Plasma cortisol response to ketoconazole administration in dogs with hyperadrenocorticism
Edward C. Feldman, DVM; David S. Bruyette, DVM; Richard W. Nelson, DVM; Thomas B. Farver, PhD
Summary: The effect of orally administered ketocon- azole on plasma cortisol concentration in dogs with hyperadrenocorticism was evaluated. Every 30 min- utes from 0800 hours through 1600 hours and again at 1800 hours, 2000 hours, and 0800 hours the fol- lowing morning, 15 clinically normal dogs and 49 dogs with hyperadrenocorticism had plasma samples obtained and analyzed for cortisol concentration. The mean (± SD) plasma cortisol concentration for the initial 8-hour testing period was highest in 18 dogs with adrenocortical tumor (5.3 ± 1.6 µg/dl), lowest in 15 control dogs (1.3 ± 0.5 µg/dl), and intermedi- ate in 31 dogs with pituitary-dependent hyperadreno- corticism (PDH; 3.4 ± 1.2 µg/dl). Results in each of the 2 groups of dogs with hyperadrenocorticism were significantly (P < 0.05) different from results in con- trol dogs, but not from each other. The same cortisol secretory experiment was performed, using 8 dogs with hyperadrenocorticism (5 with PDH; 3 with adrenocor- tical tumor) before and after administration at 0800 hours of 15 mg of ketoconazole/kg of body weight. Significant (P < 0.05) decrease in the 8-hour mean plasma cortisol concentration (0.9 ± 0.2 µg/dl) was observed, with return to baseline plasma cortisol con- centration 24 hours later.
Twenty dogs with hyperadrenocorticism (11 with PDH, 9 with adrenocortical tumor) were treated with ketoconazole at a dosage of 15 mg/kg given every 12 hours for a half month to 12 months. The disease in 2 dogs with PDH failed to respond to treatment, but 18 dogs had complete resolution of clinical signs of hyperadrenocorticism and significant (P < 0.05) re- duction in plasma cortisol responsiveness to exogenous adrenocorticotropin (ACTH). The healthy control dogs
From the Department of Veterinary Reproduction (Feld- man), the Veterinary Medical Teaching Hospital (Bruyette), and the Department of Epidemiology and Preventive Medicine (Farver), University of California, Davis, CA 95616, and the Department of Small Animal Clinics, Purdue University, West Lafayette, IN 47907 (Nelson). Dr. Bruyette’s present address is the Department of Surgery and Medicine, College of Veterinary Medicine, Kansas State University,Manhattan, KS 66502. Dr. Nelson’s present address is the Department of Medicine, School of Veterinary Medicine, University of California, Davis, CA 95616.
The authors thank Marsha S. Feldman for technical assis- tance.
Supported by contributions from Ms. Ruth Johnston.
had a mean baseline plasma cortisol concentration of 1.4 ± 0.4 g/dl and a post-ACTH cortisol concentra- tion of 10.6 ± 3.1 ug/dl. Before ketoconazole ad- ministration, all 11 dogs with PDH had a mean base- line plasma cortisol concentration of 4.4 ± 1.9 µg/dl and a post-ACTH cortisol concentration of 33.6 ± 17.6 ug/dl. The 9 dogs with adrenocortical tumor had a mean baseline plasma cortisol concentration of 4.4 ± 1.3 µg/dl and post-ACTH cortisol concentration of 28.1 + 14.1 µg/dl. After 5 days of ketoconazole administration, the post-ACTH plasma cortisol concen- tration for dogs with PDH or adrenocortical tumor was 4.0 ± 5.4 µg/dl and 6.0 ± 3.3 ug/dl, respectively. Similar responses were observed after 60, 180, and 360 days of ketoconazole treatment.
H yperadrenocorticism is a common endocrine disorder in dogs.1,2 Excess secretion of pitu- itary adrenocorticotropin (ACTH), with resultant bi- lateral adrenocortical hyperplasia (pituitary- dependent hyperadrenocorticism; PDH), is respon- sible for hyperadrenocorticism that develops in 80 to 90% of affected dogs. The disease is usually the result of ACTH secretion from an adenoma of the pars distalis or pars intermedia, and is less com- monly caused by pituitary carcinoma or hyperplasia.3 The remainder of dogs (10 to 20%) with hyperadrenocorticism have functional adreno- cortical adenoma or carcinoma.2 Dogs with PDH are usually treated with the adrenocorticolytic agent mitotane and less commonly by use of surgery or irradiation.2,4-6 Dogs with adrenocortical tumor are usually treated surgically and, less commonly, with mitotane.2,7
Ketoconazole,ª an imidazole derivative, is an antifungal agent that is active after oral adminis- tration. In addition to its antifungal activity, keto- conazole has been shown to interfere with gonadal8 and adrenal9,10 steroid synthesis through inhibition of cytochrome P-450-dependent enzymes. Similar endocrinologic effects have been observed in clin- ically normal dogs.11,12 The study reported here was undertaken to assess the effect of ketoconazole administration on plasma cortisol concentration in
ªNizoral, Janssen Pharmaceutical Inc, Piscataway, NJ.
dogs with PDH and in those with hyperadrenocor- ticism caused by a functional adrenocortical tumor.
Materials and Methods
Clinically normal dogs-Fifteen privately owned pet dogs (7 sexually intact males; 8 ovari- ohysterectomied females) were used as controls. Dogs ranged in age from 1 to 10 years, with mean (± SD) of 5.3 (± 1.8) years. Body weight was be- tween 8.5 and 42.1 kg, with a mean of 22.2 (± 10.8) kg. Dogs were not being given any med- ication. Each dog was healthy based on review of history and physical examination findings. Control dogs also had normal results of CBC, serum bio- chemical analysis and urinalysis.
Dogs with hyperadrenocorticism-Sixty pri- vately owned pet dogs (39 females; 21 males) with hyperadrenocorticism were studied. Dogs ranged in age from 4 to 16 years, with mean of 10.2 (± 2.1) years. Sixteen breeds were represented. The suspicion of hyperadrenocorticism was based on clinical signs of disease (eg, polydipsia, polyuria, polyphagia, alopecia, thin skin, and muscular weakness) and laboratory findings (eg, lymphope- nia, eosinopenia, high serum alkaline phosphatase activity and cholesterol concentration, and dilute urine specific gravity)1,2. The diagnosis of hyper- adrenocorticism was confirmed in all dogs by de- tection of an abnormal and exaggerated increase in plasma cortisol concentration 1 hour after IM administration of 0.25 mg of synthetic ACTH13,b/dog and/or inadequate suppression of the plasma cor- tisol concentration 8 hours after IV administration of dexamethasone sodium phosphate (0.01 mg/kg of body weight).14 A tentative diagnosis of func- tioning adrenocortical tumor was assigned to 22 dogs in which endogenous ACTH concentration was not detectable (< 10 pg/ml) in a randomly ob- tained morning (0800 to 1000 hours) plasma sample.15,c Unilateral or bilateral adrenocortical tumors (adenoma or carcinoma) were subsequently confirmed by results of laparotomy or necropsy in each of these 22 dogs.
Of the 60 dogs with hyperadrenocorticism, PDH was tentatively diagnosed in 38 of them. This classification was based on finding morning (be- tween 0800 and 1000 hours) endogenous ACTH concentration > 40 pg/ml in a randomly obtained sample from each of these dogs and/or suppression of plasma cortisol concentration 8 hours after IV administration of dexamethasone at a dose of 0.1 mg/kg.15 Of these 38 dogs, 16 died and had pitu- itary tumor, as well as bilateral adrenocortical hy- perplasia, identified at necropsy. The tumor was tentatively described as pars distalis tumor in 8
dogs and pars intermedia tumor in 4 dogs. The or- igin of 4 pituitary tumors could not be identified. Nine dogs with adrenocortical tumors and 11 dogs with PDH were given ketoconazole prior to any other form of treatment.
Cortisol secretion experiment-Twenty-four hours before testing began, 64 dogs (15 clinically normal; 49 with hyperadrenocorticism) were hos- pitalized, and an indwelling catheter was inserted into one external jugular vein. The dogs with hyperadrenocorticism (31 with PDH; 18 with adrenocortical tumor) had this diagnosis con- firmed at our facility, had not been treated, and had not undergone any endocrine studies in the pre- ceding 10 days. Catheterization was completed in each dog without use of anesthesia or sedation. The day after the catheter was placed, plasma cortisol concentration was assessed every 30 minutes from 0800 through 1600 hours-and at 1800, 2000, and 0800 hours the following morning. All dogs were housed in individual runs or cages, and blood samples were obtained via Iv catheter. Strict con- trol of light and dark was not attempted. All dogs were fed a commercial dog food diet (to which they were accustomed) at 0800 hours and again at 1800 hours before and during the sample collection pe- riod. Water was always available. Four dogs with hyperadrenocorticism also had endogenous ACTH concentration monitored during the sample col- lection period, in addition to assessment of plasma cortisol concentration.
Cortisol secretion experiment in dogs given keto- conazole-Prior to ketoconazole administration, 8 dogs (5 with PDH; 3 with adrenocortical tumor) had plasma cortisol concentration determined every 30 minutes from 0800 to 1600 hours, and at 1800, 2000, and 0800 hours the next morning, as previ- ously described. This same protocol was repeated the morning after initial oral administration of 15 mg of ketoconazole/kg, immediately after the 0800- hour blood sample was collected. None of these dogs had been treated previously for hyperadreno- corticism.
Adrenocorticotropin stimulation testing in dogs given long-term ketoconazole-An ACTH stimulation test was performed on 15 privately owned healthy dogs and on 60 dogs with spontaneous hyper- adrenocorticism. The long-term effect of ketocon- azole administration was assessed in 20 of the 60 privately owned dogs with hyperadrenocorticism. Dogs ranged in age from 6 to 16 years, with mean of 10.1 (± 1.8) years; 12 were female. These 20 dogs (11 with PDH; 9 with adrenocortical tumor) were given 30 mg of ketoconazole/kg, orally, divided into 2 equal doses, for 0.5 to 12 months. Other medications were not administered. An ACTH stimulation test was performed prior to initiation of ketoconazole administration and again on treat-
“Feldman EC, Tyrrell JB. Plasma cortisol and ACTH levels in spontaneous canine Cushing’s syndrome and Addison’s dis- ease (abstr), in Proceedings. 60th Annu Meet Endocr Soc 1978; 220.
ment days 5, 60, 180, and 360. In all dogs, tests were begun between 0800 and 1000 hours.
Endogenous ACTH concentration in dogs given ke- toconazole-Four dogs of the ketoconazole treat- ment group each had baseline endogenous ACTH concentration determined once before and once during the 4- to 12-month treatment period. All samples were obtained between 0800 to 1000 hours prior to any other endocrine testing.
Clinical evaluation of dogs given ketoconazole- In addition to monitoring of the endocrine vari- ables previously described, all dogs of the keto- conazole treatment group underwent clinical evaluations at 2-month intervals. Each evaluation consisted of a physical examination and obtaining information regarding the status of each clinical sign of hyperadrenocorticism (polyuria, polydip- sia, polyphagia) from the owner. Observance of any apparent side effects to ketoconazole was also considered.
Hormone assays-Blood samples obtained for plasma cortisol determination were immediately centrifuged, and the plasma was frozen. Plasma cortisol concentration was determined by use of an enzyme immunoassay.16 The assay used antiserum raised in rabbits to a cortisol 3-0 carboxymethy- loxime-bovine serum albumin immunogen and cortisol 3-0 carboxymethyloxime-horseradish per- oxidase as the label. The assay was specific for cor- tisol. Cross-reactivity to other corticosteroids was: prednisone, 6.3%; prednisolone, 9.9%; 11 a- deoxycorticosterone, 6%; cortisone, 5%; and cor- ticosterone, 0.7%. For all other steroids, including dexamethasone, cross-reactivity was lower. Corti- sol standards were prepared in ethanol to give a standard curve range of 0.1 to 250 pg/well. The intraplate coefficient of variation was 1.9%. The interplate assay precision determinations gave co- efficients of variation of 8.8% for low (0.9 µg/dl, n = 45), 7.3% for medium (18.6 µg/dl, n = 41), and 8% for high (41.2 µg/dl, n = 40) concentra- tions of cortisol in 3 pooled serum samples. The interassay coefficients of variation were 12.4, 10.5, and 6.8% for the same 3 pools of serum. The sen- sitivity of the system was 0.25 pg/well or 0.05 µg of cortisol/dl in canine plasma. Recovery was de- termined by assaying several samples to which various known amounts of cortisol had been added: average recovered was 98.8%, with range of 94 to 104%. Blood samples for endogenous ACTH assays were handled as described.17 Endogenous ACTH concentration was determined by radioimmuno- assay,18 with a reference range of 20 to 100 pg/ml established for normal dogs.17
Statistical analysis-All results are reported as mean ± SD, unless otherwise indicated. Data were analyzed by use of repeated-measures analyses of variance. Differences between group means were
evaluated, using the Student-Newman-Keul’s test. Values of P < 0.05 were considered significant.
Results
Cortisol secretion experiment-The 8-hour mean plasma cortisol concentration from samples obtained every 30 minutes (beginning at 0800 hours and ending at 1600 hours) was significantly (P < 0.05) different when each group of dogs with hyperadrenocorticism (PDH and adrenocortical tu- mor) was compared with the control group. How- ever, values in the 2 groups of dogs with hypera- drenocorticism were not significantly different from each other (Fig 1). The 8-hour mean plasma cortisol concentration for the entire period was highest in the 18 dogs with functioning adrenocor- tical tumor (5.3 ± 1.6 µg/dl), lowest in the 15 clinically normal dogs (1.3 ± 0.5 µg/dl), and intermediate in the 31 dogs with PDH (3.4 ± 1.2 µg/dl).
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Plasma was obtained for endogenous ACTH de- termination from 4 dogs (2 with adrenocortical tu- mors, 2 with PDH) every 30 minutes beginning at 0800 hours and ending at 1600 hours. The endog- enous ACTH concentration from each of the dogs with adrenocortical tumors was below the detec- tion limit of the assay (eg, <10 pg/ml). The 2 dogs with PDH had endogenous ACTH concentrations that fluctuated between 62 to 145 pg/ml and 102 to 188 pg/ml.
Cortisol secretion after the initial dose of keto- conazole-The administration of ketoconazole in the initial, orally administered dose (15 mg/kg at
0800 hours) significantly (P < 0.05) decreased the mean 8-hour serial cortisol concentration from 3.9 ± 0.5 µg/dl to 0.9 ± 0.2 ug/dl in the 3 dogs with adrenocortical tumor and in the 5 dogs with PDH. Difference was not seen by comparing re- sponse to ketoconazole in dogs with adrenocorti- cal tumors vs those with PDH. The 8-hour mean cortisol concentration in the 15 healthy dogs (1.3 ± 0.5 µg/dl) was not significantly different from the 8-hour mean post-ketoconazole cortisol concentration in the dogs with hyperadrenocorti- cism (Fig 2). Mean plasma cortisol concentration progressively returned toward pretreatment values at 1800 hours (2.6 ± 1.3 µg/dl vs 3.2 ± 1.2 µg/dl [untreated dogs]), 2000 hours (3.4 ± 2.9 µg/dl vs 3.6 ± 1.3 µg/dl [untreated dogs]), and at 0800 hours the morning after ketoconazole was admin- istered (4.2 ± 1.1 µg/dl vs 3.9 ± 1.1 µg/dl [un- treated dogs]). Prior to treatment, there was no significant difference between the 0800- to 1600- hour serial cortisol concentrations in the 31 dogs (with PDH) of the cortisol secretion experiment, compared with the 5 dogs (with PDH) of the keto- conazole experiment. There was also no significant difference between the 0800- to 1600-hour serial cortisol concentrations in the 18 dogs (with adreno- cortical tumor) of the cortisol secretion experiment and the 3 dogs with adrenocortical tumor that sub- sequently were given ketoconazole.
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Adrenocorticotropin stimulation testing after long-term ketoconazole administration-The mean baseline cortisol concentration was 1.4 ± 0.4 µg/ dl in 15 healthy control dogs. After ACTH adminis- tration, the mean cortisol concentration in control dogs was 10.6 ± 3.1 µg/dl. A post-ACTH cortisol
concentration >20.0 µg/dl (>3 SD above the refer- ence mean) was considered an abnormal response, suggestive of hyperadrenocorticism. Mean baseline plasma cortisol concentration in the 60 dogs with hyperadrenocorticism was 4.0 ± 3.3 µg/dl, and the mean post-ACTH plasma cortisol concentration was 25.2 ± 8.7 µg/dl.
The mean baseline plasma cortisol concentra- tion in the 38 dogs with PDH was 3.3 ± 3.7 µg/dl and, after ACTH stimulation, was 24.2 ± 8.0 µg/dl. Eleven dogs with PDH were treated with ketocona- zole. Before ketoconazole treatment, plasma corti- sol concentrations before and after ACTH adminis- tration were 4.4 ± 1.9 µg/dl and 33.6 ± 17.6 µg/ dl, respectively. The mean baseline and post-ACTH plasma cortisol concentrations in the dogs with PDH treated with and responding to ketoconazole were significantly (P < 0.01) lower on treatment days 5, 60, 180, and 360, compared with pretreatment values. On ketoconazole treatment day 5, the mean baseline plasma cortisol concentration in 9 dogs with PDH was 1.9 ± 2.0 µg/dl, and the post-ACTH value was 4.0 ± 5.4 µg/dl. On treatment day 60 in these 9 dogs, the mean baseline plasma cortisol concentration was 1.8 ± 1.0 µg/dl and the post- ACTH value was 6.4 ± 4.8 µg/dl. On treatment days 180 and 360, the mean baseline plasma cortisol concentration was 2.4 ± 1.1 µg/dl and 4.9 ± 0.4 µg/dl, respectively; the post-ACTH values were 5.8 ± 2.6 µg/dl and 6.0 ± 3.2 µg/dl, respectively (Fig 3). The post-ACTH plasma cortisol concentra- tion was significantly lower (P < 0.01) on treat- ment days 5, 60, 180, and 360, compared with the response to ACTH in the 15 control dogs. Of the 11 dogs with PDH given ketoconazole, treatment was discontinued in 2 because of failure to respond; 2 were euthanatized between the 180th and 360th days of treatment (see later discussion); and 5 were subsequently treated with an alternative medica- tion, 3 after 60 days and 2 after 180 days of keto- conazole administration.
The mean baseline plasma cortisol concentra- tion in the 22 dogs with adrenocortical tumor was 4.2 ± 1.4 µg/dl, and the post-ACTH value was 26.0 ± 6.4 µg/dl. The mean pre-ketoconazole ad- ministration baseline and post-ACTH plasma corti- sol concentrations in the 9 dogs with adrenocorti- cal tumor were 4.4 ± 1.3 µg/dl and 28.1 ± 14.1 ug/dl, respectively. The mean baseline and post- ACTH plasma cortisol concentrations in these 9 dogs after ketoconazole administration were signifi- cantly (P < 0.01) lower on treatment days 5, 60, 180, and 360, compared with pretreatment values. On ketoconazole treatment day 5, the mean base- line plasma cortisol concentration in these 9 dogs was 1.6 ± 1.2 µg/dl, and the post-ACTH value was 6.1 ± 3.3 µg/dl. By treatment day 60, ketocona- zole administration was continued in 7 of the 9 dogs with adrenocortical tumor, and 2 had under- gone surgical extirpation of the tumors. In the 7 dogs continuing ketoconazole treatment, the mean
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Days of Ketoconazole Treatment
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Days of Ketoconazole Treatment
A-Values in dogs with PDH. Numbers at each time point indicate number of dogs on test at that time; Rx = ketoconazole treatment
B-Values in dogs with adrenocortical tumor. Numbers at each time point indicate number of dogs on test at that time; Rx = ketoconazole treatment.
baseline plasma cortisol concentration was 0.6 ± 0.4 µg/dl, and the post-ACTH value was 2.5 ± 1.3 µg/dl. On treatment days 180 and 360, the mean baseline plasma cortisol concentration was 1.0 ± 1.2 µg/dl and 0.6 ± 0.7 µg/dl, respec- tively. After ACTH administration, values were 6.3 ± 3.1 µg/dl and 1.1 ± 0.1 µg/dl, respectively (Fig 3). After 60 days of ketoconazole administra- tion, 1 dog had surgery to remove the adreno- cortical tumor and 2 were euthanatized. Two additional dogs were euthanatized between keto- conazole treatment days 180 and 360. The post- ACTH plasma cortisol concentration was signifi- cantly (P < 0.01) lower on treatment days 5, 60, 180, and 360, compared with that in the clinically normal control dogs.
Two dogs with PDH had no endocrine response to ketoconazole. The dogs were treated for 14 and 30 consecutive days, respectively. Results of ACTH stimulation testing in these 2 dogs indicated no significant differences when comparing pretreat- ment results with those obtained during ketocon- azole administration.
Endogenous ACTH concentration during ketocon- azole treatment-Three dogs with PDH had endo- genous ACTH concentration determined over a 4- to 12-month ketoconazole treatment period; before treatment, endogenous ACTH concentration was 67, 47, and 85 pg/ml, with mean of 66.3 (± 19) pg/ml. During treatment, endogenous ACTH concentration was 83, 17, and 56 pg/ml, respectively, with mean of 52 (±33) pg/ml. At the time of the second en- dogenous ACTH assessment in each dog, remission of clinical signs of hyperadrenocorticism and sub- normal plasma cortisol response to exogenous ACTH had been observed. Endogenous ACTH deter- mination was performed serially on 1 dog with adrenocortical tumor. Change in ACTH concentra- tion (<10 pg/ml) was not seen after 9 weeks of ke- toconazole administration, despite clinical im- provement and subnormal plasma cortisol response to exogenous ACTH.
Clinical evaluation-All 9 dogs with adreno- cortical tumor and 9 of 11 dogs with PDH responded to ketoconazole. This response not only included suppression of plasma cortisol concentration, but marked and long-term improvement in clinical condition. Remission of polydipsia, polyuria, poly- phagia, and other signs of hyperadrenocorticism were observed in these 18 dogs. One of these 18 dogs had gastrointestinal side effects (anorexia, vomiting) that necessitated temporary discontinu- ation of treatment. Two dogs failed to respond to the drug, as previously mentioned.
During or after the study, 6 dogs were eutha- natized because of problems thought to be unre- lated to ketoconazole administration. One of 2 dogs with PDH that was euthanatized had heart fail- ure, and the other had renal failure. These prob- lems had been identified prior to initiation of ke- toconazole treatment. The 4 dogs with adreno- cortical tumor that were euthanatized each had tu- mor metastasis that caused cachexia, weakness, and related signs of disease. Necropsy findings confirmed the antemortem diagnosis of PDH in 2 dogs. Both dogs had bilateral adrenocortical hy- perplasia. One had solitary pars distalis adenoma, and the other had solitary pars intermedia ade- noma. The other 4 dogs had adrenocortical carci- noma confirmed at necropsy. The 5 remaining dogs with adrenocortical tumors are alive after surgical and histologic confirmation of the diagno- sis. The remaining 9 dogs with PDH are alive and well, and are being treated with ketoconazole (n = 2) or mitotaned (n =7).
Discussion
Loss of normal circadian rhythmicity of the hypothalamic-pituitary-adrenal axis is one of the hallmarks of hyperadrenocorticism in people. 18,19 Previous studies20,21 indicate that clinically normal dogs do not appear to have circadian rhythm in plasma proopiomelanocortin peptide or cortisol
dLysodren, Bristol-Myers Co, Evansville, Ind.
concentrations, although secretion is episodic, as it is in people.22,23 Dogs with PDH appear to have normal frequency of secretory episodes, but of greater amplitude, resulting in high mean daily plasma cortisol concentration. This has been doc- umented in some human patients with hyper- adrenocorticism.24 We have documented in this report that dogs with spontaneous PDH and those with cortisol-secreting adrenocortical tumor have significantly higher mean daily plasma cortisol concentration than do clinically normal dogs. The lack of circadian rhythm in clinically normal dogs allowed the use of pet dogs, those that were healthy as well as those with hyperadrenocorticism, be- cause strict control of light and dark periods was not needed.
Ketoconazole effectively blocks cortisol syn- thesis in dogs with PDH as well as in those with adrenocortical tumor. Similar results with ketocon- azole have been reported in the management of hyperadrenocorticism in people.25-30 Ketocona- zole (15 mg/kg) given to the dogs of our study re- sulted in a rapid reduction in baseline plasma cor- tisol concentration. That dose, administered twice daily, resulted in significant reduction in plasma cortisol response to exogenous ACTH. The inhibi- tion of cortisol synthesis in 18 of the dogs with hy- peradrenocorticism was sustained throughout the treatment period, as evidenced by pre- and post- cortisol responses to exogenous ACTH. Such re- sponses were below control values at treatment days 5, 60, 120, 180, and 360, and the physical characteristics and clinical signs of hyperadreno- corticism were alleviated during the same period.
Ketoconazole was an effective therapeutic drug in dogs with PDH and adrenocortical tumor. There- fore, the pathway of cortisol synthesis was inhib- ited by ketoconazole as an enzyme blocker, re- gardless of the cause of excess cortisol secretion. Little or no reduction in plasma cortisol concen- tration was observed after administration of keto- conazole at a dosage of 5/kg every 12 hours (data not shown).
As can be seen in Figure 2, the reduction in plasma cortisol concentration was rapid after ini- tial administration of ketoconazole to 8 dogs with hyperadrenocorticism. Within 30 minutes, signif- icant reduction in cortisol concentration was ob- served. The half-life of cortisol in the circulation has been estimated to be 60 to 90 minutes31; thus, the rapid action of ketoconazole was striking. The plasma cortisol concentration decreased quickly, and the effects of a single dose of ketoconazole lasted as long as 8 to 12 hours, but <24 hours.
Ketoconazole was not only an effective inhib- itor of cortisol synthesis in the short term, but over a period of months as well. Sixteen dogs with hy- peradrenocorticism were well controlled for at least 60 days. Four of these dogs were treated for at least 12 months without problems. Ketocona- zole administration was discontinued because of availability of alternative treatment (surgery and/or
mitotane administration) that was less expensive or more efficacious.
Two dogs failed to respond to ketoconazole. Ketoconazole has been shown to have unpredict- able bioavailability when administered orally to dogs and people.32 Failure to respond, therefore, could have been the result of poor intestinal absorption of ketoconazole. Assessment of thera- peutic concentration of the drug in serum or plasma may be necessary to help identify individ- uals who may not be absorbing ketoconazole from the intestinal tract. One dog developed gastrointes- tinal side effects that prompted the owner to dis- continue medication. Ketoconazole overdosing could result in hypocortisolism, with clinical signs consistent with this disease.33 Such signs include vomiting, diarrhea, anorexia, weakness, and de- pression, which were observed in the aforemen- tioned dog. The dog rapidly responded to discon- tinuation of ketoconazole administration and prednisolone treatment after which, ketaconazole was successfully reinstituted at a slightly lower dose.
A compensatory increase in endogenous ACTH concentration was seen when ketoconazole was administered in high doses to people with normal function of the hypothalamic-pituitary-adrenal axis.34 However, this compensatory increase in ACTH concentration has not been observed in peo- ple with either Cushing’s disease or Cushing’s syn- drome, despite marked reductions in their plasma and urinary cortisol concentrations.25,26,29 This would appear to be true in dogs as well, because ketoconazole had little effect on endogenous ACTH concentration in 4 affected dogs treated for 2 to 12 months. In contrast, after treatment with the adrenocorticolytic agent mitotane, a twofold in- crease in endogenous ACTH concentration has been documented in dogs with PDH.18
The mechanism(s) responsible for the lack of a compensatory increase in ACTH concentration are not known. It has been shown that the ACTH response to corticotropin-releasing factor during the administration of ketoconazole was unchanged, compared with the pretreatment response in hu- man patients with Cushing’s disease.25 This finding provides evidence against the concept that keto- conazole has an inhibitory effect at the pituitary gland level and that long-term ketoconazole treat- ment may in some way modify the hypothalamic- pituitary-adrenal axis in patients with Cushing’s disease.35 However, it has been reported that ke- toconazole inhibits corticotropin releasing factor- stimulated ACTH biosynthesis and release from rat anterior pituitary cells in vitro, through inhibition of the catalytic component of adenylate cyclase, resulting in decreased cyclic AMP generation.36,e The clinical importance of this type of inhibition of
eStalla GK, Stalla J, Hollt V. The catalytic subunit of the adenylate cylase in anterior pituitaries is inhibited by imidazole derivatives (abstr), in Proceedings. 70th Annu Meet Endocr Soc 1988; 222.
ACTH release in patients with hyperadrenocorticism is unknown.
In conclusion, the imidazole derivative keto- conazole was effective in decreasing plasma corti- sol concentration in dogs with hyperadrenocorti- cism. The drug was equally effective in dogs with PDH and in dogs with hyperadrenocorticism attrib- utable to adrenocortical adenoma or carcinoma. The effect of a single ketoconazole dose was observed within 30 minutes of administration and lasted <24 hours. Twice-daily treatment was suc- cessful in resolving clinical signs of hyperadreno- corticism in 18 of 20 dogs for at least 60 days and in 4 dogs for as long as 12 months. Two dogs failed to respond to ketoconazole, and 1 dog had evi- dence of hypocortisolism.
Our recommendations are to treat dogs with hyperadrenocorticism, using ketoconazole at a dosage of 5 mg/kg given every 12 hours for 7 days, merely to evaluate dogs for side effects such as acute hepatitis or gastritis. Although to the authors’ knowledge, such has not yet been reported in dogs, ketoconazole can cause hepatopathy in people. This may result in anorexia, lethargy, jaundice, and abnormal liver enzyme activities. If no ill effects are observed, the dosage should be increased to 10 mg/kg (q 12 h) for 14 days, and an ACTH stimula- tion test should be performed at that time. If nor- mal or exaggerated response to exogenous ACTH is observed, increase the ketoconazole dosage to 15 mg/kg (q 12 h) and continue to monitor the dog (as previously described) every 14 to 60 days.
At this time, ketoconazole treatment is princi- pally used before surgery in dogs with adrenocor- tical tumor or as the only mode of treatment, be- cause such tumors are often resistant to mitotane; in dogs that do not tolerate mitotane; and in dogs in which ketoconazole treatment is used as a diag- nostic aid. The authors are not aware of any chronic effects of ketoconazole on nonsteroid-producing body systems, as assessed by results of routine lab- oratory tests. In conclusion, ketoconazole is effec- tive for decreasing plasma cortisol concentrations in most dogs with hyperadrenocorticism.
References
1. Rijnberk A, derKineren PJ, Thijssen JHH. Spontaneous hyperadrenocorticism in the dog. J Endocrinol 1968;41:394-406.
2. Feldman EC. The adrenal cortex. In: Ettinger SJ, ed. Textbook of veterinary internal medicine. 2nd ed. Philadelphia: WB Saunders Co, 1983;1650.
3. Peterson ME, Krieger DT, Drucker WD, et al. Immu- nocytochemical study of the hypophysis in 25 dogs with pitu- itary-dependent hyperadrenocorticism. Acta Endocrinol (Copenh) 1982;101:15-24.
4. Johnston DE. Adrenalectomy via retroperitoneal ap- proach in dogs. J Am Vet Med Assoc 1977;170:1092-1096.
5. Lantz GC, Ihle SL, Nelson RW, et al. Transsphenoidal hypophysectomy in the clinically normal dog. Am J Vet Res 1988;49:1134-1142.
6. Turrel JM, Fike JR, LeCouteur RA, et al. Radiotherapy of brain tumors in dogs. J Am Vet Med Assoc 1984;184:82-86.
7. Scavelli TD, Peterson ME, Matthiesen DT. Results of
surgical treatment for hyperadrenocorticism caused by adreno- cortical neoplasia in the dog: 25 cases (1980-1984). J Am Vet Med Assoc 1986;189:1360-1364.
8. Pont A, Williams PL, Azhar DA. Ketoconazole blocks testosterone synthesis. Arch Intern Med 1982;142:2137-2140.
9. Pont A, Williams PL, Loose DS, et al. Ketoconazole blocks adrenal steroid synthesis. Ann Intern Med 1982;97:370- 372.
10. Loose DS, Kan PB, Hirst MA et al. Ketoconazole blocks adrenal steroidogenesis by inhibiting cytochrome P450- dependent enzymes. J Clin Invest 1983;71:1495-1499.
11. DeCoster R, Beerens D, Dom J, et al. Endocrinolog- ical effects of single daily dose ketoconazole administration in male beagle dogs. Acta Endocrinol (Copenh) 1984;107:275-281.
12. Willard MD, Nachreiner R, McDonald R, et al. Keto- conazole-induced changes in selected canine hormone con- centrations. Am J Vet Res 1986;47:2504-2509.
13. Feldman EC, Stabenfeldt GH, Farver TB, et al. Com- parison of aqueous-porcine ACTH with synthetic ACTH in ad- renal stimulation tests of the female dog. Am J Vet Res 1982; 43:522-524.
14. Feldman EC. Comparison of ACTH response and dexamethasone suppression as screening tests in canine hyper- adrenocorticism. J Am Vet Med Assoc 1983;182:506-510.
15. Feldman EC. Distinguishing dogs with functioning adrenocortical tumors from dogs with pituitary-dependent hy- peradrenocorticism. J Am Vet Med Assoc 1983;183:195-200.
16. Smith MC, Feldman EC. Plasma endogenous ACTH concentrations and plasma cortisol responses to synthetic ACTH and dexamethasone sodium phosphate in healthy cats. Am J Vet Res 1987;48:1719-1724.
17. Feldman EC, Bohannon NV, Tyrrell JB. Plasma adrenocorticotropin levels in normal dogs. Am J Vet Res 1977; 38:1643-1645.
18. Nelson RW, Feldman EC, Shinsako J. Effect of o,p’- DDD therapy on endogenous ACTH concentrations in dogs with hypophysis-dependent hyperadrenocorticism. Am J Vet Res 1985;46:1534-1537.
19. Liddle GW. The adrenal cortex. In: William RH, ed. Textbook of endocrinology. 6th ed. Philadelphia: WB Saunders Co, 1981;249-253.
20. Orth DN, Peterson ME, Drucker WD. Plasma immu- noreactive proopiomelanocortin peptide and cortisol in normal dogs and dogs with Cushing’s syndrome: diurnal rhythm and responses to variable stimuli. Endocrinology 1988;122:1250- 1262.
21. Kemppainen RJ, Sartin JL. Evidence for episodic but not circadian activity in plasma concentration of adrenocorti- cotrophin, cortisol and thyroxine in dogs. J Endocrinol 1984; 103:219-226.
22. Gallagher TF, Yoshida K, Roffwarg HD, et al. ACTH and cortisol secretory patterns in man. J Clin Endocrinol Metab 1973;36:1058-1068.
23. Tanaka K, Nicholson WE, Orth DN. Diurnal rhythm and disappearance half-time of endogenous plasma immuno- reactive ß-MSH (LPH) and ACTH in man. J Clin Endocrinol Metab 1978;46:883-887.
24. Van Cauter E, Retenoff S. Evidence for two subtypes of Cushing’s disease based on the analysis of episodic cortisol secretion. N Engl J Med 1985;312:1343-1349.
25. Loli P, Berselli ME, Tagliaferri M. Use of ketoconazole in the treatment of Cushing’s syndrome. J Clin Endocrinol Metab 1986;63:1365-1371.
26. Sonino N, Boscara M, Merola G, et al. Prolonged treatment of Cushing’s disease by ketoconazole. J Clin Endo- crinol Metab 1985;61:718-722.
27. Shepherd FA, Hoffert B, Evans WK, et al. Ketocona- zole use in the treatment of ectopic adrenocorticotropic hor- mone production and Cushing’s syndrome in small-cell lung cancer. Arch Intern Med 1985;145:863-864.
28. Contreras P, Alterieri E, Liberman C, et al. Adrenal rest tumor of the liver causing Cushing’s syndrome: treatment with ketoconazole preceding an apparent surgical cure. J Clin Endocrinol Metab 1985;60:21-28.
29. Angeli A, Fraira R. Ketoconazole therapy in Cushing’s disease. Lancet 1985;1:821.
30. Contreras P, Rojas A, Biagini L, et al. Regression of metastatic adrenal carcinoma during palliative ketoconazole treatment. Lancet 1985;2:151-152.
31. Ganong WF. Review of medical physiology. Los Altos, Calif: Lange Medical Publications, 1979;285-286.
32. Baxter JG, Brass C, Schentag J, et al. Pharmacokinet- ics of ketoconazole administered intravenously to dogs and orally as tablet and solution to humans and dogs. J Pharm Sci 1986;75:443-447.
33. Legendre AM, Selcer BA, Edwards DF, et al. Treat- ment of canine blastomycosis with amphotericin B and keto- conazole. J Am Vet Med Assoc 1984;184:1249-1258.
34. Trachtenberg J, Pont A. Ketoconazole therapy for advanced prostatic cancer. Lancet 1984;2:433-435.
35. Sonino N. The use of ketoconazole as an inhibitor of steroid production. N Engl J Med 1987;317:812-818.
36. Werker K, Muller OA. Ketoconazole inhibits corti- cotropic cell function in vitro. Endocrinology 1988;122:618- 623.
Effects of flunixin and flunixin plus prednisone on the gastrointestinal tract of dogs
Flunixin meglumine has been reported to induce gas- trointestinal lesions in dogs when administered at therapeu- tic dosages. We administered flunixin meglumine to dogs daily for 10 days to assess the effect of this drug on the gastrointestinal tract. We also evaluated the pos- sibility of corticosteroid potenti- ation of gastrointestinal toxico- sis by concurrent administration of prednisone to 1 group of dogs. Dogs were monitored for gas- trointestinal toxicosis by means of serial endoscopic evaluation, measurement of fecal occult blood, PCV, and total solid con- centration, and by physical ex- amination. There were 3 treat- ment groups of 5 dogs each. Group-1 dogs were given 2.2 mg of flunixin meglumine/kg of body weight daily, in 2 divided doses IM; group-2 dogs were given 4.4 mg of flunixin meglumine/kg daily, in 2 divided doses IM; and group-3 dogs were given 2.2 mg
of flunixin meglumine/kg daily, in 2 divided doses IM, plus 1.1 mg of prednisone/kg/d orally, in 2 divided doses. A fourth group of 5 dogs served as a control group.
Endoscopically visible gas- tric mucosal lesions developed in all treated dogs within 4 days of initiation of treatment. Le- sions first developed in the gas- tric pylorus and antrum, and le- sions at these sites were more severe than those observed else- where. Dogs treated with flunixin meglumine plus pred- nisone developed the earliest and most severe lesions; lesion scores in group-2 dogs were higher than those in group-1 dogs. All dogs treated had occult blood in their feces by day 5, and its presence appeared to corre- late more closely with endo- scopic findings than did physical examination findings or changes in values for PCV or total solids.
Deep ulcers were observed
in the pylorus of most treated dogs examined at necropsy on day 10. Shallow ulcers and ero- sions were in the small intestine of group-2 and -3 dogs. Capil- lary microthrombi, associated with lesions of coagulative ne- crosis of superficial epithelium, were found in the colonic and small intestinal mucosa of sev- eral dogs in groups 2 and 3, and were suggestive of vascular in- jury.
From results of this study, it was concluded that flunixin meglumine, administered at therapeutic doses, induced early gastric mucosal injury in dogs and that concurrent administra- tion of prednisone may have ex- acerbated the gastrointestinal in- jury induced by flunixin alone .- Steven W. Dow, Rodney A. W. Rosychuk, Alexander E. McCheshney, et al, Am J Vet Res, 51 (July 1990).