Use of Compounded Adrenocorticotropic Hormone (ACTH) for Adrenal Function Testing in Dogs
Serum cortisol concentrations were measured in five healthy dogs in response to five adrenocorticotropic hormone (ACTH) preparations. Cortisol concentrations were similar at time 0 (pre-ACTH) and at 30 and 60 minutes after injection of all forms of ACTH. However, at 90 and 120 minutes post-ACTH, serum cortisol concentrations were significantly lower following injection of two compounded forms of ACTH. The data showed that injection of four compounded forms of ACTH caused elevations in serum cortisol concentrations of a similar magnitude as cosyntropin in samples collected 60 minutes after administration; but concentrations at later times varied, depending on the type of ACTH used.
Introduction
The adrenocorticotropic hormone (ACTH) stimulation test is an important and useful screening test to diagnose hyper- and hypoadrenocorticism in both dogs and cats.1 In addition, the test is valuable in monitoring the effectiveness of therapy for hyperadrenocorticism.2 There are few adequate substitutes for the ACTH stimulation test in diagnosing hypoadrenocorticism or in monitoring the treatment of hyperadrenocorticism.
Two persistent problems exist with the test: namely, poor availability and the high cost of commercially available ACTH. Over the past several years, a number of approved ACTH products have been withdrawn from the market. The only ACTH product currently available is synthetic ACTH or cosyntropin.a Manufacturing issues related to production and distribution of cosyntropin make it periodically unavailable. Additionally, the cost of cosyntropin has risen significantly in the recent past.
Studies in dogs and cats have shown that lower doses of cosyntropin can reliably stimulate cortisol secretion and that the drug can be stored frozen in aliquots.3–6 These findings have permitted multiple ACTH stimulation tests to be performed from each vial of cosyntropin, thereby helping to reduce the cost of the test in small animals.7 Nevertheless, because of problems with the availability and cost of cosyntropin, several pharmacies have made compounded ACTH available to veterinarians. It is unclear whether these products are reliable and effective for use in ACTH stimulation testing. Adrenocorticotropic hormone is a peptide hormone, and its instability in plasma is well known.8 Only one pilot study has been published that examined the effectiveness of compounded ACTH in dogs.1 In that report, serum cortisol concentrations in five healthy dogs and in four dogs with clinical signs of possible hypo- or hyperadrenocorticism were measured following injection of either a compounded ACTH product or cosyntropin.1 Although no statistically significant differences in post-ACTH serum cortisol concentrations were found in the dogs, mean post-ACTH cortisol concentrations were generally lower in response to the compounded material.1
The purpose of the study reported here was to evaluate the reliability of compounded ACTH products for adrenal stimulation tests in dogs, by comparing serum cortisol concentrations after administration of four compounded substances to concentrations that were achieved after administration of cosyntropin.
Materials and Methods
Study Subjects
Five, mixed-breed, research dogs that were housed by the Division of Laboratory Animal Health at the College of Veterinary Medicine, Auburn University, were used in the study. The dogs included three intact males and two intact females that weighed between 9.1 and 11.8 kg. The females were not in proestrus or estrus at the time of the study. All dogs were considered healthy based on physical examination and routine laboratory testing (i.e., complete blood count, biochemical profile). The study was reviewed and approved by the Auburn University Institutional Animal Care and Use Committee.
Study Design
The ACTH products used in the study included cosyntropina (ACTH A) and four randomly chosen compounded forms of ACTHb,c,d,e (ACTH B through E) purchased from veterinary pharmacies. All compounded forms of ACTH were labeled as containing 40 units of ACTH per mL.
Adrenocorticotropic hormone stimulation tests were performed on five different occasions, with each test separated by 3 or 4 days.9 On each test day, the dogs were weighed, jugular blood samples were collected (time 0), and then either one of the compounded forms of ACTH was given intramuscularly in the epaxial musculature (2.2 units/kg) or cosyntropin was administered intravenously (5 μg/kg).4 Subsequent blood samples were collected at 30, 60, 90, and 120 minutes post-ACTH injection. Blood samples were allowed to clot for 2 hours and were then chilled on ice for 30 minutes, followed by centrifugation and serum collection. Each dog was given a different form of ACTH on the five test dates in order for all individual dogs to receive each ACTH product. Serum cortisol concentrations were determined using radioimmunoassay (RIA).f,10 Immunoreactive ACTH concentrations in each of the four compounded forms of ACTH were measured via a previously validated RIA that uses an antibody that recognizes amino acids 5–18 of ACTH, a region common to all forms of ACTH.10
Statistical Analysis
Cortisol concentrations measured in response to each ACTH product were compared using two-way analysis of variance (ANOVA) for repeated measures,g with the main effects being time and the ACTH product.11 The serum cortisol concentrations determined in response to cosyntropin were considered as the control for statistical purposes; P values <0.05 were considered significant. Comparisons between control values and other results were made using Dunnett’s test, and the Tukey test was used to compare values at different times within a group.g,11
Results
Figure 1 shows the mean serum cortisol concentrations in dogs following administration of the five forms of ACTH. The ANOVA indicated significant (P<0.001 for each) differences related to time, type of ACTH, and their interaction. Serum cortisol concentrations were not different when comparing mean values for the cosyntropin (ACTH A)-treated dogs to the results from the other groups at times 0, 30 minutes, and 60 minutes. However, at 90 and 120 minutes, cortisol concentrations in dogs given ACTH B or ACTH E were lower (P<0.001 for both times and both types of ACTH) than concentrations in dogs given cosyntropin (ACTH A). At 120 minutes, cortisol concentrations in dogs given ACTH D were greater (P=0.007) than the concentrations measured in dogs given cosyntropin. Concentrations at 120 minutes in dogs given ACTH D were also greater (P=0.015) than levels in dogs given ACTH D at 60 minutes.
Figures 2 through 4 illustrate the differences and the range of values measured in response to the ACTH products tested. Cortisol concentrations at 60 minutes were similar in all dogs, regardless of the ACTH products; however, at 90 minutes, greater individual variation was observed following ACTH B and ACTH E. The mean cortisol concentrations in these two groups also declined. At 120 minutes, individual variation was most apparent in response to ACTH A, ACTH B, and ACTH C. Of all the ACTH products, only ACTH D resulted in consistent and sustained serum cortisol concentrations at all the sample times. At 120 minutes, two dogs given ACTH E and one dog given ACTH B had serum cortisol concentrations below their respective values at time 0.
The compounded ACTH products varied in consistency, from a solid gel at 4°C to liquids that were thick to watery. Aliquots (50 microliters) of ACTH B to E were removed from the vials during the course of the study, serially diluted, and assayed in triplicate for immunoreactive ACTH.10 Immunoreactive ACTH concentrations varied considerably [see Table]. While the ACTH form that produced the most sustained response (ACTH D) contained the highest concentration of the hormone, the form that contained the next highest amount induced the shortest response (ACTH E). The other forms of ACTH contained considerably less immunoreactive hormone.
Discussion
The goal in ACTH stimulation testing is to provide sufficient ACTH to cause the maximal stimulation secretion of cortisol by the adrenal glands. Most dogs with hyperadrenocorticism have higher-than-normal concentrations of circulating cortisol because of their increased functional adrenal mass, while dogs with hypoadrenocorticism have lower concentrations from loss of adrenal tissue or functional atrophy. The times that maximal circulating concentrations of cortisol occur relative to the administration of ACTH may vary, depending on the form of the hormone used and its route of delivery.1,3–5 Previous studies have examined the time-dependent responses of circulating cortisol concentrations after administration of cosyntropin or ACTH gel products, as well as the effects of varying the dose of ACTH.1,3–5,12
In the study reported here, administration of four forms of compounded ACTH induced serum cortisol concentrations that were similar in degree to cosyntropin at 60 minutes following injection. However, at later times, concentrations of cortisol hormone varied considerably, depending upon the ACTH product used. Since dogs with adrenal disease were not studied, it is unknown whether the compounded forms of ACTH reliably result in higher-than-normal cortisol concentrations at 60 minutes in dogs with hyperadrenocorticism (as has been demonstrated when cosyntropin is used).3,4 Results of a previous study suggested that serum cortisol concentrations were lower (but not significantly so) when compounded ACTH was given to dogs with hyperadrenocorticism, compared with cosyntropin.1 Although there are no known scientific reasons why a maximal dose of ACTH delivered in a compounded form should be less effective than cosyntropin in dogs with hyperadrenocorticism, a comparative study using affected dogs is warranted.
While serum cortisol concentrations were similar for all forms of ACTH at 60 minutes, differences were detected at later times. Specifically, serum cortisol concentrations after administration of ACTH B and ACTH E were lower at 90 and 120 minutes than concentrations in dogs given cosyntropin. In fact, concentrations at 120 minutes in some dogs given these two forms of ACTH were near or below their respective values prior to administration of the drug. These findings are pertinent, given that the most commonly recommended protocol for ACTH response testing, when using products other than cosyntropin, is the collection of a post-ACTH sample at 120 minutes. It is conceivable that the transient response observed in some dogs following these ACTH products accounts for the previous conclusion of inactivity in some forms of ACTH.13 These data support the recommendation that veterinarians using compounded ACTH products collect several post-ACTH samples, with a minimum of 1- and 2-hour samples.
Administration of one of the ACTH products (ACTH D) resulted in a prolonged and sustained response, such that serum cortisol concentrations at 120 minutes were greater than those measured after cosyntropin. In fact, cortisol concentrations at 120 minutes were greater than those at 60 minutes in the same group. This prolonged response is similar to that previously found in dogs administered an ACTH gel product.h,5 Presumably, the prolonged presence of a maximal amount of ACTH (possibly from its continued release from the intramuscular site of injection) results in sustained secretion and gradual increases in serum cortisol concentrations.14 These findings raise the possibility that reference ranges for adrenal response tests may be influenced by the form of ACTH used and the time that post-ACTH samples are collected. It is noteworthy that all forms of ACTH, including cosyntropin, resulted in similar serum cortisol concentrations at 60 minutes post-ACTH.
Considerable variation in immunoreactive ACTH content was found in the different compounded products. Interestingly, the ACTH content did not necessarily correlate with the duration of the serum cortisol response. While the form that had the highest immunoreactive ACTH content (ACTH D) did produce the most sustained serum cortisol response, the form that contained the next highest amount (ACTH E) produced one of the most transient responses. The type or source of ACTH used in each product was not known. These findings suggested that the durations of action for compounded ACTH products in dogs vary considerably, and these variations cannot solely be accounted for by the content of immunoreactive ACTH.
Weaknesses of this study include the small number of dogs tested; the use of only healthy, laboratory-maintained dogs; and the limited number of compounded ACTH products evaluated. Future studies comparing the actions of a wider range of these products are warranted, perhaps with additional testing for possible lot-to-lot variations in activity. Studies in healthy pet dogs, as well as in dogs with possible and confirmed adrenal disease, are needed.
Conclusion
Adrenocorticotropic hormone response tests were performed in healthy dogs using four compounded formulations and cosyntropin. All ACTH products resulted in equivalent serum cortisol concentrations at 60 minutes; but at later times, cortisol concentrations varied depending on the type of ACTH used. Based on these results, veterinarians using compounded ACTH products should collect samples at a minimum of 1 and 2 hours post-ACTH administration. Further studies are warranted to examine various compounded ACTH products in a greater number of dogs, including those with adrenal disease.
Cortrosyn; Amphastar Pharmaceuticals, Rancho Cucamonga, CA 91730
Corticotropin LA Injectable Gel; Pet Health Pharmacy, Youngtown, AZ 85363
Corticotrophin LA Gel; Wedgewood Pharmacy, Swedesboro, NJ 08085
Corticotrophin LA Gel; Red Oak Drug, Red Oak, TX 75154
ACTH Solution; Meds for Vets, Sandy, UT 84070
Coat-A-Count Cortisol; Diagnostic Products Corporation, Los Angeles, CA 90045
Sigma Stat 3.1; Systat Software, Point Richmond, CA 94804
Acthar gel; Rhone-Poulenc Rorer, Collegeville, PA 19426
Acknowledgments
The authors thank Hollie Price Lee and Kathleen O’Donnell for their help in sample collection.
Citation: Journal of the American Animal Hospital Association 41, 6; 10.5326/0410368
Citation: Journal of the American Animal Hospital Association 41, 6; 10.5326/0410368
Citation: Journal of the American Animal Hospital Association 41, 6; 10.5326/0410368
Citation: Journal of the American Animal Hospital Association 41, 6; 10.5326/0410368
Mean serum cortisol concentrations in five groups of dogs immediately before (time 0) and after administration of cosyntropin (ACTH A) and four forms of compounded ACTH (ACTH B, C, D, E). Each point represents the mean value from five dogs. See text for the sources of ACTH B, C, D, and E.
Box plot showing the range of serum cortisol concentrations in dogs at 60 minutes after administration of five forms of ACTH (A, B, C, D, E). The horizontal bars define the entire range of data; the edges of the boxes show the 25th and 75th percentiles; the solid line within the box is the median; and the dotted line is the mean value.
Box plot (details in legend of Figure 2) showing the range of serum cortisol concentrations in dogs at 90 minutes after administration of five forms of ACTH (A, B, C, D, E). The asterisk (*) indicates a significant difference (P<0.001) when compared with the control group (ACTH A) given cosyntropin.
Box plot (details in legend of Figure 2) showing the range of serum cortisol concentrations in dogs at 120 minutes after administration of five forms of ACTH (A, B, C, D, E). The asterisk (*) indicates a significant difference (P<0.01 or greater) when compared with the control group (ACTH A) given cosyntropin.
