**ProHeart 6 (moxidectin)**

TABLE OF CONTENTS

[List of Tables 3](#__RefHeading___Toc93048615)

[List of Figures 4](#__RefHeading___Toc93048616)

[executive summary 5](#executive-summary)

[1.0 INTRODUCTION 8](#introduction)

[2.0 Review of Heartworm in Dogs 11](#review-of-heartworm-in-dogs)

[2.1 Life Cycle of Dog Heartworm (*Dirofilaria immitis*)
11](#life-cycle-of-dog-heartworm-dirofilaria-immitis)

[2.2 Heartworm Disease in Dogs 12](#heartworm-disease-in-dogs)

[2.3 Canine Heartworm Epidemiology in the US
13](#canine-heartworm-epidemiology-in-the-us)

[3.0 moxidectin overview 14](#moxidectin-overview)

[3.1 Pharmacology -- Mechanism of Action
14](#pharmacology-mechanism-of-action)

[3.2 Pharmacokinetics and Drug Metabolism
15](#pharmacokinetics-and-drug-metabolism)

[3.3 Toxicology 16](#toxicology)

[3.3.1 In Vitro Side-Effect Profiling
16](#in-vitro-side-effect-profiling)

[3.3.2 Single- and Repeat-Dose Toxicology Studies
17](#single--and-repeat-dose-toxicology-studies)

[3.3.3 Carcinogenicity Studies 18](#carcinogenicity-studies)

[3.3.4 Reproductive and Developmental Toxicity Studies
19](#reproductive-and-developmental-toxicity-studies)

[3.3.5 Genotoxicity Studies 19](#genotoxicity-studies)

[3.4 Experience with Oral Moxidectin in Human Volunteers
19](#experience-with-oral-moxidectin-in-human-volunteers)

[3.5 Conclusions 19](#conclusions)

[4.0 Proheart 6 overview 20](#proheart-6-overview)

[4.1 Clinical Trials with ProHeart 6 in Dogs
20](#clinical-trials-with-proheart-6-in-dogs)

[4.1.1 Heartworm 21](#heartworm)

[4.1.2 Hookworms 24](#hookworms)

[4.1.3 Safety 24](#safety)

[4.1.4 Field Studies 26](#field-studies)

[4.1.5 Exaggerated Moxidectin Overdose in Dogs
27](#exaggerated-moxidectin-overdose-in-dogs)

[4.1.6 International Studies 27](#international-studies)

[4.1.7 ProHeart 12 28](#proheart-12)

[4.1.8 Conclusion 28](#conclusion)

[4.2 Postmarketing Surveillance of Adverse Events
29](#postmarketing-surveillance-of-adverse-events)

[4.2.1 Overview 30](#overview)

[4.2.2 Review of Adverse Event Reports
31](#review-of-adverse-event-reports)

[4.2.3 Conclusions 46](#conclusions-1)

[4.3 Manufacturing Change 47](#manufacturing-change)

[4.4 Safety Profile in Dogs 49](#safety-profile-in-dogs)

[4.4.1 Methods 49](#methods)

[4.4.2 Results 51](#results)

[4.4.3 Discussion 58](#discussion)

[4.4.4 Conclusions 59](#conclusions-2)

[4.5 Field Efficacy 60](#field-efficacy)

[4.5.1 Overview 60](#overview-1)

[4.5.2 Compliance 60](#compliance)

[4.5.3 Field Efficacy from 2001 to 2004
61](#field-efficacy-from-2001-to-2004)

[4.5.4 Tuskeegee University Experience with ProHeart 6
63](#tuskeegee-university-experience-with-proheart-6)

[5.0 REFERENCES 64](#references)

[6.0 Appendices 71](#appendices)

[APPENDIX 6.1 71](#appendix-6.1)

[appendix 6.2 84](#appendix-6.2)

[APPENDIX 6.3 86](#appendix-6.3)

[APPENDIX 6.4 89](#appendix-6.4)

[APPENDIX 6.5 92](#appendix-6.5)

[APPENDIX 6.6 95](#appendix-6.6)

[]{#__RefHeading___Toc93048615 .anchor}List of Tables

[Table 4.1.1.2.1-1. Retroactive Activity of ProHeart 6 at 0.17 mg/kg
versus 4- and 6-Month Heartworm Infections
23](#table-4.1.1.2.1-1.-retroactive-activity-of-proheart-6-at-0.17-mgkg-versus-4--and-6-month-heartworm-infections)

[Table 4.1.1.2.1-2. Retroactive Activity of ProHeart 6 versus 3-month
Heartworm Infections
23](#table-4.1.1.2.1-2.-retroactive-activity-of-proheart-6-versus-3-month-heartworm-infections)

[Table 4.2.2.2.1-1. Neurologic Adverse Events -- Number of Reports
40](#table-4.2.2.2.1-1.-neurologic-adverse-events-number-of-reports)

[Table 4.2.2.2.2-1. Hematologic Adverse Events -- Number of Reports
41](#table-4.2.2.2.2-1.-hematologic-adverse-events-number-of-reports)

[Table 4.2.2.2.3-1. Hepatic Adverse Events -- Numbers of Reports
43](#table-4.2.2.2.3-1.-hepatic-adverse-events-numbers-of-reports)

[Table 4.2.2.2.4-1. Cardiac Adverse Events -- Numbers of Reports
44](#table-4.2.2.2.4-1.-cardiac-adverse-events-numbers-of-reports)

[Table 4.2.2.2.5-1. Neoplasia Adverse Events -- Numbers of Reports
45](#table-4.2.2.2.5-1.-neoplasia-adverse-events-numbers-of-reports)

[Table 4.4.1-1. Adverse Events 50](#table-4.4.1-1.-adverse-events)

[Table 4.4.2-1. Rate per 10,000 of Any Adverse Event by Treatment
Category Total Number of Encounters = 6,800,061
52](#table-4.4.2-1.-rate-per-10000-of-any-adverse-event-by-treatment-category-total-number-of-encounters-6800061)

[Table 4.4.2.1-1. Adverse Event Rate per 10,000 by Treatment Group
53](#table-4.4.2.1-1.-adverse-event-rate-per-10000-by-treatment-group)

[Table 4.4.2.1-2. Liver Adverse Events Definitions Based on Lab Measure
or Lab Abnormal Flag
54](#table-4.4.2.1-2.-liver-adverse-events-definitions-based-on-lab-measure-or-lab-abnormal-flag)

[Table 4.4.2.1-3. Potential Liver-related Adverse Events per 10,000 by
Treatment Group
55](#table-4.4.2.1-3.-potential-liver-related-adverse-events-per-10000-by-treatment-group)

[Table 4.4.2.1-4. Potential Cancer-related Adverse Events per 10,000 by
Treatment Group
56](#table-4.4.2.1-4.-potential-cancer-related-adverse-events-per-10000-by-treatment-group)

[Table 4.5.3-1. Voluntary Reports of Product Inefficacy Against Larval
Heartworm
62](#table-4.5.3-1.-voluntary-reports-of-product-inefficacy-against-larval-heartworm)

[]{#__RefHeading___Toc93048616 .anchor}List of Figures

[Figure 2.1-1. Life Cycle of Dog Heartworm
11](#figure-2.1-1.-life-cycle-of-dog-heartworm)

[Figure 4.2.2-1. Seasonality of Dispensing Heartworm Medication 2002
(All Products)
33](#figure-4.2.2-1.-seasonality-of-dispensing-heartworm-medication-2002-all-products)

[Figure 4.2.2-2. Doses Sold by Quarter
34](#figure-4.2.2-2.-doses-sold-by-quarter)

[Figure 4.2.2-3. Number of Adverse Event Reports by Quarter
35](#figure-4.2.2-3.-number-of-adverse-event-reports-by-quarter)

[Figure 4.2.2-4. Reporting Rate by Quarter
36](#figure-4.2.2-4.-reporting-rate-by-quarter)

[Figure 4.4.2-1. Doses Dispensed by Month; ProHeart 6 Only or With
Vaccine
52](#figure-4.4.2-1.-doses-dispensed-by-month-proheart-6-only-or-with-vaccine)

[Figure 4.4.2.2-1. Risk of Liver Disease by Age for ProHeart 6
57](#figure-4.4.2.2-1.-risk-of-liver-disease-by-age-for-proheart-6)

# executive summary {#executive-summary .Heading-0}

Canine heartworm infection (*Dirofilaria immitis)* occurs in many parts
of the world, including all 50 states in the US. Despite widespread
availability of monthly heartworm preventatives, the infection rate in
the US increased in the 1990s, and use of heartworm preventatives
declined. Surveys have shown that compliance (i.e., reliable monthly
treatments by owners) is problematical and a limiting factor in the
control of heartworm in the dog population.

The active ingredient in ProHeart^®^ 6 is moxidectin, a macrocyclic
lactone. Moxidectin has been thoroughly evaluated to determine its
toxicological and metabolic properties in multiple animal species and is
approved as an anthelmintic agent for use in cattle, sheep, swine,
dogs,and horses, in 70 countries. ProHeart 6 has been carefully
evaluated for the treatment of heartworms and hookworms in dogs. In
registration studies sponsored by Fort Dodge Animal Health (FDAH) (a
Division of Wyeth), the product has been demonstrated to be safe and
efficacious. Further, it appears to be well tolerated in canine breeds
that do not tolerate some of the monthly products.

In June 2001, Proheart 6 (moxidectin) was approved and launched in the
US by FDAH to prevent canine heartworm disease for 6 months, and to
treat existing larval and adult stages of the canine hookworm. Since
then, the product has also been registered in Italy, Canada, Japan,
France, Greece, Portugal, Spain, and Korea. A similar product,
ProHeart SR12, which contains approximately 3 times the amount of
moxidectin as ProHeart 6, is registered and marketed in Australia since
October 2000.

Among heartworm preventative medications for dogs, ProHeart 6 is an
innovative product. Unlike conventional products that require monthly
dosing for a minimum of 1 month prior to mosquito exposure through 1
month after mosquito exposure, in order to achieve protection against
heartworm infection over a mosquito-infestation season, 1 injection of
ProHeart 6 (moxidectin) sustained-release product provides 6 months of
protection. ProHeart 6 was specifically developed to overcome field
efficacy problems that result from poor compliance with monthly
treatments. The introduction of ProHeart 6 provided an avenue for
continuous protection against heartworm infection.

Shortly after launch of ProHeart 6, FDAH received a number of reports of
allergic-type reactions after administration. The reactions ranged from
mild and self-limiting to severe anaphylactoid reactions. Product
analysis of minor component profiles between batches of ProHeart 6
revealed a trend to lower reactions for lots with no detectable residual
solvents. At this time, FDAH was continuing to optimize the
manufacturing process. As part of this process, the decision was made
that all ProHeart products would be produced from moxidectin technical
material with no detectable solvents. After the manufacturing change was
implemented, there was a decline in the adverse-event reporting rate
from all markets.

Since the launch of the product, FDA expressed concern about the number
and seriousness of adverse event reports (AERs). The vast majority of
the AERs were submitted by FDAH based on field reports from
veterinarians and dog owners. The reports were submitted under mandatory
reporting regulations without assessment of the likelihood of
association of the AER with Proheart 6 administration. As a result of
FDA's concern, FDAH has made revisions to the product label and issued
"Dear Doctor" letters. On September 3, 2004, based on continued FDA
concerns, FDAH agreed to voluntarily recall the product from the U.S.
market. The recall prompted regulatory authorities in Canada, Australia,
Japan and Europe to further review the safety of ProHeart 6. These
authorities have allowed continued marketing of all FDAH moxidectin
products for canine heartworm control.

FDAH's postmarketing surveillance and analysis of AERs from June 2001
through August 2004 show the number of these AERs were generally
decreasing. The peak of AERs in the second quarter of each year
corresponds to peak-use periods and also appear to be decreasing over
time. Analysis of AERs for ProHeart 6 by category show that the
occurrence of injection-site reports remained low and consistent with
other injectable products in the FDAH database. Allergy AERs trended
down over time at 1.26 per 10,000 doses. Non-allergy AERs were low and
consistent over time at 1.19 per 10,000 doses (neurologic at 0.12 per
10,000 doses; hematologic at 0.09 per 10,000 doses; hepatic 0.07 per
10,000 doses; cardiac at 0.02 per 10,000 doses; neoplasia at 0.06 per
10,000 doses). When taken in context with usage, the overall rate for
AERs was low and trending down over time, up to the point of the recall.

FDAH recently sponsored a study of the safety of ProHeart 6 use in
general veterinary practice and a comparison to the monthly oral
products. The study utilized a database covering 403 full‑service
veterinary hospitals in 42 states and did not rely on voluntary
reporting. The review evaluated approximately 7 million canine office
visits with an emphasis on comparison of heartworm product safety with
and without concomitant vaccine administration. Overall, the safety
profile of ProHeart was similar to that of 2 commonly used monthly oral
products. Many of the adverse events could be attributed to concomitant
vaccine administration. The results of the study provide no support for
the withdrawal of ProHeart 6 from the market.

In conclusion, FDAH has performed additional research and further
evaluation of the ProHeart 6 database to add to the extensive safety and
toxicology database for moxidectin and formulated products. These
evaluations provide additional support for the safety and efficacy of
the product and were conducted to address questions raised by FDA.

# INTRODUCTION

Moxidectin is a semi-synthetic methoxime derivative of nemadectin that
is a fermentation product of *Streptomyces cyaneogriseus* subspecies
*noncyanogenus*. It is a pentacyclic 16-membered lactone macrolide.
Moxidectin is licensed and marketed worldwide by Fort Dodge Animal
Health (FDAH) (a Division of Wyeth) as an anthelmintic agent that causes
the paralysis and death of affected parasites in cattle, sheep, swine,
horses, and dogs. It is currently being developed in a collaboration
between Wyeth and the World Health Organization (WHO) for humans with
onchocerciasis (river blindness), a disease caused by infection with the
tissue filarial nematode *Onchocerca volvulus.*

ProHeart^®^ 6 (moxidectin) was approved in the US and launched by FDAH
in June 2001, to prevent canine heartworm (*Dirofilaria immitis)*
disease for 6 months, and to treat existing larval and adult stages of
the canine hookworms (*Ancylostoma caninum* and *Uncinaria
stenocephala*). Since then, this product has also been registered in
Italy, Canada, Japan, France, Greece, Portugal, Spain, and Korea. A
similar product, ProHeart^^ SR 12, which contains approximately 3 times
the amount of moxidectin as ProHeart 6 and provides 12 months of
protection is registered and marketed in Australia since October 2000.

Among heartworm preventative medications for dogs, ProHeart 6 is an
innovative product. Unlike conventional oral tablets or topical
applications that require monthly dosing for a minimum of 1 month prior
to mosquito exposure through 1 month after mosquito exposure, in order
to achieve protection against heartworm infection over a
mosquito-infestation season, 1 subcutaneous (SC) injection of ProHeart 6
(moxidectin) sustained-release product provides sustained 6-month
protection. The single administration of ProHeart 6 for a 6-month period
of protection eliminates the possibility of the pet owner missing 1 or
more monthly doses, which is the primary cause of lack of efficacy
associated with these heartworm medications.

ProHeart 6 has been well received by veterinary professionals and dog
owners as evidenced by its increasing market share in major markets
since launch. By the third quarter of 2004, ProHeart 6 was the number
two product in the US with a 24% market share. In Italy, it is expected
to achieve a 35% share by the end of 2004. ProHeart SR 12 is the market
leader in Australia, presently with a 47% share.

Subsequent to the product approval in June 2001, the FDA Center for
Veterinary Medicine (CVM) raised concerns over the number of reports of
adverse events associated with ProHeart 6.[^1] Further concern was
raised that "many of the reports received have involved serious,
life-threatening adverse events such as anaphylaxis, convulsions,
hematopoetic disorders, hepatopathies" and also "neurologic problems and
unusual cardiac signs." It was also stated that "Pet owners should be
advised on appropriate alternative heartworm preventatives for their
dogs." FDA requested "that the firm continue to conduct research to
determine the cause of related adverse reactions...before the product is
marketed again."

On September 3, 2004, FDAH announced that it was voluntarily ceasing
production for the US market and recalling ProHeart 6 from the US market
until resolution of FDA safety concerns, based on reports of adverse
events. Despite the voluntary recall, FDAH maintains that ProHeart 6 is
safe and efficacious with acceptable field performance. Regulatory
authorities in Canada, Australia, Japan, and Europe have allowed
continued marketing of the FDAH moxidectin products for heartworm. FDAH
supports the FDA formation of an independent Advisory Panel to review
safety data on ProHeart 6. In order to provide the Advisory Panel with
extensive analyses of available scientific data on ProHeart 6, FDAH
sought assistance from independent experts. As requested by the FDA,
additional research has been conducted to better define the adverse
reactions. FDAH anticipates that comprehensive review of available
ProHeart 6 data by the Advisory Panel will satisfactorily resolve FDA
safety concerns.

Careful evaluation of the nature and timing of the AERs identified that
allergy-based signs could be attributed to treatment and occurred
shortly after treatment. The true incidence of allergic events to
ProHeart6 is confounded because many dogs receive concurrent
vaccinations. These are recognized to trigger allergic manifestations. A
range of commonly occurring disease conditions that affect dogs is also
seen in the AERs. These appear to represent baseline occurrence of these
conditions in the canine population.

The purpose of this document is to present an overview of moxidectin and
ProHeart 6 information from non-clinical studies, clinical trials,
adverse event reporting, and analysis of safety and efficacy data for
review by the Advisory Panel. This information includes new data on
pharmacokinetics, new pharmacological investigations, new evaluation of
adverse event findings, and epidemiological information from 7.0 million
canine visits to veterinary clinics. FDAH is confident that the new
research and re-evaluation of previous data support the safety and
efficacy of ProHeart 6 and looks forward to returning this product to
the market.

# Review of Heartworm in Dogs

## Life Cycle of Dog Heartworm (*Dirofilaria immitis*)

The life cycle of the dog heartworm can be described in 3 stages as
shown.

# Figure 2.1-1. Life Cycle of Dog Heartworm {#figure-2.1-1.-life-cycle-of-dog-heartworm .Caption-Figure}

![](media/image1.wmf){width="5.625in" height="3.7284722222222224in"}

**Stage 1:** Adult female worms release tiny immature heartworms called
microfilariae into the bloodstream of the infected dog. Adult worms are
reported to live for 2 to 7 years.

**Stage 2:** The infection is spread from dog to dog by mosquitoes. When
a mosquito bites an infected dog and feeds on blood, it takes in some of
these microfilariae. Within 2 to 3 weeks, these microfilariae will
develop into a stage which can infect other dogs when they are bitten by
the same mosquito as it feeds.[^2]^,^[^3]

**Stage 3:** Following infection, the immature stages grow and develop
over 2 to 3 months in the subcutaneous (SC) tissues, muscle, and fatty
tissues of the dog. They then migrate, and from 70 to 120 days post
infection, stages of heartworm may be found in the heart or pulmonary
artery. These develop into adult heartworms which are long slender
roundworms, normally living in the right side of the heart and nearby
blood vessels. The worms may be up to 35 cm in length when mature.Error:
Reference source not found^,Error:\ Reference\ source\ not\ found^

Mature female *Dirofilaria immitis* begin releasing microfilariae into
the blood stream some 6 to 7 months after infection, thus completing the
lifecycle.

Heartworm preventative products exert their effect at Stage 3, the
tissue migrating stage.

Heartworm tests rely on detection of the parasite either at Stage 2, by
detection of circulating microfilariae, or at Stage 3, by detection of
female heartworm antigen. Heartworm antigen tests are not positive until
the infection is at least 5 months old, they are inconsistently positive
at 5 to 7 months, and are not considered to be reliably diagnostic until
the infection (with female worms present) is at least 8 months old.
These tests are highly sensitive and specific. If clinical signs raise
any doubt about the accuracy of the particular test result, the test
should be repeated, preferably with a different test kit or
laboratory.[^4]

## Heartworm Disease in Dogs

Many dogs may be infected for several months with *Dirofilaria immitis*
without showing clinical signs. This is particularly true for inactive
dogs. However, immature worms in the pulmonary artery can initiate
disease as early as 3 months after infection.[^5] Adult worms in the
heart cause inflammation of the heart lining and valves, and can
eventually lead to heart failure. The effect on blood flow can lead to
problems in other organs, particularly lungs and kidneys.

It can be difficult to know that a dog is affected in all stages of the
disease because symptoms may be slight. The dog may only be listless and
tire easily. Performance in working dogs may be affected. As the disease
progresses, there may be cough, loss of condition, and build-up of fluid
in the abdomen. Severely affected dogs may die.

Treatment of the disease can be risky, and early detection before
chronic damage occurs to heart and lungs is important. This is because
thromboembolic complications may occur, particularly in heavily infected
dogs with pulmonary arterial vascular obstruction, and especially if
congestive heart failure is present. The approved adulticide treatment
is melarsomine, trade name Immiticide, an arsenical compound. While
melarsomine is less toxic and more effective than its predecessor,
thiacetarsamide, it is administered as a deep intramuscular injection
into the lumbar muscles to reduce swelling and soreness at the injection
site. Pulmonary thrombo-embolism and/or shock may occur following
adulticide treatment even in symptomless dogs. Exercise restriction
during the recovery period (weeks) is essential to minimize
cardiopulmonary complications. Off label use of ivermectin for this
purpose has been reported, but is not recommended because of progression
of heartworm disease in the treated dogs.[^6]

## Canine Heartworm Epidemiology in the US

Heartworm infection in dogs has been diagnosed in many parts of the
world, including all 50 states of the United States. While heartworm is
considered endemic in the 48 contiguous states and Hawaii, transmission
has not been documented in Alaska, even though there has been
importation of infected dogs. It is likely that the climate is not
conducive to the maturation of infective larvae. Adequate temperature
and humidity are required both to support a suitable mosquito population
and also to provide sufficient heat for maturation to the infective
larval stage (L~3~). Laboratory studies indicate that at 80°F, 10 to 14
days are required for maturation to infective stage, this period is
longer at lower temperatures or where significant diurnal temperature
fluctuation occurs.Error: Reference source not
found^,Error:\ Reference\ source\ not\ found^

Therefore, the length of the heartworm transmission season varies with
geographical location and climatic factors. The peak months for
heartworm transmission in the northern hemisphere are July and August.
Estimates of the duration of the transmission season vary from less than
4 months in Southern Canada to essentially all year in sub-tropical
areas such as Florida and the Gulf Coast. It is believed that
transmission occurs for 6 months or less above the 37^th^ parallel.

The prevalence of heartworm in dogs varies from state to state, with
higher infection rates in southern states, and particularly higher along
the Mississippi and Ohio rivers. The number of adult heartworms present
in infected dogs has been reported to be lower in the North (Michigan)
than in the South.[^7] A survey undertaken by the American Heartworm
Society in 2001, published in 2002, reported the highest infection rates
in Texas, followed by Florida, Louisiana, North Carolina, Georgia,
Mississippi, Tennessee, South Carolina, Alabama, and Indiana. Of
significant veterinary concern, the survey reported that despite the
widespread availability of monthly heartworm preventatives, the rates of
infection with heartworm had not decreased over the previous 10
years.Error: Reference source not found^,^[^8]^,^[^9] For further
information see Section 4.5.

For the past 2 decades, heartworm prevention relied on monthly
administration of macrocyclic lactones, sometimes in combination with
other active ingredients to treat other parasites. Commonly used
products include ivermectin, milbemycin oxime, and selamectin.
Combination products include ivermectin plus pyrantel (for roundworm
control) and generics, and milbemycin oxime plus lufenuron (for flea
control). For dogs that suffered toxic effects from these compounds, the
only alternative available was daily treatment with diethyl carbamazine.

The introduction of ProHeart 6 in the US in the second half of 2001
provided 6 months of continuous protection against heartworm infection
without having to rely on monthly treatment by the dogs' owners, a major
source of lack of efficacy.

# moxidectin overview

## Pharmacology -- Mechanism of Action

Moxidectin has been shown to have activity at the γ-aminobutyric acid
(GABA)-A receptor-chloride channel complex resulting in an influx of
chloride ions and hyperpolarization of cell membranes. Hyperpolarization
causes the nerve fibers to be less excitatory and results in paralysis
and death of the parasitic organism. Another proposed mechanism of
action for moxidectin is through activity at glutamate-gated chloride
ion channels. The specificity of moxidectin for the parasite versus the
mammalian host results from 1) a low affinity for mammalian GABA-gated
chloride channels, and 2) GABA-containing neurons and receptors are
found in mammals in the central nervous system, whereas in arthropods
and nematodes these are found in the neuromuscular junctions of the
peripheral nervous system and thus are more accessible to a blood-borne
therapeutic.

In dogs, the approved oral monthly dosage of moxidectin for prevention
of heartworm is 3 μg/kg; the approved SC dosage of moxidectin as
ProHeart 6 is 0.17 mg/kg administered every 6 months. In this and the
sections that follow, dosages of ProHeart 6 or moxidectin expressed as
mg/kg refer to mg of moxidectin per kg of animal body weight.

## Pharmacokinetics and Drug Metabolism

Studies were conducted in various animal species to characterize the
absorption, distribution, metabolism, and excretion of moxidectin after
oral administration. Moxidectin was moderately absorbed with a
bioavailability of 19% in rats, and had a long serum half-life of 23 to
45 hours in rats and 8.1 days in dogs after oral gavage administration.
A single SC injection of the approved dosage of 0.17 mg/kg as ProHeart 6
to beagle dogs resulted in peak concentration in serum (C~max~) of 5.1
ng/mL, a time to peak concentration (t~max~) of 7 to10 days, an area
under the concentration-versus-time curve (AUC~0-∞~) of 217 ng•days/mL,
and an apparent elimination half‑life (t~1/2~) of approximately 35 days.
After SC injection of ProHeart 6 once every six months for a total of 6
injections, there was no evidence of alterations in pharmacokinetic
parameters or indication of accumulation. A recent study of the single
administration of moxidectin in the diet to female dogs at 45 ppm
(corresponding to approximately 1 mg/kg used in the 1-year toxicology
study) resulted in a C~max~, AUC~0-∞~, and t~1/2~ values of 290 ng/mL,
678 ng•day/mL and 8.3 days, respectively. These recent studies in the
dog, and an ongoing 28-day pharmacokinetic study in rats were designed
to further evaluate the systemic exposure to moxidectin under the
conditions of the previously conducted toxicology studies.

After oral administration, the major site of moxidectin distribution was
in fat in rats, cattle, sheep, and horses. It was eliminated largely
unmetabolized in the feces. The t~1/2~ in fat in the rat was 11.5 days,
much longer than in serum. Limited metabolism was noted in all species
and minor metabolites were identified as predominantly mono- and
di-hydroxylated moxidectin. Moxidectin did not result in significant in
vitro inhibition of cytochrome P450 isozymes, indicating that drug-drug
interactions mediated through cytochrome P450 are unlikely to occur.

P-glycoproteins (P-gps) are transmembrane proteins that transport a wide
variety of endogenous and exogenous molecules across cell membranes.
Moxidectin, similar to other macrocyclic lactones, is a substrate for
P-gps. This relationship is of clinical importance in the development of
nematode resistance to ivermectin and plays a significant role in breed
sensitivity. A mutation in the P-gp gene of ivermectin-sensitive Collie
dogs has been shown to be responsible for ivermectin-induced CNS
toxicity. Moxidectin, however, was well-tolerated by these
ivermectin-sensitive dogs (see Section 4.1.3.2). Thus, moxidectin
transport is less dependent on P-gp and subsequent toxicity is less
likely manifest due to factors which alter P-gp activity.[^10]

## Toxicology

The toxicologic profile of moxidectin administered by the oral route has
been well established. This profile is relevant to other routes of
administration because of limited metabolism of moxidectin in the body
and the long terminal half-life regardless of the route of
administration. A more detailed discussion of the pharmacokinetics and
toxicity assessment of moxidectin is presented in Appendix 6.1.

### In Vitro Side-Effect Profiling

Moxidectin and moxidectin microspheres (as present in ProHeart 6) were
recently tested in vitro for binding activity at 64 different biological
receptors. This assay is commonly used in drug discovery and development
to identify any ancillary pharmacologic activities of a molecule which
may result in undesirable biological effects. A final concentration of
10 ng/mL moxidectin was tested, which is approximately two-fold the
average C~max~ value in serum of dogs after an SC injection of ProHeart
6 at the clinical dosage of 0.17 mg/kg. The receptors tested included
those for neurotransmitters and neurotransmitter-related receptors, ion
channels, steroids, second messengers, prostaglandins, growth
factors/hormones, brain/gut peptides and enzymes. Moxidectin in either
form did not significantly inhibit the binding of appropriate
radioligands to these receptors, indicating a lack of significant,
competitive binding activity for moxidectin at the concentration
evaluated.. These results are consistent with the absence of undesirable
pharmacologic and toxic effects of moxidectin in animal studies at
plasma levels greater than those required for efficacy.

### Single- and Repeat-Dose Toxicology Studies

Single-dose toxicology studies of moxidectin were conducted to assess
effects after a single administration of large doses in the event of
accidental overdose and to assist in selection of dose levels for
subsequent repeat-dose toxicology studies. In single-dose toxicity
studies of mice and rats given moxidectin orally, the median lethal
dosage (LD~50~) values were 118 mg/kg and 42 to 78 mg/kg in male and
female mice, respectively, and 122 and 97 mg/kg in male and female rats,
respectively. After a single SC dose, LD~50~ values were 285 and 247
mg/kg in male and female mice, respectively, and \> 640 mg/kg in rats.
Common clinical signs in these studies were decreased activity, tremors,
and prostration. These studies demonstrate a large margin of safety for
ProHeart 6 since the lethal SC dose in rats is more than 3700-fold the
approved ProHeart 6 dose of 0.17 mg/kg.

Repeat-dose oral (diet) toxicity was evaluated to assess long-term
consequences of repeated, daily oral exposure to moxidectin. The
objective of these studies was to expose animals to high levels of
moxidectin to identify potential toxic effects, and to include lower
doses to assess a possible dose-response relationship and a dose without
significant adverse effects (NOAEL).

The following studies were conducted: 4-week studies in mice, rats, and
dogs; 13‑week studies in rats and dogs; and a 1‑year study in dogs.
Evaluations consisted of mortality, clinical observations, body weight,
food consumption, hematology, clinical chemistry (except mice), organ
weights, and macroscopic and microscopic examinations of organs and
tissues. Ophthalmic examinations and urinalysis were also included in
the dog studies.

In repeat-dose diet toxicity studies in rats and dogs of durations up to
2 years in mice and rats, and 1-year in dogs, no target organs of
toxicity were identified. There were no significant adverse histologic
or biochemical effects to any organ system. There were no proliferative
lesions identified in any tissue which may signal the development of
neoplasia, and no increase in tumors in 2-year studies in mice or rats.
The toxicity of moxidectin manifested at high doses in clinical signs
such as lethargy, ataxia, tremors and mortality (rodents only) with
concomitant decreases in food consumption and body weight. Such clinical
signs of hypoexcitation are consistent with an exaggerated pharmacologic
effect of moxidectin mediated via the GABA-receptor. Interim analysis at
day 21 in an ongoing 28-day diet pharmacokinetic study of moxidectin in
dogs at a concentration of 45 ppm in feed (approximately 1 mg/kg, the
NOAEL in the 1-year dog toxicity study) revealed a serum concentration
of 278.5 ng/mL 24 hours after the preceding dose in feed (ie, trough
level). This value is approximately 234-fold the AUC~0-∞~ (217
ng•days/mL) observed for moxidectin after a single SC dose in dogs of
ProHeart 6.

### Carcinogenicity Studies 

#### Mice

A 2-year carcinogenicity study was conducted in male and female mice at
diet doses of 15, 30, and 60 ppm (lowered to 50 ppm due to high
mortality at week 9). Mortality was increased in females at doses of
60/50 ppm during the last 13 weeks of the study. There were no
compound‑related findings in hematology values, organ weights, or at
macroscopic or microscopic examination. There was no evidence of
moxidectin‑related target‑organ toxicity or tumorigenicity.

#### Rats 

A 2-year carcinogenicity study was conducted in male and female rats at
diet doses of 15, 60 and 120 ppm (lowered to 100 ppm due to high
mortality in females at week 8). There were no compound-related findings
for hematology values, organ weights or at macroscopic or microscopic
examination; there was no evidence of moxidectin-related target-organ
toxicity or tumorigenicity.

### Reproductive and Developmental Toxicity Studies

Reproductive toxicity was evaluated in a rat multigeneration diet study
and in developmental studies in rats and rabbits dosed daily by oral
gavage. Based on the results from these studies, moxidectin was not
found to be a selective reproductive toxin in rats, nor a teratogen in
rats or rabbits.

### Genotoxicity Studies

Moxidectin was tested for genotoxicity in 4 in vitro and 2 in vivo
standard test systems. These assays assessed the ability of moxidectin
to induce gene mutations, chromosome damage, or increased DNA repair
which may be related to the carcinogenic potential of the test article.
Moxidectin was uniformly negative in these assays, indicating that
moxidectin is not a genotoxic compound.

## Experience with Oral Moxidectin in Human Volunteers

A study in healthy, male volunteers was conducted to assess the
pharmacokinetics and safety of moxidectin given orally as part of the
development of this compound for onchocerciasis therapy in humans.
Safety assessments indicated that moxidectin was safe and well
tolerated, with a slightly higher incidence of transient, mild, and
moderate CNS adverse events (dizziness and somnolence) as compared to
placebo. Moxidectin was safe and well tolerated in humans after single
oral doses of 3 mg to 36 mg, the highest dose evaluated.

## Conclusions

Moxidectin is a potent antiparasitic therapeutic that acts to paralyze
susceptible organisms through activity at GABA- and glutamate-gated
chloride ion channels. Moxidectin has a long half-life, distributes
predominantly to fat, shows little metabolism, and is excreted primarily
in the feces. In single-dose toxicity studies, the lethal SC dose in
rats was more than 3700-fold the efficacious dose of moxidectin given as
ProHeart 6 to dogs. In repeat-dose diet toxicity studies of durations up
to 2 years in mice and rats, and 1 year in dogs, no target organs of
toxicity were identified. There were no significant adverse histologic
or biochemical effects on any organ system. There were no proliferative
lesions identified in any tissue which may signal the development of
neoplasia, and no increase in tumors in mice or rats. The toxicity of
moxidectin manifested itself at high doses in clinical signs such as
lethargy, ataxia, tremors and mortality (rodents only) with concomitant
decreases in food consumption and body weight. Such clinical signs of
hypoexcitation are consistent with an exaggerated pharmacologic effect
of moxidectin mediated via the GABA-A-receptor. Moxidectin was not
genotoxic or carcinogenic and was without reproductive or developmental
toxicity. The highest dosage evaluated in the 1-year dog study resulted
in an estimated monthly exposure 234-fold the exposure observed after a
single SC dose in dogs of ProHeart 6. Based on the toxicology studies of
moxidectin where the dose, dosing duration, and resulting systemic
exposure to moxidectin were significantly exaggerated, clinical SC
administration of 0.17 mg/kg as ProHeart 6 to dogs is expected to be
without significant adverse effects.

# Proheart 6 overview

## Clinical Trials with ProHeart 6 in Dogs

ProHeart 6 (moxidectin) is indicated for use in dogs ≥6 months of age
for the prevention of heartworm disease caused by *Dirofilaria immitis*
and for existing larval and adult hookworm infections (*Ancylostoma
caninum* and *Uncinaria stenocephala*). The safety and efficacy of
ProHeart 6 was demonstrated during a development program that was
established in close collaboration with the FDA that included agreement
on study requirements, protocol review, in-life inspections of selected
development studies and a thorough review of data obtained during the
program. Moxidectin is well established as an anti-parasitic product for
cattle, sheep, horses, and dogs and is generally recognized as safe for
these uses in all animals. The efficacy profile of ProHeart 6 was
demonstrated in a series of dose-determination and dose‑confirmation
studies conducted in accordance with Good Clinical Practice guidelines.
The safety of the product was evaluated through a series of safety
studies in the target population as well as unique canine populations
that would receive the product. These included reproducing males and
females, dogs that had existing heartworm infections, and Collie dogs
that had a demonstrated sensitivity to ivermectin. Safety and efficacy
studies were conducted not only in laboratory Beagles but also in a
variety of breeds and cross-breeds. In the sections that follow, dosages
of ProHeart 6 expressed as mg/kg refer to mg of moxidectin per kg of
animal bodyweight.

### Heartworm

#### Dose Determination

Two (2) studies were conducted to determine the dosage of a single
injection of ProHeart 6 required to effectively prevent *Dirofilaria
immitis* infections for 6 months. Both studies had a similar
experimental design. Study animals determined to be negative for *D.
immitis* by antigen and a modified Knott's test were included in the
studies. One study (0899-C-US-1-96, Georgia) utilized Beagle dogs (12
males and 20 females) and a second study (0899-C-US-2-96, Pennsylvania)
utilized mongrel dogs (16 males and 16 females).[^11]^,^[^12] ProHeart 6
was administered as a single SC injection to dogs at a moxidectin dosage
of 0.06, 0.17, or 0.50 mg/kg. Control animals received saline. At
approximately 180 days (6 months) after treatment, all dogs were
inoculated by intravenous (IV) injection with 50 *D. immitis* L~3~
infective larvae. Infections were allowed to develop for 150 days, at
which time each animal was euthanized, necropsied, and the heart and
lungs removed for the recovery and quantification of heartworms.
Immediately after treatment with ProHeart 6 and at various times
throughout the studies, dogs were observed for adverse reaction to
treatment. Additionally, injection sites were evaluated after treatment
and throughout the study as well as histologically at necropsy.

In the Georgia study, an average of 25 worms were recovered in the
saline treated controls. No worms were recovered from any of the dogs
dosed with ProHeart 6, indicating that all dosages tested were 100%
effective in preventing heartworm disease for 6 months.

In the Pennsylvania study, an average of 36 worms were recovered from
the saline treated controls. No worms were recovered from any dog that
received ProHeart 6 at 0.17 and 0.50 mg/kg. However, 14 adult *D.
immitis* worms were found in 1 dog in the low-dose group (0.06 mg/kg).
The results of this study indicated that a ProHeart 6 dosage of 0.17
mg/kg was appropriate to prevent canine heartworm disease for a period
of 6 months.

During the dose determination studies, moxidectin concentrations were
quantified in the serum of treated dogs. Following injection with
ProHeart 6, peak moxidectin levels (approx. 5 ppb) were observed at 7 to
14 days post-treatment. At the end of the 6-month treatment period,
residual moxidectin concentrations were negligible and generally below
the limit of quantification of the assay methods. The drug did not
accumulate with repeated doses. Serum moxidectin levels evaluated after
4 treatments (6 months apart) confirmed that there was no accumulation
of moxidectin in the serum of animals after successive doses
(0899-C-US-9-98, Texas).[^13]

#### Dose Confirmation

##### Adult Dogs

The efficacy of ProHeart 6 for the prevention of heartworm disease in
dogs was confirmed in a series of studies conducted using the proposed
commercial ProHeart 6 dosage of 0.17 mg/kg. Efficacy was evaluated at 6
months post-infection, 12 months post‑infection and in dogs with
existing heartworm infections to determine retroactive efficacy. The 6-
and 12-month infection studies used similar experimental designs as the
dose-determination studies. Animals were treated and challenged with
L~3~ infective larvae at either 6 or 12 months after treatment and
necropsied for quantification of adult heartworms after the infections
had matured. For activity against existing infections (retroactive
efficacy), animals were challenged with L~3~ infective larvae at various
times prior to treatment with ProHeart 6 and necropsied after an
appropriate period for the development of microfilariae to become adult
heartworms.

ProHeart 6, administered to dogs at an SC dosage of 0.17 mg/kg, was 100%
effective for prevention of heartworm disease for 6 or 12 months
(0899-C-US-10-98, Texas).[^14] When administered to dogs at an SC dosage
of 0.17 or 0.27 mg/kg (0899-C-US-20-99, Pennsylvania), ProHeart 6 was
100% effective against development of canine heartworm disease for 12
months after treatment.[^15]

The retroactive efficacy of ProHeart 6 with existing heartworm
infections was evaluated at 4 and 6 months after challenge with L~3~
infective larvae (0899-C-US-11-98, Georgia, Table 4.1.1.2.1-1).[^16] At
0.17 mg/kg, ProHeart 6 was highly effective against 4-month-old
*D. immitis* infections (85.9%). Efficacy was even higher (97.2%)
against 4‑month-old infections, when a second injection was given 6
months after the first injection; *D. immitis* was found in only 1 of 5
animals in this group. A single injection had low efficacy against
6‑month‑old infections (24.7%). Efficacy was not improved with 3
additional treatments 6 months apart. In a second retroactive study
(0899-C-US-28-01, Georgia, Table 4.1.1.2.1-2), ProHeart 6 at 0.17 or
0.50 mg/kg was highly effective against 3‑month‑old infections (98.8 and
96.0% efficacy, respectively).[^17]

+---------------+-----------+-------------+-------------+-------------+
| # Tabl        |           |             |             |             |
| e 4.1.1.2.1-1 |           |             |             |             |
| . Retroactive |           |             |             |             |
|  Activity of  |           |             |             |             |
| ProHeart 6 at |           |             |             |             |
|  0.17 mg/kg v |           |             |             |             |
| ersus 4- and  |           |             |             |             |
| 6-Month Heart |           |             |             |             |
| worm Infectio |           |             |             |             |
| ns {#table-4. |           |             |             |             |
| 1.1.2.1-1.-re |           |             |             |             |
| troactive-act |           |             |             |             |
| ivity-of-proh |           |             |             |             |
| eart-6-at-0.1 |           |             |             |             |
| 7-mgkg-versus |           |             |             |             |
| -4--and-6-mon |           |             |             |             |
| th-heartworm- |           |             |             |             |
| infections .C |           |             |             |             |
| aption-Table} |           |             |             |             |
+===============+===========+=============+=============+=============+
| >             |           | >           |             |             |
| **Treatment** |           | **Geometric |             |             |
| >             |           | > Mean Worm |             |             |
| > **(Time     |           | > Counts    |             |             |
| > P           |           | > (Percent  |             |             |
| ost-Infection |           | >           |             |             |
| > in          |           | Efficacy)** |             |             |
| > Months)**   |           |             |             |             |
+---------------+-----------+-------------+-------------+-------------+
| **Saline**    | **Mox     | **Male**    | **Female**  | **Total**   |
|               | idectin** |             |             |             |
+---------------+-----------+-------------+-------------+-------------+
| 4 and 6       |           | 14.07       | 17.31       | 31.66       |
+---------------+-----------+-------------+-------------+-------------+
|               | 4         | 1.24\*      | 3.42\*      | 4.46\*      |
|               |           | (91.2)      | (80.3)      | (85.9)      |
+---------------+-----------+-------------+-------------+-------------+
|               | 6         | 12.09       | 11.61       | 23.85       |
|               |           | (14.1)      | (32.9)      | (24.7)      |
+---------------+-----------+-------------+-------------+-------------+
| 4, 6, 10, 12, |           | 8.97        | 7.86        | 15.24       |
| and 18        |           |             |             |             |
+---------------+-----------+-------------+-------------+-------------+
|               | 4 and     | 0.32\*      | 0.25\*      | 0.43\*      |
|               |           | (96.4)      | (96.9)      | (97.2)      |
+---------------+-----------+-------------+-------------+-------------+
| \*            |           |             |             |             |
| Statistically |           |             |             |             |
| significantly |           |             |             |             |
| different (p  |           |             |             |             |
| \<0.05) from  |           |             |             |             |
| control group |           |             |             |             |
| based on      |           |             |             |             |
| analysis of   |           |             |             |             |
| geometric     |           |             |             |             |
| means.        |           |             |             |             |
+---------------+-----------+-------------+-------------+-------------+

+-----------------+----------------------+--------------+--------------+
| # Ta            |                      |              |              |
| ble 4.1.1.2.1-2 |                      |              |              |
| . Retroactive A |                      |              |              |
| ctivity of ProH |                      |              |              |
| eart 6 versus 3 |                      |              |              |
| -month Heartwor |                      |              |              |
| m Infections {# |                      |              |              |
| table-4.1.1.2.1 |                      |              |              |
| -2.-retroactive |                      |              |              |
| -activity-of-pr |                      |              |              |
| oheart-6-versus |                      |              |              |
| -3-month-heartw |                      |              |              |
| orm-infections  |                      |              |              |
| .Caption-Table} |                      |              |              |
+=================+======================+==============+==============+
|                 | **Geometric Mean     |              |              |
|                 | Worm Counts (Percent |              |              |
|                 | Efficacy)**          |              |              |
+-----------------+----------------------+--------------+--------------+
| **Treatment**   | **Male**             | **Female**   | **Total**    |
|                 |                      |              |              |
| **(mg/kg)**     |                      |              |              |
+-----------------+----------------------+--------------+--------------+
| Saline          | 16.0                 | 22.0         | 38.3         |
+-----------------+----------------------+--------------+--------------+
| 0.17            | 0.3 (98.4)           | 0.3 (98.8)   | 0.4 (98.8)   |
+-----------------+----------------------+--------------+--------------+
| 0.50            | 0.6 (96.3)           | 1.1 (95.1)   | 1.5 (96.0)   |
+-----------------+----------------------+--------------+--------------+
| Evaluation of   |                      |              |              |
| efficacy was    |                      |              |              |
| for male dogs   |                      |              |              |
| only, where     |                      |              |              |
| adequate        |                      |              |              |
| infection was   |                      |              |              |
| observed in     |                      |              |              |
| control         |                      |              |              |
| animals.        |                      |              |              |
+-----------------+----------------------+--------------+--------------+

##### Puppies

When administered to 12-week-old small, medium and large breed puppies
at 0.17 mg/kg (0899‑C-US-30-02, Pennsylvania), ProHeart 6 reduced
heartworm infection \>99.8% compared with control puppies.[^18]
Treatment completely prevented heartworm infection in small and medium
breed puppies. One of six large breed puppies was infected with a single
worm following challenge with 50 L~3~ *D. immitis* infective larvae.

### Hookworms

Efficacy was determined to be 100% for both larval and adult stages of
*A. caninum* in three dose confirmation studies (0899-C-US-12-98, North
Carolina; 0899-C-US-15-99, Georgia; 0899‑C‑US‑16-99,
Michigan).[^19]^,^[^20]^,^[^21] Efficacy against both larval and adult
*U. stenocephala* infections was 100% in study 0899-C-US-16-99.

Three (3) additional experimental infection studies were conducted to
demonstrate the effectiveness of ProHeart 6 versus larval and adult
stages of the hookworm *U*.* stenocephala*. Studies 0899-C-US-17-99
(Michigan), 0899-C-US-18-99 (New Jersey), and 0899-C-US-19-99 (Georgia)
provided additional data that confirmed the efficacy of the product as
\>99.0%.[^22]^,^[^23]^,^[^24]

Two studies were conducted to evaluate the persistent activity of
ProHeart 12 against subsequent hookworm infections in dogs following
treatment. In this program, animals were challenged with larvae of *U.
stenocephala* and *A. caninum*. ProHeart 12 prevented infections of
these 2 species of hookworm for a period of 4 months in a study in
Georgia. In a study conducted in Michigan, ProHeart 12 prevented
infection by *U. stenocephala* for a period of 8 months and *A. caninum*
for 5 months.

### Safety

#### Healthy Dogs

The clinical and possible pathological effects were evaluated when
ProHeart 6 was administered to healthy dogs at either 1, 3 or 5 times
the recommended dosage of 0.17 mg/kg in Study 0899‑C-US-4-98
(Wisconsin).[^25] Physical examinations were conducted prior to
treatment and throughout the study. Blood and urine samples were
collected for hematology, clinical chemistry, coagulation, and
urinalysis. Dogs were also evaluated for clinical signs, food
consumption, and body weight. At the end of the study, dogs were
necropsied and evaluated for overt changes. Tissues from the control
(saline treated) and 5-times dosage groups were examined
microscopically. Dogs treated with ProHeart 6 did not demonstrate any
signs or findings associated with the possible systemic toxicity of the
drug. A single SC injection of Proheart 6 equivalent to either 1, 3, or
5 times the commercial dosage caused swelling/slight edema at the site
of injections starting within 8 hours of injection and lasting for up to
3 weeks. One dog, that received the 5-times dosage of ProHeart 6,
displayed excessive salivation after treatment on Day 78. The only overt
lesion observed was a 2.0 cm red focus at the SC injection site in
1 male at the 5-times dosage.

The safety of multiple injections of ProHeart 6 was studied in dogs
given injections at 6 monthly intervals (0899-C-US-9-98, Texas) through
2 years.Error: Reference source not found No adverse reactions to
treatment were observed following 5 injections. The injection sites were
palpated externally, then the skin and underlying muscle tissue excised
and examined. There were no gross findings at the injection sites.
Microscopically, the injected areas generally had granulomatous
panniculitis with microvacuolation (spheres) that was interpreted to be
a reaction to the injected microspheres. Three (3) of the experimental
dogs were maintained and received a total of 14 ProHeart 6 injections.
At necropsy, there were no adverse reactions attributed to test article
during this time, including no gross findings in the injection sites at
necropsy.

The safety of ProHeart 6 was demonstrated in reproducing females
(0899-C-US-3-98, Michigan) and males (0899-C-CN-1-98, Canada) at 3 times
the recommended commercial dosage (0.5 mg/kg).[^26]^,^[^27] No adverse
effects were observed in reproductive parameters of treated breeding
females or the seminal quality of treated males.

When administered to healthy 10-week-old puppies at 3 or 5 times the
recommended dosage rate of 0.17 mg/kg (0899-C-US-37-02, Michigan),
ProHeart 6 caused no physical or neurological changes, and no changes in
clinical chemistry or urinalysis parameters.[^28]

#### Ivermectin-Sensitive Collie Dogs

Some genetic lines of Collie dogs are sensitive to the administration of
ivermectin. A safety study (0899-C-US-13-98, Illinois) was conducted to
determine the safety of a single dose of ProHeart 6 at 1, 3 and 5 times
the proposed commercial SC dosage of 0.17 mg/kg.[^29] Collie dogs shown
to react to a 120 μg/kg bodyweight dosage of ivermectin (depression,
ataxia, mydriasis, and excessive salivation) were enrolled in the study.
Following treatment, dogs were observed intensively for the first 24
hours and twice daily through 21 days. There were no health conditions
suggestive of toxicity in any of the dogs treated with ProHeart 6.

#### Heartworm-Positive Dogs

The safety of ProHeart 6 in heartworm-positive dogs was evaluated in 2
studies. The first study (0899-C-US-14-98, Alabama) tested a 3 times the
proposed SC dosage in dogs with patent heartworm infections as measured
by circulating microfilarial counts and heartworm antigen.[^30] Clinical
observations, physical exams, and microfilarial counts were used to
evaluate the effects of treatment. The results demonstrated that a
dosage of 0.51 mg/kg did not cause adverse reactions in dogs with patent
heartworm infections. There were no post-treatment adverse health
effects. Reduction in microfilariae compared with controls began as
early as Day 7 post‑treatment and peaked at 99.6% on Day 21
post-treatment. There was no significant (p \<0.05) adulticidal effect
observed with ProHeart 6.

As part of the ProHeart 12 development program, the safety of this
product for dogs was evaluated in dogs that had been surgically
implanted with 20 adult heartworms via the jugular vein
(0899-C-US-39-02, Georgia).[^31] On Day 61 following implantation, dogs
were treated with ProHeart 12 at 1.5 mg/kg (approximately 9 times the
ProHeart 6 and 3 times the ProHeart 12 proposed commercial dosages).
Animals were observed twice daily for signs of toxicity with physical
and clinical examination. There were no treatment-related effects in any
of the dogs. Microfilariae counts were reduced to almost 0 at 3 weeks
after treatment, with no effect on the adult population demonstrating no
adulticidal activity in heartworm-positive dogs.

### Field Studies

The safety and efficacy of ProHeart 6 at 0.17 mg/kg was evaluated under
field conditions in dogs dosed twice at 6-month intervals
(0899-C-US-5-98, California; 0899-C-US-6-98, Texas; 0899‑C‑US-7-98,
Wisconsin; 0899-C-US-8-98, Connecticut).[^32]^,^[^33]^,^[^34]^,^[^35] A
total of 374 client-owned dogs representing 84 breeds (280 ProHeart 6
treated, 94 ProHeart oral tablet controls) completed the study. Dogs
were ≥6 months of age and were of a variety of breeds, weights, physical
condition, and were of both sexes (either intact or altered). Prior to
enrollment, animals were tested for both heartworm antigen and
microfilariae to ensure that dogs were negative for existing heartworm.
None of the 374 dogs that completed this study tested positive for
heartworms at either 3, 6, or 12 months after the initiation of ProHeart
6 treatment. The following potential adverse drug reactions were
observed (number of cases): vomiting (3), diarrhea (2), weight loss (2),
listlessness (1), seizures (1), injection site pruritus (3), and
elevated body temperature (1). Injection site evaluation revealed no
abnormalities. This level of potential reactions for the 374 dogs that
completed the study was extremely low and demonstrated the safety of the
product under field conditions. Twelve (12) ProHeart 6 animals were
euthanized or died during the 18-month study. It was determined
following thorough review of each case that these deaths could not be
attributed to treatment with ProHeart 6.

### Exaggerated Moxidectin Overdose in Dogs

FDAH markets an oral formulation of moxidectin for use in horses to
control internal parasites. This product is supplied as a syringe with a
plunger calibrated by weight so that the correct dose can be given to
horses varying in weight up to 1150 pounds.

While uncommon, dogs have been exposed to this product. Sometimes this
exposure is intentional; owners believe it is cost effective and safe to
administer the product to dogs. Occasionally dogs find discarded
syringes and chew on them or they consume gel mixed with treats intended
to entice a reluctant horse.

From 1997 to 2004, FDAH received approximately 250 reports. These cases
were characterized by a wide range of neurological manifestations. Most
(90%) of these dogs recovered to normal; the remaining 10% died. Thus,
very high doses of moxidectin may lead to neurological manifestations of
toxicity that are often reversible with no evidence of any long-term or
non‑neurological toxic effects

### International Studies

ProHeart products have been registered in a number of international
markets including Australia, Canada, the European Union (France, Greece,
Italy, Portugal, Spain), Korea, and Japan. Length of activity claims and
active ingredient concentration vary depending on the market. US data
formed the basis of each registration with supporting local data, where
required. These studies included dose-confirmation and field-efficacy
testing.

Three (3) studies (0899-C-IT-01-99, 0899-C-IT-02-99, 0899-C-IT-03-99)
were conducted in Italy to evaluate ProHeart 6 (trademark Guardian SR in
Europe) at a dosage of 0.17 mg/kg for the prevention of heartworm
disease caused by *Dirofilaria immitis* and *Dirofilaria
repens*.[^36]^,^[^37]^,^[^38] Two hundred and fifty-one (251)
client-owned dogs of various breeds (41) completed this series of
field-efficacy and safety studies. ProHeart 6 given at 6 month intervals
was 100% effective in protecting animals from heartworm infection in
this heartworm endemic area (verified by untreated animals enrolled in
the same area). No adverse reactions to treatment were reported.

In Australia, 3 studies (0899-C-AU-01-97 dose confirmation,
0899-C-AU-02-00 puppy safety, and 0899-C-AU-02-98 clinical field study)
were conducted in support of a 12-month protection product administered
at a dosage of 0.50 mg/kg.[^39]^,^[^40]^,^[^41] ProHeart SR 12 was 100%
effective in preventing heartworm disease in the laboratory
dose-confirmation study. When administered at 3 times the recommended
dose (9 times the ProHeart 6 dosage) in puppies 10 to 12 weeks of age,
no clinically apparent adverse effects or adverse injection site
reactions were observed. Breeds of dogs included Maltese cross, Lhasa
Apso cross, Fox Terrier, Staffordshire Bull Terrier, Poodle, Border
Collie, Labrador Retriever, German Shepherd and Rottweiler and
represented small, medium, and large breeds. Two hundred and ten (210)
animals representing 75 breeds completed the field clinical study. The
study demonstrated the effectiveness of ProHeart SR 12 at 0.50 mg/kg (3
times the ProHeart 6 dosage) in protecting dogs from heartworm infection
for 12 months. No adverse reactions or drug interactions were observed
in treated dogs.

### ProHeart 12

ProHeart 12, a moxidectin based product similar to ProHeart 6 is
currently in development in the US. This product, administered at an SC
dosage of 0.50 mg/kg will protect dogs from heartworm disease for a
period of 12 months and is identical to the commercial product in
Australia. A safety and field clinical program has been conducted. These
studies demonstrate the same safety profile as ProHeart 6 in reproducing
males and females, heartworm-positive dogs, ivermectin-sensitive
Collies, and a variety of canine breeds during the field evaluation
program.

### Conclusion

The efficacy and safety of Proheart 6 has been thoroughly evaluated
during the initial development program and in subsequent studies
designed to expand label claims for the product. These programs were
designed in close cooperation with the FDA and are comprehensive in
scope. ProHeart 6 has been shown to be efficacious in the protection of
dogs from heartworm disease. It is safe when administered to healthy
dogs and to unique canine populations, such as heartworm-infected or
ivermectin-sensitive dogs.

## Postmarketing Surveillance of Adverse Events

FDAH designed a postmarketing surveillance system to detect a signal of
potential drug effects and subsequently to estimate the incidence and
causality of a potential drug effect. The data used for postmarketing
surveillance generally consisted of adverse event reports (AERs).

To determine if AERs have clinical relevance, incidence is estimated and
comparisons are made with a group representative of the general
population that has not been treated with the drug. FDAH calculates
reporting rates using the number of reports divided by an estimate of
the doses sold to veterinarians for the same period. In contrast,
regulatory authorities generally do not utilize estimates of the
frequency of drug administration. Additionally veterinary
epidemiologists lack an understanding of the rate of occurrence of even
commonly observed medical problems. Thus, it is difficult in assessing
AERs to determine whether a drug is directly related to the observed
events. Even after determining that an adverse event may be likely
related to drug administration, the clinical importance of the reaction
is unknown without an understanding of its incidence. Frequent serious
reactions may warrant withdrawal of a drug from the market, while rare
reactions may require appropriate warnings to prescribers and clients.

Further confounding the interpretation of AERs is the potential for
bias. There is a considerable opportunity for extraneous eventsto
stimulate over-reporting of adverse observations in animals as possible
drug associated adverse events. For ProHeart 6 in particular, there are
additional biases that may have impacted reporting of adverse
observations. Because ProHeart 6 is a new innovative product,
veterinarians lack a frame of experience from which to judge whether or
not adverse observations are likely to be associated with the drug. The
long duration of action makes it appear plausible that adverse
observations might be associated with drug administration that occurred
several months earlier. Further, since ProHeart 6 is administered
parenterally by veterinarians and the monthly oral medicines are
administered by pet owners (often as a treat), there is a potential for
greater apparent concern with ProHeart 6. Also, veterinarians received
Dear Doctor letters discussing ProHeart 6 safety issues that may have
stimulated AERs. Lastly, there were several widely-disseminated news
reports and website postings critical of ProHeart 6 that may have
stimulated AERs.

Thus, while postmarketing surveillance of AERs is a valuable tool in
monitoring drug safety, the use of this system for regulatory decisions
is complicated by the absence of a control group, the lack of an
estimate of use, the lack of knowledge of the background rate of
systemic diseases in the canine population, and the impact of reporting
bias to stimulate over-reporting.[^42]^,^[^43]^,^[^44]

### Overview

In the subsequent sections, FDAH further evaluates AERs (1) relative to
product usage, (2) relative to likelihood that they were associated
temporally with ProHeart 6 administration, and (3) relative to
independent expert assessment of the possible causality of the event as
result of drug administration. On September 3, 2004, FDAH announced the
voluntary recall of ProHeart 6 from the US market until resolution of
FDA safety concerns. The primary concerns cited by FDA were the large
number of AERs that were attributed to ProHeart 6 and that the numbers
of these AERs were increasing in the months leading up to the recall.
Additionally, the FDA expressed concerns regarding symptoms associated
with several different body systems. These included neurologic,
hematologic, hepatic, and cardiac systems, as well as a concern
regarding neoplasia in association with ProHeart 6 administration.

FDA's website cites the submission of "more than 5500 AERs in compliance
with federal regulations (21 CFR 514.80) that require sponsors to submit
serious and unexpected AERs within 15 working days of first receiving
the information." FDAH respectfully disagrees that all of these reports
are medically serious or unexpected, or attributable to ProHeart 6. AERs
defined as serious and unexpected are submitted to FDA within 15 working
days. Those reports defined as not serious or not unexpected are
normally submitted yearly in the annual Drug Experience Report. However,
because of FDA's continuing concerns, FDAH began submitting all reports
in a 15-day window in the third quarter of 2003, including those that
were not serious and unexpected. Additionally, all events reported to
FDAH were relayed to FDA in an unfiltered manner, regardless of
causality assessment. Many AERs contain an assessment by the reporting
veterinarians that their suspicion of product involvement in the event
is low. This is particularly true for reports that were not deemed
allergic events. FDAH has also assessed many of these events as having a
low causal relationship. Thus, FDAH considers most of the AERs to be
unrelated to ProHeart treatment.

Approximately 18 million doses of ProHeart 6 have been sold with more
than 12 million doses administered. The calculated reporting rate is 2.5
reports per 10,000 doses, based on doses sold to veterinary clinics
through August of 2004. This is the equivalent of one report (without
regard to causality) per 4000 doses sold. It is recognized in a
voluntary reporting system that not all potential adverse events
associated with product use are reported. However, the trends over time
need to be considered and indicate that the rate of AERs for ProHeart 6
was low and declined over time.

Another critical element is the background rate of disease in the canine
population at large. The concerns regarding specific body systems are
addressed in detail in section 4.2.2.2. Several independent experts were
consulted and their conclusions, which are included for consideration,
strongly support the safety of ProHeart 6 and indicate that most of the
AERs were not causally-related to ProHeart 6 treatment.

One should consider the system used by FDA to assign causality to each
AER. The six point causality assessment system considers previous
experience with the drug, whether alternative etiologic candidates
exist, timing of the event, overdose, and dechallenge and rechallenge.
The system was primarily established for immediate release products.
ProHeart 6 is a 6-month sustained-release product. Sustained-release
products have not previously been evaluated using this system.
Dechallenge (drug removal from the patient's body) is not possible for
at least 6 months and FDA has acknowledged that the "challenge" and
"dechallenge" components of their scoring system are not applicable in
this instance. The modified scoring procedures to account for this fact
may have created unintended bias among their reviewers.

### Review of Adverse Event Reports

Upon receipt of an AER, the reporting party is asked a series of
questions by FDAH professionals, including patient description and
identification, previous history, date of product administration, its
dose, body location of administration, time to observation of the
adverse event, concurrent treatments employed, diagnostic testing, and
event outcome. Regardless of causality assessment by FDAH, every AER
involving an FDA regulated product received by FDAH is submitted to the
FDA.

To assist in the analysis of AERs, FDAH developed an AER categorization
system. Each AER is assigned into one or more of the following
categories:

1.  Injection site reports

2.  Allergic reports (signs consistent with an allergic response in the
    species involved, occurring less than 48 hours after product
    administration)

3.  Non-allergy systemic reports

To calculate reporting rates for evaluation of the incidence of events,
the number of reports received is divided by the number of doses sold to
veterinary clinics in the same period. The total number of vials of
product sold is converted to an estimate of doses using an average
number of doses per vial. Based on interviews with a large number of
veterinarians, FDAH determined that the average number of doses per vial
was 21. This dosage is equivalent to an 18 kg dog which is consistent
with the average weight of the population at large.

There is a significant seasonality to the pattern of use of all
heartworm preventative products. Figure 4.2.2-1 illustrates this
seasonal pattern for doses of heartworm preventatives dispensed by
veterinarians in the course of a typical year. This pattern is important
to understand, when analyzing report numbers and reporting rates, as
discussed below.

# Figure 4.2.2-1. Seasonality of Dispensing Heartworm Medication 2002 (All Products) {#figure-4.2.2-1.-seasonality-of-dispensing-heartworm-medication-2002-all-products .Caption-Figure}

![](media/image2.wmf){width="5.0in" height="2.8125in"}

Figure 4.2.2-1 demonstrates the strong seasonal use patterns of
heartworm preventative products in the US. The risk (and heightened
awareness) of heartworm infection increases as mosquito populations
either reappear after a typical northern winter, or crescendo after the
more mild winters in southern areas of the US. Therefore, there is a
large peak in use of all heartworm preventatives in April and May of the
year.

Figure 4.2.2-2 demonstrates the sales patterns of ProHeart 6. Sales are
increasing over the period. Sales peaks also occur in each year in
essentially the same periods of time as the use patterns of heartworm
products demonstrated in Figure 4.2.2.2-1.

#  {#section .Caption-Figure}

# Figure 4.2.2-2. Doses Sold by Quarter {#figure-4.2.2-2.-doses-sold-by-quarter .Caption-Figure}

![](media/image3.wmf){width="5.0in" height="3.217361111111111in"}

Figure 4.2.2-3 demonstrates the number of AERs received by FDAH
associated with ProHeart 6. The number of AERs from launch through
August of 2004 is generally decreasing. The peaks in the number of AERs
in the second quarter of each marketing year correspond to the peaks in
seasonal use and sales previously described. These peaks in AER numbers
decrease in each marketing year despite large increases in sales of
ProHeart6 shown in Figure 4.2.2-2. This seasonality in use, in
conjunction with the increasing sales trends marketing dynamics of
ProHeart 6, results in patterns of AER numbers that are difficult to
interpret without proper perspective on the number of doses
administered. Therefore reporting rates are calculated using report
numbers found in Figure 4.2.2.-3 divided by sales data found in Figure
4.2.2.-3 to create Figure 4.2.2-4.

# Figure 4.2.2-3. Number of Adverse Event Reports by Quarter {#figure-4.2.2-3.-number-of-adverse-event-reports-by-quarter .Caption-Figure}

![](media/image4.wmf){width="5.5in" height="2.4256944444444444in"}

#  {#section-1 .Caption-Figure}

# Figure 4.2.2-4. Reporting Rate by Quarter {#figure-4.2.2-4.-reporting-rate-by-quarter .Caption-Figure}

![](media/image5.wmf){width="5.502777777777778in"
height="2.6694444444444443in"}

The reporting rate shown in Figure 4.2.2-4 puts these trends into
perspective. Marketing year two produced higher reporting rates, after
which a more steady reporting rate was observed. Higher AERs were
recorded in the 2nd quarter of 2002 (refer to Figure 4.2.2-3), but
tapered off considerably following the August 2002 implementation of the
manufacturing change of zero tolerance for residual solvents in the
moxidectin used to manufacture ProHeart 6 (see Section 4.3). In the 2nd
quarter of 2002, reporting rates peak not only due to an increased
number of reports but also due to the return of large amounts of product
in 2002, as a result of outdated product sold in 2001.

Evaluation by marketing year, which eliminates reporting rate
fluctuations due to seasonality of use, reveals that the average
reporting rate for AERs is less than 4 reports per 10,000 doses sold per
year. By this same method of analysis, reports that include the death of
the patient (regardless of causality) are fewer than 0.5 reports per
10,000 doses sold per year. Additionally, there is no peak in the number
of death reports during the peaks in AERs seen in the 2nd quarter of
each year. Further, the adverse event case fatality rate associated with
ProHeart 6 reports is lower than many FDAH pharmaceuticals and similar
to case fatality rates for the FDAH canine vaccine product lines
including Duramune Max 5/4L. Thus the incidence of death does not appear
to be causally related to ProHeart 6 usage.

Most AERs occurrred within 10 days of product administration. Overall,
72% of the AERs occurred within 48 hours of administration, (17%) were
reported from 2 days to 10 days post‑administration, and 10% occurred at
11 days or later. This information suggests that a significant
percentage of reports occur in a short time frame, consistent with a
possible allergic etiology from administration of ProHeart 6 alone or in
combination with vaccines or other medication.

When the numbers of AERs were evaluated with consideration of the
seasonality of use and the increases in the numbers of doses sold over
time, the rate of reporting associated with ProHeart 6 was low and was
decreasing with time. In addition, the seriousness of reports as
estimated by case fatality rates was not increasing and was consistent
with commonly used pharmaceutical and biological products. Thus, FDAH
concludes that AERs were rare and the product should remain available as
an alternative for prevention of canine heartworm disease.

#### Analysis by Report Category

##### Injection Site Reports

The reporting rate for injection site reports through 31 August 2004 was
0.2 reports per 10,000 doses sold. The majority of the events were
self-limiting and consisted of swelling, pain and/or pruritus at the
site of administration.

The occurrence of injection site reports after the use of ProHeart 6 has
remained consistently low and the types of events reported were similar
to other injectable products in the FDAH database. For comparison, 2 of
the most widely used vaccines in the FDAH line have reporting rates of
0.1 reports per 10, 000 doses sold (Duramune Max 5/4L, a
distemper/parvo/leptospirosis combination vaccine) and 0.5 reports per
10,000 doses sold (Rabvac 3, a rabies vaccine).

##### Allergy Event Reports

The allergy report category has trended down over time. The reporting
rate through 31 August 2004 was 1.26 reports per 10,000 doses sold. The
occurrence of allergy events was most prevalent in the most popular
breeds, supporting the conclusion that there are no specific breed
sensitivities. Thirty seven percent (37%) of the reports include
concurrent vaccine administration, which likely contributed to the
reporting rate. For the vaccines previously cited, Duramune Max 5/4L has
an allergy reporting rate of 0.4 reports per 10,000 doses sold and
Rabvac 3 has an allergy reporting rate of 0.5 reports per 10,000 doses
sold.

The occurrence of allergy events are described in the product label. The
observations include facial swelling, angioedema, and urticaria, alone
or in combination with gastrointestinal (GI) signs such as vomiting or
diarrhea, anaphylaxis, or a low percentage of less common signs such as
lethargy.

##### Non-Allergy Event Reports

Non-allergy events are those not considered to be an allergic response
and not categorized as injection site. This non-allergy category
includes systemic responses (regardless of the plausibility of product
association) and may overlap with other categories. For example, an
individual event report describing injection site swelling would be
classified as an injection site report; however if fever and myalgia
also occur, it would also be categorized as a non-allergy event.
Similarly, there is an overlap in the symptoms designated as allergy and
non-allergy. Acute GI symptoms occurring within 48 hours of product
administration may be categorized as an allergic event, whereas the same
symptoms occurring at 96 hours after product administration would
typically be categorized as a non-allergy systemic event. Forty five
percent (45%) of reports categorized as non-allergy occurred within 48
hours of product administration. In many cases, some of the clinical
signs reported are consistent with allergy, while other signs vary
enough to result in the event being placed in the non-allergy category.
Therefore, FDAH concludes that a significant number of events placed in
the non-allergy category are actually allergy mediated.

As observed in the allergy group, no specific breeds appear to be
over-represented in the analysis when the population at large is
considered. The overall reporting rate for the non-allergy category
through 31 August 2004 was 1.19 reports per 10,000 doses. Concurrent
vaccine use was reported in 42% of the non allergy reports. Duramune Max
5 /4L has 0.3 reports per 10,000 doses recorded in the non-allergy group
and Rabvac 3 has 0.35 reports per 10,000 doses.

The signs reported in the non-allergy group cover a wide range, with a
large number of the signs being reported in relatively few cases. The
more commonly reported events include vomiting and/or diarrhea,
lethargy, seizures and ataxia. With the exception of ataxia, all of the
above clinical signs are included in the product label.

#### Causality Analysis of Medical Events by Selected Body Systems

Events involving specific body systems were further analyzed to
establish causality. These included events associated with neurologic
reports, hematologic reports, hepatic reports, cardiac reports, and
reports with a diagnosis of neoplasia.

Each ProHeart 6 case was individually reviewed to assess the likelihood
that the observation was the result of administration of ProHeart 6.
Medical association assessments were based on the approved International
Cooperation on Harmonization of Technical Requirements for Registration
of Veterinary Medicinal Products (VICH) pharmacovigilance draft
guidelines. These draft guidelines were developed with regulators from
Europe, Japan, and the US to provide guidance on global
pharmacovigilance harmonization. All events were assigned to the medical
association "possible" category as a starting point. When the causality
was considered "unlikely," sufficient information existed to establish
that a reported event was not likely associated with product use because
there were other more plausible explanations for the reported event. For
the analysis by body system that are presented in the sections that
follow only the medical association "possible" group was reviewed.

This medical association "possible" group was further reviewed to
distinguish between events that could potentially be associated with a
specific body system compared with those events for which it is probable
that the specific body system was involved. Therefore, for each body
system to be analyzed, 2 case definitions were developed: "potential"
and "probable." The "potential" case definition is intended to include
all clinical signs potentially reflective of an adverse effect on that
body system. The "probable" case definition is more refined to include
only those clinical signs that have a reasonable probability of being
related to that body system, and the "probable" excludes all events
classified as allergic. If any individual report included more than 1 of
the body systems evaluated, it was included in multiple evaluations. For
example, if the report involved a dog with anemia and elevated hepatic
enzymes, it was reviewed under both the hematologic and hepatic body
systems.

##### Neurologic

The analysis of neurologic events is hown in Table 4.2.2.2.1-1. The rate
of reporting is low at 0.12 reports per 10,000 doses. The difference
between the number of potential and probable neurologic events is
relatively large because events classified as allergic and events with
non-specific neurologic signs are included in the number of potential
events but not the number of probable events. Non-specific neurologic
signs are clinical signs (eg, muscle tremors) that are seen without
other clinical signs and, therefore, are not likely to include a primary
neurologic disorder.

For neurologic events, seizure and ataxia are the primary clinical signs
while other individual clinical signs each account for less than 10% of
the total in the neurologic group. As is true with each of the systems,
causality assessments are confounded by concurrent vaccine use in 47% of
the AERs. Additionally, 54% of the reported events occurred within 48
hours or less, suggesting that many may be allergic events.

+----------+------------+----------------+-----------------------------+
| # Ta     |            |                |                             |
| ble 4.2. |            |                |                             |
| 2.2.1-1. |            |                |                             |
|  Neurolo |            |                |                             |
| gic Adve |            |                |                             |
| rse Even |            |                |                             |
| ts -- Nu |            |                |                             |
| mber of  |            |                |                             |
| Reports  |            |                |                             |
| {#table- |            |                |                             |
| 4.2.2.2. |            |                |                             |
| 1-1.-neu |            |                |                             |
| rologic- |            |                |                             |
| adverse- |            |                |                             |
| events-n |            |                |                             |
| umber-of |            |                |                             |
| -reports |            |                |                             |
|  .Captio |            |                |                             |
| n-Table} |            |                |                             |
+----------+------------+----------------+-----------------------------+
| **Year** | **P        | **Probable**   | **Rate (Probable) per**     |
|          | otential** |                |                             |
|          |            |                | **10K Doses Sold**          |
+----------+------------+----------------+-----------------------------+
| 2001     | 98         | 23             | 0.05                        |
+----------+------------+----------------+-----------------------------+
| 2002     | 342        | 85             | 0.21                        |
+----------+------------+----------------+-----------------------------+
| 2003     | 235        | 69             | 0.10                        |
+----------+------------+----------------+-----------------------------+
| 2004-Aug | 211        | 68             | 0.12                        |
+----------+------------+----------------+-----------------------------+
| Total    | 886        | 245            | 0.12                        |
+----------+------------+----------------+-----------------------------+

The "probable" neurologic reports were reviewed by Dr. Alexander
deLahunta D.V.M., PhD., diplomate of the American College of Veterinary
Internal Medicine, specialty neurology and honorary member of the
American College of Veterinary Pathologists (Appendix 6.2). FDAH
randomly selected about 75% of the neurologic AERs for his review. Dr.
deLahunta noted that seizures were the most common clinical sign
reported in the cases, which is consistent with the general observations
in the practice of clinical neurology. He also commented that the most
common cause of seizures in dogs is idiopathic epilepsy. Dr. deLahunta
concludes his assessment with the following statement: "The extensive
toxicologic studies performed by FDAH clearly support the safety of
ProHeart 6 at the dose injected in these dogs. There is no rational
scientific reason to believe that this product played any role in the
post-injection clinical signs reported in these case summaries."

##### Hematologic Events

The analysis of hematologic events is shown in Table 4.2.2.2.2-1. The
rate of reporting is low at 0.09 reports per 10,000 doses. As is true
with each of the systems, causality assessments are confounded by
concurrent vaccination in 49% of the AERs.

+-----------+-------------+--------------+----------------------------+
| # Tabl    |             |              |                            |
| e 4.2.2.2 |             |              |                            |
| .2-1. Hem |             |              |                            |
| atologic  |             |              |                            |
| Adverse E |             |              |                            |
| vents --  |             |              |                            |
| Number of |             |              |                            |
|  Reports  |             |              |                            |
| {#table-4 |             |              |                            |
| .2.2.2.2- |             |              |                            |
| 1.-hemato |             |              |                            |
| logic-adv |             |              |                            |
| erse-even |             |              |                            |
| ts-number |             |              |                            |
| -of-repor |             |              |                            |
| ts .Capti |             |              |                            |
| on-Table} |             |              |                            |
+-----------+-------------+--------------+----------------------------+
| **Year**  | **          | **Probable** | **Rate (Probable) per 10K  |
|           | Potential** |              | Doses Sold**               |
+-----------+-------------+--------------+----------------------------+
| 2001      | 75          | 13           | 0.03                       |
+-----------+-------------+--------------+----------------------------+
| 2002      | 212         | 63           | 0.16                       |
+-----------+-------------+--------------+----------------------------+
| 2003      | 172         | 48           | 0.07                       |
+-----------+-------------+--------------+----------------------------+
| 2004-Aug  | 139         | 60           | 0.10                       |
+-----------+-------------+--------------+----------------------------+
| Total     | 598         | 184          | 0.09                       |
+-----------+-------------+--------------+----------------------------+

FDAH consulted with Dr. Alan Rebar, a diplomate of the American College
of Veterinary Pathologists with a specialty in clinical pathology, to
review all potential hematologic events (Appendix 6.3). Dr. Rebar was
provided with clinical case summaries and all available clinical
laboratory data for all the clinical cases identified by FDAH criteria
as probable hematologic or hepatic adverse events to ProHeart 6. (The
review of the hepatic events follows in Section 4.2.2.2.3.)

In contrast to FDAH's assessment (Table 4.2.2.2.2-1), Dr. Rebar
determined that 151 of the AERs he reviewed should be categorized as
probable hematologic AERs. Particular emphasis was placed on potential
toxic-induced hematologic events: immune mediated hemolysis (IMHA),
immune mediated thrombocytopenia (ITP), Heinz body hemolysis, and bone
marrow hypoplasia /aplasia (IMMD). Of these, only IMHA, ITP and/or IMMD
were identified among the 151 hematologic reports.

Dr. Rebar also evaluated the 151 probable hematologic events to
determine which may have been immune mediated. Using a conservative case
definition he determined that 79 could be immune mediated. In many of
the 79 cases, however, not enough data were provided to establish a
definitive diagnosis of IMHA, ITP or IMMD. For example, in many cases,
blood films were not evaluated for the presence of spherocytes, direct
antiglobulin tests (DAT, Coombs' tests) were not run, and blood films
were not scanned to confirm an instrument reported thrombocytopenia.

Of the 79 cases determined to be potentially immune mediated, 76 cases
were determined to be IMHA/ITP. Of these, 53 cases occurred after the
first injection of ProHeart 6, and 26 occurred after 2 or more
injections. Of the 53 cases occurring following a single ProHeart 6
injection, only 23 (those occurring 8 to 30 days post-injection) were
considered possibly related to ProHeart 6. Fourteen (14) occurred too
soon following ProHeart 6 injection (0 to 7 days) to have allowed
sufficient antibody to form against ProHeart 6 to precipitate an
immune-mediated hematologic event. Thirteen (13) occurred after 30 days
or more, too long an interval to suggest a likely role for ProHeart 6
injection.

Of the remaining 23 cases, it is not possible to rule out a role for
ProHeart 6, although IMHA and ITP are relatively common disorders in all
breeds of dogs.

Of the 26 cases occurring following multiple injections of ProHeart 6,
twenty two (22) (those occurring between 0 and 30 days post ProHeart 6
injection) were considered possibly related to ProHeart 6. The 10 cases
occurring within 7 days post-injection are relatively unlikely to be
related to ProHeart 6 injection but are included because of potential
anamnestic immune response to multiple injections.

Three (3) cases of apparent IMMD were identified. The cause of the IMMD
in each case could not be determined from the diagnostic data available.
Two (2) cases occurred after the first dose of ProHeart 6; the first
occurred at 7 days post administration and the second occurred at more
than 30 days post administration. The third case occurred more than 30
days after the dog had received its fourth dose of ProHeart 6. The
timing of all 3 events indicates that they were unlikely to be related
to ProHeart 6 administration.

In summary, 45 cases of hematologic AERs may have been induced by
ProHeart 6. However, since more than 18 million doses of ProHeart 6 have
been sold it is possible that these findings represent the normal
baseline incidence in canines.

##### Hepatic Body System

The analysis of hepatic events is shown in Table 4.2.2.2.3-1. The rate
of reporting is low at 0.07 reports per 10,000 does sold. As is true
with each body system, causality assessments are confounded by
concurrent vaccine use in 38% of the AERs.

+----------+------------+-------------+-------------------------------+
| # Table  |            |             |                               |
| 4.2.2.2. |            |             |                               |
| 3-1. Hep |            |             |                               |
| atic Adv |            |             |                               |
| erse Eve |            |             |                               |
| nts -- N |            |             |                               |
| umbers o |            |             |                               |
| f Report |            |             |                               |
| s {#tabl |            |             |                               |
| e-4.2.2. |            |             |                               |
| 2.3-1.-h |            |             |                               |
| epatic-a |            |             |                               |
| dverse-e |            |             |                               |
| vents-nu |            |             |                               |
| mbers-of |            |             |                               |
| -reports |            |             |                               |
|  .Captio |            |             |                               |
| n-Table} |            |             |                               |
+----------+------------+-------------+-------------------------------+
| **Year** | **P        | *           | **Rate (Probable) per**       |
|          | otential** | *Probable** |                               |
|          |            |             | **10K Doses Sold**            |
+----------+------------+-------------+-------------------------------+
| 2001     | 36         | 12          | 0.03                          |
+----------+------------+-------------+-------------------------------+
| 2002     | 125        | 48          | 0.12                          |
+----------+------------+-------------+-------------------------------+
| 2003     | 109        | 49          | 0.07                          |
+----------+------------+-------------+-------------------------------+
| 2004-Aug | 118        | 47          | 0.08                          |
+----------+------------+-------------+-------------------------------+
| Total    | 388        | 156         | 0.07                          |
+----------+------------+-------------+-------------------------------+

One hundred (100) probable hepatic AERs were reviewed by Dr. Rebar. This
number of 100 probable hepatic reports is different from the total
probable hepatic reports in Table 4.2.2.2.3.-1 because Dr. Rebar removed
56 reports that he determined were not probable hepatic events.
Forty-three (43) cases of possible primary liver disease were identified
among the 100 AERs evaluated. Fifty-seven (57) cases were probably not
primary hepatic events, and the mild non-specific elevations in hepatic
enzyme(s) were thought to be due to anorexia and/or stress.

Of the remaining 43 cases, 42 occurred between 0 and 3 days post-
injection; again, however, a causal relationship with ProHeart 6 cannot
be ruled out. However, since more than 18 million doses of ProHeart 6
have been sold it is possible that these findings represent the normal
baseline in the canine population.

Pathology reports from all the probable hepatic AERs that contain any
pathology reports (15 cases) were reviewed by Dr. Keith Harris D.V.M.,
Assistant Vice President Pathology and Bioresources, Wyeth Research and
diplomate of the American College Veterinary Pathologists. There was no
pattern of liver pathology indicative of a common toxicological agent in
all or a subset of the 15 cases (other than corticosteroids in two
cases). Only one case out of the 15 exhibited histomorphologic changes
consistent with a direct acting hepatotoxin that could be temporally
related to ProHeart 6 administration. Pathology reports from 15 cases
with liver disease were reviewed. There was no obvious pattern of liver
pathology that would indicate toxicity common to all or a subset of the
15 cases (other than corticosteroids in two cases). Only one case out of
the 15 exhibited histomorphologic changes consistent with a direct
acting hepatotoxin that could be temporally related to ProHeart 6
administration. The acute hepatocellular necrosis described in this
particular case is a non-specific finding and could have been caused by
a number of different toxicants.

##### Cardiac Events

The analysis of cardiac events is seen in Table 4.2.2.2.4-1. The
reporting rate is low at 0.02 reports per 10,000 doses.

+----------+------------+-------------+------------------------------+
| # Table  |            |             |                              |
| 4.2.2.2. |            |             |                              |
| 4-1. Car |            |             |                              |
| diac Adv |            |             |                              |
| erse Eve |            |             |                              |
| nts -- N |            |             |                              |
| umbers o |            |             |                              |
| f Report |            |             |                              |
| s {#tabl |            |             |                              |
| e-4.2.2. |            |             |                              |
| 2.4-1.-c |            |             |                              |
| ardiac-a |            |             |                              |
| dverse-e |            |             |                              |
| vents-nu |            |             |                              |
| mbers-of |            |             |                              |
| -reports |            |             |                              |
|  .Captio |            |             |                              |
| n-Table} |            |             |                              |
+----------+------------+-------------+------------------------------+
| **Year** | **P        | *           | **Rate (Probable) per**      |
|          | otential** | *Probable** |                              |
|          |            |             | **10K Doses Sold**           |
+----------+------------+-------------+------------------------------+
| 2001     | 100        | 7           | 0.02                         |
+----------+------------+-------------+------------------------------+
| 2002     | 254        | 15          | 0.04                         |
+----------+------------+-------------+------------------------------+
| 2003     | 186        | 10          | 0.02                         |
+----------+------------+-------------+------------------------------+
| 2004-Aug | 153        | 15          | 0.02                         |
+----------+------------+-------------+------------------------------+
| Total    | 693        | 47          | 0.02                         |
+----------+------------+-------------+------------------------------+

Concurrent vaccination confounds the assessment of the cardiac events.
Forty-nine percent (49%) of the cases classified as cardiac events
included concurrent vaccination. Sixty percent (60%) of the reports
occurred within 48 hours of ProHeart 6 administration. The timing of
these reports is not indicative of a direct cardiac effect. Many events
appear to overlap as non-typical allergic events, as observed for the
other body systems, so that an indirect effect associated with an
allergic event cannot be ruled out.

Dr. Keith Harris DVM, Assistant Vice President Pathology and
Bioresources Wyeth Research, and diplomate of the American College of
Veterinary Pathologists, reviewed the histopathological slides from 2
cases of cardiac necrosis thought to be associated with ProHeart 6
administration. Histopathologic evaluation of tissue specimens from the
2 dogs identified in an FDA presentation revealed cardiac pathology
secondary to uremia in a Boxer dog and chronic heart disease that
clearly predated ProHeart 6 administration in a Labrador Reteiever. The
two IDEXX pathologists who had originally indicated that these cases
might be associated with ProHeart 6 subsequently concurred with this
interpretation.

Based on these data, including timing of the events and
histopathological assessments, FDAH concludes that there is no causal
relationship between ProHeart 6 and these cardiac events.

##### Neoplasia Events

The analysis of neoplasia cases is shown in Table 4.2.2.2.5-1. The rate
of reporting is low at 0.06 reports per 10,000 doses.

+------------+--------------------+-----------------------------------+
| # Table    |                    |                                   |
| 4.2.2.2.5- |                    |                                   |
| 1. Neoplas |                    |                                   |
| ia Adverse |                    |                                   |
|  Events -- |                    |                                   |
|  Numbers o |                    |                                   |
| f Reports  |                    |                                   |
| {#table-4. |                    |                                   |
| 2.2.2.5-1. |                    |                                   |
| -neoplasia |                    |                                   |
| -adverse-e |                    |                                   |
| vents-numb |                    |                                   |
| ers-of-rep |                    |                                   |
| orts .Capt |                    |                                   |
| ion-Table} |                    |                                   |
+------------+--------------------+-----------------------------------+
| **Year**   | **Potential**      | **Rate per 10K Doses Sold**       |
+------------+--------------------+-----------------------------------+
| 2001       | 7                  | 0.01                              |
+------------+--------------------+-----------------------------------+
| 2002       | 38                 | 0.09                              |
+------------+--------------------+-----------------------------------+
| 2003       | 41                 | 0.06                              |
+------------+--------------------+-----------------------------------+
| 2004-Aug   | 36                 | 0.06                              |
+------------+--------------------+-----------------------------------+
| Total      | 130                | 0.06                              |
+------------+--------------------+-----------------------------------+

Dr Philip Bergman, D.V.M, MS, PhD, diplomate American College of
Veterinary Internal Medicine, Oncology (Appendix 6.4) reviewed all 130
cases involving a diagnosis of neoplasia, regardless of medical
association. A summary of Dr. Bergman's comments follows.

The vast majority of ProHeart 6-related cancers occurred within 21 days
or less of ProHeart 6 administration. Veterinary oncologists agree that
at least 6 to 8 weeks are required to develop a tumor after exposure to
a carcinogen. Therefore, cases that were diagnosed with cancer within a
3 to 4 week period after ProHeart 6 administration likely had a tumor
present before ProHeart 6 administration. There was not a relationship
between additional doses of ProHeart 6 administered and increasing
reports of neoplasia. This lack of a dose-response relationship with
repeated ProHeart 6 administration further supports the conclusion that
ProHeart 6 is not responsible for canine tumor induction. This is
further supported by the moxidectin nonclinical toxicology studies
showing no evidence of carcinogenicity (see Section 3.3.3 and Appendix
6.1).

Dr. Bergman's review of the 130 cases revealed that 100 cases were
clearly not associated with ProHeart 6 administration. Twenty eight (28)
cases did not allow the immediate exclusion of an association to
ProHeart 6 administration. However the association was deemed unlikely
due to 1 or more of the following factors: 1) a time period from product
administration to observation of the neoplasia was too short to be
considered a causal relationship; 2) most of the reports revealed no
association between the site of administration and the location of the
neoplasia; 3) there was a broad cross section of neoplasia types, rather
than a predilection for a specific tumor type, as would be expected in a
product-specific effect; and 4), the types were representative of those
seen in the general dog population. There were 2 cases in which a
relationship to ProHeart 6 administration could not be ruled out.

### Conclusions

Based on the in-depth analysis described in this summary section, it is
the conclusion of FDAH that ProHeart 6 is a safe and effective product
for the prevention of canine heartworm disease. The overall reporting
rate for AERs was low and generally trended down over time to the time
of the recall. At that time, the frequency and severity of AERs was not
increasing. Analysis of specific body and organ systems support the
conclusion that the AER clinical finding were generally not causally
related to ProHeart 6 administration and appeared to occur at a rate
consistent with naturally occurring disease in the canine population.

## Manufacturing Change

ProHeart 6 was launched in the US in June 2001. Shortly after launch,
FDAH received a number of reports of allergic-type reactions after
administration. The reactions reported ranged from mild and
self-limiting (eg, urticaria, itching at injection site, an episode of
vomiting or diarrhea) to severe anaphylactoid reactions.

A working group of FDAH and Wyeth immunology experts was convened to
investigate the possible causes, and outside experts were retained to
assist. There were 2 aspects of the investigations: (1) field reports
and their follow-up, and (2) product analysis for any abnormalities,
contaminants, and/or reactive components.

A "cluster effect" was observed where some practices reported several
adverse events, while others in close proximity with usage patterns of
ProHeart 6 did not report reactions. This was observed in both the US
and Australia. Visits were made to practices to evaluate product
storage, handling, and administration, and veterinarians and dog owners
were interviewed to try to identify any predisposing factors. No age or
breed predispositions were found, nor any interactions with concurrent
treatment with other veterinary therapeutics or biologicals.

Extensive investigations into reactor dogs (dogs which showed allergic
reactions) found no evidence that the allergies were IgE (immunoglobulin
E) or IgG (immunoglobulin G) mediated (L. Gershwin, Department of
Pathology, Microbiology and Immunology, School of Veterinary Medicine,
University of California, Davis, and R. Schultz, Professor and Chair,
School of Veterinary Medicine, Department of Pathological Sciences,
University of Wisconsin, Madison \[see Appendix 6.5\]). The reactions
appeared to be idiosyncratic and mediated either by histamine or
complement.[^45]^,^[^46]^,^[^47] Attempts to induce reactions in dogs
using passive cutaneous allergy testing after sensitization with serum
from reactor dogs were unsuccessful.[^48]^,^[^49]

Extensive testing of all raw materials and formulated products used in
manufacture of ProHeart 6 showed that all were within quality
specifications. Tests for 16 trace metals did not reveal any
contamination. Quality Assurance audits were conducted at the
manufacturing sites of glyceryl tristearate (Germany) and hydroxypropyl
methylcellulose (US) to evaluate the potential for cross contamination
during or after manufacturing. No potential problems were found.
Cleanout validation was repeated at the formulation plant to confirm
that no carryover from other manufacturing processes could
occur.[^50]^,^[^51]^,^[^52]

Both in the US and Australia, differences were found in reaction rates
between batches, so investigations focused on trying to identify any
batch differences. Thin layer chromatography (TLC) analyses detected no
unknown components either in moxidectin technical material or in
finished product. However, evaluation of minor component profiles
between batches revealed a trend to lower reactions for lots with no
detectable residual solvents. Coincident with this investigation, FDAH
was optimizing the manufacturing process and initiated a manufacturing
change to produce moxidectin with no detectable solvents.

Although additional studies were done to better understand the cause of
the allergic-type reactions, no conclusive findings were generated.
Nevertheless, there has been a decline in the adverse event reporting
rate from all markets since the manufacturing change was implemented.

## Safety Profile in Dogs

In August of 2004, FDAH requested a review of the safety of ProHeart 6
use in general veterinary practice and a comparison of its safety
profile with that of commonly used oral monthly heartworm drugs by Dr.
Larry Glickman VMD, DrPh, FACE, Professor of Epidemiology and Public
Health and Head, Section of Clinical Epidemiology, School of Veterinary
Medicine, Purdue University (Appendix 6.6). This section summarizes the
experience with ProHeart 6 by Banfield the Pet Hospital™ veterinarians
nationwide who had administered 735,654 doses of ProHeart 6 to dogs from
January 1, 2002 through August 31, 2004.

### Methods

The source of data for this analysis was the medical records of Banfield
the Pet Hospital™. Banfield was founded in 1955 in Oregon to deliver
primary health care to companion animals, and by 2005 will operate a
national network of 440 full-service veterinary hospitals in 42 states.
Banfield practices employ more than 900 full and part-time
veterinarians, have over 1.4 million active patients, and conduct
approximately 50,000 patient visits per week. The Banfield database is
paperless and contains over 8 million patient records in electronic
format. A quality assurance team consisting of veterinarians and
veterinary technicians regularly monitors the safety profile of all
medications, vaccines, and procedures used by Banfield veterinarians and
tracks the incidence of diseases that are preventable by these vaccines
or drugs.

All encounters (office visits) were characterized as being associated
with ProHeart 6, 2 oral monthly heartworm preventative drugs (Heartworm
Preventative 1 or Heartworm Preventative 2), vaccine, or none of these
exposures. Adverse events (AEs) of interest included liver disease,
neurological disease, ocular disease, immune-mediated disease, allergic
reaction, death, cancer (mast cell, lymphosarcoma, and histiocytoma),
cardiovascular disease, anaphylaxis, or inflammatory bowel disease. The
specific diseases or laboratory findings comprising each of these AEs
based on Banfield computer codes are shown in Table 4.4.1-1. Each
encounter was then evaluated for potential AEs over the following 30
days. Of these encounters, 275,189 that occurred from August 1 to August
31, 2004, were excluded from analysis because they lacked a full 30-day
followup interval. This followup period was terminated early if a dog
had another exposure to ProHeart 6, either of the 2 monthly heartworm
preventatives, a vaccine, or died. Except in the case of death, a new
followup period was initiated after this new exposure. The incidence of
AERs, expressed as either the number of AEs per 10,000 encounters or the
number of AE per 10,000 days at risk following an exposure, were
calculated for the following exposure groups: no exposure, vaccine
alone, ProHeart 6 with or without vaccine, and Heartworm Preventative 1
or Heartworm Preventative 2 with or without vaccine. Formal testing to
identify statistically significant differences in the AE rates between
exposure groups was generally not done for the univariate analyses due
to the very large sample size within each exposure group. That is, the
power to detect statistically significant differences was so high for
common events that even very small differences in event rates were
likely to be statistically different at p \< 0.001. However, the same
was not necessarily true for less common events. For this reason, the
focus was placed on the clinical relevance of differences in the AE
rates and the 95% confidence intervals of the odds ratios in
multivariate analysis.

# Table 4.4.1-1. Adverse Events {#table-4.4.1-1.-adverse-events .Caption-Table}

![](media/image6.wmf){width="6.250694444444444in"
height="4.170833333333333in"}

For the purposes of this study, it was assumed that oral monthly
heartworm preventatives had been administered by owners on the same day
of the encounter in which they had been dispensed. In contrast, ProHeart
6 and vaccines were assumed to have been administered during the same
office encounter indicated in the medical record. Also, a search for
potential AEs was limited to the 30 days immediately following an
encounter of interest. The potential consequences of these assumptions
would be to underestimate the incidence of AEs associated with oral
monthly heartworm drugs, compared with those associated with ProHeart 6
or vaccines, because it was not certain if, or when, the monthly
heartworm preventative dispensed for a dog was actually given to the dog
by its owner. That is, some of the oral heartworm preventatives
dispensed by Banfield veterinarians were probably never given to the
dogs, yet AEs were calculated as if these drugs had been administered.

### Results

From January 1, 2002 to August 31, 2004, there were 6,800,061 dog visits
or encounters at 403 Banfield hospitals. The following number of
encounters was associated with the administration of: ProHeart 6
(735,654), Heartworm Preventative 1 (411,082), Heartworm Preventative 2
(18,405), or vaccine (2,230,202), (Table 4.4.2-1). In addition, there
were 5,634,016 encounters during which no heartworm preventative was
administered or dispensed. Heartworm preventatives were often
administered or dispensed at the same time as a vaccine. The proportion
of encounters associated with vaccination was 62.9% for Proheart 6,
59.9% for Heartworm Preventative 1, and 65.1% for Heartworm Preventative
2. In contrast, vaccines were only administered during 26.4% of the
encounters for which no heartworm preventative was given. That is, these
dogs may have presented with signs of disease for which diagnostic tests
were performed and other drugs administered. The number of doses of
ProHeart 6 administered monthly by Banfield veterinarians increased over
time since January 2002 and a higher number of doses was administered
each year during the peak time of mosquito activity, namely from March
through September (Figure 4.4.2-1).

# Table 4.4.2-1. Rate per 10,000 of Any Adverse Event by Treatment Category Total Number of Encounters = 6,800,061 {#table-4.4.2-1.-rate-per-10000-of-any-adverse-event-by-treatment-category-total-number-of-encounters-6800061 .Caption-Table}

![](media/image7.wmf){width="5.991666666666666in" height="1.90625in"}

# Figure 4.4.2-1. Doses Dispensed by Month; ProHeart 6 Only or With Vaccine {#figure-4.4.2-1.-doses-dispensed-by-month-proheart-6-only-or-with-vaccine .Caption-Figure}

![](media/image8.png){width="5.999305555555556in"
height="3.7270833333333333in"}

#### Univariate Analyses

ProHeart 6 was associated with a higher rate of liver-related AE
compared with either of the 2 monthly heartworm preventatives,
regardless of whether a vaccine was administered or not, while Heartworm
Preventative 1 was associated with the highest death rate (Table
4.4.2.1-1). In general, vaccine administration was associated with an
increased rate of liver disease for dogs receiving ProHeart 6, but not
for dogs on monthly heartworm preventative. The rates of all other
diseases or conditions except for cancer were similar among all of the
heartworm preventatives, whether given orally or parenterally.

# Table 4.4.2.1-1. Adverse Event Rate per 10,000 by Treatment Group {#table-4.4.2.1-1.-adverse-event-rate-per-10000-by-treatment-group .Caption-Table}

![](media/image9.wmf){width="6.5in" height="4.276388888888889in"}

The rate of potential liver-related AEs was examined further on the
basis of a clinical diagnosis only, an increase in the serum
concentration of a liver-associated enzyme or bilirubin, or any
combination of a clinical diagnosis plus an abnormal laboratory finding
(Table 4.4.2.1-2). ProHeart 6 when administered either with or without a
vaccine, was associated with a higher rate of any liver-related clinical
diagnosis, enzyme ALT, bilirubin, or a combination of a clinical
diagnosis plus any abnormal laboratory test result (Table 4.4.2.1-3).
However, in an analysis based on the rate of potential AEs per 10,000
days at risk, the rate of liver disease was comparable between ProHeart
6 and the 2 monthly heartworm preventatives, while the rate was highest
for dogs that received no heartworm drug. It should be noted that the
mean days at risk per encounter for ProHeart 6 was 29.2 compared with
27.2 for Heartworm Preventative 1. This could lead to a slight
underestimation of the AE rate associated with Heartworm Preventative 1
compared with ProHeart 6. The liver-related AEs appeared to occur with
similar frequency during days 0 to 2, 3 to14, and 15 to 30, following
the exposures of interest.

# Table 4.4.2.1-2. Liver Adverse Events Definitions Based on Lab Measure or Lab Abnormal Flag {#table-4.4.2.1-2.-liver-adverse-events-definitions-based-on-lab-measure-or-lab-abnormal-flag .Caption-Table}

![](media/image10.wmf){width="5.996527777777778in"
height="2.040277777777778in"}

# Table 4.4.2.1-3. Potential Liver-related Adverse Events per 10,000 by Treatment Group  {#table-4.4.2.1-3.-potential-liver-related-adverse-events-per-10000-by-treatment-group .Caption-Table}

![](media/image11.wmf){width="6.502083333333333in"
height="1.9590277777777778in"}

Vaccine administration was associated with a markedly increased rate of
allergic reactions for dogs on ProHeart 6 as well as for dogs on monthly
heartworm preventative

(Table 4.4.2.1-1). The rate of allergic reactions per 10,000 days at
risk was similarly increased for vaccinated dogs compared with dogs
receiving any one of the heartworm preventative drugs or those receiving
no heartworm drug. Unlike liver-related AEs, allergic reactions occurred
more commonly during days 0 to 2 following an encounter of interest.

There was no apparent association of immune-mediated or cardiovascular
events with any of the heartworm preventatives or with vaccines, and the
rate of these AEs was relatively low (Table 4.4.2.1-1).

The rate of mast-cell tumor, lymphosarcoma, or histiocytoma was
generally \< 3 AEs per 10,000 encounters (Table 4.4.2.1-4). ProHeart 6,
whether administered alone or with a vaccine, was associated with a
slight (\~ 2 per 10,000 encounters), but higher rate of mast-cell tumors
compared with administration of vaccines alone or any of the monthly
heartworm preventatives. The rate of mast cell tumors per 10,000 days at
risk was similarly elevated in dogs receiving ProHeart 6 compared with
those receiving a vaccine or any one of the monthly heartworm
preventative drugs. However, compared with dogs that had received 5
doses of ProHeart 6, there was no statistically significant difference
in the age-adjusted risk of developing mast cell tumor for dogs that had
received 1, 2, 3, or 4 doses of ProHeart 6 from Banfield veterinarians.
That is, all of the odds ratios included 1.0. There was also no apparent
dose-response relationship between ProHeart 6 dose number and mast cell
tumor risk. However, it is not known if any dogs had previously received
ProHeart 6 from a non-Banfield veterinarian.

# Table 4.4.2.1-4. Potential Cancer-related Adverse Events per 10,000 by Treatment Group {#table-4.4.2.1-4.-potential-cancer-related-adverse-events-per-10000-by-treatment-group .Caption-Table}

![](media/image12.wmf){width="6.415972222222222in"
height="2.2263888888888888in"}

#### Multivariate Analyses

To control for potential confounding effects and to identify
interactions between the variables, multivariate logistic regression
models were constructed that included the following variables: heartworm
preventative, vaccine, age, weight, non-steroidal anti-inflammatory
(NSAID), steroid, and ProHeart 6 dose number. In the liver disease
model, steroid use was associated with a 25% increased risk while
ProHeart 6 was associated with a 15% reduction in risk. Each additional
dose of ProHeart 6 was associated with an 8% reduction in liver disease
risk. There was evidence of a strong interaction (effect modification)
between age and ProHeart 6. Upon further examination of the relationship
between ProHeart 6 dose number and age, the risk of liver disease
increased with age, regardless of the exposure group. Using the best-fit
equation generated from the logistic regression model, the relationship
between the risk of liver disease associated with ProHeart 6 use was
graphed as a function of age (Figure 4.4.2.2-1). ProHeart 6
administration was associated with a decreased risk of liver disease in
dogs \< 4 years of age, whereas there was an increased risk in dogs \> 4
years of age.

# Figure 4.4.2.2-1. Risk of Liver Disease by Age for ProHeart 6 {#figure-4.4.2.2-1.-risk-of-liver-disease-by-age-for-proheart-6 .Caption-Figure}

![](media/image13.wmf){width="5.498611111111111in"
height="3.9069444444444446in"}

> Risk (1yr old) = 0.854\*1.043 = 0.89; Risk(7yr old) =
> 0.853\*1.043\*\*7 = 1.14
>
> Risk (age yrs) = OR(Proheart6) \* OR(Proheart6\*age
> interaction)\*\*age

In the allergic reaction multivariate logistic regression model,
ProHeart 6, Heartworm Preventative 1, vaccines, NSAIDS, and
glucocorticoids, were all associated with an increased risk of allergic
reactions; vaccine had the greatest affect. However, each additional
dose of ProHeart 6 did not further increase the risk of allergic events.

Multivariate models for the risk of cancer indicated that ProHeart 6 was
associated with a modest increase in the risk of mast cell tumor. It is
not clear why NSAIDs were associated with a 423% increase in the risk of
mast-cell tumor. Glucocorticoids were associated with a 182% increased
risk of lymphosarcoma, probably because glucocorticoids are used as a
treatment for this cancer. None of the heartworm preventatives were
associated with the risk of histiocytoma.

In the multivariate model for the risk of death, only Heartworm
Preventative 1 was associated with an increased risk (23%), whereas
ProHeart 6 was associated with a 71% reduction in risk of death. There
was no positive relationship between the number of ProHeart 6 doses
administered and risk of death (odds ratio = 0.910).

#### Temporal Trends in Event Rates

The rate of cancer, deaths, liver disease, and allergic reactions, were
evaluated by quarter of the year for ProHeart 6 and Heartworm
Preventative 1, both with and without vaccines, to determine if AEs
associated with ProHeart 6 increased during the 3^rd^ quarter of 2004,
as suggested by the FDA. No evidence was found to suggest that AE rates
associated with either ProHeart 6 or Heartworm Preventative 1, when used
without a vaccine, increased in the 3^rd^ quarter of 2004. In contrast,
there was an increase in the 3^rd^ quarter of 2004 in the rate of
allergic events associated with either ProHeart 6 or Heartworm
Preventative 1 when administered with a vaccine. Therefore, the
increased rate of allergic events may be explained by 1 or more vaccines
new to the market in mid-2004 that were used by Banfield veterinarians
and that caused an increased rate of allergic reactions when
administered with either ProHeart 6 or Heartworm Preventative 1 compared
with administration of either of these heartworm preventatives without a
vaccine.

### Discussion

The results of these analyses involving almost 7 million dog encounters
at Banfield clinics in the US did not reveal any clinically significant
increase attributed to ProHeart 6 use in the risk of liver-related AEs,
neurological disease, ocular disease, immune-mediated disease,
cardiovascular disease, anaphylaxis, inflammatory bowel disease, or
death, when compared with 2 commonly used monthly oral heartworm
preventatives. While ProHeart 6 was associated with an increased rate of
some liver-related AEs in the univariate analyses, this increased rate
was not found in either the days at risk or in the multivariate
analysis. In contrast, ProHeart 6, monthly heartworm preventatives, and
vaccines were all associated with a clinically significant increased
risk of allergic reactions in both univariate and multivariate analyses,
especially during the first few days post-exposure.

The only potential AE studied that was independently associated with an
increased risk following ProHeart 6 use in dogs was mast cell tumor.
However, the absolute magnitude of the risk of mast-cell tumor
associated with ProHeart 6 alone (2.1 events per 10,000 doses
administered) or ProHeart 6 plus vaccine (1.9 events per 10,000 doses
administered) was small. In addition, there was no statistically
significant dose response relationship between the risk of mast cell
tumor and the cumulative number of doses of ProHeart 6 that a dog had
received. Compared with the rate of mast cell tumor in dogs that had
received a vaccine alone (1.2 events per 10,000 doses administered), the
rate of mast cell tumor in dogs that had received ProHeart 6 plus a
vaccine (1.9 events per 10,000 doses administered) does not appear to be
clinically significant. There is no known mechanism that would explain
how ProHeart 6 or any other heartworm preventative induces or promotes
mast cell tumor in dogs, especially within 30 days of its
administration.

Evidence was presented to show that the number of AEs associated with
ProHeart 6 use increased in the 3^rd^ quarter of 2004 compared with
previous quarters. However, this increase primarily involved allergic
reactions, was also observed in dogs that had received monthly oral
heartworm preventatives, and was restricted to dogs that simultaneously
received a vaccine. These findings suggest that any observed increase in
adverse events associated with heartworm preventatives in the 3^rd^
quarter of 2004, were likely due to one or more vaccines that were given
with heartworm preventative drugs. Since nearly two-thirds of the
heartworm preventative drugs studied were administered simultaneously or
in close proximity with a vaccine, and since vaccines are generally
associated with a higher rate of allergic reactions than are heartworm
preventative drugs, AE reports involving administration of ProHeart 6 or
other heartworm preventative drugs must be interpreted cautiously and
take into account a dog's vaccine history.

### Conclusions

In summary, the safety profile of ProHeart 6 appears similar to that of
2 commonly used monthly oral heartworm preventatives. The results of
this study provide no support for the withdrawal of ProHeart 6 from the
veterinary market. Lack of compliance is recognized as a common reason
for lack of efficacy for both human and veterinary drugs. Since the
likelihood of exposure of dogs to *Dirofiliaria immitis* infected
mosquitoes is common during many months of the year, drugs are needed
that prevent heartworm infection for an extended period of time, thus
obviating the need for owners to remember to retreat their dog. The
results of this study indicate the risks associated with ProHeart 6 use
are few and similar to that for monthly oral heartworm preventatives
that only offer protection from heartworm infection for a period of 30
days.

## Field Efficacy

### Overview

While the clinical efficacy of ProHeart 6 has been established in
controlled laboratory and field studies, evaluation of efficacy under
conditions of commercial use after launch can be complicated by many
confounding factors. These include heartworm testing protocols, the age
at which dogs commence a ProHeart 6 program, and timing of change from
other prophylaxis to ProHeart 6.

Products for heartworm prophylaxis have been available in veterinary
medicine for decades, initially as daily oral treatments; later, a range
of monthly oral and topical treatments were introduced with ivermectin,
selamectin, or milbemycin oxime as the active ingredient.Error:
Reference source not
found^,Error:\ Reference\ source\ not\ found,Error:\ Reference\ source\ not\ found^
Some breeds of dogs exhibit toxic signs at doses of ivermectin and/or
milbemycin oxime that are well tolerated in most other breeds, and a
genetic basis for this sensitivity has been identified.Error: Reference
source not found^,^[^53]^,^[^54]^,^[^55]^,^[^56]^,^[^57]^,^[^58]^,^[^59]
Moxidectin can be used safely in these breeds based on FDAH clinical
studies.

### Compliance

A high level of efficacy is found with these therapeutic options in
controlled studies. However, under field conditions, an increasing
incidence of heartworm infections in dogs in the US in the 1990s was
reported with approximately 240,000 dogs testing heartworm positive in
2001. Surveys of dog owners have shown that compliance (ie, reliable
administration of monthly treatments by owners) is problematical.
Despite reminder systems such as calendar stickers, a survey in the US
in 2000 found that \>80% of participants had failed on multiple
occasions to give their dogs the monthly preventative on the indicated
day, and about one-third of participants completely missed the monthly
dosage. Approximately one-fifth of the participants had missed giving
their dogs the monthly oral heartworm preventative and then stopped
altogether.[^60] Another survey conducted in 2001 found that only 55% of
dog-owning households in the US were using heartworm prevention, which
was down from a high of 66% in 1998, despite heartworm having been
diagnosed in all states.[^61] Similarly, Yabsley et al. reported an
increased prevalence of heartworm in shelter dogs in South Carolina in
1999-2000 (12.7%) versus 1991-1992 (8.7%).[^62] Compliance with dosing
schedules is a limiting factor in the control of heartworm infections in
the dog population.

Several years ago, Cummings et al. demonstrated that clinic compliance
failure is generally higher than predicted by individual hospitals
before their records are examined.[^63] A veterinary practice survey
conducted by Fort Dodge veterinary technicians at Michigan State in 2004
showed a similar picture.[^64] Compliance was better with ProHeart 6
than with the other heartworm preventatives studied. Average compliance
was 58% for ProHeart 6 (11 clinics), 45% for Heartgard Plus (the most
widely used preventative)(16 clinics), 50% for Sentinel (6 clinics), and
37% for Interceptor (3 clinics).

Moxidectin is a poor substrate for P glycoprotein compared with other
macrocyclic lactones.Error: Reference source not found^,^[^65]^,^[^66]
It can be used safely in ivermectin sensitive Collies. It is effective
in the face of ivermectin resistance in nematodes. Additionally, recent
work has demonstrated that with use of moxidectin, unlike some other
compounds, animals are not more susceptible to infection with filariid
parasites after product withdrawal.[^67] This provides an additional
safety factor, if owners are late in returning for treatment. Moxidectin
has retroactive activity against *D. immitis* larval stages for 3
months, again providing a safety factor, if owners are late in returning
for treatment.

### Field Efficacy from 2001 to 2004

Field experience with ProHeart 6 in the US is limited to the time period
from June 2001 to August 2004. There is a time-lag between infection and
the first possible heartworm diagnosis of minimally 5 to 6 months.
Therefore, the field evaluation of ProHeart 6 efficacy essentially spans
the years 2002 to mid 2004. Proving "lack of efficacy" of any heartworm
preventative product in client-owned dogs is inherently difficult,
unless the dog is started on a prevention program at 6‑ to 8-weeks of
age, remains on the product year-round for life and clinic records
indicate the owner acquired the recommended number of doses. With orally
or topically administered products, one is seldom confident the owner
properly administered all of the doses. Owner compliance is greatly
improved with ProHeart 6, but switching products tends to complicate the
picture. To prove with reasonable confidence that a new product is
completely effective, the dog must be tested prior to switching products
and within 4 months later. If the dog is heartworm positive during this
4-month period, the original product failed. If the dog is positive
during the next few months, it is virtually impossible to determine
whether the dog was infected before or after switching products.
Voluntary reports of product inefficacy to FDA are summarized in Table
4.5.3-1.

+-------------+----------+----------+----------+----------------------+
| # T         |          |          |          |                      |
| able 4.5.3- |          |          |          |                      |
| 1. Voluntar |          |          |          |                      |
| y Reports o |          |          |          |                      |
| f Product I |          |          |          |                      |
| nefficacy A |          |          |          |                      |
| gainst Larv |          |          |          |                      |
| al Heartwor |          |          |          |                      |
| m {#table-4 |          |          |          |                      |
| .5.3-1.-vol |          |          |          |                      |
| untary-repo |          |          |          |                      |
| rts-of-prod |          |          |          |                      |
| uct-ineffic |          |          |          |                      |
| acy-against |          |          |          |                      |
| -larval-hea |          |          |          |                      |
| rtworm .Cap |          |          |          |                      |
| tion-Table} |          |          |          |                      |
+=============+==========+==========+==========+======================+
| **Active**  | **2001** | **2002** | **2003** | **2003 Market        |
|             |          |          |          | Share**              |
+-------------+----------+----------+----------+----------------------+
| IVM         | 16       | 108      | 13       | 6%                   |
+-------------+----------+----------+----------+----------------------+
| IVM+PYR     | 21       | 195      | 137      | 37%                  |
+-------------+----------+----------+----------+----------------------+
| MILB        | 0        | 0        | 748      | 21%                  |
+-------------+----------+----------+----------+----------------------+
| MILB+LUF    | 0        | 0        | 347      | 8%                   |
+-------------+----------+----------+----------+----------------------+
| SEL         | 16       | 1162     | 888      | 4%                   |
+-------------+----------+----------+----------+----------------------+
| ProHeart 6  | 0\*      | 70       | 196      | 24%                  |
+-------------+----------+----------+----------+----------------------+
| \*          |          |          |          |                      |
| indicates   |          |          |          |                      |
| year of     |          |          |          |                      |
| launch      |          |          |          |                      |
|             |          |          |          |                      |
| IVM =       |          |          |          |                      |
| ivermectin; |          |          |          |                      |
| PYR =       |          |          |          |                      |
| pyrantel;   |          |          |          |                      |
| MILB =      |          |          |          |                      |
| milbemycin  |          |          |          |                      |
| oxime; LUF  |          |          |          |                      |
| =           |          |          |          |                      |
| lufenuron;  |          |          |          |                      |
| SEL =       |          |          |          |                      |
| selamectin  |          |          |          |                      |
+-------------+----------+----------+----------+----------------------+

The 2003 market share information is indicative of the numbers of dogs
treated and provides context when interpreting the significance of the
number of reports. When ProHeart 6 is compared with the market leaders
the adjusted efficacy rates for 2003 (when market share information is
available) are equivalent or lower than ivermectin plus pyrantel, or
milbemycin.

The overall reporting rate for ProHeart 6 is low at 1 report per 41,000
doses through marketing year 2003. Further, only 10% have a history that
justifies classification as a potential efficacy concern. The majority
are thought to be associated with a misunderstanding of the heartworm
life cycle in relation to testing procedures and limitations. When
potential dosing errors, administration errors, and mixing and storage
errors are considered, the low reporting rate of failures in efficacy
appears to be within acceptable parameters.

FDAH guarantees the efficacy of ProHeart 6 as a preventative for
heartworm disease. This program encourages veterinarians to contact the
company about potential concerns related to efficacy. When received,
these reports are submitted to the FDA unfiltered. Based on unfiltered
reports and doses sold, regardless of whether FDA or FDAH reports are
considered, ProHeart 6 delivers a high level of efficacy under field use
conditions.

### Tuskeegee University Experience with ProHeart 6

Tuskegee University is located in Macon County, in eastern Alabama. It
is a teaching hospital and unlike some universities which are
predominantly referral centers, Tuskegee operates largely as a community
practice. Most pets in the area receive their primary care at this
hospital, so it is reflective of the population of dogs receiving
preventative care, rather than specialist or tertiary care levels.

#### Retrospective Study: Tuskegee University School of Veterinary Medicine

The effectiveness of heartworm preventatives in this endemic area was
studied during the period of 2000-2004 (unpublished data).[^68] In 2001,
97.7% of the dogs on a heartworm preventative received an oral product,
while 1.0% received ProHeart 6. By 2004, 82.9% received an oral product
and 17.1% received ProHeart 6. During this time, the percentage of
heartworm-positive dogs dropped from 14.0% (of 898) to 5.8% (of 833),
and the percentage of dogs treated with Immiticide dropped from 5.2 to
3.0. More importantly, at no time was any dog receiving ProHeart 6
diagnosed as heartworm-positive, while dogs on the oral products
accounted for 15.8%, 29.4%, and 50.0% of the dogs that received a first,
second, or third Immiticide treatment, respectively. This study also
reviewed owner compliance in a limited number of dogs that received
Immiticide treatment. Of the 100 dogs studied, 61.5% of the dogs
receiving ProHeart 6 were up-to-date on purchasing their heartworm
product, while only 17.2% of those on oral products were compliant.
Furthermore, it is known that 100% of the dogs on ProHeart 6 received
their full treatment, whereas the number of oral doses administered is
not known.

While this study represents a limited number of dogs, it supports the
position that ProHeart 6 is highly effective in preventing heartworm
infection, and along with increased owner compliance, has led to a
decrease in the number of dogs requiring Immiticide treatment.

These results indicate that ProHeart 6 introduction into the canine
heartworm preventative market is helping to achieve the desired goals.
By overcoming the problems with owner compliance with monthly dosing,
ProHeart 6 not only protects the individual dog to which it is
administered, but also provides the veterinary professional with an
important medicine to reduce the prevalence of heartworm in the canine
population.

# REFERENCES

#   {#section-2 .unnumbered}

# Appendices

# APPENDIX 6.1 {#appendix-6.1 .Heading-0}

**Moxidectin Overview**

**Pharmacology -- Mechanism of Action**

The mechanism of action of moxidectin is multifaceted and continues to
be evaluated in laboratory studies. Moxidectin has been shown to have
activity at the γ-aminobutyric acid (GABA)-A receptor-chloride channel
complex resulting in an influx of chloride ions and hyperpolarization of
cell membranes^1^. This hyperpolarization causes the nerve fibers to be
less excitatory and results in paralysis and death of the parasitic
organism. The specificity of moxidectin for the parasite versus the
mammalian host results from 1) this compound having low affinity for
mammalian GABA-gated chloride channels^2^, and 2) the observation that
GABA-containing neurons and receptors are found in mammals in the
central nervous system, whereas in arthropods and nematodes these are
found in the neuromuscular junctions of the peripheral nervous system
and thus are more accessible to a blood-borne therapeutic. Another
proposed mechanism of action for moxidectin is through activity at
glutamate-gated chloride ion channels also resulting in paralysis and
death of the organisms^3,4^.

In dogs, the approved, oral monthly dosage of moxidectin for prevention
of heartworm is 3 μg/kg; the approved subcutaneous (sc) dosage of
moxidectin as ProHeart 6 is 0.17 mg/kg administered every 6 months.

Pharmacokinetics and Drug Metabolism

A series of studies was conducted in various animal species to
characterize the absorption, distribution, metabolism, and excretion of
moxidectin after oral administration. In rats, moxidectin was absorbed
at a moderate rate, with a mean time to peak concentration (t~max~) of
4.8 hours^5^. The bioavailability of moxidectin was moderate at 19% and
the apparent terminal half-life (t~1/2~) was long (22.9 to 44.6 hours).
After intravenous (IV) administration in rats, the clearance of
moxidectin was low and the steady-state volume of distribution (Vd~ss~)
was high, indicating that the compound is widely distributed to tissues.
In beagle dogs after an oral dose of 90 μg/kg moxidectin in tablet form,
the peak concentration (C~max~) was 29.8 ng/mL with a t~max~ of 8 hours
and a serum half-life of 8.1 days^6^. A single sc injection of the
approved dosage of 0.17 mg/kg moxidectin as ProHeart 6 to beagle dogs
resulted in a serum C~max~ of 5.1 ng/mL, a t~max~ of 7 to10 days, an
AUC~0-∞~ of 217 ng•day/mL, and a half-life of approximately 35 days^6^.
Moxidectin did not accumulate in the serum of dogs after injection with
ProHeart 6 once every 6 months for a total of 6 injections^6^. A recent
study of the administration of moxidectin once in the diet to female
dogs at 45 ppm (corresponding to approximately 1 mg/kg as used in the
1-year toxicology study) resulted in a Cmax, AUC~0-∞~, and half-life
values of 290 ng/mL, 678 ng•day/mL and 8.3 days, respectively.
Administration in the diet at this same level for 21 days to dogs
resulted in a plasma concentration of 278 ng/mL approximately 24 hours
after the previous feeding (ie, trough level). The previous 2 ongoing
diet pharmacokinetic studies in the dog, and an ongoing 28-day
pharmacokinetic study in rats by the diet route were initiated because
of FDA concerns regarding the safety of ProHeart 6, and to document the
high degree of systemic exposure to moxidectin achieved in the
toxicology studies conducted by the diet route.

After oral administration in rats, the major site of moxidectin
distribution was fat^5^. The t~1/2~ in fat was 11.5 days. Moxidectin
represented the major component of total radioactivity in tissues and
feces, and was primarily eliminated through feces (60% to 91% of the
recovered dose over 7 days)^5^. Studies in other species such as cattle,
sheep and horses have confirmed the distribution of moxidectin primarily
to fat and the fecal route of excretion^7^. Six (6) metabolites were
isolated from rat liver and fecal samples, none of which accounted for
more than 10% of the radioactivity in tissue samples collected from
animals 7 days after dosing^5^. Therefore, these metabolites are not of
toxicologic concern because of the low levels observed. Similarly
limited metabolism was noted in cattle, sheep and horses^7^ and in rat
and human liver microsomes^8^ where the metabolites were characterized
as hydroxylations at various positions.

In human liver microsomes, there was no significant inhibition of
selected cytochrome P450 (CYP) enzyme activities (CYP2A6, CYP2C8,
CYP2C19, CYP2D6, and CYP3A4) at the highest substrate concentration used
(100 μM)^9^. Human CYP enzymes were used in this study since they are
the best characterized. Based on plasma concentrations at efficacious
doses and the high substrate concentrations, clinical metabolic
drug-drug interactions for all the CYPs tested are unlikely to occur.

P-glycoproteins (P-gps) are transmembrane proteins which transport a
wide variety of endogenous and exogenous molecules across cell
membranes. Moxidectin, as do other macrocyclic lactones, acts as a
substrate for P-gps^10^. This mechanism has been found to be of clinical
importance in the development of nematode resistance to ivermectin and
plays a significant role in breed sensitivity^11^. A mutation in the
P-gp gene of ivermectin-sensitive Collie dogs has been shown to be
responsible for ivermectin-induced CNS toxicity^12^. Moxidectin,
however, was well-tolerated by these ivermectin-sensitive dogs.
Moxidectin transport, therefore, is less dependent on P-gp and
subsequent toxicity is less likely to be altered by factors which alter
P-gp activity.

Toxicology

The toxicologic profile of moxidectin administered by the oral route has
been well established. This profile is relevant to other routes of
administration because of limited metabolism of moxidectin in the body
and an understanding of its pharmacokinetics by different routes of
administration. The toxicologic program for moxidectin was reviewed
during the 45^th^ meeting of the Joint Expert Committee on Food
Additives^13^ and was subsequently published^7^. In addition, this
toxicology program was deemed sufficient by regulatory authorities to
proceed with Phase 1 trials of oral moxidectin in normal, human
volunteers in preparation for efficacy studies in people in countries
where onchocerciasis is endemic. The toxicology studies were conducted
in accordance with Good Laboratory Practice (GLP) regulations.

**In Vitro Side-Effect Profiling**

Moxidectin and moxidectin microspheres (as present in ProHeart 6) were
recently tested in vitro for binding activity at 64 different biological
receptors^14^, based on some of the safety concerns raised by the FDA.
This assay is commonly used in drug discovery and development to
identify any ancillary pharmacologic activities of a molecule which may
result in undesirable biological effects. A final concentration of 10
ng/mL moxidectin was tested, which is approximately two-fold the average
C~max~ value in serum of dogs after a subcutaneous injection of ProHeart
6 at the clinical dosage of 0.17 mg/kg. The receptors tested included
those for neurotransmitters and neurotransmitter-related receptors, ion
channels, steroids, second messengers, prostaglandins, growth
factors/hormones, brain/gut peptides and enzymes. Moxidectin in either
form did not significantly inhibit the binding of appropriate
radio-ligands to these receptors, indicating a lack of binding activity
for moxidectin. These results are consistent with the absence of
undesirable pharmacologic and toxic effects of moxidectin in animal
studies at plasma levels many fold those required for efficacy.

**Single-Dose Studies**

Single-dose toxicology studies of moxidectin were conducted to assess
effects after a single administration of large doses in the event of
accidental overdose and to assist in selection of dose levels for
subsequent repeat-dose toxicology studies. In single-dose toxicity
studies of mice and rats given moxidectin orally, the median lethal
dosage (LD~50~) values were 118 mg/kg in male mice^15^, 42 to 78 mg/kg
in female mice^16,17^, and 122 and 97 mg/kg in male and female rats^18^,
respectively. After a single sc dose, LD~50~ values were 285 and 247
mg/kg in male and female mice^19^, respectively, and \> 640 mg/kg in
rats^20^. Common clinical signs in these studies were decreased
activity, tremors, and prostration. The decreased toxicity with sc
dosing is likely the result of a lower C~max~ and daily exposure, albeit
of longer duration, as compared to oral gavage dosing. These studies
demonstrate a large margin of safety for ProHeart 6 in which the
approved sc dose in the dog is 0.17 mg moxidectin/kg, less than
3700-fold the lethal sc dose in the rat.

Field results from the accidental and sometimes intentional overexposure
of dogs orally to moxidectin likewise demonstrate a large margin of
safety. Oral overdoses occur when a product intended for use in horses
is administered to dogs. In these cases, the dogs can receive up to a
63-fold higher dose orally (ie, 10.7 mg/kg) than the 0.17 mg
moxidectin/kg when given sc as ProHeart 6, but in most cases the actual
dose is not known. From 1997 to 2004, FDAH received approximately 250
such cases. In all cases the events were characterized by a wide range
of neurological symptoms. In those cases where FDAH was informed, the
dogs had typical hematology or serum panels. In 90% of these cases, the
animals fully recovered whereas the remainder died. These cases
demonstrate that neurological symptoms induced by high, oral doses of
moxidectin in dogs are typically survivable without any nonneurologic
toxic effects.

**Repeat-Dose Studies**

Repeat-dose oral (diet) toxicity was evaluated to assess more long-term
consequences of repeated, daily oral exposure to moxidectin. The
objective of these studies was to dose the animals high enough to
identify potential toxic effects, and to include lower doses so as to
assess a dose-response relationship and identify a dose without
significant adverse effects (i.e., No-Observed-Adverse-Effect-Level;
NOAEL).

The following studies were conducted: 4-week studies in mice, rats, and
dogs; 13‑week studies in rats and dogs, and a 1‑year study in dogs.
Moxidectin was administered in the diet providing for a continuous
exposure for the duration of each study, which is a significant
exaggeration of the exposure of dogs to one sc dose of 0.17 mg
moxidectin/kg as ProHeart 6 every 6 months. Evaluations consisted of
mortality, clinical observations, body weight, food consumption,
hematology, clinical chemistry (except mice), organ weights, and
macroscopic and microscopic examinations of organs and tissues.
Ophthalmic examinations and urinalysis were also included in the dog
studies.

**Mice**

In the 4-week study in mice, doses in the diet were 33.7, 75, 100, 125,
and 150 ppm^21^. Mortality occurred at [\>]{.underline} 75 ppm.
Microscopic examination did not reveal the cause of death in these
animals. Clinical observations were tremors, hypersensitivity to touch,
and urine-stained fur at [\>]{.underline} 75 ppm. Body weights and
body-weight gains were decreased at [\>]{.underline} 100 ppm. There were
no other compound-related effects. Based on mortality, the
no-observed-adverse-effect-level (NOAEL) was 33.7 ppm (6.9 mg/kg/day).

**Rats**

In the 4-week study in rats, doses in the diet were 100, 200, 400, and
600 ppm^22^. Mortality occurred at [\>]{.underline} 200 ppm and resulted
from anorexia based on microscopic examination. Clinical observations
included ataxia, tremors, salivation, piloerection, and diuresis at
[\>]{.underline} 200 ppm, as well as decreased body weight, body-weight
gain, and food consumption. There were no other compound-related
findings in rats that survived the study. Based on mortality, the NOAEL
was 100 ppm (12.2 mg/kg/day).

In the 90-day study in rats at diet doses of 25, 50, 100 and 150 ppm,
mortality occurred for 3 females at the highest dose, 150 ppm^23^.
Microscopic examination did not reveal the cause of death for these
animals. Clinical observations included hypersensitivity to touch at 100
ppm, and lethargy, anorexia, aggressive behavior, loss of righting
reflex, ataxia, tremors, and urine-stained coat at 150 ppm. Body weight
and body-weight gain were decreased in males at 100 ppm or more and in
females at 150 ppm. Food consumption was decreased in males and females
at 150 ppm. Adrenal gland and kidney weights were increased in females
at [\>]{.underline} 100 ppm, but there were no compound-related
macroscopic or microscopic findings in these or any other organs or
tissues. The NOAEL was 50 ppm (3.9 mg/kg/day).

**Dogs**

In the repeat-dose toxicity studies in beagle dogs, ophthalmic
examinations and urinalysis were also included for evaluation of
potential toxic effects.

In the 4-week study, there was no mortality at doses in the diet of 20,
80 and 160 ppm^24^. The dose of 160 ppm was toxic, based on debilitating
clinical observations, and the dose was reduced to 50 ppm. Clinical
observations included tremors, languid appearance, ataxia, emesis, and
mydriasis at [\>]{.underline} 80 ppm. Testes weights were decreased at
[\>]{.underline} 80 ppm. Microscopic findings were decreased
spermatogenic activity at [\>]{.underline} 80 ppm and decreased colloid
in the thyroid gland in males at 80 ppm. These findings may have been
related to variations in age of maturation (study dogs were 5 to
6 months old at study termination) and were not seen in the 1‑year
toxicity study in dogs. Based on the testicular findings, the NOAEL in
this 4‑week study was 20 ppm (0.8 mg/kg/day).

In the 90-day toxicity study in dogs at diet doses of 10, 30 and 60 ppm,
there was no mortality and clinical observations included lacrimation at
[\>]{.underline} 10 ppm, languid appearance and tremors at 10 and 60
ppm, thin appearance at [\>]{.underline} 30 ppm, and slight salivation
and slight ataxia at 60 ppm^25^. A 60 ppm, alanine aminotransferase
(ALT) was increased in 1 male and 1 female, and serum alkaline
phosphatase was increased in 3 males and 3 females compared with
pretreatment and/or control values (increases were small in magnitude
and not toxicologically significant). However, there were no
compound-related macroscopic or microscopic changes in the liver or any
other tissues or organs. Based on the absence of toxicologically
significant findings at any tested concentration, the NOAEL in this
90-day study was 60 ppm (1.6 mg/kg/day).

In the 1-year toxicity study in dogs at diet doses of 10, 20 and 45 ppm,
there was no mortality^26^. Decreased ovarian weights were observed at
45 ppm; however, the absolute weights were within reference or historic
control ranges and the decreases were not considered to be biologically
important. There were no compound-related effects on clinical chemistry
or hematology parameters, and no compound‑related microscopic findings.
Based on the absence of toxicologically significant findings at any
tested concentration, the NOAEL in this study was 45 ppm
(1.1 mg/kg/day).

Interim analysis at day 21 in an ongoing 28-day diet pharmacokinetic
study of moxidectin in dogs at a concentration of 45 ppm in feed
(approximately 1 mg/kg, the NOAEL in the 1-year dog toxicity study)
revealed a serum concentration of 278.5 ng/mL 24 hours after the
preceding dose in feed (ie, trough level). This concentration is not yet
at steady state, but would correspond to a daily AUC of 278.5 ng•d/mL
which would represent the minimal daily exposure to moxidectin for most
of the duration of the 1-year toxicology study in dogs, or an AUC of
50826 ng.day/mL over a 6-month period, the retreatment interval for
ProHeart 6.. This value is approximately 234-fold the AUC ~0-∞~ (217
ng•d/mL) observed for moxidectin after a single sc dose in dogs of
ProHeart 6.

**Carcinogenicity Studies**

**Mice**

A 2-year carcinogenicity study was conducted in male and female mice at
diet doses of 15, 30, and 60 ppm (lowered to 50 ppm due to high
mortality at week 9)^27^. Mortality was increased in females at doses of
60/50 ppm during the last 13 weeks of the study. There were no
compound‑related findings in hematology values, organ weights, or at
macroscopic or microscopic examination. There was no evidence of
moxidectin‑related target‑organ toxicity or tumorigenicity.

**Rats**

A 2-year carcinogenicity study was conducted in male and female rats at
diet doses of 15, 60 and 120 ppm (lowered to 100 ppm due to high
mortality in females at week 8)^28^. There were no compound-related
findings for hematology values, organ weights or at macroscopic or
microscopic examination; there was no evidence of moxidectin-related
target-organ toxicity or tumorigenicity.

**Reproductive and Developmental Toxicity Studies**

Reproductive toxicity was evaluated in developmental studies of rats^29^
and rabbits^30^ dosed orally by gavage and in pilot^31^ and
definitive^32^ multigeneration diet studies. Maternal toxicity was
evident at [\>]{.underline}10 mg/kg/day in the rat developmental study,
125 ppm (calculated dosage averaging between 10.9 and 12.0 mg/kg/day) in
the rat pilot multigeneration study, and at [\>]{.underline}5 mg/kg/day
in the rabbit developmental study; the toxicity consisted primarily of
decreased body weight and/or food consumption. At maternally toxic
dosages (≥ 10 mg/kg/day) in the rat developmental study only, there were
statistically significant increases in the number of fetuses with
malformations and/or variations, largely reflective of increases in
cleft palate and reversible delays in ossification. There was decreased
fetal and/or pup survival at 10 mg/kg/day in the rabbit developmental
study and at doses of [\>]{.underline}10 ppm (calculated maternal dosage
of ≥ 0.8 mg/kg/day) in the rat pilot 1‑generation and 3‑generation
studies. The reproductive NOAELs were 5 mg/kg/day in both the rat and
rabbit developmental studies and was 5 ppm (0.4 mg/kg/day) in the rat
3‑generation study. Therefore, moxidectin was not considered teratogenic
in these species. This conclusion is consistent with that of the FDA
Center for Veterinary Medicine, which concluded that moxidectin is
neither a selective developmental toxicant nor a teratogen in rats or
rabbits.

**Genotoxicity Studies**

Moxidectin was tested for genotoxicity in 4 in vitro and 2 in vivo
universally recognized test systems. These assays assessed the ability
of moxidectin to caus gene mutations, chromosome damage, and increased
DNA repair which may be related to the carcinogenic potential of the
test article. Moxidectin was negative in the following assays for
genotoxicity: bacterial reverse mutation assay in *Salmonella
typhimurium* (strains TA98, TA100, TA1535, TA1537, and TA1538) and
*Escherichia coli* (WP‑2 uvrA‑)^33^; mammalian cell mouse lymphoma
(L5178Y thymidine kinase) assay^34^; chromosome aberration assay in
Chinese hamster ovary cells^35^; unscheduled DNA synthesis assay in
primary rat hepatocytes^36^; in vivo micronucleus assay in mouse bone
marrow cells^37^; and the in vivo chromosome aberration assay in rat
bone marrow cells^38^. The in vivo studies were conducted using the oral
route of administration. The results from these studies indicate that
moxidectin is not a genotoxic compound.

**Experience with Oral Moxidectin in Human Volunteers**

A study in healthy, male volunteers was conducted to assess the
pharmacokinetics and safety of moxidectin given orally as part of the
development of this compound for treatment of onchocerciasis in
humans^39^. A total of 37 subjects in this study were treated with
single doses of 3 mg to 36 mg (approximately 50 μg/kg to 600 μg/kg). The
t~1/2~ ranged from 19.9 to 37.4 days, the C~max~ at 36 mg was 296 ng/mL,
and the distribution of moxidectin was extensive, as indicated by a
large apparent volume of distribution. The C~max~ observed for humans
dosed orally with 36 mg moxidectin was 59-fold the C~max~ observed in
dogs dosed sc with the clinical dosage of 0.17 mg/kg moxidectin as
ProHeart 6, and exposure (based on AUC) was approximately 6-fold the
exposure in dogs administered ProHeart 6.

There was no significant relationship between the overall number of
adverse events and the dose of moxidectin administered. Safety
assessments indicated that moxidectin was safe and well tolerated, with
a slightly higher incidence of transient, mild, and moderate central
nervous system adverse events (dizziness and somnolence) as compared to
placebo. There were no clinically significant changes in vital signs,
clinical chemistries, physical examinations or electrocardiograms. The
conclusion was that moxidectin was safe and well tolerated in humans
after single, oral doses of 3 mg to 36 mg.

**Conclusions**

Moxidectin is a potent antiparasitic therapeutic that acts to paralyze
susceptible organisms through activity at GABA- and glutamate-gated
chloride ion channels. Moxidectin has a long half-life, a high volume of
distribution (predominantly distributing to fat), shows little
metabolism, and is excreted primarily in the feces. These
characteristics of moxidectin appear constant across the mammalian
species studied, including humans. In single-dose toxicity studies, the
lethal sc dose in rats was more than 3700-fold the efficacious dose of
moxidectin given as ProHeart 6 to dogs. In repeat-dose diet toxicity
studies in mice,rats, and dogs of durations up to 2-years in mice and
rats, and 1-year in dogs, no target organs of toxicity were identified.
There were no significant adverse histologic or biochemical effects on
any organ system including the cardiovascular, gastrointestinal,
hepatobiliary, genitourinary and central nervous systems. There were no
proliferative lesions identified in any tissue which may signal the
development of neoplasia, and no increase in tumors in mice or rats. The
toxicity of moxidectin manifested itself at high doses in clinical signs
such as lethargy, ataxia, tremors and mortality (rodents only) with
concomitant decreases in food consumption and body weight. Such clinical
signs of hypoexcitation are expected and are consistent with an
exaggerated pharmacologic effect of moxidectin mediated via the
GABA-receptor. Moxidectin was not genotoxic or carcinogenic, and was
without reproductive or developmental toxicity. The NOAEL in the rat
2-year study was 12.2 mg/kg/day; the NOAEL in the dog 1-year study was
1.1 mg/kg/day.

Based on ongoing pharmacokinetic studies in the dog, a single dose of
moxidectin in the diet at approximately 1 mg/kg (NOAEL in the 1-year dog
study), when extrapolated to infinity, results in an overall exposure
over 3-fold that of a single SC dose of ProHeart 6. Repeated dosing at
this level resulted in an estimated daily exposure at least 1.3-fold the
total exposure observed after a single SC dose in dogs of ProHeart 6
(AUC~1day~ of 278.5 versus AUC~0-∞~ of 217 ng•days/mL, respectively).
The total exposure over the duration of the 1-year study, therefore,
would be approximately 365 times the daily AUC, or more than 450-fold a
single SC dose of ProHeart 6. Moxidectin was also found safe in a human
clinical trial after a single oral dose of 36 mg, which resulted in a
C~max~ and AUC approximately 59- and 6-fold, respectively, that was
observed in dogs dosed SC with the clinical dosage of 0.17 mg/kg as
ProHeart 6.

Based on the toxicology studies of moxidectin where the dose, dosing
duration, and resulting systemic exposure to moxidectin were
significantly exaggerated without inducing significant adverse effects,
the single, clinical sc administration of 0.17 mg moxidectin/kg as
ProHeart 6 to dogs would be predicted to be without significant adverse
effects.

**References**

1.  Ingle D, Wood IB. Princeton, NJ, Fort Dodge Company Report, 1990.

2.  Cole LM, Casida JE. GABA-gated chloride channel: binding site for
    4-ethynyl-4-n-\[2,3-^3^H~2~\] propylbicycloortho benzoate
    (\[^3^H\]EBOB) in vertebrate brain and insect head. *Pesticide
    Biochemistry and Physiology*. 1992;44:1-8.

3.  Fisher MH. Structure-activity relationships of the avermectins and
    milbemycins. *American Chemical Society Symposium Series,
    Phytochemicals for Pest Control*. 1997;17:220-238.

4.  Paiement J-P, Leger C, Ribeiro P, Prichard RK. *Haemonchus
    contortus:* Effects of glutamate, ivermectin and moxidectin on
    inulin uptake activity in unselected and ivermectin-selected adults.
    *Experimental Parasitology.* 1999;92:193-198.

5.  Wu S-S. Moxidectin (CL 301,423): Absorption, distribution,
    excretion, and metabolism of carbon‑14 labeled CL 301,423 in the rat
    (report amendment 01). American Cyanamid Report Number PD‑M, volume
    28‑33.01, 1995.

6.  Barton W, Rulli RD. Final report of a three year study with
    moxidectin canine SR injectable. Fort Dodge Animal Health Report
    Number GASD 07‑21.00, 2000.

7.  Rock DW, DeLay RL, Gliddon MJ. Chemistry, pharmacology and safety:
    moxidectin. In: Macrocyclic Lactones in Antiparasitic Therapy,
    Vercruysse J, Rew RS eds. CABI Publishing, Wallingford, Oxon, UK,
    2002, pp 75-96.

8.  Shilling A, Hasan Y. Moxidectin: IC~50~ determination for the
    inhibition of cytochrome P450 enzymes in human liver microsomes
    (Protocol 03_1744). Wyeth RPT-51894, 2003.

9.  Shilling A, Young-Sciame R, Wang J. Moxidectin: metabolic stability
    and metabolism in rat and human liver microsomes (Protocol 03_1745).
    Wyeth RPT‑51895, 2003.

10. Roulet A, Bousquet-Meloub A, Concordet D, Dupuy J, Lespine A,
    Alvinerie M, Pineau T. Transport studies of macrocyclic lactones in
    P-glycoprotein-recombinant cells are consistent with *in
    vivo*-measured bioavailability parameters in several species.
    *WAAVP* 2003;19:254.

11. Huang, Y-J, Prichard RK. Identification and stage-specific
    expression of two putative P-glycoprotein coding genes in
    *Onchocerca volvulus.* *Molecular and Biochemical Parasitology.*
    1999;102:273-281.

12. Roulet A, Puel O, Gesta S, Lepage JF, Drag M, Soll M, Alvinerie M,
    Pineau T. MDRI-deficient genotype in Collie dogs hypersensitive to
    the P-glycoprotein substrate ivermectin. *European J Pharmacol.*
    2003;460:85-91.

13. Woodward K. Moxidectin. Toxicological evaluation of certain
    veterinary drug residues in food. *World Health Organization,*
    Geneva. pp 27-50.

14. NovaScreen Biosciences Corporation, 2005, report in progress.

15. Fischer JE. Oral LD~50~ study in the albino mouse with AC 301,423
    technical. American Cyanamid Toxicology Report Number A90‑45.01,
    1996.

16. Fischer JE. Oral LD~50~ study in the female albino mouse with AC
    301,423 technical. American Cyanamid Toxicology Report Number
    A91‑44, 1991.

17. Fischer JE. Oral LD~50~ study in the female albino mouse with AC
    301,423. American Cyanamid Toxicology Report Number A91‑43, 1991.

18. Fischer JE. Oral LD~50~ study in the albino rat with AC 301,423.
    American Cyanamid Toxicology Report Number A90‑35, 1990.

19. Fischer JE. Subcutaneous LD~50~ study in the albino mouse with AC
    301,423. American Cyanamid Toxicology Report Number A90‑74, 1990.

20. Fischer JE. Subcutaneous LD~50~ study in the albino rat with AC
    301,423. American Cyanamid Toxicology Report Number A90‑65, 1990.

21. Fischer JE. AC 301,423: a 28‑day mouse feeding study. American
    Cyanamid Toxicology Report Number AX89‑3, 1989.

22. Fischer JE. AC 301,423: a 28‑day rat feeding study. American
    Cyanamid Toxicology Report Number AX88‑1, 1988.

23. Fischer JE. AC 301,423: a 13‑week rat feeding study. American
    Cyanamid Toxicology Report AX89‑1, 1989.

24. Schulze GE. 28 Day range-finding dietary study in purebred beagle
    dogs with AC 301,423. Study conducted by Hazleton Laboratories
    America, Inc., Vienna, VA. HLA Study Number 362‑197, 1989.

25. Schulze GE. 91 Day dietary toxicity study in purebred beagle dogs
    with AC 301,423. Study conducted by Hazleton Laboratories America,
    Inc., Vienna, VA. HLA Study Number 362‑198, 1989.

26. Schulze GE. One‑year dietary toxicity study in purebred beagle dogs
    with AC 301,423. Study conducted by Hazleton Washington, Inc.,
    Vienna, VA. HWA Study 362‑200. American Cyanamid Protocol Number
    971‑88‑175, 1991.

27. Goldenthal EI. Chronic dietary toxicity and oncogenicity study with
    AC 301,423 in mice. Study conducted by International Research and
    Development Corporation, Mattawan, MI. Study Number 141‑031.
    American Cyanamid Protocol Number 971‑89‑155, 1992.

28. Zoetis T. Chronic dietary toxicity and oncogenicity study with AC
    301,423 in rats. Study conducted by Hazleton Washington, Inc.,
    Vienna, VA. HWA Study 362‑202. American Cyanamid Protocol Number
    971‑89‑156, 1992.

29. Lochry EA. An oral developmental toxicity study with AC 301,423 in
    rats. Study conducted by Argus Research Laboratories, Perkasie, PA.
    Argus Report Identification Number 101‑007. American Cyanamid
    Protocol Number 971‑89‑139, 1989.

30. Hoberman AM. A developmental toxicity (embryo-fetal toxicity and
    teratogenicity) definitive study with AC301,423 in rabbits. Study
    conducted by Argus Research Laboratories, Inc., Horsham, PA. Argus
    Protocol Number 101‑006. American Cyanamid Protocol Number
    971‑88‑158, 1989.

31. Schroeder RE. A pilot one-generation (two litters) reproduction
    study with AC 301,423 to rats. Study conducted by Bio/dynamics,
    Inc., East Millstone, NJ. Project Identification Number 88‑3388.
    American Cyanamid Protocol Number 971‑88‑176, 1991.

32. Schroeder RE. A three-generation (two litters) reproduction study
    with AC 301,423 to rats. Study conducted by Bio/dynamics, Inc., East
    Millstone, NJ. Project Identification Number 89‑3496. American
    Cyanamid Protocol Number 971‑89‑163, 1992.

33. Traul KA. Evaluation of CL 301,423 in a bacterial/microsome
    mutagenicity test. American Cyanamid Study Number 89‑02‑01, 1990.

34. Pant KJ. Evaluation of AC 301423 in the L5178Y TK+/‑ mouse lymphoma
    mutagenesis assay with colony size evaluation in the presence and
    absence of induced rat liver S‑9 with a confirmatory study. Study
    conducted by SITEK Research Laboratories, Rockville, MD. SITEK Study
    Number 0504‑2400. American Cyanamid Protocol Number 9971‑98‑147,
    1999.

35. Xu J. Test for chemical induction of chromosome aberration in
    cultured Chinese hamster ovary (CHO) cells with and without
    metabolic activation with AC 301423. Study conducted by SITEK
    Research Laboratories, Rockville, MD. SITEK Study Number 0504‑3110.
    American Cyanamid Study Number 971‑98‑131, 1999.

36. Curren RD. Unscheduled DNA synthesis in primary rat hepatocytes with
    AC 301,423. Study conducted by Microbiological Associates, Inc.,
    Rockville, MD. Project ID T9090.380025. American Cyanamid Protocol
    Number 971‑89‑176, 1990.

37. Xu J. In vivo test for chemical induction of micronucleated
    polychromatic erythrocytes in mouse bone marrow cells. Study
    conducted by SITEK Research Laboratories, Rockville, MD. SITEK Study
    Number 0504‑1521. American Cyanamid Study Number 971‑98‑132, 1999.

38. Sharma RK. Evaluation of moxidectin in the in vivo chromosome
    aberration assay in rat bone marrow cells. American Cyanamid Study
    Number 89‑14‑002, 1990.

39. Cotreau MM, Warren S, Ryan JL, Fleckenstein L, Vanapalli S, Brown
    KR, Rock D, Chen C-Y, Schwertschlag US. The antiparasitic
    moxidectin: Safety, tolerability and pharmacokinetics in humans. *J
    Clinical Pharmacology* 2003;43:1108-1115.

# appendix 6.2 {#appendix-6.2 .Heading-0}

![](media/image14.wmf){width="5.996527777777778in"
height="7.434027777777778in"}

![](media/image15.wmf){width="5.979861111111111in"
height="5.365277777777778in"}

# APPENDIX 6.3 {#appendix-6.3 .Heading-0}

**REBAR, ALAN H., DVM, PhD, DACVP 1/05**

**[EDUCATION]{.underline}**

DVM, 1973, Purdue University

Ph.D. (Clinical Pathology), 1975, Purdue University

Internship/Residency (Pathology), 1976, Purdue University

Diplomate -- American College of Veterinary Pathologists (Clinical
Pathology), 1978

**[EMPLOYMENT]{.underline}**

July 1996 -- Present Dean, School of Veterinary Medicine, Purdue
University

July 1989 -- June 1996 Associate Dean for Research, School of Veterinary
Medicine, Purdue University

July 1995 -- June 1996 Head, Veterinary Pathobiology, School of
Veterinary Medicine, Purdue University

July 1993 -- June 1995 Interim Department Head, Veterinary Pathobiology,
School of Veterinary Medicine, Purdue University,

July 1987 -- June 1995 Acting Director of Continuing Education, School
of Veterinary Medicine, Purdue University

July 1987 -- July 1989 Director of Research Programs Development, School
of Veterinary Medicine, Purdue University

1986 -- Present Director, Veterinary Cytology Resource Center, Purdue
University,

July 1983 -- Present Professor of Clinical Pathology, School of
Veterinary Medicine, Purdue University

July 1979 -- July 1987 Co-Director of Clinical Pathology Laboratory,
School of Veterinary Medicine, Purdue University

July 1979 -- June 1983 Associate Professor of Clinical Pathology, School
of Veterinary Medicine, Purdue University

Sept. 1977 -- July 1979 Clinical Pathologist, Lovelace Inhalation
Toxicology Research Institute, Albuquerque, New Mexico

Jan. 1976 -- Aug. 1977 Director of Clinical Pathology Laboratory, School
of Veterinary Medicine, Purdue University

Jan. 1976 -- Aug. 1977 Assistant Professor of Clinical Pathology, School
of Veterinary Medicine, Purdue University

Sept. 1974 -- Dec. 1975 NIH Postdoctoral Fellow. (Ultrastructural
studies on cobalt and isoproterenol induced cardiomyopathy in swine.)
School of Veterinary Medicine, Purdue University,

Sept. 1972 -- Aug. 1974 Graduate Instructor, Veterinary Pathology,
School of Veterinary Medicine, Purdue University

May 1973 -- Sept. 1973 Staff Veterinarian, Colonial Oaks Animal
Hospital, Gainesville, Florida

**[HONORS]{.underline}**

Indiana Veterinarian of the Year Award. Presented by the Indiana
Veterinary Medical Association, 2002.

The Waltham Award given in recognition of outstanding activities or
contributions by a veterinarian that have resulted in the improvement of
the well-being companion animals in the international veterinary
community. Presented by the American Animal Hospital Association, 2001.

The Gaines Cycle Fido Award for outstanding contributions to small
animal medicine and surgery. Presented by the American Animal Hospital
Association, 1994.

Distinguished Alumnus Award, School of Veterinary Medicine, Purdue
University, 1990.

**[REPRESENTATIVE PUBLICATIONS (more than 115 overall)]{.underline}**

1.  Herbert, R.A.,Stegelmeier, B.S., Gillett, **Rebar, A.H**., Carlton,
    W.W., Singh, G., and Hahn, F.F.: *Plutonium-induced proliferative
    lesions and pulmonary epithelial neoplasms in the rat:
    immunohistochemical and ultrastructural evidence for their origin
    from type II pneumocytes*. Vet Pathol 31(3):366-374, 1994.

2.  Stegelmeier, B.L., Gillett, N.A., Hahn, F.F., **Rebar, A.H**., and
    Kelly, G.: *Expression of transforming growth factor alpha and
    epidermal growth factor receptor in rat lung neoplasms induced by
    plutonium-239.* Radiat-Res, Nov, 140(2):191-8, 1994.

3.  Lipscomb, T.P., Harris, R.K., **Rebar, A.H.**, Ballachey, B.E., and
    Haebler, R.J.: *Pathology of Sea Otters.* In: Marine Mammals and the
    Exxon Valdez. Academic Press, San Diego, CA. 1994.

4.  Reagan, W.J. and **Rebar, A.H**., *Platelet Dysfunction.* In:
    Textbook of Veterinary Internal Medicine. W.B. Saunders Company,
    Philadelphia, PA. 1994.

5.  Skowronek, L.A., LaFranco, L., Stone-Marschat, M.A., Burrage, T.G.,
    **Rebar, A.H.**, and Laegreid, W.W.. *Clinical Pathology and
    Hemostatic Abnormalities in Experimental African Horsesickness*. Vet
    Pathol 32:112-121, 1995.

6.  **Rebar, A.H.,** Lipscomb, T.P., Harris, R.K., and Ballachey, B.E.
    *Clinical and Clinical Laboratory Correlates in Sea Otters Dying
    Acutely in Rehabilitation Centers Following the Exxon Valdez Oil
    Spill*. Vet Pathol 32(4):346-350, 1995.

7.  **Rebar, A.H.**, Metzger, F.: The Veterinary CE Advisor\--*Clinical
    pathology for small-animal practitioners: Interpreting the
    hemogram*. Veterinary Medicine 90(6) (Suppl.):1-12, 1995.

8.  Snipes, M.B., Barnett, Harkema, J.R., Hotchkiss, J.A., **Rebar,
    A.H.**, Reddick, L.J.: *Specific Biological Effects of an Anti-Rat
    PMN Antiserum Intraperitoneally Injected into F344/N Rats*. Vet Clin
    Pathol 24(1), 11-17. 1995.

9.  **Rebar, A. H.**, Thrall, M. A.: *Blood Film Evaluation: Cytology of
    Circulating Blood Cells.* Vet Tech 16(9), 578-586, 607, 1995.

10. **Rebar, A.H.**, Metzger, F.: The Veterinary CE Advisor\--*Clinical
    Pathology for Small-animal Practitioners: Profiling the Urinary
    System*. Vet Med 90(11) (Suppl.):1-16; 1995.

11. Christian, J.A., **Rebar, A.H.**, Boon, G.D., Low, P.S.
    *Methodological considerations for the use of canine [in
    vivo]{.underline} aged biotinylated erythrocytes to study RBC
    senescence.* Experimental Hematology 24(1):82-8, 1996 Jan.

12. **Rebar, A.H.**, Metzger, , F.: The Veterinary CE
    Advisor\--*Clinical Pathology for Small-animal Practitioners:
    Laboratory Evaluation of the Liver.* Vet Med 91(9)
    (Suppl.):1-12;1996

13. **Rebar, A.H.**, Section Editor, *Hematology and Immunology.* In:
    The 5 Minute Veterinary Consult, Tilley, L., and Smith, F., ed.,
    Williams and Wilkins, Media, PA. 1997.

14. **Rebar, A.H.,** *Metabolic Anemias (Anemias with Spiculated Red
    Cells)*. In: The 5 Minute Veterinary Consult, Tilley, L., and Smith,
    F., ed., Williams and Wilkins, Media, PA. 1997.

15. Christian, J.A., **Rebar, A.H.**, *Anemia, Regenerative*. In: The 5
    Minute Veterinary Consult, Tilley, L., and Smith, F., ed., Williams
    and Wilkins, Media, PA. 1997.

16. **Rebar, A.H.**, *Anemia, Nuclear Maturation Defect (Anemia,
    Megaloblastic)*. In: The 5 Minute Veterinary Consult, Tilly, L., and
    Smith, F., ed., Williams and Wilkins, Media, PA. 1997.

17. **Rebar, A.H.**, *Hemogram Interpretation for Dogs and Cats*,
    Ralston Purina Company, St. Louis, MO, The Gloyd Group, Inc., 1998.

18. **Rebar, A.H.**, Boon, G.D., and Christian, J.A., *Biochemical
    Profiling in the Dog and Cat. A Case Oriented Approach*. Ralston
    Purina Company, St. Louis, MO, The Gloyd Group, Inc. 1999.

19. **Rebar, A.H.**, MacWilliams, P.S., Feldman, B.F., Metzger, F.L.,
    Pollock, R.V.H., Roche, J., *A Guide to Hematology in Dogs and
    Cats*. Teton NewMedia, Jackson, WY. 2002.

20. Giger, U., **Rebar, A.H.**, Feldman, B.F., *Using White Blood Cell
    Information More Effectively: A Logical Approach* as part of
    Hematology Symposium. A Supplement to Compendium on Continuing
    Education for the Practicing Veterinarian, Vol. 25, No. 9(A),
    September 2003.

21. Thrall, M.A., Baker, D.C., Campbell, T.W., DeNicola, D., Fettman,
    M.J., Lassen, E.D., **Rebar, A.H.**, Weiser, G., *Hematology and
    Clinical Chemistry*, Lippincott Williams & Wilkins, Baltimore, MD,
    2004.

22. Thrall, M.A., Baker, D.C., Campbell, T.W., DeNicola, D., Fettman,
    M.J., Lassen, E.D., **Rebar, A.H.**, Weiser, G., *Clinical Case
    Presentations for Veterinary Heamtology and Clinical Chemistry*,
    Lippincott Williams & Wilkins, Baltimore, MD, 2005.

# APPENDIX 6.4 {#appendix-6.4 .Heading-0}

+---------------------------------+------+---+---------+---+---------------+
| GRANT NUMBER:                   |      |   |         |   |               |
+---------------------------------+------+---+---------+---+---------------+
|                                 |      |   |         |   |               |
+---------------------------------+------+---+---------+---+---------------+
| **BIOGRAPHICAL SKETCH**         |      |   |         |   |               |
|                                 |      |   |         |   |               |
| Provide the following           |      |   |         |   |               |
| information for all ***new***   |      |   |         |   |               |
| key personnel.                  |      |   |         |   |               |
|                                 |      |   |         |   |               |
| Copy this page for each person. |      |   |         |   |               |
+---------------------------------+------+---+---------+---+---------------+
| NAME                            |      | P |         |   |               |
|                                 |      | O |         |   |               |
| Philip J. Bergman, DVM, MS,     |      | S |         |   |               |
| PhD, DACVIM                     |      | I |         |   |               |
|                                 |      | T |         |   |               |
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+---------------------------------+------+---+---------+---+---------------+
| EDUCATION/TRAINING              |      |   |         |   |               |
+---------------------------------+------+---+---------+---+---------------+
| INSTITUTION AND LOCATION        | DE   |   | YEAR(s) | F |               |
|                                 | GREE |   |         | I |               |
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+---------------------------------+------+---+---------+---+---------------+
| University of Arizona - Tucson, |      |   | Un      | M |               |
| AZ                              |      |   | dergrad | i |               |
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+---------------------------------+------+---+---------+---+---------------+
| Colorado State University -     | DVM, |   | 19      | V |               |
| Fort Collins, CO                | BS   |   | 86-1990 | e |               |
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| Manhattan, KS                   |      |   | 90-1991 | m |               |
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|                                 |      |   |         | R |               |
|                                 |      |   |         | e |               |
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|                                 |      |   |         | n |               |
|                                 |      |   |         | c |               |
|                                 |      |   |         | y |               |
+---------------------------------+------+---+---------+---+---------------+
| M.D. Anderson Cancer Center -   | PhD  |   | 19      | C |               |
| Houston, TX                     |      |   | 94-1999 | a |               |
|                                 |      |   |         | n |               |
|                                 |      |   |         | c |               |
|                                 |      |   |         | e |               |
|                                 |      |   |         | r |               |
|                                 |      |   |         | B |               |
|                                 |      |   |         | i |               |
|                                 |      |   |         | o |               |
|                                 |      |   |         | l |               |
|                                 |      |   |         | o |               |
|                                 |      |   |         | g |               |
|                                 |      |   |         | y |               |
+---------------------------------+------+---+---------+---+---------------+

**[Professional Positions:]{.underline}**

1991. Rotating Small Animal Intern, Kansas State University, Manhattan,
      KS

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1994. Comparative Oncology Residency, Colorado State University, Fort
      Collins, CO

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```
1999. Cancer Biology Fellow, M.D. Anderson Cancer Center, Houston, TX

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```
1997. Staff Oncologist, Gulf Coast Veterinary Specialists, Houston, TX

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```
1999. American Cancer Society Physician Research Training Fellow, M.D.
      Anderson Cancer Center, Houston

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```
2000. Chair, ACVIM Forum, Oncology Subspecialty

1999-Present Head, Donaldson-Atwood Cancer Clinic, The Animal Medical
Center, New York, NY

2000-2002 President-Elect, Veterinary Cancer Society

2001-Present Director, Flaherty Comparative Oncology Laboratory

2002-Present Adjunct Associate Faculty Member, Memorial Sloan-Kettering
Cancer Center & Sloan-Kettering Cancer Institute

2002-2004 President, Veterinary Cancer Society

**[Honors and Awards:]{.underline}**

1989. R. Barry Prynn Memorial Scholarship. *For excellence in
      neurology.*

1990. AAHA Senior Student Award. *For excellence in small animal
      medicine and surgery.*

1993 William K. Riddell Memorial Scholarship. *For imminent success &
broad impact in the biomedical research field.*

1999 R.E. \"Bob\" Smith Fellow, Department of Cell Biology, M.D.
Anderson Cancer Center.

> AACR Symposium, Molecular Biology in Clinical Oncology. Invited
> Participant (Aspen, CO).

1997 & 1998 UT-Houston Graduate School of Biomedical Sciences Travel
Award

1996 -- 1999 American Cancer Society Physician Research Training
Fellowship (PRTA #40)

> AACR Clinical Trials Symposium. Invited participant (Vail, CO)

2001 Jean Holzworth Keynote Address, Angell Memorial Animal Hospital

2001 Japanese Veterinary Cancer Society Keynote Address

2002 Adjunct Associate Faculty, Memorial Sloan-Kettering Cancer Center &
Sloan-Kettering Cancer

> Institute

2003 World Small Animal Veterinary Association Hill's Award for
Excellence in Veterinary Healthcare

**Bibliography:**

1.  **Bergman PJ**, Withrow SJ, Straw RC, et al. Canine Infiltrative
    Lipoma: 16 Cases (1981-1992). *J Amer Vet Med Assoc*
    1994;205:322-324.

2.  **Bergman PJ**, Bruyette DS, Coyne BE, et al. Paraneoplastic
    Clinical Peripheral Neuropathy Associated with Canine Insulinoma.
    *Prog Vet Neuro* 1994;5:57-62.

3.  Ogilvie GK, Moore AS, Chen C, Ciekot PA, Atwater SW, **Bergman PJ**,
    Walters LM. Toxicoses associated with the administration of
    mitoxantrone to dogs with malignant tumors: A dose escalation study.
    *J Amer Vet Med Assoc* 1994;205:570-573.

4.  Ogilvie GK, Atwater SW, Ciekot PA, **Bergman PJ**, et al. Incidence
    of anaphylaxis associated with the intramuscular administration of
    L-asparaginase to 81 dogs with cancer: 1989-1991. *J Am Anim Hosp
    Assoc* 1994;30:62-65.

5.  **Bergman PJ**, Ogilvie GK. Drug Resistance and Cancer Therapy.
    *Compend Contin Educ* 1995;17:549-558.

6.  Macy DW, **Bergman PJ.** Vaccine-induced sarcomas in cats. *Feline
    Practice* 1995;23:24-27.

7.  Gupta KP, Ward NE, Gravitt KR, **Bergman PJ**, O\'Brian CA. Partial
    Reversal of Multidrug Resistance in Human Breast Cancer Cells by an
    N-myristoylated Protein Kinase C- Pseudosubstrate Peptide. *J Biol
    Chem* 1996;271:2102-2111.

8.  **Bergman PJ**, MacEwen EG, Kurzman IL, et al. Amputation and
    Carboplatin for Treatment of Dogs with Osteosarcoma (48 Cases). *J
    Vet Intern Med* 1996;10:76-81.

9.  **Bergman PJ**, Ogilvie GK, Powers BE. Monoclonal Antibody C219
    Immunohistochemistry in canine Lymphoma: Predictive Ability and
    Sequential Analysis. *J Vet Intern Med* 1996;10:354-359.

10. **Bergman PJ**, Gravitt KR, Ward NE, Gupta KP, O\'Brian CA. An
    N-myristoylated Protein Kinase C- Pseudosubstrate Peptide that
    Partially reverses Multidrug Resistance in Human Breast Cancer Cells
    is not a P-glycoprotein Substrate. *Cancer Chemotherapy
    Pharmacology* 1997;40:453-456.

11. McCaw DL, Miller MA, **Bergman PJ**, Withrow SJ, Moore AS, Knapp DW,
    Fowler D, Johnson JC. Vincristine therapy for mast cell tumors in
    dogs. *J Vet Intern Med* 1997;11:375-378.

12. **Bergman PJ**, Gravitt KR, Ward NE, Beltran P, Gupta KP, O'Brian
    CA. Potent induction of human colon cancer cell uptake of
    chemotherapeutic drugs by N-myristoylated protein kinase C-alpha
    (PKC-alpha) pseudosubstrate peptides through a
    P-glycoprotein-independent mechanism. *Inv New Drugs*
    1997;15:311-318.

13. **Bergman PJ.** Etiology of feline vaccine-associated sarcomas:
    history and update. Vaccine-Associated Feline Sarcoma Symposium. *J
    Amer Vet Med Assoc* 1998;213:1424-1425.

14. **Bergman PJ.** Advances in the Treatment of Feline
    Vaccine-Associated Sarcomas. *Adv Small Anim Med Surg* 2000.

15. O'Brian CA, Stewart JR, Ward NE, **Bergman PJ**. Regulatory
    mechanisms governing protein kinase C signaling. *Electr J Pathol
    Histol* 2000;6:1-10.

16. Rocha TA, Mauldin GN, Patnaik,AK, **Bergman PJ**. Prognostic Factors
    in Dogs with Urinary Bladder Carcinoma. *J Vet Intern Med*
    2000;14:486-490.

17. Kovak JR, Ludwig LL, Noone KE, **Bergman PJ**, et al. The use of
    thoracoscopy to diagnose pleural effusion of ambiguous etiology. *J
    Am Vet Med Assoc* 2002;221(7):990-994.

18. Charney SC, **Bergman PJ**, Hohenhaus AE, McKnight JA. Risk factors
    for sterile hemorrhagic cystitis in dogs with lymphoma receiving
    cyclophosphamide with or without concurrent administration of
    furosemide: 216 cases (1990-1996). J Am Vet Med Assoc
    2003;222(10):1388-1393.

19. **Bergman PJ**, McKnight J, Novosad A, Charney S, Farrelly J, Craft
    D, Sadelain M, Wulderk M, Jeffers Y, Hohenhaus AE, Segal N, Gregor
    P, Engelhorn M, Riviere I, Houghton AN, Wolchok JD. Long term
    survival of dogs with advanced malignant melanoma following DNA
    vaccination with xenogeneic human tyrosinase: A phase I trial. Clin
    Cancer Res 2003;9:1284-1290.

20. **Bergman PJ**. Clinical techniques in small animal molecular
    oncology. Clin Tech Small Anim Pract 2003;18:88-91.

21. Simpson AM, Ludwig LL, Newman SJ, **Bergman PJ**, Hottinger HA,
    Patnaik AK. Canine cutaneous mast cell tumors: A prospective study
    of surgical margins. *J Am Vet Med Assoc* 2004;224(2):236-240.

22. Farrelly J, Denman DL, Hohenhaus AE, Patnaik AK, **Bergman PJ**.
    Hypofractionated radiation therapy of oral melanoma in five cats.
    *Vet Radiol Ultras* 2004;45(1):91-94.

23. Newman SJ, **Bergman PJ**, Williams B, Scase T, Craft D.
    Characterization of Spindle Cell Component of Ferret (Mustela
    putorius furo) Adrenal Cortical Neoplasms -- Correlation to Clinical
    Parameters and Prognosis. *Comp & Vet Oncol* 2004;2(3):113-124.

24. Patnaik AK, Newman SJ, Scase T, Erlandson RA, Antonescu C, **Bergman
    PJ**. Canine hepatic neuroendocrine carcinoma: An
    immunohistochemical study of 10 cases. *Vet Pathol* 2005, in press.

25. Jakubiak MJ, Zenger E, Siedlecki CT, Matteucci ML, Bruskiewicz KA,
    Rohn DA, **Bergman PJ**. Laryngeal, laryngotracheal and tracheal
    masses in cats: 27 cases (1998-2003). *J Am Anim Hosp Assoc* 2005,
    in press.

26. Winston J, Craft DM, Scase TJ, **Bergman PJ**. Immunohistochemical
    detection of Her-2/neu expression in spontaneous feline mammary
    tumors. *Vet Comp Oncol* 2005, in press.

27. McAbee KP, Ludwig L, Newman S, **Bergman PJ**. Cutaneous
    Hemangiosarcoma in 18 cats. *J Am Anim Hosp Assoc* 2005, in press.

28. Allenspach K, Grone A, Doherr MG, **Bergman PJ**, Gaschen F.
    P-glycoprotein expression in lamina propria lymphocytes of duodenal
    biopsies in dogs with inflammatory bowel disease. *J Vet Intern Med*
    2005, submitted.

29. Charney SC, **Bergman PJ**, McKnight JA, Farrelly J, Novosad CA,
    Leibman NF, Camps-Palau MA. Evaluation of intracavitary mitoxantrone
    and carboplatin for treatment of carcinomatosis, sarcomatosis, and
    mesothelioma, with or without malignant effusions: A retrospective
    analysis of 12 cases (1997-2002). *Vet Comp Oncol* 2004, in press.

30. Novosad CA, **Bergman PJ**, O'Brien M, Charney SC, et al (VCOG).
    Retrospective evaluation of adjuvant chemotherapy for the treatment
    of feline mammary gland adenocarcinoma. *JAVMA* 2004, submitted.

# APPENDIX 6.5 {#appendix-6.5 .Heading-0}

![](media/image16.wmf)

![](media/image17.wmf){width="5.509027777777778in" height="7.6875in"}

# APPENDIX 6.6 {#appendix-6.6 .Heading-0}

![](media/image18.wmf){width="5.345833333333333in"
height="7.5472222222222225in"}

![](media/image19.wmf)

![](media/image20.wmf)

![](media/image21.wmf){width="5.603472222222222in"
height="8.05486111111111in"}

![](media/image22.wmf){width="5.351388888888889in"
height="7.811805555555556in"}

[^1]: FDA September 3, 2004 Press Release.

[^2]: McCall et al., 2004, "Recent Advances in Heartworm Disease",
    Veterinary Parasitology, Vol. 125, 105-130.

[^3]: Guerrero, et al., 2002, "The Use of Macrocyclic Lactones in the
    Control and Prevention of Heartworm and Other parasites in Dogs and
    Cats", Chapter 10, 353-369 in Macrocyclic Lactones in Antiparasitic
    Therapy, ISBN-0-85199-617-5.

[^4]: McCall, et al., 2001, "Evaluation of the Performance of Canine
    Heartworm Antigen Test Kits Licensed for Use by Veterinarians and
    Canine Heartworm Antigen Tests Conducted by Diagnostic Laboratories"
    in Recent Advances in Heartworm Disease, Symposium 2001, Babewia,
    IL, American Heartworm Society, 97-104.

[^5]: Roundtable discussion 2001, "Rethinking Prevention and Treatment
    Practices for Canine Heartworm Infection", Veterinary Healthcare
    Communications, Lenexa, Kansas , 2-15.

[^6]: Venco, et al., 2004, "Efficacy of Long-term Monthly Administration
    of Ivermectin on the Progress of Naturally Acquired Heartworm
    Infections in Dogs", Veterinary Parasitology, Vol. 124, 259-268.

[^7]: Kaiser and Williams, 2004, "*Dirofilaria immitis*: worm burden and
    pulmonary artery proliferation in dogs from Mighigan (United
    States). Veterinary Parasitology 124 (1-2), 125-129.

[^8]: American Heartworm Society Website (cited August 17, 2004)
    <http://www.heartwormsociety.org/MediaRelease.html>

[^9]: Verdon, DR, "Heartworm Survey: No Change in 10 Years", DVM
    Magazine, July 2002.

[^10]: Prichard, R., 2004, Pers. Comm.

[^11]: 0899-C-US-1-96, GASD 05-10.00, Prophylactic Activity of
    Moxidectin-SR Injectable Formulation Against *Dirofilaria immitis*
    Infections in Beagles

[^12]: 0899-C-US-2-96, GASD 05-14.00, Prophylactic Activity of
    Moxidectin-SR Injectable Formulation Against *Dirofilaria immitis*
    Infections in Mongrel Dogs.

[^13]: 0899-C-US-9-98, GASD 07-14.00, Three Year Study with Moxidectin
    Canine SR Injectable.

[^14]: 0899-C-US-10-98, GASD 07-03.00, Twelve Month Prophylactic
    Activity of Moxidectin Canine SR Injectable Formulation against
    *Dirofilaria immitis* Infections in Beagles.

[^15]: 0899-C-US-20-99, GASD 09-37.00, Dose Determination of the Twelve
    Month Prophylactic Activity against *Dirofilaria immitis* Infections
    in Mongrels with the Canine SR Injectable Formulation.

[^16]: 0899-C-US-11-98, GASD 08-09.00, Evaluation of Retroactive and
    Adulticidal Activity of Moxidectin Canine SR Injectable Formulation
    Against *Dirofilaria immitis* Infections in Beagles.

[^17]: 0899-C-US-28-01, GASD 09-30.00, Evaluation of Retroactive
    Activity of Moxidectin Canine SR Injectable Formulation Against
    Three-month Old *Dirofilaria immitis* Infections in Beagles.

[^18]: 0899-C-US-30-02, GASD 11-48.00, The Six Month Prophylactic
    Activity of ProHeart 6 (Moxidectin Canine SR Injectable) against
    *Dirofilaria immitis* Infections Administered to 12 Week Old
    Puppies.

[^19]: 0899-C-12-98, GASD 06-34.00, Efficacy of Moxidectin Canine SR
    Injectable against Canine Nematodes and Ticks.

[^20]: 0899-C-15-99, GASD 06-33.00, Efficacy of Moxidectin Canine SR
    Injectable against Experimental Hookworm Infections in Dogs in
    Georgia.

[^21]: 0899-C-16-99, GASD 06-31.00, Efficacy of Moxidectin Canine SR
    Injectable against Experimental Hookworm Infections in Dogs in
    Michigan.

[^22]: 0899-C-US-17-99, GASD 07-22.00, Efficacy of Moxidectin Canine SR
    Injectable Against Experimental Infections of Hookworms and
    Whipworms in Dogs in Michigan.

[^23]: 0899-C-US-18-99, GASR 06-01.00, Efficacy of Moxidectin Canine
    Sustained Release (SR) Injectable Against Larval/Immature Stages of
    Experimental *Trichuris vulpis* (Whipworms), *Ancylostoma caninum*
    and *Uncinaria stenocephala* (Hookworms) Infections of Dogs in New
    Jersey.

[^24]: 0899-C-US-19-99, GASD 07-04.00, Efficacy of Moxidectin Canine SR
    Injectable against Experimental Infections of *Uncinaria
    stenocephala* in Dogs in Georgia.

[^25]: 0899-C-US-4-98, GASD 06-15.00, Three Month Target Animal Safety
    Study (Toxicity) with Moxidectin Canine SR (Sustained Release)
    Injectable Formulation.

[^26]: 0899-C-US-3-98, GASD 06-23.00, A Reproductive Study of Moxidectin
    Canine SR Injectable in Female Beagle Dogs Following Subcutaneous
    Injection.

[^27]: 0899-C-CN-1-98, GASD 06-11.00, Effect of Moxidectin Sustained
    Release (SR) Injectable Solution on the Seminal Quality of Breeding
    Beagles.

[^28]: 0899-C-US-37-02, GASD 10-47.00, Safety Evaluation Study of
    ProHeart 6 (moxidectin canine sustained release injectable) in Ten
    Week Old Puppies.

[^29]: 0899-C-US-13-98, GASD 06-21.00, Clinical Observations from the
    Administration of Moxidectin Canine Sustained Release Injectable in
    Ivermectin-Sensitive Dogs.

[^30]: 0899-C-US-14-98, GASD 06-16.00, Clinical Observations from the
    Administration of Moxidectin Canine Sustained Release Injectable in
    Heartworm Positive Dogs.

[^31]: 0899-C-US-39-02, GASD 11-04.00, Clinical Observations Following
    the Administration of ProHeart 12 (Moxidectin Canine Sustained
    Release Injectable) Given at 3x to Dogs with Implanted Adult
    Heartworm (*Dirofilaria immitis*) Infections in Georgia.

[^32]: 0899-C-US-5-98, GASD 06-37.00, Safety and Efficacy Evaluation of
    Moxidectin Canine Sustained Release Injectable Formulation for the
    Prevention of Heartworm Disease in Dogs Under Conditions of Field
    Use.

[^33]: 0899-C-US-6-98, GASD 06-38.00, Safety and Efficacy Evaluation of
    Moxidectin Canine Sustained Release Injectable Formulation for the
    Prevention of Heartworm Disease in Dogs Under Conditions of Field
    Use.

[^34]: 0899-C-US-7-98, GASD 06-40.00, Safety and Efficacy Evaluation of
    Moxidectin Canine Sustained Release Injectable Formulation for the
    Prevention of Heartworm Disease in Dogs Under Conditions of Field
    Use.

[^35]: 0899-C-US-8-98, GASD 06-39.00, Safety and Efficacy Evaluation of
    Moxidectin Canine Sustained Release Injectable Formulation for the
    Prevention of Heartworm Disease in Dogs Under Conditions of Field
    Use.

[^36]: 0899-C-IT-01-99, GASD 08-04.00, Field Efficacy and Safety of
    Moxidectin Sustained Release Injectable in the Control of Infection
    Caused by the Nematodes *Dirofilaria immitis* and *Dirofilaria
    repens* and by Gastrointestinal Nematode in NW of Italy.

[^37]: 0899-C-IT-02-99, GASD 08-02.00, Field Efficacy and Safety of
    Moxidectin Sustained-Release Injectable in the Control of Infection
    Caused by the Nematode *Dirofilaria immitis* and *Dirofilaria
    repens* and by Gastrointestinal Nematode in Northern Italy.

[^38]: 0899-C-IT-03-99, GASD 08-06.00, Field Efficacy and Safety of
    Moxidectin Sustained-Release Injectable in the Control of Infection
    Caused by the Nematodes *Dirofilaria immitis* and *Dirofilaria
    repens* and by Gastrointestinal Nematode in the Centre of Italy.

[^39]: 0899-C-AU-01-97, GASD 06-42.00, GASD 06-42.01, Twelve Month
    Prophylactic Activity of 10% Moxidectin-SR Injectable Formulation
    Against *Dirofilaria immitis* in Mature Mixed Breed Dogs.

[^40]: 0899-C-AU-02-00, GASD 08-10.00, Safety Overdose Study of
    Moxidectin 10% Sustained Release Injectable in Dogs at 12 Weeks of
    Age.

[^41]: 0899-C-AU-02-98, GASD 06-45.00, Safety and Efficacy Evaluation of
    Moxidectin Sustained Release Injectable Formulation for the
    Prevention of Heartworm Disease in Dogs Under Conditions of Field
    Use (12 Month Results).

[^42]: Borden K. Postmarketing surveillance of drugs in the clinical
    research process in the pharmaceutical industry edited by Matoren,
    Marcel Dinker Publisher. 1984.

[^43]: Arrowsmith JB, Anello C. A view from a regulatory agency. In: BL
    Strom, ed. *Pharmacoepidemiology*. Churchill Livingstone; 1989.

[^44]: Carson JL, et al. Screening for unknown effects of newly marketed
    drugs. In: BL Strom, ed. *Pharmacoepidemiology*. Churchill
    Livingstone; 1989.

[^45]: Report GASD 11-16.00, "An Evaluation of Canine Sera for
    Immunoglobulins (IgE or IgG) to ProHeart 6 Components Using an ELISA
    Test System", July 2004.

[^46]: Final Report on Allergic Reactivity to ProHeart, L. Gershwin,
    DVM, Ph.D., January 2003.

[^47]: Gershwin, 2001, "Immunoglobulin E-mediated hypersensitivity in
    food-producting animals," Veterinary Clinics of North America, Vol.
    17(3), 4:599-619.

[^48]: Report GASD 11-17.00, "Intradermal Skin Testing with ProHeart 6
    and ProHeart 6 Components in Dogs Experiencing ProHeart 6
    Hypersensitivity Reactions", July 2004.

[^49]: Interim Study Summery, 0899-C-02-04-RES, "The Effect of MTM
    Residual Solvents on Intradermal Skin Tests in Dogs with no History
    of ProHeart 6 Sensitivity", October 2004.

[^50]: Supplier Quality Audit Report, Dow Chemical Co.,
    Hydroxypropylmethyl Cellulose, October 2001.

[^51]: Audit Report, Sasol (glyceryl tristearte), June 2002.

[^52]: Report Number 12387, "ProHeart 6 Cleaning Validation Report",
    July 2002.

[^53]: Mealey, et al., 2001, "Ivermectin Sensitivity in Collies is
    Associated with a Deletion of the MDR 1 Gene", Pharmacogenetics,
    Vol. 11(8), 727-733.

[^54]: Mealey and Bentjen, 2003, "Sequence and Structural Analysis of
    the Presumed Downstream Promoter of the Canine MDR 1 Gene."
    Veterinary and comparative Oncology, Vol. 1(1), 30-35.

[^55]: Mealey, et al., 2003, "Increased Toxicity of
    P-glycoprotein-Substrate Chemotherapeutic Agents in a Dog with the
    MDR 1 Deletion Mutation Associated with Ivermectin Sensitivity."
    Journal of the American Veterinary Association, Vol. 233(10),
    1453-1455.

[^56]: Nelson, et al., 2003, "Ivermectin Toxicity in an Australian
    Shepherd Dog with the MDR 1 Mutation Associated with Ivermectin
    Sensitivity in Collies." Journal of Veterinary Internal Medicine,
    Vol. 17(3), 354-356.

[^57]: Neff, et al., 2004, "Breed Distribution and History of Canine MDR
    1-1 Delta, a Pharmacogenetic Mutation that Marks the Emergence of
    Breeds from the Collie Lineage." Proceedings of the National Academy
    of Sciences of the United States of America, Vol. 101(32),
    11725-11730.

[^58]: Hugnet et al., 2004, "Frequency of the Mutant MRD 1 Allele
    Associated with Multidrug Sensitivity in a Sample of Collies from
    France." Journal of Veterinary Pharmacology and Therapeutics, Vol.
    27(4), 227-229.

[^59]: Roulet A., Bousquet-Meloub A, Concordet D., Dupuy J., Lespine A.,
    Alvinerie M., Pineau T, 1999, "Transport Studies of Macrocyclic
    Lactones in P-glycoprotein-recombinant Cells are Consistent with *in
    vivo-*measured Bioavailability Parameters in Several Species. WAAVP,
    Vol. 19, 254.

[^60]: Knowledge Systems and Research, Inc., 2000, Qualitative Research
    Report.

[^61]: American Animal Hospital Association, 2003, "The Path to
    High-Quality Care, Practical Tips for Improving Compliance", ISBN
    1-58326-048-X.

[^62]: Yabsley, et al., 2004, "Filarial Worm Infections in Shelter Dogs
    and Cats from Northwestern South Carolina, USA" Comparative
    Parasitology, Vol. 71, 154-157.

[^63]: Cummings, et al., 1995, "Evaluataion of Veterinary Dispensing
    Records to Measure "clinic compliance" with Recommended Heartworm
    Prevention Programs." Proceedings of the Heartworm Symposium,
    American Heartworm Society, 183-186.

[^64]: Table of Practice Compliance Rates, 36 Clinics in Michigan.

[^65]: Sartor et al., 2004, "Loperamide toxicity in a Collie with the
    MDR 1 Mutation Associated with Ivermectin Sensitivity." Journal of
    Veterinary Internal Medicine, Vol. 18(1):117-118.

[^66]: Roulet A., Puel O, Gesta S, Lepage JF, Drag M, Soll M, Alvinerie
    M., Pineau T, 2003, "MDRI-Deficient Genotype in Collie Dogs
    Hypersensitive to the P-glycoprotein Substrate Ivermectin. European
    Journal of Pharmacology, Vol. 460, 85-91.

[^67]: Njongmeta, et al, 2004, "Cattle Protected from Onchocerciasis by
    Ivermectin are Highly Susceptible to Infection After Drug
    Withdrawal," International Journal for Parasitology, Vol. 34(9),
    1069-1074.

[^68]: Retrospective Study on Heartworm Prevention, Tuskegee University,
    2004.