Naturally occurring hyperadrenocorticism is a wellrecognized

Evaluation of twice-daily lower-dose trilostane treatment administered orally in dogs with naturally occurring hyperadrenocorticism Edward C. Feldman, dvm, dacvim Objective To evaluate effectiveness and incidence of adverse reactions to twice-daily lower-dose oral administration of trilostane in the treatment of dogs with naturally occurring hyperadrenocorticism (NOH). Design Clinical trial. Animals 47 dogs with NOH. Procedures 47 dogs were treated orally with trilostane (0.21 to 1.1 mg/kg [0.1 to 0.5 mg/ lb], q 12 h). All dogs were reevaluated at 2 weeks and 2 months, 38 dogs at 6 months, and 28 dogs at 1 year of treatment. Results 9 of 47 dogs had an adrenocortical tumor causing NOH, and all had good responses after 2 months (mean trilostane dosage, 0.89 mg/kg [0.40 mg/lb], q 12 h). All successfully underwent surgical adrenal tumor extirpation. Thirty-eight dogs had pituitarydependent hyperadrenocorticism (PDH); 15 dogs did not require a dose increase during the study, and at each of 4 reevaluations, 10 of 15, 13 of 15, 14 of 15, and 11 of 11 had a good response. Twenty-three dogs with PDH had their dose or frequency of trilostane administration increased during the study. Mean trilostane dosage at 1-year reevaluation in dogs with a good response was 1.7 mg/kg (0.8 mg/lb), twice daily, or 1.1 mg/kg, 3 times daily. At each of 4 reevaluations, 17 of 23, 14 of 23, 17 of 23, and 13 of 17 dogs with PDH had a good response. Five dogs became ill because of trilostane-induced adverse effects, but only 1 required hospitalization. Conclusions and Clinical Relevance Administration of initial lower doses of trilostane to dogs with NOH is effective. (J Am Vet Med Assoc 2011;238:1441 1451) Naturally occurring hyperadrenocorticism is a wellrecognized endocrine disorder in dogs. It is estimated that approximately 85% of dogs with hyperadrenocorticism have PDH because of secondary bilateral adrenocortical hyperplasia and that 15% have ATH as a result of a cortisol-secreting adrenocortical adenoma or carcinoma. 1,2 In dogs with either PDH or ATH, clinical signs are the result of chronic excesses in circulating glucocorticoids. At present, one of the most common medical treatments for dogs with NOH is administration of mitotane (o,p -DDD); its use has been recommended for more than 3 decades. 3 The effectiveness of mitotane in dogs with ATH is variable. Clinical signs are controlled in about 80% of dogs with PDH treated with this drug. 1,4 6 Treatment with mitotane is, however, associated with potential adverse effects and disadvantages, such as transient hypoadrenocorticism, permanent mineralocorticoid and glucocorticoid deficiencies, drug intolerance, and relapses. 4 6 The most promising new drug for treating dogs with NOH is trilostane. a Trilostane is a 4α, 5-epoxysteroid competitive inhibitor of the 3β-hydroxysteroid From the Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616. Supported in part by donations from the late Ruth Johnston, San Mateo, Calif. The author thanks Dr. Richard W. Nelson for technical assistance. Address correspondence to Dr. Feldman (ecfeldman@ucdavis.edu). ATH LDDST NOH PDH UCCR Abbreviations Adrenocortical tumor associated hyperadrenocorticism Low-dose dexamethasone suppression test Naturally occurring hyperadrenocorticism Pituitary-dependent hyperadrenocorticism Urine cortisol-to-urine creatinine concentration ratio dehydrogenase-isomerase enzyme system that, following oral administration, actively interferes with metabolic pathways in which progesterone is involved and blocks the synthesis of end products, including cortisol and aldosterone. 7 9 Decades ago, 3 to 4 daily doses of trilostane were typically administered to humans with NOH, generally with poor results. 9 11 However, in dogs with PDH, the efficacy of trilostane administered once daily is reported to be similar to that of mitotane, and trilostane has been effective in diminishing hypercortisolemia in dogs with functional adrenocortical tumors. 12 16,b,c Currently, the dosage of trilostane recommended by the manufacturer for use in dogs is approximately 3 to 6 mg/kg (1.36 to 2.73 mg/lb) given orally once each day. Most investigations of this drug s treatment in dogs have involved this once-daily dose protocol. Interestingly, in some dogs with either good or poor control of NOH, the UCCR 24 hours after once-daily trilostane administra- JAVMA, Vol 238, No. 11, June 1, 2011 Scientific Reports 1441

tion is increased, suggesting that trilostane does not consistently suppress circulating cortisol concentration for as long as 24 hours. 17 In dogs for which there is an inadequate duration of effect, a twice-daily dosing regimen has been recommended. 13,15,17 In a review of 8 studies 13 19,d involving 191 dogs treated once daily with trilostane, 42 (22%) dogs became sufficiently ill to have their adverse effects reported. Usually, the adverse effects included anorexia, vomiting, diarrhea, and weakness. At least 6 of the 191 (3.1%) dogs were described as having died of the effects of trilostane. In the manufacturer s package insert 20 for trilostane, it is reported that 8 healthy dogs given 3 to 5 times the recommended maximum dosage of 6.7 mg/kg, twice daily, died between 23 and 46 days of daily dosing. In this same insert, results of a US field study on 107 treated dogs are reported, indicating that 2 dogs had adrenal necrosis and subsequent adrenal rupture because of the effects of trilostane and that 1 of these 2 dogs died. It is also reported that 2 dogs developed hypoadrenocorticism, a permanent condition for 1 of the 2 dogs. Further, it is reported that additional adverse reactions were observed in 93 dogs; many of these reactions were likely due to decreases in circulating concentrations of glucocorticoids or mineralocorticoids secondary to trilostane administration. This resulted in at least 4 of those 93 dogs being permanently removed from the drug. Thus, in the short-term, 97 of 107 dogs had adverse reactions, many of which were attributable to trilostane. In this same insert, it is stated that in a longterm follow-up of the US field study dogs, an additional 3 dogs were described as having died, possibly related to trilostane. 20 In 4 previously published reports, the dosages of trilostane administered orally once daily at the end of the study periods were 5.3 to 50 mg/kg (2.41 to 22.73 mg/lb), 15 4.4 to 16 mg/kg (1.82 to 7.27 mg/lb), 14 1.9 to 8.2 mg/kg (0.86 to 3.73 mg/lb), 17 and a mean of 3.2 to 11.4 mg/kg (1.45 to 5.18 mg/lb). 13 In another study 21 to evaluate twice-daily oral administration of trilostane in dogs, adverse reactions attributable to glucocorticoid or mineralocorticoid deficiencies were detected in 11 of 44 (25%) treated dogs, and the mean dosage at the end of the study period was 3.2 mg/kg, twice daily (total daily dose, 6.4 mg/kg [2.91 mg/lb]). In a study 19 that evaluated twice-daily low-dose oral administration of trilostane in dogs, 8 and 15 of 22 dogs had a good response after 1 to 2 weeks and 4 to 8 weeks of treatment, respectively. Adverse reactions attributable to glucocorticoid or mineralocorticoid deficiencies were detected in 2 of the 22 (9%) dogs, and the mean dosages at the end of the study period were 1.9 mg/kg, twice daily (total daily dose, 3.8 mg/kg [1.73 mg/lb]), in 13 dogs and 1.3 mg/kg (0.59 mg/lb), 3 times daily (total daily dose, 3.9 mg/kg [1.77 mg/lb]), in 3 dogs. 19 On the basis of the observation of a rapid but transient response (< 12 hours) to a low dose of trilostane 19 and also on the basis of the incidence of serious adverse effects caused by trilostane as reported by several authors and the company distributing trilostane, 13 21,d investigation of a lower-dose regimen was warranted. Further, on the basis of the experience of 1 group that used a twice-daily treatment regimen 21 and previously reported results on the duration of trilostane effect, 19 twice-daily administration of trilostane appears warranted. The purpose of the study reported here was to evaluate the effects of twice-daily lower-dose trilostane treatment administered orally in dogs with NOH. Materials and Methods Animals The study included dogs that were initially evaluated at the Veterinary Medical Teaching Hospital of the University of California-Davis, from April 2007 through December 2008. All dogs were enrolled in the study with the informed consent of their owners. The diagnosis of NOH was suspected from a review of historical data and physical examination results. Each dog had polyuria as an owner concern, and each had at least 4 of the following 6 clinicopathologic findings: high serum alkaline phosphatase activity, high serum alanine aminotransferase activity, hypercholesterolemia, low BUN concentration or BUN concentration near the lower reference limit (low-normal value), urine specific gravity < 1.020, and microbial growth on bacteriologic culture of urine. None of the dogs had a BUN concentration > 25 mg/dl, and none had received prior treatment for NOH. Each dog underwent abdominal ultrasonography. e For each dog, results of at least 2 of 3 screening tests (ie, ACTH stimulation test, LDDST, or assessment of UCCR) were consistent with NOH. The diagnosis of PDH was made if a dog had at least 2 of the following 3 diagnostic test results: an LDDST result indicative of PDH, ultrasonographic evidence of 2 relatively equal-sized adrenal glands, or plasma concentration of endogenous ACTH > 45 pg/ml. 22 27 The diagnosis of ATH was made if all of the following were applicable: abnormal LDDST result, a low or undetectable plasma concentration of endogenous ACTH, ultrasonographic evidence of an adrenal mass, and histologic confirmation of an adrenocortical tumor (adenoma or carcinoma) following examination of adrenal mass tissue removed during celiotomy. 1 Treatment and assessments Initial trilostane dosage for each dog was 0.2 to 1.1 mg/kg (0.1 to 0.5 mg/lb) administered orally every 12 hours. Capsules provided by the manufacturer (30 and 60 mg) were used whenever possible. If needed, capsules with the calculated dose were prepared by a compounding pharmacy using trilostane purchased from the manufacturer. Each owner of a dog enrolled in the study agreed to return the dog for reevaluation after 1 to 2 weeks of treatment, then at 6 to 7 weeks after the first reevaluation (approx 2 months after initiation of treatment), 15 to 17 weeks after the second reevaluation (approx 6 months after initiation of treatment), and 24 to 28 weeks after the third evaluation (approx 1 year after initiation of treatment). Dogs with a diagnosis of PDH must have been treated for at least 6 months after trilostane initiation or until illness caused discontinuation of treatment or death. If a dog with ATH was scheduled to undergo surgery for extirpation of an adrenal gland tumor, owners agreed to return the dog for reevaluation at the same intervals as for dogs with PDH or until surgery, whichever came first. All dogs with ATH must have been treated with trilostane for at least 2 months for inclusion. 1442 Scientific Reports JAVMA, Vol 238, No. 11, June 1, 2011

On the mornings of each reevaluation, owners were instructed to obtain a free-catch urine sample from the dog, if possible, for analysis. Owners were also asked to feed their dog within an hour after urine sample collection and to administer trilostane at that morning s meal (usually between 7 am and 9 am). Dogs were to be brought to the hospital about 3 hours after trilostane administration, at which time an ACTH stimulation test would be started and completed. Urinalysis and UCCR assessments were to be performed on the urine sample collected by the owner. If adverse reactions were observed during the first year of treatment, at times other than planned reevaluations, the event was assigned to the temporally closest of the 4 planned reevaluations. Some dogs, after 6 months of treatment, were returned to the referring veterinarian for continued monitoring of treatment. Data from dogs monitored outside our hospital were not included in this study. At each reevaluation, owners were questioned about their dog s general well-being, changes in clinical signs, and any adverse effects of treatment. Also, at each evaluation, a physical examination and an ACTH stimulation test were completed. A urinalysis was performed if owners were able to bring in a urine sample. For each dog at each visit, response to treatment was classified by owners as good, poor, or ill. A good response was defined as resolution of or marked improvement in clinical signs related to hyperadrenocorticism (ie, reduction in or absence of polydipsia, polyuria, polyphagia, and panting and an increase in muscle strength and activity; hair regrowth was not among the goals of treatment). A poor response was defined as persistence of unacceptable clinical signs of hyperadrenocorticism (eg, polyuria). Dogs were considered ill if signs of anorexia, vomiting, diarrhea, unusual listlessness, or unusual weakness were present. The trilostane dose or administration frequency was altered, as needed, on the basis of owner observations, physical examination findings, or test results. When possible, each dog was monitored by the same clinician throughout the study. Whenever there was discordance between test results and the owner s opinion, the latter was given greater weight in determining whether a change in trilostane dose or administration frequency was warranted. As an aid to resolution of clinical signs without causing illness, treatment was directed, in part, to achieve post-acth stimulation serum cortisol concentration 1.5 and 5.5 µg/dl. If a dog had a post-acth stimulation serum cortisol concentration < 1.5 µg/dl and no adverse clinical signs, the trilostane dosage was either unchanged or decreased at the discretion of the veterinarian. No change in trilostane dosage was made if a dog s response to treatment was classified as good and test results supported that determination. If a dog s response to treatment was classified as poor and its post- ACTH stimulation serum cortisol concentration was > 5.5 µg/dl at the first reevaluation, the dosage of trilostane was maintained or increased at the discretion of the veterinarian; if these findings were present at the second or third reevaluation, the dose of trilostane was typically increased by 10% to 50%. If a dog s response to treatment was classified as poor and its post-acth stimulation serum cortisol concentration was 5.5 µg/dl, it was usually recommended to the owner that the frequency of trilostane administration be increased (q 8 h) while each dose was unchanged or decreased. If a dog was described by an owner as ill, trilostane administration was discontinued until the dog was considered well (usually 2 to 5 days) and then recommenced after reduction of each dose by 25% to 50%. If possible, a blood sample was collected from ill dogs for hematologic and serum biochemical analyses (including BUN, potassium, and sodium concentrations) and a urine sample was collected for urinalysis. If an owner could not bring an ill dog to the hospital, it was suggested that those assessments be made by a local veterinarian. Endocrine tests For ACTH stimulation tests, blood samples (2 ml each) were collected before and 1 hour after IM administration of synthetic ACTH f (0.25 mg) for measurement of serum cortisol concentration. Serum cortisol concentration > 22 µg/dl (607 nmol/l) at 1 hour after administration of ACTH was considered consistent with NOH. g For the LDDST, blood samples (2 ml each) were collected before and 4 and 8 hours after IV administration of dexamethasone (0.01 mg/kg [0.005 mg/lb]) for determination of serum cortisol concentration. Serum cortisol concentration > 1.4 µg/dl at 8 hours after dexamethasone administration was considered consistent with naturally occurring PDH or ATH. 1,28 Serum cortisol concentrations < 1.4 µg/dl at 4 hours or < 50% of basal concentration at 4 or 8 hours after dexamethasone administration were considered consistent with PDH. 1,28 Values of UCCR 13.5 X 10 6 were considered consistent with a diagnosis of NOH. 29 Hormone assays Blood samples obtained for determination of plasma concentrations of endogenous ACTH were collected, stored, and assayed as previously described. 1 Serum and urine cortisol concentrations were measured by use of a commercial cortisol radioimmunoassay h that has been validated for use in dogs. 28 The analytic sensitivity of this radioimmunoassay was 0.3 µg/dl (8.3 nmol/l). Statistical analysis Data were analyzed by use of a commercially available statistical program. i To compare findings between groups, paired Student t tests as well as 1-way ANOVAs were performed as indicated. The Bonferroni method was used for post hoc analysis to adjust for multiple comparisons. A value of P < 0.05 was considered significant. Results are presented as mean ± SD. Results Dogs Forty-seven dogs with NOH were included in the study. The dogs were 3 to 15 years old (mean, 10.40 ± 2.26 years). Mean weight of the dogs was 20.7 ± 12.7 kg (45.5 ± 27.9 lb; range, 4.9 to 42.1 kg [10.8 to 92.6 lb]). Included were 38 dogs with PDH and 9 dogs with ATH. Mean ages of the dogs with ATH (10.44 ± 1.42 years) and the dogs with PDH (10.34 ± 2.85 years) were not significantly different. Mean body weights of the dogs with ATH (19.43 ± 12.78 kg [42.75 ± 28.12 lb]; range, 8.1 to 38.4 kg [17.82 ± 84.48 lb]) and the dogs with PDH (20.42 ± 10.44 kg [44.92 ± 22.97 lb]; range, 4.9 to 42.1 kg [10.78 ± 92.62 lb]) were not significantly different. JAVMA, Vol 238, No. 11, June 1, 2011 Scientific Reports 1443

Clinical signs and endocrine screening test results The most common clinical signs initially reported by the owners of the 47 dogs were polyuria (n = 47), polydipsia (45), polyphagia (40), lethargy or weakness (35), panting (27), abdominal distention (24), and dermatologic abnormalities (eg, alopecia, thin skin, or hyperpigmentation; 21). Mean urine specific gravity before treatment was 1.009 (median, 1.009). Among the 47 dogs, 39 had abnormal results of 2 endocrine screening tests and 8 had abnormal results of all 3 endocrine screening tests. Results were abnormal in 15 of 33 ACTH stimulation tests, 43 of 43 assessments of UCCR, and 43 of 44 LDDSTs. Treatment evaluation of all 47 dogs Mean initial dosage of trilostane for all 47 dogs with NOH was 0.86 mg/kg (0.39 mg/lb, q 12 h). The mean initial dosage of trilostane for the ATH dogs (0.89 ± 0.16 mg/kg [0.40 ± 0.07 mg/lb], q 12 h) was not significantly different from the initial dosage of trilostane for the PDH dogs (0.84 ± 0.22 mg/kg [0.38 ± 0.10 mg/lb], q 12 h). The goal of treatment for each dog with ATH was to minimize the incidence of adverse reactions associated with anesthesia and surgical removal of an adrenocortical tumor commonly encountered in dogs with NOH. Each of the 9 ATH dogs successfully underwent adrenal tumor extirpation 8 to 10 weeks after beginning trilostane treatment. The goal of treatment for the PDH dogs was long-term resolution of clinical signs. Therefore, response to trilostane treatment in the dogs with ATH was arbitrarily evaluated separately from the response to treatment in the dogs with PDH. Treatment evaluation of dogs with ATH Initial dosage of trilostane in the 9 dogs with ATH was 0.89 ± 0.16 mg/kg, twice daily, with a range of 0.54 to 1.1 mg/ kg (0.25 to 0.50 mg/lb), twice daily. Treatment findings among all 9 dogs with ATH at each reevaluation were assessed (Table 1). At the first reevaluation, treatment response was good in 4 dogs and poor in 5 dogs. Seven of the 9 dogs with ATH underwent a pretreatment ACTH stimulation test (serum cortisol concentration, 20.9 ± 13.4 µg/dl; range, 5.3 to 42.4 µg/dl), and all had a lower post-acth stimulation serum cortisol concentration at first reevaluation. All 9 dogs underwent ACTH stimulation testing at the first reevaluation. For 3 dogs with a good response and 3 dogs with a poor response, serum cortisol concentrations after ACTH were < 5.5 µg/dl; a post-acth stimulation serum cortisol concentration 5.5 and < 12.5 µg/dl was detected in 1 dog with a good response and in 2 dogs with a poor response. The dose and frequency of trilostane administration were not changed in any of the 9 dogs. At the second reevaluation, all 9 owners classified the treatment response of their dog as good. Seven of the 9 dogs had post-acth stimulation serum cortisol concentration < 5.5 µg/dl, and 2 had a concentration of 8.0 and 9.6 µg/dl. All 9 dogs had a post-acth stimulation serum cortisol concentration at the second reevaluation that was lower than the result at the first reevaluation. All 9 dogs, after the second reevaluation, had an adrenocortical tumor successfully extirpated (4 adrenocortical adenomas and 5 adrenocortical carcinomas), and for each dog, trilostane was permanently Table 1 Summary of trilostane dose and frequency of administration, post-acth stimulation serum cortisol concentration, UCCR value, and urine specific gravity in 9 dogs with NOH due to an adrenocortical tumor at each of 2 reevaluations following initiation of treatment. Reevaluation* Variable 1 2 All dogs No. of dogs 9 9 Mean ± SD dose every 12 h (mg/kg) 0.89 6 0.16 0.89 6 0.16 Range of doses every 12 h (mg/kg) 0.54 1.1 0.54 1.1 Dogs with good response No. of dogs 4 9 Mean ± SD dose every 12 h (mg/kg) 0.94 6 0.05 0.89 60.16 No. of dogs with post-acth SSCC # 5.5 µg/dl (range [µg/dl]) 3 (2.1 5.3) 7 (0.5 5.0) No. of dogs with post-acth SSCC. 5.5 µg/dl (range [µg/dl]) 1 (12.4) 2 (8.0 9.6) Mean UCCR (X 10 6 [range]) 32 (12.1 60.6) 14.9 (8.0 30.1) Mean urine specific gravity (range) 1.022 (1.015 1.028) 1.028 (1.014 1.037) Dogs with poor response No. of dogs 5 Mean ± SD dose every 12 h (mg/kg) 0.85 6 0.21 No. of dogs with post-acth SSCC # 5.5 µg/dl (range [µg/dl]) 3 (2.1 3.7) No. of dogs with post-acth SSCC. 5.5 µg/dl (range [µg/dl]) 2 (7.9 11.4) Mean UCCR (X 10 6 [range]) 18.5 (12.4 27.3) Mean urine specific gravity (range) 1.006 (1.004 1.009) At each visit, response to treatment was classified by owners as good or poor; no dog was considered ill. *Reevaluations were completed 1 to 2 weeks after initiation of trilostane treatment (first reevaluation) and 4 to 8 weeks after the first reevaluation (second reevaluation). Urine was obtained 1 hour prior to the morning trilostane administration. The ACTH stimulation tests were completed 3 to 4 hours after trilostane administration. A good response to treatment was defined as resolution of or marked improvement in clinical signs related to hyperadenocorticism (ie, absence of polydipsia, polyuria, polyphagia, and panting and an increase in muscle strength and activity; hair regrowth was not among the short-term goals of treatment). A poor response to treatment was defined as persistence of unacceptable clinical signs of hyperadrenocorticism (eg, polyuria). = Not applicable. SSCC = Stimulation serum cortisol concentration. To convert mg/kg to mg/lb, divide by 2.2. 1444 Scientific Reports JAVMA, Vol 238, No. 11, June 1, 2011

discontinued following surgery. Clinical signs of NOH have not recurred in any dog. Treatment evaluation of dogs with PDH Initial dosage of trilostane in the 38 dogs with PDH was 0.84 ± 0.22 mg/kg, twice daily, with an interval of 0.21 to 1.1 mg/kg (0.1 to 0.5 mg/lb), twice daily. The initial dosage of trilostane for the dogs with PDH was not significantly different from the initial dosage of trilostane in the dogs with ATH (0.89 ± 0.16 mg/kg, q 12 h). Treatment findings among all 38 dogs with PDH at each reevaluation were assessed, and after reviewing the data, the dogs were arbitrarily separated into 3 groups: dogs whose dose and frequency of trilostane administration did not change or decreased during the study period (PDH group 1), dogs that were maintained on a twicedaily dosing schedule but whose dose increased during the study period (PDH group 2), and dogs whose dosing schedule changed from twice daily to 3 times daily during the study period (PDH group 3). The mean body weights for the 3 groups of dogs were not significantly different. The mean initial dosage of trilostane for each of the 3 groups was not significantly different (0.83 ± 0.26 mg/kg [0.38 ± 0.12 mg/lb], q 12 h; 0.84 ± 0.20 mg/kg [0.38 ± 0.09 mg/lb], q 12 h; and 0.86 ± 0.24 mg/kg [0.39 ± 0.11 mg/lb], q 12 h; respectively), nor was the initial dose for any group significantly different from the initial dose for the dogs with ATH. Treatment evaluation of PDH group 1 dogs Initial dose of trilostane did not change or it decreased during the study in 15 dogs with PDH (PDH group 1 dogs). The mean initial dosage was 0.83 ± 0.26 mg/kg, twice daily, with an interval of 0.21 to 1.1 mg/kg, twice daily. Treatment findings among all 15 PDH group 1 dogs at each reevaluation were assessed (Table 2). At the first reevaluation, treatment response was good in 10 dogs and poor in 5 dogs. Ten of the 15 dogs underwent a pretreatment ACTH stimulation test (post-acth stimulation serum cortisol concentration, 22.9 ± 13.7 µg/dl; range, 9.1 to 50 µg/dl), and all 10 had a lower post-acth stimulation serum cortisol concentration at first reevaluation. All 15 dogs underwent ACTH stimulation testing at the first evaluation. The post-acth stimulation serum cortisol concentration was < 5.5 µg/dl in 8 of 10 dogs with a good response (< 1.5 µg/dl in 1 of those 8 dogs) and in 0 dogs with a poor response. The dosage of trilostane was decreased from 1.0 to 0.8 mg/kg (0.45 to 0.36 mg/ lb), twice daily, in the dog with a post-acth stimulation serum cortisol concentration < 1.5 µg/dl. At the second, third, and fourth reevaluations, 13 of 15, 14 of 15, and 11 of 11 owners, respectively, classi- Table 2 Summary of trilostane dose and frequency of administration, post-acth stimulation serum cortisol concentration, UCCR value, and urine specific gravity in 15 PDH group 1 dogs with NOH at each of 4 reevaluations following initiation of treatment. Reevaluation* Variable 1 2 3 4 All dogs No. of dogs 15 15 15 11 Mean ± SD dose every 12 h (mg/kg) 0.83 6 0.26 0.81 6 0.25 0.74 6 0.29 0.73 6 0.32 Range of doses (mg/kg) 0.21 1.1 0.21 1.1 0.21 1.1 0.21 1.1 Dogs with good response No. of dogs 10 13 14 11 Mean ± SD dose every 12 h (mg/kg) 0.81 6 0.29 0.81 6 0.27 0.74 6 0.30 0.73 6 0.32 No. of dogs with post-acth 8 (1.3 5.5) 10 (0.2 5.2) 13 (1.5 5.1) 10 (1.5 4.5) SSCC # 5.5 µg/dl (range [µg/dl]) No. of dogs with post-acth 2 (8.3 8.6) 3 (5.9 7.4) 1 (6.0) 1 (9.4) SSCC. 5.5 µg/dl (range [µg/dl]) Mean UCCR (X 10 6 [range]) 26.9 (9.4 71) 17.8 (9.1 35) 12.7 (6.9 21.4) 11.6 (7.1 21.4) Mean urine specific gravity (range) 1.014 (1.008 1.025) 1.022 (1.009 1.046) 1.027 (1.014 1.037) 1.028 (1.012 1.035) Dogs with poor response No. of dogs 5 Mean ± SD dose every 12 h (mg/kg) 0.86 6 0.19 No. of dogs with post-acth 0 SSCC # 5.5 µg/dl No. of dogs with post-acth 5 (8.0 19.0) SSCC. 5.5 µg/dl (range [µg/dl]) Mean UCCR (X 10 6 [range]) 22.9 (11.7 32.8) Mean urine specific gravity (range) 1.007 (1.005 1.011) Ill dogs No. of dogs 2 1 Mean ± SD dose every 12 h (mg/kg) 0.84 (0.74 0.94) 1.0 No. of dogs with post-acth 1 (0.2) 1 (0.8) SSCC # 5.5 µg/dl (range [µg/dl]) No. of dogs with post-acth 1 (6.2) SSCC. 5.5 µg/dl (range [µg/dl]) Mean UCCR (X 10 6 [range]) 23.5 (18 29.1) 27 Mean urine specific gravity (range) 1.010 (1.009 1.010) 1.016 Trilostane dose in each dog remained static or decreased during the study period. *Reevaluations were completed 1 to 2 weeks after initiation of trilostane treatment (first reevaluation), 4 to 8 weeks after the first reevaluation (second reevaluation), approximately 16 weeks after the second reevaluation (third reevaluation), and approximately 6 months after the third evaluation (fourth reevaluation). Dogs were considered ill if signs of anorexia, vomiting, diarrhea, unusual listlessness, or unusual weakness were present. See Table 1 for remainder of key. JAVMA, Vol 238, No. 11, June 1, 2011 Scientific Reports 1445

fied the treatment response of their dog as good and no owner classified the response as poor. Two owners at the second reevaluation and 1 owner at the third reevaluation described their dog as ill. Combining results from these 3 reevaluations, 32 of 38 dogs described as having a good response had post-acth stimulation serum cortisol concentrations < 5.5 and > 1.5 µg/dl, 1 had a concentration < 1.5 µg/dl, and 5 had concentrations > 5.5 and < 9.5 µg/dl. The trilostane dosage was decreased from 0.8 to 0.6 mg/kg (0.36 to 0.27 mg/lb) twice daily in the dog with a post-acth stimulation serum cortisol concentration < 1.5 µg/dl. Each of the 3 ill dogs was described as anorexic and unusually lethargic, 2 had been vomiting and weak, none had physical examination abnormalities, none had serum electrolyte abnormalities, and none required hospitalization. The ill dogs had post- ACTH stimulation serum cortisol concentrations of 0.2, 0.8, and 6.2 µg/dl, and each had its trilostane treatment stopped for 4 or 5 days, during which time each improved clinically, and then was placed back on trilostane at 50% of its original dosage (0.35, 0.50, and 0.50 mg/kg [0.16, 0.23, and 0.23 mg/lb], q 12 h, respectively). All 3 dogs received these dosages through the fourth reevaluation, and all were described as having a good response at the fourth reevaluation. Eleven dogs had no change in trilostane dose or frequency of administration. After the third reevaluation, 4 dogs were placed under the care of their referring veterinarians, all with good responses to trilostane treatment. Treatment evaluation of PDH group 2 dogs Initial dose of trilostane increased but without a change in the twice-daily frequency of administration during the study period in 16 dogs with PDH (PDH group 2 dogs). The mean initial trilostane dosage was 0.84 ± 0.20 mg/ kg, twice daily, with a range of 0.47 to 1.0 mg/kg (0.21 to 0.45 mg/lb). Treatment findings among all 16 PDH group 2 dogs at each reevaluation were assessed (Table 3). At the first reevaluation, treatment response was good in 14 dogs and poor in 2 dogs. Eleven of these 16 dogs underwent a pretreatment ACTH stimulation test (serum cortisol concentration, 27.3 ± 13.8 µg/dl; range 13.1 to 50 µg/ dl), and all 11 had a lower post-acth stimulation serum cortisol concentration at first reevaluation. All 16 dogs underwent ACTH stimulation testing at the first evaluation. The post-acth stimulation serum cortisol concentration was > 5.5 µg/dl in all 16 dogs. The dose of trilostane was increased 25% to 50% in 13 dogs (including both dogs with a poor response) and was unchanged in 3. At the second, third, and fourth reevaluations, 11 of 16, 12 of 16, and 7 of 11 owners, respectively, classified the treatment response of their dog as good. At these same reevaluations, 5 of 16, 4 of 16, and 1 of 11 owners, respectively, classified the response as poor. Three owners at the Table 3 Summary of trilostane dose and frequency of administration, post-acth stimulation serum cortisol concentration, UCCR value, and urine specific gravity in 16 PDH group 2 dogs with NOH at each of 4 reevaluations following initiation of treatment. Reevaluation* Variable 1 2 3 4 All dogs No. of dogs 16 16 16 11 Mean ± SD dose every 12 h (mg/kg) 0.84 6 0.20 1.18 6 0.33 1.75 6 0.82 2.0 6 1.21 Range of doses (mg/kg) 0.47 1.0 0.73 1.5 0.75 3.0 0.75 4.0 Dogs with good response No. of dogs 14 11 12 7 Mean ± SD dose every 12 h (mg/kg) 0.81 6 0.20 1.17 6 0.36 1.9 6 0.84 1.7 6 1.0 No. of dogs with post-acth SSCC 0 3 (4.2 5.3) 9 (1.2 4.8) 7 (3.1 5.5) # 5.5 µg/dl (range [µg/dl]) No. of dogs with post-acth SSCC 14 (6.7 18.6) 8 (6.6 14.5) 3 (6.2 9.2) 0. 5.5 µg/dl (range [µg/dl]) Mean UCCR (X 10 6 [range]) 35.2 (12.2 79.2) 27.5 (8 61) 17.6 (7.2 27.4) 18.4 (7.1 29.5) Mean urine specific gravity (range) 1.012 (1.006 1.016) 1.013 (1.006 1.019) 1.022 (1.015 1.039) 1.023 (1.014 1.032) Dogs with poor response No. of dogs 2 5 4 1 Mean ± SD dose every 12 h (mg/kg) 1.0 (1.0) 1.2 6 0.28 1.3 6 0.69 1.26 No. of dogs with post-acth SSCC 0 0 2 (4.2 4.8) 1 (4.8) # 5.5 µg/dl (range [µg/dl]) No. of dogs with post-acth SSCC 2 (6.4 14.7) 5 (6.9 14.5) 2 (7.1 10.6) 0. 5.5 µg/dl (range [µg/dl]) Mean UCCR (X 10 6 [range]) 41.5 (41 42) 46.9 (28 63) 39.9 (27 54) 54 Mean urine specific gravity (range) 1.005 1.008 1.006 1.001 (1.004 1.006) (1.004 1.011) (1.001 1.014) Ill dogs No. of dogs 3 Mean ± SD dose every 12 h (mg/kg) 2.86 6 1.62 No. of dogs with post-acth SSCC 3 (0.4 3.7) # 5.5 µg/dl (range [µg/dl]) No. of dogs with post-acth SSCC 0. 5.5 µg/dl Mean UCCR (X 10 6 [range]) 16.4 (7.2 31) Mean urine specific gravity (range) 1.028 (1.021 1.039) Trilostane dose in each dog increased over the study period, but the frequency of administration remained twice daily. See Tables 1 and 2 for key. 1446 Scientific Reports JAVMA, Vol 238, No. 11, June 1, 2011

fourth reevaluation described their dog as ill. Combining results from these 3 reevaluations, 18 of 30 dogs described as having a good response had post-acth stimulation serum cortisol concentrations < 5.5 and > 1.5 µg/dl, 1 had a concentration < 1.5 µg/dl, and 11 had concentrations > 5.5 and < 14.6 µg/dl. Three of 10 dogs with a poor response had post-acth stimulation serum cortisol concentrations < 5.5 and > 1.5 µg/dl, and 7 dogs with a poor response had post-acth stimulation serum cortisol concentrations > 5.5 and < 14.6 µg/dl. The 3 ill dogs had post-acth stimulation serum cortisol concentrations of 0.4, 0.6, and 3.7 µg/dl. The trilostane dose was increased from 25% to 50% in 11 dogs (including the 3 whose dose was unchanged after the first evaluation) and unchanged in 5 after the second reevaluation. After the third reevaluation, the trilostane dose was decreased 25% in 1 dog with a good response and a post-acth stimulation serum cortisol concentration < 1.5 µg/dl after having a 50% dose increase at the previous reevaluation. Eleven dogs with a good response had no change in trilostane dose after the third reevaluation, 3 dogs with a poor response had their dose increased 25% to 50%, and 1 dog was taken off trilostane and placed on an alternative drug because of continuing polydipsia and polyuria despite having a post-acth stimulation serum cortisol concentration < 5.5 µg/dl (this owner elected not to continue using a recommended 3-times-daily administration). After the third reevaluation, 4 dogs, each with a good response to treatment, were returned to their referring veterinarian for continued care. One ill dog, about 1 year after initiation of treatment, had signs of anorexia and lethargy, had a post-acth stimulation serum cortisol concentration of 3.7 µg/dl, was identified as having a large pituitary mass on magnetic resonance imaging, and was euthanized at the owner s request. This large pituitary mass was not considered an adverse reaction to trilostane on the basis of a previous study, 30 in which results indicated that a reduction of serum cortisol in dogs with PDH did not contribute to pituitary tumor growth. One dog, about 10 months after initiation of treatment, was receiving 1.0 mg/kg of trilostane twice daily and developed anorexia, vomiting, and lethargy. This dog had no physical examination abnormalities, was not hospitalized, had trilostane discontinued, and improved after several days, and the owner refused to place the dog back on the drug. One dog, about 11 months after initiation of treatment, was receiving 3.6 mg/kg (1.64 mg/ lb) of trilostane twice daily and developed clinical signs similar to those of the previous dog. On physical examination, the dog was weak and dehydrated. This dog had hyperkalemia and hyponatremia documented, was hospitalized and treated with parenteral fluids, improved after several days, and was returned to the owner, who refused to place the dog back on trilostane. Treatment evaluation of PDH group 3 dogs Initial twice-daily frequency of trilostane administration was increased to 3 times daily during the study period in 7 dogs with PDH (PDH group 3 dogs). Their mean initial dosage was 0.85 ± 0.24 mg/kg (0.39 ± 0.11 mg/ lb) twice daily with a range of 0.48 to 1.1 mg/kg (0.22 to 0.50 mg/lb) twice daily. Treatment findings among all 7 PDH group 3 dogs at each reevaluation were assessed (Table 4). At the first reevaluation, treatment response Table 4 Summary of trilostane dose and frequency of administration, post-acth stimulation serum cortisol concentration, UCCR value, and urine specific gravity in 7 PDH group 3 dogs with NOH at each of 4 reevaluations following initiation of treatment. Reevaluation* Variable 1 2 3 4 All dogs No. of dogs 7 7 7 6 Mean ± SD dose every 12 h (mg/kg) 0.85 6 0.24 1.11 6 0.36 5.75 6 6.01 Range of doses every 12 h (mg/kg) 0.48 1.1 0.48 1.5 1.5 10.0 Mean dose every 8 h (mg/kg) 2.55 6 3.67 1.07 6 0.43 Range of doses every 8 h (mg/kg) 0.48 10.0 0.48 1.5 Dogs with good response No. of dogs 3 3 5 6 Mean ± SD dose every 12 h (mg/kg) 0.74 6 0.26 1.27 6 0.25 Mean dose every 8 h (mg/kg) 1.05 6 0.38 1.07 6 0.43 No. of dogs with post-acth SSCC 0 2 (1.9 3.9) 4 (0.7 4.8) 6 (1.3 5.4) # 5.5 µg/dl (range [µg/dl]) No. of dogs with post-acth SSCC 3 (7.2 10.7) 1 (7.8) 1 (8.1) 0. 5.5 µg/dl (range [µg/dl]) Mean UCCR (X 10 6 [range]) 31.1 (1 dog) 22.5 (9.3 37.1) 12.6 (9.7 16.4) 14.5 (9.7 23.7) Mean urine specific gravity (range) 1.010 (1.006 1.014) 1.018 (1.011 1.032) 1.024 (1.014 1.034) 1.025 (1.018 1.034) Dogs with poor response No. of dogs 4 4 2 0 Mean ± SD dose every 12 h (mg/kg) 0.94 6 0.20 0.99 6 0.42 1.5 Mean dose every 8 h (mg/kg) 10.0 No. of dogs with post-acth 0 0 0 SSCC # 5.5 µg/dl No. of dogs with post-acth SSCC 4 (7.1 41.3) 4 (5.8 9.2) 2 (5.9 14.3). 5.5 µg/dl (range [µg/dl]) Mean UCCR (X 10 6 [range]) 24.6 (17.9 34) 27.1 (24.7 31.8) (19.2 32.4; 2 dogs) Mean urine specific gravity (range) 1.007 (1.006 1.010) 1.008 (1.004 1.014) (1.004 1.010; 2 dogs) The frequency of trilostane administration increased from twice daily to 3 times daily in each dog during the study period. See Tables 1 and 2 for key. JAVMA, Vol 238, No. 11, June 1, 2011 Scientific Reports 1447

was good in 3 dogs and poor in 4 dogs. Five of these 7 dogs underwent a pretreatment ACTH stimulation test (serum cortisol concentration, 24.2 ± 8.9 µg/dl; range, 17.2 to 39 µg/dl), and 4 of the 5 had a lower post-acth stimulation serum cortisol concentration at first reevaluation. All 7 dogs underwent ACTH stimulation testing at the first evaluation, and the post-acth stimulation serum cortisol concentration for all was > 5.5 µg/dl (13.8 ± 12.3 µg/dl; range, 7.1 to 41.3 µg/dl). The dose of trilostane was increased 25% to 50%, but the frequency of administration remained twice daily in 5 dogs (including the 4 dogs with a poor response) and unchanged in 2. At the second, third, and fourth reevaluations, 3 of 7, 5 of 7, and 6 of 6 owners, respectively, classified the treatment response of their dog as good. At these same reevaluations, 4 of 7, 2 of 7, and 0 of 6 owners, respectively, classified the response as poor. Combining results from these 3 reevaluations, 10 of 14 dogs described as having a good response had post-acth stimulation serum cortisol concentrations < 5.5 and > 1.5 µg/dl, 2 had concentrations of 0.7 and 1.3 µg/dl, and 2 had concentrations of 7.8 and 8.1 µg/dl. All 6 dogs with a poor response had post-acth stimulation serum cortisol concentrations > 5.5 and < 14.4 µg/dl. After the second reevaluation, the dosage of trilostane was not changed from 1.5 mg/kg (0.68 mg/lb), twice daily, in 1 dog; was increased about 50% from 1.0 to 1.47 mg/kg (0.45 to 0.67 mg/lb), twice daily, in 1 dog; and was increased to 3-times daily administration in 5 dogs (range, 0.48 to 1.5 mg/kg, q 8 h). After the third reevaluation, 4 dogs with a good response, all receiving trilostane 3 times daily, had no change in trilostane dose or frequency of administration (range, 0.48 to 1.5 mg/kg, q 8 h). One dog with a good response had the trilostane dosage decreased from 1.0 to 0.8 mg/kg, 3 times daily. One dog with a poor response had the frequency of trilostane administration increased from 1.47 mg/kg, twice daily, to 3 times daily. One dog progressively had its dosage of trilostane increased during the interceding 4 months from 1.5 mg/kg, 3 times daily, to 10 mg/kg (4.5 mg/lb), 3 times daily. That dog was removed from the study after consistently having a poor response (severe polyuria) and was treated with surgical bilateral adrenalectomy. Evaluation of owner opinion, ACTH stimulation test result, UCCR, and urine specific gravity When post-acth stimulation serum cortisol concentrations were related with owner opinions at each reevaluation, 20 dogs classified as having a good response at first reevaluation had post-acth stimulation serum cortisol concentrations > 5.5 µg/dl, and 14 dogs classified as having a good response at second reevaluation had post-acth stimulation serum cortisol concentrations > 5.5 µg/dl, but only 5 and 1 dogs, respectively, had similar results at the third and fourth reevaluations. The post-acth stimulation serum cortisol concentration < 5.5 µg/dl was an arbitrary goal. In 122 of 160 reevaluations for all 47 dogs, owners reported marked improvement or complete resolution of clinical signs. In 82 of those 122 reevaluations, the post-acth stimulation serum cortisol concentration was < 5.5 µg/dl, but similar results were noted in only 3 dogs classified as having a poor response. The 5.5 µg/dl cutoff value, therefore, was relatively specific but was not sensitive because 40 of the 122 dogs with a good response had post-acth stimulation serum cortisol concentrations > 5.5 µg/dl. However, if the goal had been increased to a serum cortisol concentration < 6.5 µg/dl, only 4 of 40 reevaluations in which a good response was reported by the owner and in which a dog had a post-acth stimulation serum cortisol concentration > 5.5 µg/dl would have been added, while 3 of the 32 dogs with a poor response would also be included. If the goal had been increased to < 9.0 µg/dl, 106 of the 122 reevaluations classified as good would be included, but 19 of the 32 reevaluations on dogs classified as having a poor response would also be included. Thus, the interpretation becomes more sensitive but less specific as the ACTH stimulation test result is increased. Neither the UCCR nor urine specific gravity was as reliable as the ACTH stimulation test results in helping determine whether a dose or frequency of administration should be changed. Of 104 UCCR values from dogs described as markedly improved or in which clinical signs had completely resolved, 41 (39%) were within reference limits (< 13.5). Of 26 UCCR values from dogs described as continuing to have clinical signs of NOH, 2 were < 13.5. Of 4 UCCR values from dogs described as ill, 1 value was < 13.5. Therefore, most NOH dogs with continuing clinical signs had UCCR results above the reference range, but this is true of 61% of the dogs described as markedly improved or in which clinical signs had resolved. Of 99 urine specific gravities from dogs described as having a good response to treatment, 50 were 1.020 and 49 were < 1.020. Of 27 urine specific gravities from dogs described as having a poor response to treatment, all were < 1.020. Thus, a urine specific gravity < 1.020 supports the concept that a dog may be continuing to show clinical signs, but this same result is seen in almost half the dogs thought to have undergone marked improvement or whose clinical signs fully resolved. Discussion With rare exceptions, NOH in dogs is neither a rapidly progressive nor a life-threatening condition that requires a rapid response to treatment. Typically, NOH is a chronic progressive disease that ultimately causes unacceptably bothersome clinical signs (eg, polyuria, polyphagia, weakness, and panting) in affected dogs. A primary goal in treating dogs with NOH is to reduce risk associated with anesthesia and surgery in dogs with ATH or to achieve resolution of signs of PDH, as perceived by the owners. Most owners of dogs with PDH are willing to allow a sufficient period of treatment (weeks to months) to achieve resolution of clinical signs. However, treatment may cause adverse effects that are more worrisome than signs of the disease, including death or severe illness requiring hospitalization. If their pet recovers from an episode of illness, some owners refuse to consider any further treatment (as happened in 3 dogs in this study) because of their 1448 Scientific Reports JAVMA, Vol 238, No. 11, June 1, 2011

concerns that adverse effects could recur, incurring further expense and, perhaps, similar or more serious consequences. Therefore, if possible, treatment of NOH should be benign with respect to associated adverse effects, yet effective within a reasonable time period in minimizing or resolving clinical signs. Trilostane effectively blocks the 3β-hydroxysteroid dehydrogenase-isomerase enzyme system in dogs. This, in turn, inhibits synthesis and secretion of cortisol that occur autonomously from an adrenocortical tumor or secondary to autonomous secretion of ACTH from a pituitary tumor that, in turn, stimulates synthesis and secretion of cortisol from adrenal cortices. In either condition, by decreasing serum cortisol concentrations in affected dogs, clinical signs of NOH can be diminished or eliminated. Most published reports 13,14,19,21 on the use of trilostane state that some dogs require trilostane administration more frequently than once per day to resolve clinical signs. There is also a percentage of treated dogs that become ill or die as a direct adverse reaction to trilostane. It has been documented that apparent glucocorticoid or mineralocorticoid deficiency or development of adrenal gland necrosis is associated with trilostane administration. 31,32 It also appears possible that, in some dogs, trilostane may more effectively block the synthesis of mineralocorticoid than glucocorticoid. This would explain how severe clinical signs (dehydration, weakness, hyponatremia, and hyperkalemia) could develop in a dog that becomes ill during trilostane treatment despite having pre- and post-acth stimulation serum cortisol concentrations that may not be as low as the values in other dogs that seem to be underdosed or that have responded well. 33 The incidence of worrisome adverse effects in our early experiences of trilostane treatment of dogs with NOH, in reports from authors at other institutions, and in our previous report, 19 which included a twice-daily low-dose dosing schedule, suggested that a twice-daily lower-dose treatment plan was worthy of investigation. The goal of the present study was to control clinical signs associated with NOH while reducing the incidence and severity of adverse effects. 13 19,31,32,d One of 47 dogs in this study developed worrisome clinical signs secondary to development of a large pituitary tumor, a problem that likely developed independent of trilostane administration. Despite quite low doses of trilostane administered to the dogs in this study, 5 appeared to have adverse reactions to the drug, although unrelated conditions may have been the cause of these illnesses. Four of the 5 dogs with worrisome adverse effects were being given trilostane at dosages well below manufacturer recommendations (0.74, 0.94, 1.0, and 1.0 mg/kg [0.34, 0.43, 0.45, and 0.45 mg/lb], q 12 h). One dog was being given 3.6 mg/kg, twice daily, when the illness was considered sufficiently severe to require hospitalization and parenteral fluid therapy. On the 1 hand, it was disappointing to have 5 of 47 (10.6%) trilostane-treated dogs develop adverse effects. However, no dog died, and only 1 had an episode of illness requiring in-hospital treatment out of 160 reevaluations. Both the incidence and severity of adverse effects were less in the dogs in this study than those reported in other studies or by the manufacturer in its insert. 13 21,d Veterinarians who elect to use trilostane for treatment of NOH should be aware that adverse effects caused by the drug can occur in dogs receiving recommended or less than recommended doses. Further, such adverse effects can occur at any time after treatment has been initiated. Trilostane-induced adverse effects were documented after about 2 months of treatment in 2 dogs, after about 6 months of treatment in 1 dog, and after about a year of treatment in 2 dogs. Veterinarians should consider using low initial trilostane doses, such as those reported herein, to minimize the incidence and severity of such adverse effects. The initial dosage of trilostane given to all 47 dogs was lower than that reported in any study to date, to the author s knowledge. In our previous low-dose trilostane study, 19 22 dogs with NOH were initially given 0.5 to 2.5 mg/kg (0.23 to 1.14 mg/lb, q 12 h; mean, 1.4 mg/ kg [0.64 mg/lb]). In that study, no dog required more than 2.8 mg/kg (1.27 mg/lb) of trilostane per treatment to control clinical signs of NOH. In the present study, the initial dosage given to 47 dogs with NOH ranged from 0.21 to 1.1 mg/kg, twice daily (mean, 0.86 mg/kg). It was assumed that the final dose necessary to control clinical signs in these dogs would be well in excess of these initial doses. However, all 9 ATH dogs responded well to the initial dose used in this study, and 15 of the 38 dogs with PDH (PDH group 1 dogs) responded well or were overdosed by similar initial doses. Further, 8 dogs whose twice-daily dose increased over the course of the study (PDH group 2 dogs) and 3 dogs given trilostane 3 times daily (PDH group 3 dogs) never received as much as the lowest (3 mg/kg/d) manufacturer s recommended initial daily dose. Thus, 35 of the 47 (74%) study dogs never received as much as the lowest initial manufacturer s recommended total daily dose of trilostane. Remembering that the manufacturer s recommended initial dosage is 3 to 6 mg/kg, once daily, it is valuable to note that only 4 of 47 dogs were given > 3 mg/ kg of trilostane per treatment. One of these latter 4 dogs responded well to 3.8 mg/kg, twice daily; 1 dog receiving 4.0 mg/kg (1.82 mg/lb), twice daily, developed a large pituitary tumor (independent of trilostane) and was euthanized; 1 dog given 3.6 mg/kg, twice daily, became ill with adverse effects requiring hospitalization; and 1 dog failed to respond to trilostane despite being given dosages as high as 10 mg/kg, 3 times daily. None of the dogs with ATH required an increase in dose, and all owners reported good responses to trilostane. These results suggested that adrenocortical tumors are quite sensitive to trilostane and that dogs with this condition, treated in preparation for surgery, should be given doses at or below those used herein. If surgery is not planned for any dog with ATH and if trilostane is the only treatment anticipated to be used in an attempt to gain long-term resolution of clinical signs, it is assumed that the dose used should be at or below those used herein. Results indicating that trilostane may be superior to mitotane in the treatment of dogs with ATH are supportive of future studies that would compare the efficacy of these 2 drugs in preoperative treatment. The results of the present study indicated that trilostane is both potent and effective in dogs at doses JAVMA, Vol 238, No. 11, June 1, 2011 Scientific Reports 1449

less than those recommended by the manufacturer and reported in the veterinary medical literature. In our previous report, 19 it was hypothesized that the safety of twice-daily low-dose trilostane treatment might have been improved if, at the first reevaluation, fewer dogs had had a post-acth stimulation serum cortisol concentration 5.5 µg/dl, none of the dogs had had a good response to treatment, and the dosage needed by each dog to achieve resolution of clinical signs had been determined over a period of several months by slow adjustment of the dose and frequency of administration. Such a slow-adjustment protocol could result in treatment of most dogs with a dose just above an established threshold that achieves the response desired, thereby reducing risk of adverse drug effects. The increase in time needed to achieve such a response would be balanced by a decrease in the incidence of adverse effects. Indeed, none of the 47 dogs in the study reported herein became ill in the first 2 weeks of treatment, and perhaps consistent with this result, only 14 of the 47 dogs had post-acth stimulation serum cortisol concentration 5.5 µg/dl at the 10- to 14-day (first) reevaluation. Owner opinions are not necessarily consistent. Marked improvement in the opinion of one owner may not be so to another. Improvement in the first few weeks of treatment may not be seen as improvement after several months. Owner expectations did appear to change with duration of treatment. It appeared that owners reported a good response early in the course of treatment with minor improvements but were more critical in their assessment as treatment progressed. Closer agreement in owner observations and ACTH stimulation test results does occur with time. Despite these caveats, it would appear that owner opinion, coupled with the ACTH stimulation test result, provides the best opportunity for recognizing which doses and frequency of administrations appear satisfactory versus those that should be changed. It is suggested that the goals of a post-acth stimulation serum cortisol concentration 5.5 µg/dl and a urine specific gravity > 1.020 be retained, but that owner opinion remain key in evaluating treatment success or failure. Dogs with a post-acth stimulation serum cortisol concentration > 5.5 µg/dl and an owner who continues to observe clinical signs should have the twice-daily dose increased. Dogs with a post-acth stimulation serum cortisol concentration 5.5 µg/dl and an owner who continues to observe clinical signs should be considered for a change from 2 to 3 treatments/d. It would appear that there is no advantage in performing the relatively expensive UCCR evaluation on each reevaluation. 34 However, reevaluating urine from dogs being treated for NOH for specific gravity is relatively inexpensive, may provide information of interest, and would allow each sample to also be assessed for glucose, protein, or evidence of infection. Such parameters have potential value in the continuing assessment of dogs being treated long-term with trilostane. a. Vetoryl, Arnolds Veterinary Products Ltd, Harlescott, Shrewsbury, Shropshire, England. b. Hurley K, Sturgess K, Cauvin A, et al. The use of trilostane for the treatment of hyperadrenocorticism in dogs (abstr). J Vet Intern Med 1998;12:210. c. Eastwood JM, Elwood CM, Hurley KJ. Trilostane treatment of four dogs with functional adrenocortical neoplasia (abstr), in Proceedings. 11th Eur Soc Intern Med Cong 2001;185. d. Eastwood JM, Elwood CM. Prolonged hypoadrenocorticism in five dogs treated with trilostane for pituitary dependent hyperadrenocorticism (PDH) (abstr), in Proceedings. Br Small Anim Vet Assoc Cong 2003;527. e. HDI 5000, ATL Ultrasound, Philips Medical Systems Co Inc, Bothell, Wash. f. Cortrosyn, Amphastar Pharmaceuticals Inc, Rancho Cucamonga, Calif. g. Endocrinology Laboratory, University of California-Davis, Davis, Calif. h. Coat-a-Count cortisol assay, Diagnostic Products Corp, Los Angeles, Calif. i. SPSS for Windows, version 15.0, SPSS Inc, Chicago, Ill. References 1. Feldman EC. Distinguishing dogs with functioning adrenocortical tumors from dogs with pituitary-dependent hyperadrenocorticism. J Am Vet Med Assoc 1983;183:195 200. 2. Reusch CE, Feldman EC. Canine hyperadrenocorticism due to adrenocortical neoplasia. Pretreatment evaluation of 41 dogs. J Vet Intern Med 1991;5:3 10. 3. Schechter RD, Stabenfeldt GW, Ling GV, et al. Treatment of Cushing s syndrome in the dog with an adrenocorticolytic agent (o,p -DDD). J Am Vet Med Assoc 1973;162:629 639. 4. Feldman EC, Nelson RW, Feldman MS, et al. Comparison of mitotane treatment for adrenal tumor versus pituitarydependent hyperadrenocorticism in dogs. J Am Vet Med Assoc 1992;200:1642 1647. 5. Kintzer PP, Peterson ME. Mitotane (o,p -DDD) treatment of 200 dogs with pituitary-dependent hyperadrenocorticism. J Vet Intern Med 1991;5:182 190. 6. Hertog E, Braakman JCA, Teske E, et al. Results of non-selective adrenocorticolysis by o,p -DDD in 129 dogs with pituitarydependent hyperadrenocorticism. Vet Rec 1999;144:12 17. 7. Potts GO, Creange JE, Hardong HR, et al. Trilostane, an orally active inhibitor of steroid biosynthesis. Steroids 1978;32:257 267. 8. Komanicky P, Spark RF, Melby JC. Treatment of Cushing s syndrome with trilostane (WIN 24,540), an inhibitor of adrenal steroid biosynthesis. J Clin Endocrinol Metab 1978;47:1042 1051. 9. Robinson DT, Earnshaw RJ, Mitchell R, et al. The bioavailability and metabolism of trilostane in normal subjects, a comparative study using high pressure liquid chromatographic and quantitative cytochemical assays. J Steroid Biochem 1984;21:601 605. 10. Winterberg B, Vetter W, Groth H, et al. Primary aldosteronism: treatment with trilostane. Cardiology 1985;77:117 121. 11. Dewis P, Anderson DC, Bu lock DE, et al. Experience with trilostane in the treatment of Cushing s syndrome. Clin Endocrinol 1983;18:533 540. 12. Wenger M, Sieber-Ruckstuhl NS, Müller C, et al. Effect of trilostane on serum concentrations of aldosterone, cortisol, and potassium in dogs with pituitary-dependent hyperadrenocorticism (Erratum published in Am J Vet Res 2004;65:1562). Am J Vet Res 2004;65:1245 1250. 13. Neiger R, Ramsey I, O Conner J, et al. Trilostane treatment of 78 dogs with pituitary-dependent hyperadrenocorticism. Vet Rec 2002;150:799 804. 14. Ruckstuhl NS, Nett CS, Reusch CE. Results of clinical examinations, laboratory tests, and ultrasonography in dogs with pituitary-dependent hyperadrenocorticism treated with trilostane. Am J Vet Res 2002;63:506 512. 15. Braddock JA, Church DB, Robertson ID, et al. Trilostane treatment in dogs with pituitary-dependent hyperadrenocorticism. Aust Vet J 2003;81:600 607. 16. Eastwood JM, Elwood CM, Hurley KJ. Trilostane treatment of a dog with functional adrenocortical neoplasia. J Small Anim Pract 2003;44:126 131. 17. Bell R, Neiger R, McGrotty Y, et al. Study of the effects of once daily doses of trilostane on cortisol concentrations and responsiveness to adrenocorticotrophic hormone in hyperadrenocorticoid dogs. Vet Rec 2006;159:277 281. 1450 Scientific Reports JAVMA, Vol 238, No. 11, June 1, 2011

18. Neiger R, Ramsey I. Trilostane treatment of canine hyperadrenocorticism, in Proceedings. 20th Am Coll Vet Intern Med Forum 2002;544 546. 19. Vaughan M, Feldman EC, Hoar BR, et al. Evaluation of twicedaily, low-dose trilostane treatment administered orally in dogs with naturally occurring hyperadrenocorticism. J Am Vet Med Assoc 2008;232:1321 1328. 20. Vetoryl [package insert]. Overland Park, Kan: Dechra Veterinary Products, 2008. 21. Alenza DP, Arenas C, Lopez ML, et al. Long-term efficacy of trilostane administered twice daily in dogs with pituitary-dependent hyperadrenocorticism. J Am Anim Hosp Assoc 2006;42:269 276. 22. Feldman EC. Effect of functional adrenocortical tumors on plasma cortisol and corticotropin concentrations in dogs. J Am Vet Med Assoc 1981;178:823 826. 23. Feldman EC. Comparison of ACTH response and dexamethasone suppression as screening tests in canine hyperadrenocorticism. J Am Vet Med Assoc 1983;182:506 510. 24. Kantrowitz BM, Nyland TG, Feldman EC. Adrenal ultrasonography in the dog. Vet Radiol 1986;27:15 21. 25. Pennick DG, Feldman EC, Nyland TG. Radiologic features of canine hyperadrenocorticism caused by autonomously functioning adrenocortical tumors: 23 cases (1978 1986). J Am Vet Med Am Assoc 1988;192:1604 1608. 26. Voorhout G, Rijnberk A, Sjollema BE, et al. Nephrotomography and ultrasonography for the localization of hyperfunctioning adrenocortical tumors in dogs. Am J Vet Res 1990;51:1280 1285. 27. Barthez PY, Nyland TG, Feldman EC. Ultrasonographic evaluation of the adrenal glands in dogs. J Am Vet Med Assoc 1995;207:1180 1183. 28. Feldman EC, Nelson RW, Feldman MS. Use of low- and highdose dexamethasone tests for distinguishing pituitary-dependent from adrenal tumor hyperadrenocorticism in dogs. J Am Vet Med Assoc 1996;209:772 775. 29. Feldman EC, Mack RE. Urine cortisol:creatinine ratio as a screening test for hyperadrenocorticism in dogs. J Am Vet Med Assoc 1992;200:1637 1641. 30. Bertoy EH, Feldman EC, Nelson RW, et al. One-year follow-up evaluation of magnetic resonance imaging of the brain in dogs with pituitary-dependent hyperadrenocorticism. J Am Vet Med Assoc 1996;208:1268 1273. 31. Reusch CE, Sieber-Ruckstuhl N, Wenger M, et al. Histological evaluation of the adrenal glands of seven dogs with hyperadrenocorticism treated with trilostane. Vet Rec 2007;160:219 224. 32. Chapman PS, Kelly DF, Archer J, et al. Adrenal necrosis in a dog receiving trilostane for the treatment of hyperadrenocorticism. J Small Anim Pract 2004;45:307 310. 33. Peterson ME, Kintzer PP, Kass PH. Pretreatment clinical and laboratory findings in dogs with hypoadrenocorticism: 225 cases (1979 1993). J Am Vet Med Assoc 1996;208:85 91. 34. Galac S, Buijtels JJCWM, Kooistra HS. Urinary corticoid: creatinine ratios in dogs with pituitary-dependent hyperadrenocorticism during trilostane treatment. J Vet Intern Med 2009;23: 1214 1219. From this month s AJVR Effect of topical vapocoolant spray on response to arthrocentesis and intravenous catheterization in unsedated horses Cathrine T. Fjordbakk and Henning A. Haga Objective To assess the efficacy of a commercially available topical vapocoolant spray in reducing responses to arthrocentesis of the middle carpal (MC) and metacarpophalangeal (MCP) joints and jugular vein catheterization in unsedated horses. Animals 8 healthy research horses. Procedures Arthrocentesis of both MC and MCP joints and bilateral jugular vein catheterization were performed in each horse. Immediately prior to skin penetration, 1 randomly selected MC joint, MCP joint, and jugular vein were sprayed with a vapocoolant liquid (intervention product), and the contralateral MC joint, MCP joint, and jugular vein were sprayed with water (placebo). An observer blinded to the type of spray treatment used evaluated the horses responses to needle or catheter placement procedures by use of a 6-point categorical scale and a 100-mm visual analog scale. Results Responses evaluated via visual analog scale were significantly reduced after application of the intervention product, compared with responses after application of the placebo, for the MC and MCP joints; no difference in responses to jugular vein catheterization was detected between the 2 treatments. Conclusions and Clinical Relevance Vapocoolant spray was safe and effective in reducing horses responses to arthrocentesis. The use of such products prior to joint injections may reduce procedural nociception and pain anticipation in unsedated horses and may improve the safety of personnel performing such procedures. (Am J Vet Res 2011;72:746 750) June 2011 See the midmonth issues of JAVMA for the expanded table of contents for the AJVR or log on to avmajournals.avma.org for access to all the abstracts. JAVMA, Vol 238, No. 11, June 1, 2011 Scientific Reports 1451