episode" occurs until 'Dog' stops circling for anv reason (for example: if someone intenupts him). If he immediateiy starts circling again, that is a new episode.

AI1 the information that you provide will be treated in confidence.

Dr. Caroline Hewson DVM Department of Population Medicine University of Guelph Guelph, Ontario NIG 2W1

Tel: (5 19) 824-4120 extension 4472 Q - 1 a) Please state today's date and aie time now.

Today's date:

Time now: am 1 pm

b) Are you the same person who scored 'Dog' last time? (Ciicle your answer. If today is the very frst day of the study, cin=Ie 'Not applicable')

Yes No Not applicable

Q - 2 a) Please think back to this time yesterday. Since that time, how many hours have spent with 'Oog' so that vou could have noticed if he yas circling?

hours

Go to part (c) below

b) NOT APPLICABLE

c) During the hours that you indicated in 2(a), approximately how many individual episodes of circling did you see?

episodes ...... If vour answer is 0,go straight to Question 4 on page 3. OTHERWISE go to Question 3 on page 2. What was the Wpical length of the circling episodes that saw? (Please specrfy minutes or seconds)

When you saw 'Dog' circling, did you or anyone else intempt him? (Cide one number).

1 'Dog' was intempted every time I saw him cirding 2 'Dog' was interrupted iilrnost every time I saw him circling 3 'Dog' was intemipted some of the times when I saw him circling 4 'Dog' was interrupted a few of the times when I saw him circling 5 'Dog' was never intempted when I saw him circling

What was the length of the lonaest episode that saw? (Please specrfy minutes or seconds)

How did that episode come to an end? (Cideone number)

1 'Dog' stopped circling on his own - I am not aware that anyone or anything intemipted hirn 2 Somebody intempted 'Dogr and made him stop circling 3 Somethina else intempted 'Dog' (For example: a car drove up to the house) and he stopped circling

What was the length of the shortest episode that vou saw? (Please spectfy minutes or seconds)

How did that episode come to an end? (Circle one number)

1 'Dog' stopped circling on his own - I am not aware that anyone or anything intempted him 2 Sornebody intempted 'Dog' and made him stop circling 3 Somethina else intempted 'Dog' (For example: a car drove up to the house) and he stopped circling

Go to Question 4 Q - 4 Situations which rnake 'Dog' circle, are listed in the left-hand column of the table below. We need to know the foltowing things about each situation: (a) Has the situation occurred since this tirne yesterday? (middle column) (b) If the situation hais occurred, did see 'Dog' circling then? (right hand column) For each situation, please answer the questions by encircling the appropriate response. Two examples are given: -1: -1: The situation did not occur (answer to (a) is '&:'(6) does not apply). -le 2: The situation occurred (answer to (a) is '-7 but you did not see 'DoQ' circling then (answer to (b)

- (a) Has the situation occurred (b) If the situation hâa since this the yesterday? (Circle occurred, did ypy see 'DoQ' youranswer. II'Yes', go to (b) in the circling then? (Cimle your next column) answer) -le 1: SITUATION X Yes @ ~otsure Yes No Not sure

-e 2: SITUATION Y @ No Not sure Yes Not sure ------

1. 'Dog' was groomed outside Yes O Not sure Yes No Not sure

2. 'Oog' played with you Yes No Not sure Yes No Not sure 3. 'Dog' played on his own Yes No Not sure Yes No Not sure 4. 'DoQ'was scolded or disciplined Yes No Not sure Yes No Not sure

5. You prepared ' Dog's' food Yes No Not sure Yes No Notsure Q - 5 Understindably, orniers sometimes filfiustrated or wom0edby their dog's compulsive behaviout. We would like to ask if you have had similar feelings at times during the fast 24 hours. PIease indcate yourresponse to each of the following statements, by circling one answer in each case.

At times durinu the last 24 hours mv

has made me Extremely Very Moderately A Iittie Not at al1 feel worned womed womied wo rried w O rried womed

has made me Extremely Very Moderately A IÏttfe Not at all feel frustrated frustrated frustrated fnistrated fnistrated frustrated

has made me Extremely Very Moderately A Iittle Not at al1 feel angry angrY angrY WV angV aWY

has Extremely Very Moderately A little Not at al1 prevented much me from enjoying his Company

You have finished. THANK YOU! APPENDIX 8 Checklist osed by veterinarïans to evaluate dogs with compulsive disorder, pnor to enrollment in a clinical trial of clomipramine

Ciinical Trial of Clorniparnine (Dr. Caroline Hewson, Ontario Veterinary College; Tel (519) 824- 41 20; ext 4472; Fax (519) 7633117)

Veterinary Evaluation Sheet

Owner: Dog's name

1. Dog's bodyweig ht: please specw kilograms or pounds li. History and physical exam

ITEM YES Please check appropriate answer on the nght Please attach details 1 i) History of seizures? 1 1 1) ii) History of cardiac disease? 1 1 1 iii) History of liwr disease? 1 1 iv) History of glaucoma? v) History of diabetes rnellitus? 11 vi) Is the dog on thyroid hormone? 1 1 -- vii) 1s the dog on medication for anything at II present? I viii) Has the dog ever had clomipramine (Anafranil)? ix) To your knowledge, have anv behavioural drugs been prescribed for the dog during the past 4 weeks {e-g. phenobaibital, Vaiium (diazepam), Rivotril (clonazepam), îïavii (amitriptyline)}

x) Physical exam: Evidence of glaucoma? xi) Physical exam: Evidenœ of cardiac disease? I I xii) Femoral pulse rate p- min

II xiii) Heart rate p. min iii. Blood and urine f - BLOOD (Send to 'Laboratoty') URINE (Test in the chic)

i) 1 mL in EDTA (lavender-top) 1Specfic gravity: ii) 2 rn L serum (red-top. Spin down if 2. Giucoser possible) * DO NOT USE HEPARIN

iii) 1 rnL in fiuoride oxalate (grey-top. " Check that dog hasnY eaten within îast 12 hours " L

StGNED: DVM DATE: APPENDIX 9 Part of the randomisation list for a placebo-controlled clinical trial of clomip ramine in dogs with compulsive disorder.

The trial had a crossover design, wïth balanced treatment allocation

Behavioural Category 1: TAIL-CHASING GERMAN SHEPHERDS

FIRST SECOND OVVNER'S NAME PAIR # CASE # TREATMENT TREATMENT

C 1 Placebo 1 02 Placebo Dmg

O3 1 Drug 1 Placebo 1 - -- 04 Placebo DW

05 Placebo DW Dnig Placebo

O7 Dnig Placebo 08 Placebo DWJ

09 Placebo Dw Io Dnig Placebo

l2 Dnig Placebo APPENDIX 10. Information provided to owners of dogs with compulsive disorder, about how to unmask treatment allocation in a double- blind, randomised controlled clinical tnal of clomipramine

'Dog Surname' (Case number 00): Instructions on how to find out what treatment he (she) is receiving, in the event of a GENUINE emergency

1. Telephone the Ontario Veterinary College Teaching Hospital: (519) 823- 8830 (available Monday - Friday, 24 hours. Available on Saturday and Sunday, 9am - 9pm EST)

2. Tell the receptionist that 'Dog' is enrolled in Dr. Hewson's clinical trial and that you need to find out what treatrnent he(she) is receiving

3. The receptionist will want to know i) your name ii) 'Dog's' name iii) the treatment 'Dog' is on at present - that is. A or B. iv) when 'Dog' started that treatment

m Piease note that you should only foliow this procedure in a aenuine emergency (for example, if your dog or a child afe al1 the capsules). The study will be ut~atlvdevalued if you find out your dog's treatment just out of curiosity.

At the end of the study, you will be told the order in which your dog received the treatrnents.

Thank you for your co-operation

Dr. Caroline Hewson, Dept of Population Medicine, Ontario Veterinary College, Guelph, Ontario. Tel: (519) 824 4120 ext 4472; fax (519) 763 3 117 APPENDIX 11. Calendar used by owners of dogs with compulsive disorder, during a randomised, placebo - controlled, double-blind clinical trial of clomiprarnine (3rng/kg q 12 h).

Before sending the calendar to the owner, Ît was dated according to the time when the dog was enrolled in the trial.

DOSE 1 : give the dog the morning dose of treatrnent DOSE 2: give the dog the evening dose of treatment SCORE: use a questionnaire to record the frequency, duration and context of the dog's compulsive behaviour. This questionnaire is shown in Appendix 7.

Owners were asked to check off the above daily, as each task was performed. THE CANIFCE-T Dr. Caroline Hemon DW, Ontario Veterinary Colleqe. Universitv- of Gueloh. Telephone: 519 82Wf 20 e-kension 4472

Manday

DOSE 1 - DOSE 1 - SCORE - SCORE - SCORE - SCORE - SCORE - DOSE 2 - I DOSE 2 - DOSE 1 - DOSE 1 - DOSE 1- DOSE 1 - DOSE 1 - IDOSE 1 - DOSE 1 - DOSE 2 - DOSE 2 - DOSE 2 - DOSE 2 - DOSE 2 - 00SE2- 1 DOSEZ-

DOSE 1 - DOSE 1 - DOSE 1- OOSE 1 - DOSE 1- DOSE 1 - 1 DOSE 1 - DOSE 2 - DOSE 2 - DOSE 2 - DOSE 2 - DOSE 2 - DOSE 2 - 1 DOSE 2-

DOSE 1 - DOSE 1 - DOSE 1 - DOSE 1 - DOSE 1 - DOSE 1 - DOSE 1 - DOSE 2 - DOSE 2 - DOSE 2 - DOSE 2 - DOSE 2 - DOSE--r- 2 - DOSE 2 -

DOSE 1 - 3OSE 1 - DOSE 1 - DOSE 1 - DOSE 1 - DOSE 2 - >OSE 2 - 30SE 2 - DOSE 2 - 30SE 2 - SCORE - SCORE - SCORE - SCORE - SCORE -

DOSE 1 - DOSE 1 - DOSE 2 -

OOSE 1 - 30SEI 1 - DOSE 1 - DOSE 2 -

DOSE 1 - )OSE 1 - 1 DOSE 1 - DOSE 2 -

DOSE 1 - DOSE 2 -

SCORE- APPENDIX 12 Information supplied to the veterinarians attending dogs with compulsive disorder dunng a randomised, placebo-controlled, double-blind, clinical trial of clomipramine

INFORMATION ABOUT THE CLlNlCAL TRIAL OF CLOMIPRAMINE AS A TREATMENT FOR COMPULSIVE DISORDERS IN DOGS

Dr. Caroline Hewson DVM, Department of Population Medicine. Ontario Veterinary College. Guelph, Ontario. Tel: 51 9 824 4120 extension 4472 ; fax 763 31 17.

Study Design

Dogs receive both clomiprarnine and placebo. in random order. The trial is double-blind: neither the owner nor I nor you know which treatment a dog is receiving. If any of us identify the treatment that a dog is receiving, the dog must be dropped from the study. The dose rate of the treatments is 3mgkg q 12 houn (- a total of 6 mgkg each day).

IN A GENUINE EMERGENCY, to find out what treatment a dog is receiving:

Call the OVC Teaching Hospital (519 823-8840.24 hours) and tell the receptionist that the dog is in my study. The receptionist will also want to know the ownets name and the dog's Case Number. narne, breed and behaviour. Please do not identify the dog's treatment except in a genuine emergency (e-g. a child consumes the treatment or the dog consumes several capsules at once).

IMPORTANT-bit is necessary to idenbify a freatment, piease do not tell me what it is. If furfher advice is needed following 'unblinding: please cal1 Dr. Andrew Luescher (519 824-4120, ext 4054) or Dr. Pefer Conion (ext 4950).

Concurrent medication during the clinical trial:

Behavioural druas and sedatives (e-g. acepromazine for thunderston phobia) should not be given dun'ng the study because they may have unpredictable effects and they will confound the effects of the test treatments. Dogs that require sedatives or behavioural drugs dunng the study will no longer be eligible and will have to be dropped. General anaesthesia: Dogs that are anaesthetised during the study will have to be dropped ffom it. Non-essential surgery should be postponed until after the study.

Self mutilatory conditions: see p.4

Reasons to ldentify Treatrnent during the Study

It should not be necessary to identify treatrnent during the study. The reasons for this are as follows:

1. Placebo: is an inactive substance.

II. Clomipramine: a. What is it? It is a tricyclic antidepressant (TCA) with serotonin-reuptake blocking effects. It also has norepinephrine-reuptake blocking effects and is anti- histaminic and anti-muscarinic. It is made by Ciba-Geigy.

b. Contraindications of clomipramine in man: liver disease, hyperthyroidism, diabetes rnellitus, epilepsy, glaucoma and cardiac disease. Only dogs that are 'normal' on clinical examination + CBC and profile are enrolled in the study.

c. In a one-year toxicity study by Ciba-Geigy, groups of 6 dogs were given daily doses of 12.5 mgkg, 50 mg/kg and 100 mglkg respectively: one dog died on the highest dose and another produced fewer sperm. No other adverse effects were reporteci.

d. Experience with clomipramine in a laboratory study: In 1993 1 conducted a laboratory study of clomipramine in 6 mixed-breed male dogs. Each dog was on 3 mgkg clomipramine per os daily for 6 weeks. i) Anaesthesia: All the dogs were anaesthetised on 4 occasions, using atropine-acepromazine, thiopentone and isoflurane-nitrous oxide. No adverse reactions were seen. ii) FCG. CBC and Profile: ECGs were taken regularly and CBC and profiles were done at the beginning and end of the 6-week period. No abnormalities were detected using these tests. iii) Anthelrnintics: the dogs received praziquantel and oxfendazole twice. at the manufacturers' recommended doses with no adverse effects. e. Experience with clomipnmine in dogs at the OVC Behaviour Clinic: i) Vornitinq if the drug is given on an empty stomach. ii) Diarrhoea ii) Constipation and dry mouth are reporteci side-effects in hurnans iii) Sedation (sleeping more -> -> ataxia) occur if the dose is too high for an individual dog.

f. Drug interactions in HUMANS (see attached): 0 Cimetidine: a liver enzyme inhibitor. lncreases the half- Ife of TCAs. ii) TCAS will displace warfarin from plasma protein -> warfarin toxicity iii) When TCAs are oiven wÏth neurolepticç or anti- cholineraic druos (atropine. acepromazine) hyperexcitation may occur (not our experience in the laboratory study). iv) Anaesthetia: Increased risk of arrhythmias and hypotension (not our experience in the laboratory study V) Svmpathomirnetics: hypertension/arrhythmia with adrenaline and noradrenaline. Local anaesthetics seem to be safe.

WHAT TO DO IN THE EVENT OF SlDE EFFECTS

" Please alwavs contact Dr. Hewson (519 8244120 ext 4472) if the owner reports side effects. If I am out of the office and it is urgent. contact Dr. Luescher (ext 4054) or Dr. Conlon (ext 4950).

* If we have to reduce the dose of treatment i rnay need to prepare a new set of capsules. Please do not alter the dog's dose without consulting me.

The following are suggestions re how to approach possible side-effects. (Note that these are suggestions only and are not intended to supplant your clinical judgement. The suggestions are made because it would be helpfùl if al1 dogs wlh side effects were dealt with in a reasonably consistent way). a) sedation: defined as some çombination of i) not alert - 'sleepy'; droopy eyelids; not responsive to stimuli such as going for a walk I seeing a cat I being in the vet clinic. ii) ataxia ii) sleeping alot Rule out other causes. If none, monitor for five days. if sîill sedated affer that contact Dr. Hewson to discuss reducing the dose.

b) constipation: defined as not defaecating for more than two days, in the absence of dyschezia. Rule out other causes. Consider laxatives and monitor progress.

C) vomiting: Rule other causes. Check what is in vomit - grass-bile; digests?; the capsules?? . If vomits on daily basis withn 30 mins of giving freatment, ensure giving wm enough food. If continues to vomit at same frequency despite giving treatment wm food, contact Dr. Hewson.

d) diarrhoea: Rule out other causes. Typalliative matment and monitorpmgress. if persists, contact Dr. He wson. e) urinary retention: to date we have only seen this in cats. Rule out other causes. If none contact Dr. Hewson to discuss reducing the dose. e) deterioration of a self-mutilatory condition (e-g. lick granuloma; tail-chasing Shepherd that attacks its tail). Provide whatever ancillary freatment you feel is appropriate: - If possible, topical treabnent should be attempted fint (Topagen spray; Synotic-banamine; mild corfcostemÏd cream). - if a collar or bandages are used the owner should be instructed to remove them for hvo hours each day, at a time when the owner can monitor whether the dog is showing the selfimutilatory behaviour. - If systemic treatment is indicafed, please provide it Ho wever, glease avoid depot corticostemids- Dogs that are given depot cotticostemid injections wilf have to be dmpped fiam the study. - please avoid surgery if possible (because sedative/anaesthetic may affect behaviour).

Thank you for your support. APPENDIX 13 The results of statistical modebbuilding (ordinal regression. proportional odds model, stepwise selection) in a randornised placebo-controlled clinical trial of clomipramine in dogs with compulsive disorder.

The criücal level of significance for accepting parameten in the model was 0.1. The experirnent is described in Chapter 7.

Abbreviations:

- Cumulative OR: cumulative odds ratio. Estimate of the odds of canine compulsive disorder being 'Bettef with clomipramine (3mgkg q 12h PO for 4 weeks) compared to placebo - Order: treatment sequence - Duration: time for which the dog had been exhibiting the behaviour while owned by the curent owner - CD2: acral lick dematitis - C03: miscellaneous behavioun (the referent, CD1, was locomotory behavioun)

Question 3

Modef Score test for -2 Log L Variable Cumulative OR Wald f P proportional odds (P) (95 % confidence assurn~tion intewal)

Order 4-88 (1-60 - 14.90) Breed 2.43 (0-73- 8-05)

3 0.81 9 101-73 (O. 0493)

Order 5.12 (1.65 - 15.88) Breed 2-58 (0.75 - 8.88) CD2 1.30 (0.32 - 5.23) CD3 0-71 (0.21 - 2.44)

8 0548 102.23 (O. 0067) Question 15

Model Score test for -2 Log L Variable Cumulative OR Wald xL P proportional odds (P) (95 % confidence assumption intentai)

1 0.0001 104.55 Order 3.17 (1.08 - 9.24) 4-44 0.035 (O. 103) Breed 1.26 (0.4 - 4-05} 0-16 0.693

2 0.257 100-47 Order 3.60 (1-1 9 - 10.89) 5.13 0.024 (0.01 34) Duration 0-97 (0.94 - 0.99) 4.32 0.038

96.1 9 Order 4.000 (1 -27- 12.6) 5-63 0.018 (0.01 18) Duration 0-96 (0-94 - 0.99) 4.25 0.039 CD2 3.67 (0.78 - 17.18) 2-73 0.099 CD3 0.86 (0.23 - 3.22) 0,048 0.83 APPENOUC 14 Descriptive data on 75 dogs diagnosed with canine compulsive disorder by expert opinion- The diagnostic procedure is described in Chapter 5.

Dag Breed CD Sex Age Duration of CD Dog had CD (ys) with present when owner owner (rnos) got dog?

American Bull Temer Tail chase No American Cocker Spaniel Chew fur No Arnerican Cocker Spaniel Spin in tight cirde Yes Bearded Collie Li& seIf No Border Collie Jumping No Bouvier de Flandres Spin in tight Urde No Cairn Terrier LigM btion No Cavaiier King Charles Lick self No Spaniel Chinese Crested tick self Don't know Dachshund flank-su& Dont know Dalrnatian Amal tick dematitis No Dalrnatian Frozen body position No Doberman Pinscher Acral lick dematitis No Doberman Pinscher Acral lick dermabios No Doberman Pinscher AcraI lick dermatitis No Doberman Pinscher Flank-suck Yes Doberman Pinscher Flank-suck Don't know Dobeman Pinscher Spin in tight arcle Don't know Doberman Pinscher Spin in tight arde Doberman Pinscher Spin in tight circie Don't know English Bull Terrier Flank-su& No English Bull Terrier iick self No Engiiih Bull Terrier Tail chase No English Bull Terrier Tail diase Don't know English Bull Temer Tai[ chase No English Bull Temer Tail diase Don't know English Springer Spaniel Acral lick dermatitis No English Spflnger Spaniel Li& seif No Flataated Retriever Potydipsia No Flatded Retriever Running in a cirde Don7 know German Shepherd Amal lick dematitis No GemnShepherd Acrai Iick derniatitis No German Shepherd Running in a cide No Dog Breed CD Sex Age Duration of CD with Had CD when (yrs) present owner owner got dog?

Geman Shepherd Running in a urde M 25 Geman Shepherd Running in a arde M (c) 2 Don't know Geman Shepherd Tai1 chase M 125 No German Shepherd Tai1 chase F 3 No German Shepherd Tail chase F (n) 2 No Geman Shep herd Tail chase M 3 No Irish Setter Acral lick dematitis F (n) 5 No Jack Russell Temer Jumping M (cl 2 Yes Jack Russell Temer Spin in tight cirde F (n) 3 No Keeshund Am1Iick dematitis F 3 No Labrador Retriever AmlIidcdemaüüs M(c) 2 No Labrador Retriever Li& seif F (n) 2 No Miniature Poodle Li& seif M (c) 8 Yes Miniature Schnauzer "Check" rear end F (n) 9 No Miniature Schnauzer "Check" rearend M 3 Miniature Schnauzer "Check" rearend M 2 No Miniature Schnauzer "Check" rearend M (c) 9 No Miniature Schnauzer "Check" rear end M (c) 3 No Miniature Schnauzer "Check" rear end M (c) 1-5 Don't know Neapolitan Bull Mastiff tick self M (cl 7 No Old English Sheepdog Pace up & down F (n) 2 Don't know Porneranian Air- bite F 3 Don't know Pomeranian Tail chase M (c) 2 Yes pug tick seff M (cl 4 Don't know Rottweiler îick self M (c) 4.5 No Rottweiler Chase shadows F 1.75 No Rottweiler Chase shadows M 0.75 No Shetland Sheepdog Spinintightarde M(c) 5.5 Yes Sh ïTzu Spin in tight circle M (c) 1-5 Yes Standard Poodle Running in a circle F (n) 1-5 No Standard Poodle Running in a circle M (c) 6 Yes Toy Poodle "Check" rearend M 3 Don7 know Dog Breed CD Sex Age Ouration of CD with Had CD when owner (yTS) present owner (rnos) got dog? 66 Cross-bred Acral Iick dermatitis F (n) 1-5 6 No Acral Iick dermatitis Dont know Acral Iick dematitis No Acral lidc dennatitis No Acral lick dermatitis No Air-bite Air-bite Dont know Pace up & down No Spin in tight cirde Don? know Spin in tight arcle No APPLlEO i IWGE. lnc