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The Treatment of Adrenal Cortical Disease in Ferrets with 4.7-mg Deslorelin Acetate Implants
Robert A. Wagner, VMD, Mark R. Finkler, DVM, Kellie A. Fecteau, PhD, and Tim E. Trigg, PhD

Abstract Thirty pet ferrets with adrenocortical disease (ACD) of varying severity and duration were evaluated for response to a single administration of a slow release 4.7 mg deslorelin acetate implant. Clinical response to deslorelin was monitored via a physical examination performed every 3 to 4 months. Adrenal ultrasound measurements were taken every 3-4 months until clinical relapse. At clinical relapse, duration of symptom suppression and adrenal size and growth were determined. Administration of a single 4.7 mg implant of deslorelin acetate resulted in signicant decreases in the clinical signs and hormonal concentrations associated with ACD. Within 14 days post-implant, vulvar swelling, pruritus, sexual behaviors and aggression decreased or disappeared. Hair re-growth was evident by 4-6 weeks post implant. Within two months post deslorelin implant, plasma concentrations of steroid hormones decreased: mean estradiol concentration decreased 28%; 17-hydroxyprogesterone levels decreased 89% and androstenedione levels decreased 88%. The response to a single 4.7 mg implant of deslorelin acetate was transitory. The mean SD time to recurrence of clinical signs was 17.6 5.0 months (range, 8.0-30.0 months). Repeated ultrasound measurements revealed no statistical difference in size of the adrenals (right or left) before, during the months of deslorelin implant and at clinical relapse. Slow release 4.7 mg deslorelin implants can effectively be used to temporarily eliminate the clinical signs and reduce steroid hormone concentrations in ferrets with ACD. This dose of deslorelin does appear to inuence adrenal tumor growth causing a decrease in adrenal size in some ferrets, and mild enlargement of adrenal glands in most ferrets with 2 of 30 implanted animals developing large tumors before clinical relapse. The long-term effect of treatment with deslorelin on adrenal tumor pathology requires additional investigation. At this time, surgical removal of the adrenal tumor remains the only curative treatment; however, 4.7 mg deslorelin implants are useful in the long-term management of ACD hormone-induced sequelae and may be as effective assurgical management. Copyright 2009 Elsevier Inc. All rights reserved. Key words: ferret; adrenocortical disease; adrenal tumor growth; hyperadrenocorticism

From the Division of Laboratory Animal Resources, Pittsburgh, PA, USA, Roanoke Animal Hospital, Roanoke, VA USA, Department of Comparative Medicine (Clinical Endocrinology Service), College of Veterinary Medicine, The University of Tennessee, Knoxville, TN USA, and Peptech Animal Health, Macquarie Park, NSW, Australia. Address correspondence to: Robert A. Wagner, VMD, Division of Laboratory Animal Resources, 3500 Terrace St, S1049 BST, University of Pittsburgh, Pittsburgh, PA USA 15261. E-mail: bwagner@pitt.edu. 2009 Elsevier Inc. All rights reserved. 1557-5063/09/1802-$30.00 doi:10.1053/j.jepm.2008.11.003

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Deslorelin Treatment of ACD in Ferrets

147 than those in sexually intact dogs.15 It is theorized that this continuous LH stimulus possibly plays a role in adrenocortical tumor formation in the dog.15 Certain strains of inbred and genetically engineered mice have been known to develop adrenal tumors within weeks to months after neutering, and prolonged elevation of LH is a prerequisite for such neoplastic transformation.16-19 A recent article described the similar histological changes and molecular markers shared by ferrets with ACD and these strains of mice.14 In short, neutering leads to an increase in circulating LH, which induces the ectopic expression of LH receptor (LHR) within adrenal tissue, triggering differentiation of adrenal tissue to the gonadal phenotype and culminating in the production of sex steroid hormones. An alternative theory on ACD formation in ferrets postulates that only an initial (not persistent) LH release after neutering is needed for progression of the disease.20-22 Instead, this LH surge in the presence of a tumor suppressor gene allows anaplasia to continue, going from hyperplasia to adenoma to adenocarcinoma. Indeed, recent research has detected a genetic marker (GATA-4) of anaplasia in ferret adrenocortical tumors.23 Similar molecular markers have been found in certain strains of mice that develop ACD after gonadectomy.24 GnRH analogs, at pharmacologic doses, suppress production and/or release of the LH and FSH by down regulating GnRH receptors at the pituitary.25 One GnRH agonist, leuprolide acetate, has been shown to reduce specic hormone production and eliminate hormone effects in ferrets with adrenal disease.26 Deslorelin acetate is another GnRH agonist that can be formulated into a long-acting, slow-releasing drug. In one study, deslorelin implants were shown to be effective in blocking estrus in jills for at least 1 year, with no side effects.27 When intact male ferrets were treated with deslorelin implants, testosterone levels were markedly suppressed, and plasma levels of LH and FSH were below normal (compared with placebotreated ferrets).10 A single 3-mg deslorelin implant was shown to eliminate clinical signs and reduce steroid sex hormone levels in ferrets with ACD for 13.7 3.5 months, although the implant did not appear to inuence tumor growth.28 A higher dose of deslorelin in a slow-release implant form could possibly prolong the clinical effect, presumably by prolonged suppression of LH. In the current study, a 4.7-mg deslorelin implant was used to determine its effect on clinical signs, sex steroid hormone levels, and tumor growth in ferrets with conrmed ACD.

drenal cortical disease (ACD) in domestic ferrets (Mustela putorius furo) is a common problem in neutered, middle-aged to older ferrets. The pathology varies from nodular hyperplasia to adenoma to adenocarcinoma, any of which can secrete various sex steroid hormones including estradiol, 17-hydroxyprogesterone, and androstenedione. Excess secretion of these hormones eventually leads to an apparent diminished quality of life. Common clinical signs associated with ACD include vulvar swelling in neutered females and bilaterally symmetrical alopecia in both sexes. Less common signs include pruritus (which can be intense), sexual activity or aggression, stranguria, and hind limb weakness. Males can develop prostatic cysts, prostatitis, and urethral obstruction. Females can develop a stump pyometra. Bone marrow suppression associated with chronic exposure to elevated estradiol levels can lead to anemia and thrombocytopenia. Occasionally, tumors continue to grow locally and can become space-occupying masses, impinging on other abdominal organs and vessels. Adenocarcinomas have the potential to invade adjacent tissues, metastasize, become necrotic and rupture.1-4 The high prevalence of ACD in pet ferrets has been associated with neutering at an early age (approximately 5 weeks in the United States). Delaying neutering until 6 months of age or older (as in The Netherlands) merely delays the age at time of diagnosis of ACD in a linear fashion.5 It has been speculated that the high incidence of ACD may be due to chronic stimulation of the adrenal cortex by the pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH).1,6 Sex steroid hormones are primarily produced in 2 locations: the gonads and the adrenal glands. Gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus stimulates release of pituitary gonadotropins LH and FSH. It is interesting that LH receptors are found not only in gonadal tissue but also in both normal and neoplastic adrenal tissue of ferrets, mice, and humans.6-9 In ferrets, these LH receptors have been shown to be functional because they secrete adrenal androgens in response to injection of a GnRH agonist.6 A hormonal ux occurs after neutering in a number of species. Plasma LH and FSH levels increase in both male and female ferrets after surgical neutering.10,11 Mice, rats, guinea pigs, and hamsters also develop a signicant increase in gonadotropin levels after neutering.12-15 In all 5 of these species, neutering is associated with sex steroidproducing adrenal tumors.6,12-15 In the spayed bitch and the castrated male, serum LH and FSH levels are 10 times higher

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Materials and Methods

Thirty pet ferrets with ACD of varying severity and duration were evaluated for response to a single administration of a slow-release 4.7-mg deslorelin acetate implant (Suprelorin; Peptech Animal Health Pty Limited, NSW, Australia). The ferrets were 3 to 6.5 years old (median age, 5 years) and weighed 0.63 to 1.54 kg. Fourteen ferrets were female and 16 were male; all animals had been castrated or spayed before 2 months of age. Three ferrets that had previously undergone left adrenalectomy for the treatment of ACD and had signs of relapse were included in the study. The diagnosis of ACD was conrmed in the ferrets on the basis of clinical ndings and results of a plasma hormone prole (Clinical Endocrinology Service, The University of Tennessee, College of Veterinary Medicine, Knoxville, TN USA). Clinical ndings included alopecia, pruritus, swollen vulva in females, increased sexual behavior, and aggression. A subjective assessment of the severity of clinical signs throughout the investigation was performed by comparing each ferret with a clinically normal ferret. Severity of alopecia was estimated (100% representing full normal pelage and 0% representing whole body alopecia) at every assessment and compared with the initial assessment. Pruritus, vulvar size, sexual behavior, and aggression were evaluated as normal, decreased, no change, or increased from the initial assessment and from the previous assessment. Clinical response to deslorelin was monitored via a physical examination performed every 3 to 4 months. At each examination, clinical evaluations and scoring of the severity of clinical signs in affected animals were performed. Ultrasound assessment and measurements of adrenal glands and deep abdominal palpation of the adrenal glands were taken at this time. Before treatment, blood was collected for plasma hormone analyses and ultrasound examination of adrenal glands was performed; these procedures were performed on the day of subcutaneous administration of the deslorelin implant. After implant treatment, blood was collected for plasma hormone analyses at 3 to 4 months in 18 of the 30 ferrets. Ultrasound examinations and abdominal palpation were performed every 3 to 4 months until relapse of clinical signs was noted. Plasma samples obtained were analyzed for estradiol, androstenedione, and 17-hydroxyprogesterone concentrations. Plasma hormone concentrations at 3 to 4 months after implant were averaged. Each ferret was treated with one 4.7-mg deslorelin acetate implant (Suprelorin; Peptech Animal Health Pty Limited), which was inserted subcutaneously via

a single-use syringe and a 14-gauge needle at the dorsal aspect of the base of the neck. The injection site was then sealed with a veterinary tissue adhesive. Dosages ranged from 3.05 to 7.46 mg/kg. The implant is manufactured by extruding deslorelin with a matrix from low-meltingpoint lipids and biological surfactant. The implant is 10 mm long and 2.3 mm in diameter and releases doses of 1 g/d for periods of 1 year in dogs (Suprelorin; Peptech Animal Health Pty Limited). The in vivo release rate in ferrets has not been determined. Implants can be removed surgically. Steroid hormone concentrations were determined in plasma for estradiol, androstenedione, and 17-hydroxyprogesterone. Plasma hormone concentrations were measured via commercially available radioimmunoassays validated for use in ferrets (Clinical Endocrinology Service, The University of Tennessee, College of Veterinary Medicine, Knoxville, TN USA). Plasma hormone concentrations before deslorelin treatment were compared with reference ranges as follows: estradiol, 30 to 180 pmol/L; androstenedione, 1 to 15 nmol/L, and 17-hydroxyprogesterone, 0.05 to 0.8 nmol/L (Clinical Endocrinology Service, The University of Tennessee, College of Veterinary Medicine, Knoxville, TN). These values of plasma hormone concentrations before deslorelin treatment were used in conjunction with clinical signs to conrm a diagnosis of ACD.

Statistical Methods
Plasma hormone concentrations were measured in samples obtained before and 3 to 4 months after deslorelin treatment. Pretreatment and posttreatment hormone concentrations were averaged; these mean values were used in a repeated measures analysis to compare plasma hormone values before treatment and after treatment. The model contained the variable stage (before treatment and after treatment), with ferret being considered as the experimental unit. Results were log transformed before statistical analysis, and data are presented as geometric means SEM. This transformation was necessary to meet the assumption of normality and homogeneity of variance. In addition, the means (before treatment and after treatment) were tested against the upper reference limit by use of a t test. A multivariate repeated measures analysis of variance was used to evaluate differences in right and left adrenal length and width before and 3 to 6, 9 to 12, 15 to 18, and 21 to 24 months after deslorelin treatment. Statistical analyses were performed with SPSS (SPSS, Inc., Chicago, IL USA). Data are presented as mean SEM. A value of P .05 was considered signicant.

Deslorelin Treatment of ACD in Ferrets

149 SD time to recurrence of clinical signs was 17.6 months (range, 8.0-30.0 months). 5.0

Results
Clinical Findings
Before treatment, all 30 ferrets had clinical signs consistent with ACD. The most common ndings were bilateral, symmetric, truncal alopecia in males and females and vulvar swelling of at least 2 months duration in females. No ferrets had palpably enlarged adrenal glands at the time of administration of the deslorelin implant. No adverse effects from deslorelin implants were noted during the study. Twelve of 30 ferrets had varying degrees of pruritus before treatment; the severity of the pruritus was greatly decreased in all 12 animals by 2 weeks after treatment. Sexual behaviors or aggression observed in 8 ferrets before treatment were greatly decreased or eliminated by 2 weeks after treatment. Fourteen ferrets had a swollen vulva before treatment; 10 to 14 days after treatment vulvar turgidity was decreased, and by 6 weeks after treatment all 14 ferrets had a normal-appearing vulva. Prostatitis was diagnosed in 5 of 16 ferrets (determined on the basis of clinical signs and results of urinalysis). These ferrets were treated orally with trimethoprim sulfamethoxazole (30 mg/kg, every 12 hours, for 21 days). All 5 ferrets were managed successfully during the study period and showed no signs of prostatitis relapse. By 6 weeks after treatment, most ferrets had regrowth of hair. Twenty-eight of 30 ferrets initially had 40% to 60% pelage; by 8 weeks after treatment, these ferrets had 90% to 100% coat cover. The remaining 2 ferrets initially had 40% and 50% pelage; by 12 weeks after treatment, these ferrets had 70% to 80% pelage, which was maintained throughout the study period. Four ferrets had thin, incomplete regrowth of the hair on the tail. Twenty-ve ferrets had relapse of clinical signs after deslorelin treatment. Five ferrets were euthanized before clinical relapse; 2 ferrets had hepatic carcinoma, 1 had un-controllable insulinoma, 1 had renal failure, and 1 had a large, necrotic adrenal tumor. The mean

Plasma Hormone Assay Results

Before deslorelin treatment, 18 of 30 ferrets had plasma hormone analyses performed and were consistent with a diagnosis of ACD. In 16 of 18 ferrets, at least 2 of the 3 hormones assessed were greater than the upper reference limit. After implantation, 4 of 18 ferrets had plasma hormone concentrations near or slightly above the upper reference limits for estradiol. Compared with values before treatment, mean plasma 17-hydroxyprogesterone concentrations decreased signicantly after treatment in all but 1 ferret. All plasma androstenedione concentrations were signicantly decreased after treatment compared with pretreatment values. Within 3 to 4 months after deslorelin implant, mean plasma concentrations of steroid hormones decreased: estradiol concentration decreased 32%; 17-hydroxyprogesterone levels decreased 91%; and androstenedione levels decreased 93%. Geometric mean SEM plasma hormone concentrations before and 3 to 4 months after treatment were calculated (Table 1). Pretreatment and posttreatment mean plasma estradiol concentrations in the ferrets were 179.54 1.12 pmol/L and 130.35 1.07 pmol/L, respectively. After treatment, estradiol concentration was signicantly (P .016) lower than the value before treatment and below the upper reference limit of 180 pmol/L (Clinical Endocrinology Service, The University of Tennessee, College of Veterinary Medicine, Knoxville, TN). Pretreatment and posttreatment mean plasma androstenedione concentrations in the ferrets were 41.68 1.07 nmol/L and 4.81 1.19 nmol/L, respectively. After treatment, androstenedione concentration was signicantly (P .001) lower than the value before treatment and below the upper reference limit of 15 nmol/L (Clinical Endocrinology Service, The University of Tennessee, College of Veterinary Medicine, Knoxville, TN). Pretreatment and

Table 1. Effect of deslorelin (4.7-mg implant) on hormone concentrations* in ferrets with adrenal cortical disease
_Hormone Estradiol (pmol/L) Androstenedione (nmol/L) 17-OH Progesterone (nmol/L) Pretreatment 179.54 41.68 2.27 1.12 1.07 1.31 Posttreatment 130.35 4.81 0.24 1.07 1.19 1.15
SEM.

P Value .016 .001 .001

*Hormone concentrations were log transformed prior to statistical analysis. Values provided are the geometric means Pre-treatment and post-treatment hormone concentrations are signicantly different (P .05).

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Table 2. Right adrenal gland size (cm) in ferrets before (0) and during (mo) deslorelin treatment
Months* 0 3-6 9-12 0 15-18 0 21-24 Observations (N) 18 18 18 15 15 11 11 Length Mean ( SEM) 0.73 0.85 0.89 0.78 0.77 0.84 1.30 0.05 0.12 0.16 0.07 0.06 0.07 0.34 Width Mean ( SEM) 0.62 0.79 0.78 0.69 0.65 0.69 0.98 0.04 0.19 0.21 0.04 0.06 0.06 0.25 P Value .54 .54 .54 .63 .63 .50 .50

*Ferrets were grouped based on time of measurement. There were no signicant differences (P .05) in adrenal gland size before and during the months of deslorelin treatment.

posttreatment mean plasma 17-hydroxyprogesterone concentrations in the ferrets were 2.27 1.31 nmol/L and 0.24 1.15 nmol/L, respectively. After treatment, 17-hydroxyprogesterone concentration was signicantly (P .001) lower than the value before treatment and below the upper reference limit of 0.8 nmol/L (Clinical Endocrinology Service, The University of Tennessee, College of Veterinary Medicine, Knoxville, TN).

.63); left adrenal size before and after treatment was not signicantly different (P .83). Four ferrets adrenals shrank in size, and 24 ferrets adrenals increased in size an average of 15% before clinical relapse. Two of 30 (6%) implanted animals developed large tumors ( 1 cm) before clinical relapse.

Discussion
This study measured the efcacy and safety of a longacting deslorelin therapy to treat ACD or alleviate its symptoms in ferrets of both sexes. One important aspect of this study was to monitor adrenal size via ultrasound examination to determine deslorelin implant effect on adrenal growth. The study also reports on the hormonal changes associated with the treatment, which have been speculated to be associated with chronic LH and FSH stimulation of the adrenal cortex.

Adrenal Ultrasound Measurement Results

All 30 ferrets had pretreatment and posttreatment adrenal measurements. Two ferrets missed 1 adrenal measurement interval, and 4 ferrets missed 2 adrenal measurement intervals. Adrenal length and width before and 3 to 6, 9 to 12, 15 to 18, and 21 to 24 months after deslorelin treatment are presented in Tables 2 and 3. Right adrenal gland size before and after treatment was not signicantly different (P

Table 3. Left adrenal gland size (cm) in ferrets before (0) and during (mo) deslorelin treatment
Months* 0 3-6 9-12 0 15-18 0 21-24 Observations (N) 18 18 18 15 15 11 11 Length Mean ( SEM) 0.68 0.73 0.66 0.60 0.61 0.55 0.57 0.03 0.04 0.03 0.03 0.40 0.03 0.05 Width Mean ( SEM) 0.56 0.55 0.55 0.59 0.57 0.61 0.59 0.04 0.03 0.04 0.04 0.05 0.05 0.04 P Value .08 .08 .08 .83 .83 .76 .76

*Ferrets were grouped based on time of measurements. There were no signicant differences (P .05) in adrenal gland size before and during the months of deslorelin treatment.

Deslorelin Treatment of ACD in Ferrets

151 some ferrets. Tumors that are initially hormone dependent can progress over time to a hormone-resistant state and may grow regardless of the removal of stimulatory hormones.38 In such instances, down regulation of GnRH receptors will not prevent further tumor growth. The effectiveness of deslorelin in ferrets with specic types of adrenal gland disease and its effect on tumor growth need to be further evaluated. The time to relapse of the 25 ferrets to clinical ACD was quite variable though not unexpected, as similar results have been shown in many species including dogs of both sexes.30,31 The cause is unknown; however, quality control technology used on the implants is thorough and release from individual implants is consistent. Similar variability has been seen in other studies,29 so the possibility of within-species variation in response to treatment cannot be discounted. Repeat implantation was not the focus in this study, but 7 ferrets were reimplanted after clinical relapse and successfully maintained clinical remission. Slow-release 4.7-mg deslorelin implants can effectively be used to temporarily eliminate the clinical signs and reduce steroid hormone concentrations in ferrets with ACD. This dose of deslorelin does appear to inuence adrenal growth, causing a reduction in adrenal size in some ferrets and mild enlargement of adrenal glands in most ferrets. Only 2 of 30 implanted animals developed large tumors before clinical relapse. The long-term effect of treatment with deslorelin on adrenal tumor pathology requires additional investigation. At this time, surgical removal of the adrenal tumor remains the only curative treatment; however, 4.7-mg deslorelin implants are useful in the long-term management of ACD hormone-induced sequelae and may be as effective as surgical management.

In many species, GnRH agonists delivered continuously in small doses over long periods of time suppress the reproductive endocrine system,29 preventing production of pituitary (LH and FSH) and gonadal hormones (estradiol and progesterone in females and testosterone in males). The observed effects are similar to those after ovariectomy or castration, but are reversed after the hormone content of the implant is depleted. To date, trials with deslorelin acetate have focused primarily on domestic dogs,30 although there has been one controlled study in domestic cats32 and preliminary data from a number of exotic species.33-35 These investigations have not detected any adverse effects, although weight gain was seen occasionally, as is often the case after castration or ovariectomy. In all cases where reversibility was tested, reproductive function was fully restored.30,31 Recent results from tests of another GnRH agonist with prepubertal domestic cats found that estrous cycles occurred after treatment ended. Treatment of ferrets in this study was shown to be clinically effective and safe in all animals. This is in agreement with the data obtained from dogs.31 The effects of the drug were impressive and, after 2 weeks, purititis and vulvar swelling were much reduced in the affected animals. With the latter, swelling had disappeared in 6 weeks. This is consistent with the rapid reduction in circulating sex steroid hormones seen after treatment in this study and in studies with dogs.31 The reduction or disappearance of sexual behavior over 14 days is also indicative of a rapid reduction of circulating gonadotrophins as seen in most mammals treated with long-acting GnRH agonists. Although the concentrations of LH were not measured in the present study, the elimination of ACD clinical signs suggests that LH concentrations were decreased below the threshold necessary to stimulate adrenal production of sex hormones. Reduction in LH after deslorelin acetate administration has been seen in dogs36 and bulls.37 The further reduction of steroid sex hormones over 3 to 4 months indicates the effectiveness of deslorelin in reducing these hormones to below the upper reference level for ACD. The effect on the one ferret that maintained its high level of 17 hydroxyprogesterone is unknown but may be related to dose and individual sensitivity to the GnRH analog. There was no signicant growth or enlargement of adrenal gland size, suggesting that the direct or indirect effects of deslorelin acetate controls adrenal mass or adrenal growth as well as adrenal hormone activity. Deslorelin did not control adrenal growth in all ferrets, suggesting that the effect is lost after some time in

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