Introduction
Ocular surface inflammation and anterior uveitis are commonly encountered conditions in veterinary medicine that have the potential to be both painful and vision-threatening. Treatment of these conditions often requires anti-inflammatory medications that can be administered topically, by local injection (subconjunctival, intracameral, or intravitreal), or systemically.
The administration route for anti-inflammatory medications is determined by the severity and location of the inflammation. Topical ophthalmic glucocorticoid drugs and NSAIDs are most commonly used to control inflammation in the anterior segment of the eye. Topical administration is often the preferred route of treatment because of ease of application, high local drug concentration, and minimal systemic adverse effects.1 Glucocorticoid drugs are more commonly used than NSAIDs for their greater anti-inflammatory and immunosuppressive effects mediated through action on the intracytoplasmic glucocorticoid receptor.2 A number of ophthalmic glucocorticoid drug preparations are available with varying potency, concentration, and degree of corneal penetration.1,3
When topical ophthalmic medications are applied, they remain within the conjunctival sac for approximately 10 seconds to several minutes depending on the vehicle used, volume instilled, blinking rate, and degree of reflex tearing elicited.4,5 A small portion (< 5% to 10%) of topically applied ophthalmic medications enters the eye after absorption via corneal and, to a lesser extent, noncorneal (conjunctival or scleral) routes.3,6–10 Most of the medication is eliminated through the nasolacrimal duct.5,11 Following topical application of ophthalmic medications, a portion is absorbed systemically via the conjunctiva and/or the nasal, oral, or gastrointestinal mucosa after the medication passes through the nasolacrimal system.9–15 When absorbed via the ocular, oral, or nasal mucous membranes, medications do not undergo first-pass metabolism in the liver but instead have direct delivery into systemic circulation.9,16 A portion of an eyedrop is also wasted via spillage over the periocular skin (where there is presumably no to little systemic absorption), especially for volumes that exceed the volumetric capacity of the ocular surface.17,18
Systemic absorption of topically applied ophthalmic glucocorticoid drugs has been evaluated in several species including dogs,19 horses,20 rabbits,21,22 and humans.23–25 Although systemic absorption is incomplete, systemic adverse effects including the development of iatrogenic hyperadrenocorticism, suppression of the hypothalamic pituitary adrenal axis (HPAA), and glucose dysregulation have been reported following topical application of ophthalmic glucocorticoid drugs.26,27
Since topical ophthalmic glucocorticoid drugs are commonly prescribed for dogs with ocular surface inflammation and anterior uveitis, knowledge of the systemic absorption of these medications is important due to the potential risk of systemic side effects. The primary aim of this study was to establish the systemic absorption of 2 commercially available ophthalmic glucocorticoid drugs (prednisolone acetate 1% and neomycin polymyxin B dexamethasone 0.1% ophthalmic suspensions) in healthy dogs. We hypothesized that there would be low, but detectable, plasma concentrations of prednisolone and dexamethasone following topical ophthalmic application.
A secondary objective of this study was to evaluate the potential impact of sampling site on plasma drug concentrations. Previous studies28–30 evaluating plasma drug concentrations following oral mucosal administration have found increased drug levels in samples obtained from the jugular vein when compared to samples obtained from peripheral veins. Since the jugular veins drain the mucosa from the conjunctiva, nose and oral cavity,31 we hypothesized that there would be increased plasma drug concentrations in jugular samples when compared with peripheral venous samples.
A tertiary objective of this study was to evaluate for suppression of the HPAA following topical administration of ophthalmic glucocorticoid drugs in healthy dogs. We hypothesized there would be a significant decrease in plasma cortisol concentrations from baseline following topical ophthalmic application of both prednisolone and dexamethasone.
Materials and Methods
Animals
Twelve purpose-bred adult Beagles were enrolled in this study. None of the dogs had received topical or systemic glucocorticoid drugs for at least 1 month prior to enrollment in the study. All dogs were housed in a climate-controlled environment with a 12-hour light-dark cycle. Dogs were individually housed in separate enclosures for the duration of the study. All protocols were reviewed and approved by the Kansas State University Institutional Animal Care and Use Committee.
Examination
Prior to enrollment, each dog underwent a complete physical and ophthalmic examination performed by both a board-certified veterinary ophthalmologist (AJR) and a resident in a veterinary ophthalmology training program (MME). The ophthalmic examination included a neuro-ophthalmic examination (menace response, dazzle reflex, direct and indirect pupillary light reflexes, and palpebral reflex), measurement of aqueous tear production with Schirmer tear test strips (Intervet Inc), fluorescein staining (Akorn Inc), rebound tonometry (Tono-Vet; Icare Finland), slit-lamp biomicroscopy (SL-17 portable slit-lamp biomicroscope; Kowa Co), and binocular indirect ophthalmoscopy (binocular indirect ophthalmoscope; Welch Allyn Distributors) following pharmacologic (tropicamide 1% ophthalmic solution; Akorn Inc) dilation. All dogs had normal ophthalmic and physical examinations prior to inclusion in the study.
Acclimation
The dogs were given a 3-day acclimation period prior to the treatment phase of the study. During this acclimation period, each dog was administered 1 drop of preservative-free artificial tear solution (Refresh Plus® preservative-free lubricant eye drops; Allergan Inc) to each eye, 3 times daily.
Topical ophthalmic drug administration
The dogs were randomly assigned via coin toss to receive either 1% prednisolone acetate ophthalmic suspension (Pacific Pharma Inc) or neomycin polymyxin B 0.1% dexamethasone ophthalmic suspension (Sandoz Inc) with 6 dogs in each treatment group. After vigorous shaking of the dropper bottle for approximately 10 seconds, 1 drop of the assigned treatment was applied to each eye 4 times a day (6 PM, 12 AM, 6 AM, and 12 PM) for a total of 14 days (days 1 to 14) by the same investigator (MME). The dogs were allowed to blink normally after instillation of the eyedrops.
Sample collections
Following the acclimation period (day 0), peripheral venous blood samples (5 mL) were collected from each dog for measurement of plasma drug (prednisolone or dexamethasone) and cortisol concentrations, serving as baseline prior to the treatment phase of the study (Supplementary Figure S1). Peripheral venous blood samples (3 mL) were collected from either the cephalic or saphenous vein of each dog 0.25 hours following instillation of the 12 PM treatment on days 1, 7, and 14 of the study period to determine the plasma concentrations of prednisolone or dexamethasone. Six dogs (3 from each treatment group) had jugular venous blood samples (3 mL) collected for plasma drug concentration measurement immediately following the 0.25-hour peripheral blood collection on days 1, 7, and 14 to assess the effects of sample site on plasma drug concentrations. In the remaining 6 dogs (3 from each treatment group), peripheral venous samples (3 mL at each time point) were collected 0.5 hours after the 12 PM treatment on days 1 and 7, and 0.5, 1, 3, and 6 hours after the 12 PM treatment on day 14 for plasma drug concentration measurements. All dogs had peripheral venous samples collected 0.25 hours following the 12 PM treatment on day 14 for plasma cortisol measurements. All blood samples obtained for plasma drug concentrations were collected in lithium heparin tubes. Following centrifugation and plasma separation, samples were frozen and stored in a –80 °C freezer until analyzed.
Plasma drug analysis (prednisolone and dexamethasone)
Plasma preparation for prednisolone and dexamethasone measurement included adding 0.1 mL of plasma (incurred sample, standard, or quality control) with 0.1 mL of methanol, 0.1 mL of internal standard (IS) solution at 50 ng/mL, and 0.3 mL of 4% phosphoric acid in water. The internal standards included either prednisolone-d8 or dexamethasone-d5 (Toronto Research Chemicals). The mixture was vortexed and then applied to 96 well solid phase extraction plates (Oasis PRIME HLB µ-elution plates; Waters Corp) and positive pressure was applied using nitrogen. The wells were then washed with 0.3 mL of deionized water, followed by 20% methanol in deionized water. The samples were then eluted using 0.05 mL of acetonitrile:methanol (9:1) into a clean collection plate. Deionized water with 0.2% formic acid (0.05 mL) was then added to the collection plate and sealed with a cap mat. The injection volume was 0.005 mL.
Plasma concentrations of prednisolone and dexamethasone were quantified by liquid chromatography-mass spectrometry (Acquity H UPLC and a TQ-S triple quadrupole mass spectrometer; Waters Corporation). The mobile phase consisted of A: 0.1% formic acid in deionized water and B: acetonitrile using a gradient starting at 90% A to 60% A at 5 minutes, then to 0% A at 6 minutes and returning to 90% A at 8 minutes with a flow rate of 0.5 mL/min. Separation was achieved using a 10 X 2.1-mm, 1.8-µM C18 column (Eclipse Plus C19; Agilent Technologies) maintained at 55 °C. The retention times for prednisolone and dexamethasone were 2.74 and 3.42 minutes, respectively. Detection was performed by positive electrospray ionization using multiple reaction monitoring (MRM). The MRM transitions for prednisolone and the prednisolone-d8 solution were m/z 361.4–307.2, 325.2, 147.1, and 369.5–332.3, respectively. The MRM transitions for dexamethasone and dexamethasone-d5 were m/z 393.4–373.2, 355.2, 237.1 and 398.5–378.2.
The prednisolone calibration curve was linear from 0.1 to 250 ng/mL and had a R2 value of 0.999. Four quality controls were prepared at 0.5 ng/mL, 1.5 ng/mL, 15 ng/mL, and 150 ng/mL with intraday precisions (n = 3) of 8.8%, 8.6%, 3.4%, and 3.9% respectively and interday precisions (n = 6) of 13.4%, 12.1%, 7.2%, and 5.7% respectively. The interday accuracies (n = 6) at 0.5, 1.5, 15 and 150 ng/mL were 83.2%, 100.7%, 107.4%, and 98.7% respectively. The dexamethasone calibration curve was linear from 0.1 to 250 ng/mL and had also a R2 value of 0.999. Four quality controls were prepared at 0.15, 1.5, 15, and 150 ng/mL with intraday precisions (n = 3) of 8.0%, 4.9%, 2.8%, and 3.1% respectively and interday precisions (n = 6) of 10.2%, 7.6%; 2.8% and 3.7% respectively. The interday accuracies (n = 6) at 0.15, 1.5, 15, and 150 ng/mL were 96.2%, 100.5%, 104.1%, and 103.9% respectively.
Pharmacokinetic analysis
Pharmacokinetic analysis of plasma prednisolone and dexamethasone was performed using commercially available software (Excel PK functions; Microsoft Corp) using noncompartmental methods. The following parameters were generated or calculated: area under the concentration-versus-time curve (AUC) from 0 to 6 hours (AUC0-6h), which was determined with the linear trapezoidal rule; and the maximum plasma concentration (Cmax) and time to Cmax (tmax), which were both determined directly from the data. Other pharmacokinetic variables evaluated were the elimination rate constant (λz) and elimination half-life (t1/2).
Plasma cortisol analysis
Plasma cortisol concentrations were analyzed by use of a chemiluminescence analyzer (Immulite 1000; Siemens) with a normal reference range of 20 to 160 nmol/L. Sensitivity of the cortisol assay was 5.5 nmol/L.
Statistical analysis
Statistical analyses were performed to compare the drug concentrations obtained in relation to time (in hours and days) and sampling site (peripheral vs jugular). Normality of the data was assessed using the Shapiro-Wilk test. The independent group t test was performed to compare weight and age between treatment groups. Due to violation of the normality assumption for several parameters and the small sample size, nonparametric repeated-measures ANOVA (Friedman test) and post-hoc Dunn test were used to determine differences in plasma drug concentrations between the different days, time points, and collection sites. The Wilcoxon matched-pairs signed rank test was used to compare paired jugular and peripheral drug concentrations and paired 0.25-hour and 0.5-hour peripheral drug concentrations when data from all days were combined (days 1, 7, and 14). This same test was also performed to compare cortisol concentrations at the end of the 14-day treatment period with baseline values for both treatment groups. The Mann Whitney test was used to compare the change in cortisol concentrations over the course of the study between dexamethasone and prednisolone.
Statistical analyses were performed using commercially available software (GraphPad Prism 9; GraphPad Software Inc). Values of P < 0.05 were considered significant for all comparisons. Data are presented as median (range). A post-hoc power analysis using an online post-hoc power calculator (Clincalc; https://clincalc.com/Stats/Power.aspx) with α = 0.05 and power = 0.80 was performed for each comparison.
Results
Animals
The 12 dogs included 8 castrated males and 4 spayed females with a median (range) age and weight of 6.9 years (6.9 to 7.6 years) and 10.8 kg (9.7 to 14.0 kg), respectively. There was no statistical difference in age (P = 0.07) or weight (P = 0.10) between treatment groups.
Systemic absorption
Plasma prednisolone concentrations—No detectable concentrations of prednisolone were found in samples collected at baseline. Prednisolone was detected in plasma of all treated dogs at all measured time points and sampling sites on day 1 (median and range, 40.65; 10.20 to 74.00 ng/mL), day 7 (26.10; 13.90 to 45.00 ng/mL), and day 14 (19.40; 6.20 to 47.00 ng/mL). The combined median (range) plasma prednisolone concentration from all sampling sites and time points on days 1, 7, and 14 was 24.80 ng/mL (6.20 to 74.00 ng/mL). Peripheral plasma concentrations of prednisolone 0.25-hour post-treatment on days 1 (30.25; 10.20 to 48.80 ng/mL), 7 (17.80; 13.90 to 28.80 ng/mL), and 14 (18.00; 13.80 to 45.20 ng/mL) were not significantly (P ≥ 0.14) different.
When each day was tested individually, there was no difference (P ≥ 0.28) in peripheral prednisolone concentrations and their paired jugular concentrations for any day. When all days (days 1, 7, and 14) were combined, jugular prednisolone concentrations (44.10; 22.10 to 74.00 ng/mL) were significantly (P = 0.008) higher than peripheral prednisolone concentrations (19.60; 10.20 to 48.80 ng/mL; Figure 1). The median (range) jugular plasma concentrations of prednisolone relative to peripheral plasma concentrations were 154% (97% to 324%) with 6/9 paired measurements being > 115%. All measurements < 115% belonged to the same dog. On day 14, 1 peripheral plasma concentration (45.20 ng/mL) was slightly greater than the paired jugular plasma concentration (44.07 ng/mL), but within the expected analytical variability (± 15%); all other peripheral plasma concentrations were less than their paired jugular plasma concentration.
Median and range of paired peripheral (black circles) and jugular (white circles) plasma prednisolone (left side) and dexamethasone (right side) concentrations 0.25 hours after topical ophthalmic application of 1% prednisolone acetate ophthalmic suspension (1 drop/eye, q 6 h) or neomycin polymyxin B 0.1% dexamethasone 1% ophthalmic suspension (1 drop/eye, q 6 h), respectively (n = 3 Beagles/treatment group) on days 1, 7, and 14; and all days combined (All). *The jugular concentration was significantly (P < 0.05) higher than the paired peripheral concentration.
Citation: American Journal of Veterinary Research 83, 4; 10.2460/ajvr.21.04.0059
There was no difference between paired 0.25-hour and 0.5-hour post-treatment peripheral plasma concentrations of prednisolone on days 1, 7, or 14 (P ≥ 0.57). When the paired 0.25-hour and 0.5-hour post-treatment peripheral plasma samples were combined for all 3 days, the 0.5-hour post-treatment plasma prednisolone concentrations (28.20; 19.40 to 48.70 ng/mL) were significantly higher (P = 0.008) than the 0.25-hour post-treatment plasma concentrations (19.50; 13.80 to 48.00 ng/mL); Figure 2).
Median and range of paired peripheral plasma drug concentrations 0.25 hours and 0.5 hours after the treatments of Figure 1. *The peripheral 0.25-hour concentration was significantly (P < 0.05) higher than paired peripheral 0.5-hour concentration. See Figure 1 for key.
Citation: American Journal of Veterinary Research 83, 4; 10.2460/ajvr.21.04.0059
On day 14, the peripheral plasma concentrations were significantly (P = 0.045) higher at the 1-hour post-treatment time point (24.40; 20.40 to 27.90 ng/mL) compared to the 6-hour post-treatment time point (7.30; 6.20 to 7.70 ng/mL), but there were no other significant differences in plasma prednisolone concentrations (Figure 3).
Median and range plasma drug concentrations over time after the treatments of Figure 1. *Value was significantly (P < 0.05) different, compared with the 6-hour post-treatment plasma concentration. See Figure 1 for key.
Citation: American Journal of Veterinary Research 83, 4; 10.2460/ajvr.21.04.0059
The combined median (range) peripheral plasma prednisolone concentration from all time points on days 1, 7, and 14 was 20.4 ng/mL (6.2 to 48.8 ng/mL).
Plasma dexamethasone concentrations—No detectable concentrations of dexamethasone were found in samples collected at baseline. Dexamethasone was detected in plasma of all treated dogs at all measured time points on day 1 (2.60; 1.40 to 17.70 ng/mL), day 7 (2.75; 1.20 to 12.80 ng/mL), and day 14 (1.90; 0 to 9.90 ng/mL). On day 14, 1 dog did not have dexamethasone detected in the jugular sample collected immediately following the 0.25-hour post-treatment peripheral blood draw, but dexamethasone was detected in the peripheral sample. The combined median (range) plasma dexamethasone concentration from all sampling sites and time points on days 1, 7, and 14 was 2.30 ng/mL (0 to 17.70 ng/mL). Peripheral plasma concentrations of dexamethasone 0.25-hour post-treatment on days 1 (2.35; 1.40 – 2.70 ng/mL), 7 (2.15; 1.20 to 3.30 ng/mL), and 14 (2.05; 0.80 to 3.00 ng/mL) were not significantly (P ≥ 0.43) different.
When each day was tested individually, there was no difference (P ≥ 0.11) in peripheral dexamethasone concentrations and the paired jugular concentrations for any day. When all days (days 1, 7, and 14) were combined, jugular dexamethasone concentrations (7.10; 0 to 17.70 ng/mL) were significantly (P = 0.008) higher than paired peripheral dexamethasone concentrations (2.20; 0.80 to 2.90 ng/mL); Figure 1). The median (range) jugular plasma concentrations of dexamethasone relative to peripheral plasma concentrations were 331% (162% to 1298%), with 8/9 paired measurements being > 115%. The measurement < 115% had a peripheral plasma dexamethasone concentration of 0.8 ng/mL but did not have a measurable jugular plasma dexamethasone concentration.
No significant (P ≥ 0.28) difference in paired 0.25-hour and 0.5-hour post-treatment peripheral plasma concentrations of dexamethasone was identified on days 1, 7, or 14. When the paired 0.25-hour post-treatment peripheral and 0.5-hour post-treatment peripheral plasma samples were combined for all 3 days, the 0.5-hour post-treatment peripheral plasma concentrations of dexamethasone (2.50; 1.95 to 3.45 ng/mL) were significantly (P = 0.035) higher than the 0.25-hour post-treatment peripheral plasma concentrations (2.40; 1.50 to 2.85 ng/mL) with a median difference of 0.4 ng/mL (Figure 2).
On day 14, the peripheral plasma concentrations of dexamethasone were significantly (P = 0.0195) higher at the 0.5-hour post-treatment time point (2.30; 1.10 to 3.70 ng/mL) compared to the 6-hour post-treatment time point (0.70; 0.20 to 1.20 ng/mL). There were no other significant differences in plasma dexamethasone concentrations (Figure 3).
The combined median (range) peripheral plasma dexamethasone concentration from all time points on days 1, 7, and 14 was 2.05 ng/mL (0.2 to 4.2 ng/mL).
Pharmacokinetic analysis
Following the final topical ophthalmic application of prednisolone or dexamethasone on day 14, pharmacokinetic parameters were determined for 3 dogs in each treatment group (Supplementary Table S1). For prednisone, geometric mean values for AUC0-6h, tmax, Cmax, λΖ, and t1/2 were 94.1 ng•h/mL, 0.79 hours, 24.2 ng/mL, 0.246 hours–1, and 2.81 hours, respectively. For dexamethasone, these values were 6.06 ng•h/mL, 0.50 hours, 2.08 ng/mL, 0.210 hours–1, and 3.29 hours, respectively.
Plasma cortisol concentrations
Plasma cortisol levels (median; range) were significantly lower than baseline following treatment with both prednisolone acetate 1% (baseline: 37.50; 18.00 to 44.00 nmol/L vs day 14 of prednisolone treatment: 14.50; 14.00 to 31.00 nmol/L; P = 0.03) and dexamethasone 0.1% (baseline: 50.50; 40.00 to 77.00 nmol/L vs day 14 dexamethasone treatment: 14.00; 14.00 to 19.00 nmol/L; P = 0.03) ophthalmic suspensions. The percentage reduction of cortisol from baseline ranged from 22% to 70% and 65% to 82% for the prednisolone and dexamethasone treated groups, respectively.
Post hoc power analysis
Post hoc power analysis was performed for all data comparisons. All comparisons except for serial peripheral plasma prednisolone concentrations on day 14 compared with the 6-hour time point, combined jugular compared with the paired combined 15-minute peripheral dexamethasone concentrations, and plasma cortisol levels were underpowered (power < 0.8).
Discussion
This study demonstrated that systemic glucocorticoid drug absorption occurs after topical 1% prednisolone acetate and neomycin polymyxin B 0.1% dexamethasone ophthalmic suspensions are administered to both eyes of healthy dogs 4 times daily for 14 days. This study also identified discrepancies in plasma drug concentrations based on blood collection site (jugular vs peripheral), and significant decreases in endogenous cortisol concentrations after 2 weeks of treatment with both of these ophthalmic glucocorticoid drug suspensions.
Plasma prednisolone concentrations have been previously evaluated in dogs after various routes of administration, including topical ophthalmic19 and oral.32 In a recent publication evaluating the tear film pharmacokinetics and systemic absorption of topically applied 1% prednisolone acetate ophthalmic suspension in healthy dogs, the plasma prednisolone concentration ranged from 3.9 to 34.0 ng/mL 10 to 15 minutes following application of 1 (35 µL) or 2 drops (70 µL) to each eye 4 times daily for 3 days.19 Similarly, in the present study, the plasma concentrations of prednisolone 15-minutes post-treatment on days 1, 7, and 14 ranged from 10.20 to 48.80 ng/mL. Slight differences in plasma concentrations could be due to interdog variability, lack of dose uniformity in drop size, differences in composition of the commercial drug preparations, or methods used to determine plasma concentrations. While it would have been more precise to administer each drop with a micropipette as was performed in the Sebbag study,19 this was not done in the present study in an effort to closely mimic the clinical setting. Both studies utilized generic formulations of prednisolone acetate 1% ophthalmic suspension, and there are variable reports33,34 on the dose uniformity of different formulations of prednisolone acetate ophthalmic suspension. One study33 concluded that generic prednisolone acetate had highly variable drug concentrations ranging from 7% to 231.5% of the declared concentration, while less variability was seen with branded prednisolone acetate (20.5% to 181.4%). A second study34 did not appreciate this variability as long as bottles containing the suspension were shaken for 5 seconds prior to dispensing as done in the present study.
The pharmacokinetics following oral administration of prednisolone in healthy dogs have also been described.32,35 The mean AUC from time 0 to infinity (AUC0-∞) following oral administration of prednisolone at 1 mg/kg and 2 mg/kg in Beagles was 937.1 ng•h/mL and 2090.3 ng•hr/mL, respectively. In the present study, the geometric mean AUC0-6h (equivalent to AUC0-∞ because prednisolone is at steady state based on prednisolone t1/2 of 1.7 hours following intravenous administration of prednisolone sodium succinate36 and 2.81 hours in the present study) was 94.1 ng•h/mL.
Given that the AUC represents the total drug exposure and should be equivalent to dose, the AUC in the present study was compared with the AUC reported in previous studies32,35 following oral administration of prednisolone to determine the dose equivalence. The dogs in the present study had an equivalent prednisolone dose of 94.1/937.1 or 0.1 × 1 mg/kg and 94.1/2,090.3 or 0.045 X 2 mg/kg (prednisolone doses from published studies32,35). This would give a relative drug exposure comparable to 0.09 to 0.1 mg/kg oral prednisolone. Given that the topical ophthalmic treatments were provided 4 times daily in the present study, the topical dose would be equivalent to 0.36 to 0.4 mg/kg/d of oral prednisolone, close to what is considered a physiologic dose (0.1 to 0.3 mg/kg/d)37 of prednisolone. The half-life of prednisolone in this study was longer than the 2 mg/kg oral study35 (2.8 hours vs 1.5 hours), but comparable to the 1 mg/kg oral study.32 The variability between the studies could be due to random variation, interdog differences, concentration dependent differences, differences in absorption rate or differences in study design.
To the authors’ knowledge, this is the first study to report the systemic absorption of dexamethasone following topical ophthalmic application in dogs. While the plasma concentrations were low, all but 1 sample were above the lower limit of quantification (0.1 ng/mL). The peripheral plasma concentrations of dexamethasone 15-minutes post-treatment ranged from 0.8 to 3.3 ng/mL. Systemic absorption of topically applied ophthalmic dexamethasone has been evaluated in several other species including horses,20 humans,23 and rabbits.22 Dexamethasone was detected in horse serum and urine 10 to 15 minutes following topical application of 1% dexamethasone ophthalmic ointment (100 mg) to 1 eye 4 times daily for 8 consecutive days, and serum concentrations ranged from 0.10 to 0.49 ng/mL.20 Following frequent application of topical ophthalmic dexamethasone disodium phosphate (an aqueous solution) in humans prior to vitrectomy, systemic absorption was low, with a mean of 0.7 ng/mL from 3 to 101 minutes following topical application.23 Venous blood concentrations of dexamethasone following topical ophthalmic, IN, and IV administration of 0.5% dexamethasone-cyclodextrin in rabbits was found to be similar at 20 to 30 ng/g, regardless of the route of administration.22
Various factors must be considered when comparing the systemic dexamethasone concentrations following ophthalmic administration in these different studies. Factors inherent to different species which may impact the systemic drug concentration include the overall body size and blood volume, the volumetric capacity of the conjunctival sac, tear film turnover rate, blink rate, and length of the nasolacrimal duct. Additionally, various ophthalmic preparations, concentrations, and volumes were utilized in the different studies which can impact the rate of absorption and drug contact time.
The pharmacokinetics following IV dexamethasone administration in healthy dogs have also been described. The mean AUC following IV administration of dexamethasone at 0.01 mg/kg and 1 mg/kg in dogs was 2,060 ng•min/mL and 155,956 ng•min/mL, respectively.38,39 In the present study, the geometric mean AUC0-6hr following topical ophthalmic administration of dexamethasone was 6.06 ng•hr/mL (277 ng•min/mL). The dogs in the present study had an equivalent dexamethasone dose of 0.13 X 0.01 mg/kg and 0.0018 X 1 mg/kg (dexamethasone doses from published studies38,39). This would give a relative exposure comparable to intravenous dexamethasone administration at 0.0013 to 0.0018 mg/kg. Given that the topical ophthalmic treatments were provided 4 times daily in the present study, the topical dose would be equivalent to 0.0052 to 0.0072 mg/kg/d of IV dexamethasone. The t1/2 of dexamethasone in our study was shorter than the 0.01 mg/kg IV study38 (approx 2 hours vs 3 hours), but comparable to the 1-mg/kg IV study.39
In the present study, when comparing the peripheral plasma concentrations of prednisolone and dexamethasone following topical ophthalmic suspension administration, the median concentration of prednisolone (20.4 ng/mL) was 9.95 times the median concentration of dexamethasone (2.05 ng/mL). Given the concentration of 1% prednisolone acetate ophthalmic suspension is 10-fold that of the neomycin polymyxin B 0.1% dexamethasone ophthalmic suspension, these results suggested near equivalent systemic absorption of both topical ophthalmic steroid medications. The conclusion that there is near equivalent systemic bioavailability relies on several assumptions, including uniform drop volume and consistency and equal volume of distribution.
A previous study28 evaluating plasma drug concentrations following sublingual administration in dogs found the jugular sample to have a 4.3-fold higher Cmax and 2.2-fold higher AUC compared with the saphenous vein, raising concerns for using the jugular vein as a site of sampling for pharmacokinetic studies after transmucosal routes in the head region. In the present study, the jugular concentrations overestimated the peripheral venous concentrations by a median of 154% for prednisolone and 331% for dexamethasone. This finding supports the conclusion that sampling from the jugular vein can overestimate the systemic bioavailability of a substance that has been absorbed through the mucous membranes of the head.
In a clinical setting, treatment of anterior uveitis can be several months in duration, and in some cases, medication may be necessary indefinitely. Additionally, concurrent treatment with systemic NSAIDs may be indicated, and if there is significant systemic absorption of topical glucocorticoid drugs, an adverse drug reaction could occur. The degree to which different ophthalmic glucocorticoid drugs are absorbed systemically may influence a veterinarian’s decision on which medications to use to treat anterior uveitis, especially in small dogs with hyperadrenocorticism or preexisting liver disease. Suppression of the HPAA has been reported following long term use of 0.05% difluprednate ophthalmic emulsion40 and 1% prednisolone acetate ophthalmic suspension for 2 weeks in dogs.41,42 Topical application of 0.1% dexamethasone suspension 4 times daily to both eyes in Beagles also resulted in adrenal suppression as well as histopathologic changes in the liver.43 Reversible iatrogenic hyperadrenocorticism has been caused by the use of a topical ophthalmic glucocorticoid medication in a dog27 and in humans.44,45 Unlike in dogs,46 topical ophthalmic glucocorticoid drugs have been found to significantly increase blood glucose levels in diabetic humans26,47 undergoing cataract surgery.
Consistent with previous reports,41–43 the cortisol levels in the present study were significantly (P = 0.03) decreased from baseline following 4 times daily treatment with 1% prednisolone acetate and 0.1% dexamethasone ophthalmic suspensions for 2 weeks. The percentage reduction of cortisol from baseline ranged from 22% to 70% and 65% to 82% for the prednisolone- and dexamethasone-treated groups, respectively. Due to the high degree of structural similarity between prednisolone and cortisol, there is approximately 49% cross-reactivity of the cortisol assay (Immulite 1000®; Siemens) with prednisolone, potentially underestimating the degree of cortisol suppression in that treatment group. The clinical significance of the reduced cortisol concentrations and suppression of the HPAA in the present study is difficult to assess as dynamic testing to stimulate production of adrenocorticotropic hormone or cortisol were not performed. Partial adrenal suppression, characterized by decreased plasma cortisol concentrations with an intact HPAA response to metyrapone tartrate, has been documented following 6 weeks of topical ophthalmic 0.1% dexamethasone sodium phosphate in humans.48 A study49 evaluating the utility of baseline cortisol measurements for the diagnosis of hypoadrenocorticism found that baseline cortisol concentration of ≤ 22 nmol/L have a sensitivity of 96.9% and specificity of 95.7% for hypoadrenocorticism in dogs. In the present study, 16.7% of the baseline cortisol samples were ≤ 22 nmol/L. Alternatively, 91.7% of the cortisol samples were ≤ 22 nmol/L following 2 weeks of 4 times daily treatment with either topical ophthalmic 1% prednisolone acetate or neomycin polymyxin B 0.1% dexamethasone ophthalmic suspensions.
The results of this study should be interpreted in view of a few limitations. The sample size was low, particularly for evaluation of sample site comparisons and pharmacokinetic parameters, making the study prone to type II error. Additionally, while blood was sampled from the different sites within the shortest time possible for paired jugular and peripheral samples, blood sampling was not actually simultaneous. For the paired peripheral and jugular samples, the peripheral venous sample was always obtained immediately prior to the jugular sample. The slight delay in obtaining the jugular blood sample could explain the 2 jugular samples which had lower plasma drug concentrations than the paired peripheral sample. Alternatively, those samples in which the jugular concentration was lower were within the range of normal analytical variation in plasma drug concentrations. Given that dexamethasone and prednisolone are both relatively small molecules with low molecular weights (392 g/mol and 360 g/mol, respectively), they are readily absorbed through the conjunctiva and nasal mucosa typically leading to early peak plasma drug concentrations around 10 minutes.17 Given that the tmax for prednisolone in the present study was 0.79 hours, the plasma concentrations at the 0.25 and 0.5 hour time points may have underestimated the degree of systemic absorption following topical ophthalmic administration of prednisolone.
Another limitation was that the study was performed in ophthalmologically healthy dogs and may not be representative of clinical patients with anterior segment inflammation. Dogs with acute conjunctivitis can have reflex tearing with a significant increase in tear volume and tear turnover rate which may decrease precorneal drug retention.50 Additionally, conjunctivitis can lead to the breakdown of the blood-tear barrier allowing for serum albumin to leak into the tear film which can lead to decreased bioavailability of protein-bound medications.51–53
Additional limitations of this study were the method by which suppression of the HPAA was assessed and lack of a negative control group. While there was a significant decrease in cortisol concentrations for both treatment groups, measurement of basal plasma cortisol concentrations alone has limitations as an indicator of adrenal suppression.54 In healthy dogs, serum cortisol concentrations fluctuate episodically throughout the day and can occasionally decrease to subnormal values. Several studies55–60 have been performed evaluating basal plasma or serum cortisol concentrations in normal dogs with a wide range of reported values (24.8 to 85.5 nmol/L,60 < 3 to 77.5 ng/mL,56 2.29 to 28.20 ng/mL,55 16.3 to 27.7 ng/mL,57 9.4 to 37 ng/mL,58 6 to 28.5 ng/mL59). An adrenocorticotropic hormone–stimulation test ideally would have been performed to determine the integrity of the HPAA.
The results of this study demonstrated that prednisolone and dexamethasone are detectable in the plasma of healthy dogs following topical ophthalmic administration 4 times per day. Treatment of both eyes with 1 drop of 1% prednisolone acetate ophthalmic suspension 4 times daily led to plasma concentrations equivalent to 0.36 to 0.4 mg/kg/d of oral prednisolone, close to a physiologic dose (0.1 to 0.3 mg/kg/d) and approaching an anti-inflammatory dose (0.5 to 1 mg/kg/d) of prednisolone. Blood samples collected for evaluation of systemic absorption of medications following topical ophthalmic administration should be obtained from peripheral veins so as to not overestimate the systemic bioavailability of a medication. Findings also indicated that topical ophthalmic administration of 1% prednisolone acetate and neomycin polymyxin B 0.1% dexamethasone ophthalmic suspensions administered 4 times daily for 2 weeks leads to a significant decrease in endogenous cortisol levels. Additional research is needed to evaluate the systemic absorption of topical ophthalmic prednisolone acetate and dexamethasone in dogs with ocular surface inflammation or anterior uveitis.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org
Acknowledgments
Funded by a Mark Derrick Canine Research Grant, Kansas State University College of Veterinary Medicine. The authors declare that there were no conflicts of interest.
The authors thank Dr. Demitria Vasilatis for assistance with statistical analysis and Jennifer Klingele, CVT, Cora Farley, and Drs. Jordan Roberts, Megan Cullen, Morgan Johnson, and Abigail Sturbaum for assistance with data collection.
References
- 1. ↑
Holmberg BJ, Maggs DJ. The use of corticosteroids to treat ocular inflammation. Vet Clin North Am Small Anim Pract. 2004;34(3):693–705. doi:10.1016/j.cvsm.2003.12.007
- 2. ↑
Sulaiman RS, Kadmiel M, Cidlowski JA. Glucocorticoid receptor signaling in the eye. Steroids. 2018;133:60–66. doi:10.1016/j.steroids.2017.11.002
- 3. ↑
McGhee CNJ. Pharmacokinetics of ophthalmic corticosteroids. Br J Ophthalmol. 1992;76(11):681–684. doi:10.1136/bjo.76.11.681
- 4. ↑
Sebbag L, Allbaugh RA, Wehrman RF, et al. Fluorophotometric assessment of tear volume and turnover rate in healthy dogs and cats. J Ocul Pharmacol Ther. 2019;35(9):497–502. doi:10.1089/jop.2019.0038
- 5. ↑
Krishna N. Rate of lacrimal excretion of ophthalmic vehicles. Am J Ophthalmol. 1968;65(6):942–944. doi:10.1016/0002-9394(68)92234-4
- 6. ↑
Ahmed I, Patton TF. Importance of the noncorneal absorption route in topical ophthalmic drug delivery. Invest Ophthalmol Vis Sci. 1985;26(4):584–587.
- 7.
Schoenwald RD, Deshpande GS, Rethwisch DG, Barfknecht CF. Penetration into the anterior chamber via the conjunctival/scleral pathway. J Ocul Pharmacol Ther. 1997;13(1):41–59. doi:10.1089/jop.1997.13.41
- 8. ↑
Burstein NL, Anderson JA. Corneal penetration and ocular bioavailability of drugs. J Ocul Pharmacol. 1985;1(3):309–326. doi:10.1089/jop.1985.1.309
- 9. ↑
Farkouh A, Frigo P, Czejka M. Systemic side effects of eye drops: a pharmacokinetic perspective. Clin Ophthalmol. 2016;10:2433–2441. doi:10.2147/OPTH.S118409
- 10. ↑
Ramsay E, del Amo EM, Toropainen E, et al. Corneal and conjunctival drug permeability: systematic comparison and pharmacokinetic impact in the eye. Eur J Pharm Sci. 2018;119:83–89. doi:10.1016/j.ejps.2018.03.034
- 11. ↑
Anderson JA. Systemic absorption of topical ocularly applied epinephrine and dipivefrin. Arch Ophthalmol. 1980;98(2):350–353. doi:10.1001/archopht.1980.01020030346024
- 12.
Ohman L, Edqvist L-E, Johnansson EB. Absorption of topically applied hydrocortisone from the eye of the rhesus monkey. Acta Ophthalmol (Copenh). 1982;60(1):106–112. doi:10.1111/j.1755-3768.1982.tb05786.x
- 13.
Lanuza R, Rankin AJ, KuKanich B, Meekins JM. Evaluation of systemic absorption and renal effects of topical ophthalmic flurbiprofen and diclofenac in healthy cats. Vet Ophthalmol. 2016;19:24–29. doi:10.1111/vop.12295
- 14.
Hsu KK, Pinard CL, Johnson RJ, Allen DG, Kukanich BK, Nykamp SG. Systemic absorption and adverse ocular and systemic effects after topical ophthalmic administration of 0.1% diclofenac to healthy cats. Am J Vet Res. 2015;76(3):253–265. doi:10.2460/ajvr.76.3.253
- 15. ↑
Griggs AN, Yaw TJ, Haynes JS, Ben-Shlomo G, Tofflemire KL, Allbaugh RA. Bioavailability and biochemical effects of diclofenac sodium 0.1% ophthalmic solution in the domestic chicken (Gallus gallus domesticus). Vet Ophthalmol. 2017;20(2):171–176. doi:10.1111/vop.12387
- 16. ↑
Vaajanen A, Vapaatalo H. A single drop in the eye—effects on the whole body? Open Ophthalmol J. 2017;11(1):305–314. doi:10.2174/1874364101711010305
- 17. ↑
Urtti A, Salminen L. Minimizing systemic absorption of topically administered ophthalmic drugs. Surv Ophthalmol. 1993;37(6):435–456. doi:10.1016/0039-6257(93)90141-s
- 18. ↑
Sebbag L, Kirner NS, Allbaugh RA, Reis A, Mochel JP. Kinetics of fluorescein in tear film after eye drop instillation in Beagle dogs: does size really matter? Front Vet Sci. 2019;6:457. doi:10.3389/fvets.2019.00457
- 19. ↑
Sebbag L, Kirner NS, Wulf LW, Mochel JP. Tear film pharmacokinetics and systemic absorption following topical administration of 1% prednisolone acetate ophthalmic suspension in dogs. Front Vet Sci. 2020;7:571350. doi:10.3389/fvets.2020.571350
- 20. ↑
Spiess BM, Nyikos S, Stummer E, Sahin A, Naegeli H. Systemic dexamethasone concentration in horses after continued topical treatment with an ophthalmic preparation of dexamethasone. Am J Vet Res. 1999;60(5):571–576.
- 21. ↑
Janes RG, Stiles JF. The penetration of cortisol into normal and pathologic rabbit eyes. Am J Ophthalmol. 1963;56(1):84–90. doi:10.1016/0002–9394(63)91306–0
- 22. ↑
Sigurdsson HH, Konrádsdóttir F, Loftsson T, Stefánsson E. Topical and systemic absorption in delivery of dexamethasone to the anterior and posterior segments of the eye. Acta Ophthalmol Scand. 2007;85(6):598–602. doi:10.1111/j.1600-0420.2007.00885.x
- 23. ↑
Weijtens O, Schoemaker RC, Romijn FPHTM, Cohen AF, Lentjes EGWM, van Meurs JC. Intraocular penetration and systemic absorption after topical application of dexamethasone disodium phosphate. Ophthalmology. 2002;109(10):1887–1891. doi:10.1016/s0161-6420(02)01176-4
- 24.
Steelman J, Kappy M. Adrenal suppression and growth retardation from ocular corticosteroids. J Pediatr Ophthalmol Strabismus. 2001;38(3):177–178.
- 25. ↑
Baba S, Mishima H, Okimoto M, Miyachi Y. Plasma steroid levels and clinical effects after topical application of betamethasone. Graefes Arch Clin Exp Ophthalmol. 1983;220(5):209–214. doi:10.1007/BF02308076
- 26. ↑
Bahar I, Rosenblat I, Erenberg M, et al. Effect of dexamethasone eyedrops on blood glucose profile. Curr Eye Res. 2007;32(9):739–742. doi:10.1080/02713680701573704
- 27. ↑
Murphy CJ, Feldman EC, Bellhorn R. Iatrogenic Cushing’s syndrome in a dog caused by topical ophthalmic medications. J Am Anim Hosp Assoc. 1990;26:640–642.
- 28. ↑
Sohlberg E, Halldin MM, Annas A, et al. The impact of the site of blood sampling on pharmacokinetic parameters following sublingual dosing to dogs. J Pharmacol Toxicol Methods. 2013;67(1):1–4. doi:10.1016/j.vascn.2012.10.004
- 29.
Hedges AR, Pypendop BH, Shilo Y, Stanley SD, Ilkiw JE. Impact of the blood sampling site on time-concentration drug profiles following intravenous or buccal drug administration. J Vet Pharmacol Ther. 2014;37(2):145–150. doi:10.1111/jvp.12075
- 30. ↑
Messenger KM, Davis JL, Lafevers DH, Barlow BM, Posner LP. Intravenous and sublingual buprenorphine in horses: pharmacokinetics and influence of sampling site. Vet Anaesth Analg. 2011;38(4):374–384. doi:10.1111/j.1467-2995.2011.00613.x
- 31. ↑
Hermanson JW, de Lahunta A, Evans HE. The veins. In: Hermanson JW, de Lahunta A, Evans HE, eds. Miller and Evans’ Anatomy of the Dog. 5th ed. Elsevier; 2020:1170–1227.
- 32. ↑
Van der Heyden S, Croubels S, Gadeyne C, et al. Influence of P-glycoprotein modulation on plasma concentrations and pharmacokinetics of orally administered prednisolone in dogs. Am J Vet Res. 2012;73(6):900–907. doi:10.2460/ajvr.73.6.900
- 33. ↑
Stringer W, Bryant R. Dose uniformity of topical corticosteroid preparations: difluprednate ophthalmic emulsion 0.05% versus branded and generic prednisolone acetate ophthalmic suspension 1%. Clin Ophthalmol. 2010;4(1):1119–1124. doi:10.2147/OPTH.S12441
- 34. ↑
Marlowe ZT, Davio SR. Dose uniformity of loteprednol etabonate ophthalmic gel (0.5%) compared with branded and generic prednisolone acetate ophthalmic suspension (1%). Clin Ophthalmol. 2014;8:23–29. doi:10.2147/OPTH.S55004
- 35. ↑
Nam A, Kim SM, Jeong JW, Song KH, Koo TS, Seo KW. Comparison of body surface area-based and weight-based dosing format for oral prednisolone administration in small and large-breed dogs. Pol J Vet Sci. 2017;20(3):611–613. doi:10.1515/pjvs-2017-0076
- 36. ↑
Ekstrand C, Pettersson H, Gehring R, Hedeland M, Adolfsson S, Lilliehöök I. Prednisolone in dogs—plasma exposure and white blood cell response. Front Vet Sci. 2021;8:666219. doi:10.3389/fvets.2021.666219
- 37. ↑
Blois S, Mathews KA. Anti-inflammatory therapy. In: Ettinger SJ, Feldman EC, Côté E, eds. Textbook of Veterinary Internal Medicine: Diseases of the Dog and Cat. 8th ed. Elsevier; 2017:1782–1793.
- 38. ↑
Greco DS, Brown SA, Gauze JJ, Weise DW, Buck JM. Dexamethasone pharmacokinetics in clinically normal dogs during low- and high-dose dexamethasone suppression testing. Am J Vet Res. 1993;54(4):580–585.
- 39. ↑
Toutain PL, Alvinerie M, Ruckebusch Y. Pharmacokinetics of dexamethasone and its effect on adrenal gland function in the dog. Am J Vet Res. 1983;44(2):212–217.
- 40. ↑
Quantz KR, Anderson A, Harman CD, et al. Localized alopecia and suppression of hypothalamic pituitary adrenal axis in canines following treatment with difluprednate 0.05% ophthalmic emulsion (Durezol®). In: Proceedings of the ACVO 51st Annual Conference & Trade Show. American College of Veterinary Ophthalmologists; 2020:60.
- 41. ↑
Eichenbaum JD, Macy DW, Severin GA, Paulsen ME. Effect in large dogs of ophthalmic prednisolone acetate on adrenal gland and hepatic function. J Am Anim Hosp Assoc. 1988;24(6):705–709.
- 42. ↑
Roberts SM, Lavach JD, Macy DW, Severin GA. Effect of ophthalmic prednisolone acetate on the canine adrenal gland and hepatic function. Am J Vet Res. 1984;45(9):1711–1714.
- 43. ↑
Glaze MB, Crawford MA, Nachreiner RF, Casey HW, Nafe LA, Kearney MT. Ophthalmic corticosteroid therapy: systemic effects in the dog. J Am Vet Med Assoc. 1988;192(1):73–75.
- 44. ↑
Fukuhara D, Takiura T, Keino H, Okada AA, Yan K. Iatrogenic Cushing’s syndrome due to topical ocular glucocorticoid treatment. Pediatrics. 2017;139(2):e20161233.doi:10.1542/peds.2016-1233
- 45. ↑
Ozerdem U, Levi L, Cheng L, et al. Systemic toxicity of topical and periocular corticosteroid therapy in an 11-year-old male with posterior uveitis. Am J Ophthalmol. 2000;130(2):240–241. doi:10.1016/s0002-9394(00)00501-8
- 46. ↑
Rankin AJ, Kukanich KS, Schermerhorn T, et al. Evaluation of diabetes mellitus regulation in dogs treated with ophthalmic preparations of prednisolone acetate versus diclofenac sodium. Am J Vet Res. 2019;80(12):1129–1135. doi:10.2460/ajvr.80.12.1129
- 47. ↑
Kymionis GD, Panagiotoglou T, Tsilimbaris MK. The effect of intense, short-term topical dexamethasone disodium phosphate eyedrops on blood glucose level in diabetic patients. Ophthalmologica. 2007;221(6):426–429. doi:10.1159/000107505
- 48. ↑
Krupin T, Mandell AI, Podos SM, Becker B. Topical corticosteroid therapy and pituitary-adrenal function. Arch Ophthalmol. 1976;94(6):919–920. doi:10.1001/archopht.1976.03910030459003
- 49. ↑
Gold AJ, Langlois DK, Refsal KR. Evaluation of basal serum or plasma cortisol concentrations for the diagnosis of hypoadrenocorticism in dogs. J Vet Intern Med. 2016;30(6):1798–1805. doi:10.1111/jvim.14589
- 50. ↑
Sebbag L, Soler EA, Allbaugh RA, Mochel JP. Impact of acute conjunctivitis on ocular surface homeostasis in dogs. Vet Ophthalmol. 2020;23(5):828–833. doi:10.1111/vop.12804
- 51. ↑
Sebbag L, Moody LM, Mochel JP. Albumin levels in tear film modulate the bioavailability of medically-relevant topical drugs. Front Pharmacol. 2020;10:1560. doi:10.3389/fphar.2019.01560
- 52.
Sebbag L, Allbaugh RA, Weaver A, Seo YJ, Mochel JP. Histamine-Induced Conjunctivitis and breakdown of blood–tear barrier in dogs: a model for ocular pharmacology and therapeutics. Front Pharmacol. 2019;10:752. doi:10.3389/fphar.2019.00752
- 53. ↑
Page L, Allbaugh RA, Mochel JP, Peraza J, Bertram M, Sebbag L. Impact of diurnal variation, sex, tear collection method, and disease state on tear protein levels in dogs. Vet Ophthalmol. 2020;23(6):994–1000. doi:10.1111/vop.12840
- 54. ↑
Schlaghecke R, Kornely E, Santen RT, Ridderskamp P. The effect of long-term glucocorticoid therapy on pituitary-adrenal responses to exogenous corticotropin-releasing hormone. N Engl J Med. 1992;326(4):226–230.
- 55. ↑
Spencer KB, Thompson FN, Clekis T, Lorenz MD. Adrenal gland function in dogs given methylprednisolone. Am J Vet Res. 1980;41(9):1503–1506.
- 56. ↑
Johnston SD, Mather EC. Canine plasma cortisol (hydrocortisone) measured by radioimmunoassay: clinical absence of diurnal variation and results of ACTH stimulation and dexamethasone suppression tests. Am J Vet Res. 1978;39(11):1766–1770.
- 57. ↑
Richkind M, Edqvist LE. Peripheral plasma levels of corticosteroids in normal Beagles and Greyhounds measured by a rapid competitive protein binding technique. Acta Vet Scand. 1973;14(5):745–757. doi:10.1186/BF03547402
- 58. ↑
Becker MJ, Helland D, Becker D. Serum cortisol (hydrocortisone) values in normal dogs as determined by radioimmunoassay. Am J Vet Res. 1976;37:1101–1102.
- 59. ↑
Chen CL, Kumar M, Willard M, Liao T. Serum hydrocortisone (cortisol) values in normal and adrenopathic dogs as determined by radioimmunoassay. Am J Vet Res. 1978;39(1):179–181.
- 60. ↑
Kemppainen RJ, Sartin JL. Evidence for episodic but not circadian activity in plasma concentrations of adrenocorticotrophin, cortisol and thyroxine in dogs. J Endocrinol. 1984;103(2):219–226. doi:10.1677/joe.0.1030219
