Evidence for Ototopical Glucocorticoid-Induced Decrease in Hypothalamic-Pituitary-Adrenal Axis Response and Liver Function
http://www.100md.com
内分泌学杂志 2005年第7期
Leipzig University, Institute of Pharmacology, Pharmacy and Toxicology (G.A., F.R.U.), and Institute of Physiological Chemistry (J.G.), 04103 Leipzig, Germany
Address all correspondence and requests for reprints to: Dr. Getu Abraham, Leipzig University, Institute of Pharmacology, Pharmacy, and Toxicology, An den Tierkliniken 15, D-04103 Leipzig, Germany. E-mail: gabraham@rz.uni-leipzig.de.
Abstract
To clarify whether ototopical glucocorticoid treatment is associated with impaired hypothalamic-pituitary-adrenal axis (HPA) activity and altered hepatic metabolism, one commercially available dexamethasone-containing ointment was tested. At present, very little is known about the effects of ototopical glucocorticoid treatment on HPA and liver function. Ten beagle dogs received two daily therapeutic doses of dexamethasone (0.6 mg/ear) in the outer auditory canal for 21 d in a single-blind, placebo-controlled study. Resting cortisol concentrations were assessed before, during, and after treatment using an RIA system. Adrenal function and HPA feedback sensitivity were measured by a standard dose (250 μg) ACTH stimulation test. Serum biochemical and hematological parameters were measured, whether ototopical glucocorticoids affect hepatic function was studied, and blood cell counts were made. Ototopical dexamethasone treatment induced a marked suppression (to about 100%) of resting plasma cortisol concentrations below the placebo effect (P < 0.0001) within the first 11 d, and these remained reduced during the entire treatment period up to d 19. As well, an ACTH stimulation test found a markedly reduced rise in plasma cortisol concentrations (P = 0.0004). Concomitantly, significant increases in serum activities of alkaline phosphatase, -glutamyl transferase, alanine transaminase, and aspartate transaminase were detected. Moreover, we found a significant reduction in differential leukocyte counts of eosinophils and lymphocytes, whereas neutrophils increased. Although cortisol levels and hematological parameters returned to baseline 7 d after treatment cessation, liver enzyme activities remained elevated. In conclusion, these findings suggest that after ototopical application, dexamethasone is sufficiently absorbed from the auditory canal to suppress HPA function as well as to alter metabolic and hemopoietic profiles. Thus, in long-term treatment of otitis externa or media, the systemic adverse suppression of HPA has to be considered in relation to stress exposure, whereas changes in serum enzyme activities may not be interpreted as hepathopathy.
Introduction
OTOTOPICAL GLUCOCORTICOID formulations, with or without an antimicrobial or antimycotic agent, have commonly been used for the treatment of acute or chronic otitis externa or acute otitis media with otorrhea commonly in humans (children). Although topical corticosteroid therapy seemed to be more beneficial by minimizing the degree of the well-known systemic bioactivity than oral or parenteral glucocorticoids, it is now becoming increasingly accepted that such forms of application of glucocorticoid-containing drugs are associated with systemic adverse effects at the clinically recommended doses. Despite this widespread argument, however, even ototopical therapy with topical glucocorticoids such as dexamethasone might lead to transcutaneous systemic absorption and, hence, to any total systemic adversative burden, but this remains to be evaluated.
Experimental findings have emphasized over the past years the role of topical glucocorticoids as relevant triggering factors for many systemic alterations, such as suppression of the hypothalamic-pituitary-adrenal axis (HPA), depression of leukocyte numbers, alteration in hepatic enzyme activities, and changes in bone metabolism and growth (1). For example, when using inhaled glucocorticoids for the treatment of asthma (for review, see Ref. 2) or topical glucocorticoids for atopic dermatitis (3, 4) and nasal rhinosinusitis (5), a detectable suppression of the HPA could be demonstrated. A decreased responsiveness of the adrenal gland is recognized, most likely as sensitive and reproducible indices of the systemic adverse effects of glucocorticoids (6, 7). For this effect on the HPA, most studies have focused on assessing endogenous cortisol production level in plasma or serum, which is suppressed by exogenous glucocorticoids, as a reliable and suitable method of measurement (8, 9). Moreover, an integrated cortisol determination in plasma after an ACTH stimulation test at high dose (250 μg) is regarded to also be sensitive for detecting adrenocortical suppression, for example, in patients taking long-term inhaled corticosteroids (10). However, the mode and magnitude of such iatrogenic adrenal suppression, measured as depressed levels of cortisol after exogenous glucocorticoid treatment, are supposed to be associated with excessive high dose and long-term administration of glucocorticoids, which might even result in reversible manifestations of Cushing’s syndrome (11). In asthmatic patients, it has been shown that prolonged treatment with excessive glucocorticoids caused chronic suppression of the HPA and secondary adrenocortical insufficiency, which persisted for months or more than a year after discontinuation of treatment (7, 12), but information is lacking regarding the duration or the effects of suppression after ototopical administration of antiinflammatory doses. In contrast, considerable debate continues about the clinical significance of basal nonstimulated cortisol concentrations, because although these offer an indication of the relative systemic activity of inhaled corticosteroids at the recommended therapeutic doses, they presumably do not indicate the degree of adrenal reserve in response to various stress factors. Hence, an ACTH stimulation test might offer an assessment of impaired HPA negative feedback based on the measurement of cortisol levels.
Supporting this, a few studies have examined the biosensitivity of HPA to dermal topical glucocorticoids, which could be mediated by transcutaneous absorption in humans and experimental animals (3, 13, 14), in part with conflicting results. Patients with atopic dermatitis may have decreased plasma cortisol levels without therapeutic intervention (15). Rupprecht et al. (16) found that subjects with atopic dermatitis display impaired suppression of the HPA by glucocorticoids, which seemed to be a phenomenon of glucocorticoid resistance. In contrast, in healthy subjects and experimental animals, topical glucocorticoids suppressed endogenous cortisol release; however, this was found to be dose related and dependent on the route and duration of treatment, usually applied for a long period of time at higher doses (17). Such conditions have, however, failed to be considered during the use of ear drops containing antiinflammatory components such as dexamethasone.
Although the results of previous studies have indicated that oral or parenteral glucocorticoids alter hepatic functions, little information exists concerning the induction of serum liver enzyme activities during topical treatment with glucocorticoids in general, and even less is known about this process specifically during therapy with ototopical glucocorticoids. In this context, a unique spectrum of morphological and biochemical changes to the liver has been reported that is markedly reflected in serum by high activities of several liver enzymes, such as alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and -glutamyl transferase (GGT) (18). These enzymes are often used as markers to test the state of integrity of liver cell membranes, perhaps referred as liver dysfunction, either as a result of drug application or any disease-related hepatic disorders. Accordingly, a more pronounced progressive increase has been described for hepatic ALP and GGT activities after oral or parenteral glucocorticoids and in hepatic diseases (19, 20). However, the results of studies evaluating the extent to which glucocorticoids, after ototopical (or dermal) administration, affect these enzymes and how long these effects persist after cessation of treatment have been rarely presented.
Because previous studies have shown that exogenous topical (inhaled or dermal) glucocorticoids, usually at high doses, induce complete suppression of cortisol secretion, which is controlled by HPA, partly through a negative feedback mechanism, we investigated in this study the use of an ototopical dexamethasone-containing preparation at recommended doses to detect changes in HPA function (responsiveness and recovery), by measuring resting cortisol levels compared with ACTH-stimulated cortisol release. Moreover, we assessed serum hepatic enzyme activities and hematological parameters during a twice-daily application of dexamethasone-containing ear drops for 21 d. We compared both basal and post-ACTH cortisol levels after administration of a placebo and a pharmacologically active agent, dexamethasone, in the same animal.
Materials and Methods
Animals
Animal experiments were approved by the local ethical committee for animal welfare and were carried out in accordance with the guidelines of the German law relating to animal welfare.
In these studies a total of 10 adult beagle dogs of mixed gender (four males and six females) between 3 and 4 yr of age, weighing 11–16 kg, were enrolled. The animals were housed in solid-bottom cages with an outlet, three and four dogs per cage, respectively; they were fed commercial diet food and received water ad libitum. Animals were clinically healthy and received no steroid therapy for at least 3 months before this experiment, except for routine vaccination and deworming. Otoscopic examination showed no symptoms of inflammatory disorders of the external auditory canal. Each dog served as its own control.
Experimental design and blood sampling
A single-blind, nonrandomized, placebo-controlled, repeated measure design was used with washout periods of 1 wk. Three days before any treatment (d 0), a complete physical and special otoscopic examination, the baseline complete blood cell count and serum biochemical analysis, as well as determination of basal plasma cortisol levels before and after ACTH stimulation test were performed in all dogs. Moreover, in this pretreatment period, both ears (left and right) were cleaned with dry soft tissues. Dogs had an initial placebo run, a 5-d period during which 20 drops of placebo (0.60 g placebo ointment) were administered in the left ear twice daily at 0800 and 1800 h. Thereafter, dogs received an ototopic drug combination in recommended therapeutic doses containing dexamethasone-21-acetate (1 mg/g), neomycin sulfate (5 mg/g), and clotrimazole (10 mg/g), two doses for 21 d each: 20 drops (0.60 g ointment, 60 μg/kg body weight dexamethasone) of dexa-methasone-containing ear gel twice daily (total daily dose, 1200 μg) in the right ear canal at 0800 and 1800 h. Correspondingly, dogs were given daily vs. twice daily 20 drops (0.60 g) of a nonactive agent containing placebo formulation while receiving dexamethasone-containing ear drops. With a 1-wk washout period the whole study was designed to take about 40 d. Clinical and otoscopic examinations were carried out every morning before drug application during the treatment phase. Blood samples for the analysis of total blood cell count, serum biochemical profile (including urea, electrolytes, and liver function tests), and plasma cortisol levels were collected from cephalic venipuncture in EDTA- or lithium-heparin-displaced tubes (Sarstedt, Hamburg, Germany) at different time points: on d 0 (baseline), d 5 (placebo phase), d 11 and 19 (during treatment with dexamethasone and placebo in parallel for 21 d), and d 33 (7 d after last application of pharmacologically active substances). For determination of endogenous cortisol concentrations, all blood samples from each animal were collected frequently at 0800 h during these five periods. On similar days, a response challenge test to a standard dose (250 μg) synthetic ACTH was performed. Serum and plasma samples were obtained after centrifugation of whole blood (4000 x g, 10 min, 4 C), and the specimens were then aliquoted in 1.5-ml polypropylene tubes and stored at –20 C until analysis.
ACTH stimulation test
To assess the feedback sensitivity of the HPA, adrenocortical suppression and responsiveness after application of ototopical dexamethasone-containing formulations in the external auditory canal was studied by a serial determination of baseline plasma cortisol levels and the cortisol concentrations after ACTH challenge test. All samples for cortisol determination were obtained at 0800 h and immediately before and 1 h after iv administration of an ACTH bolus (250 μg). Adrenocortical function was assessed on d 0 (before drug treatment), 5 (after placebo), 12 (during topical glucocorticoid application), and 33 (after drug withdrawal). ACTH was administered in the morning 1 h before the usual time of treatment.
Plasma cortisol determination
Plasma cortisol was measured with a slight modification of the methods described by Abraham et al. (21) using an RIA kit with commercially available [3H]cortisol (specific activity, 50–90 Ci/mmol; Amersham Biosciences, Freiburg, Germany). All assays were performed in duplicate in a blind fashion by a separate technician. In brief, perturbing components were separated by precipitating the blood plasma with absolute alcohol. For this purpose, 100 μl thawed blood plasma were mixed with 900 μl ethanol (99.98%) and centrifuged for 15 min at 4 C. Thereafter, 100 μl supernatant were transferred to new incubation tubes, and samples were dried overnight at 37 C in a drying closet. After drying the samples, 100 μl phosphate buffer were added to each sample. Samples were incubated after the addition of aliquots of 100 μl [3H]cortisol at a final concentration of 1.4–2.5 nM (125 nCi/ml) and 100 μl polyclonal antibody (raised in rabbits against cortisol) at final volumes of 300 μl for 4 h at 4 C. Nonspecific binding was determined in the presence of phosphate buffer instead of the antibody. Separation of protein-bound [3H]cortisol from unbound steroid was achieved by adsorption of the free steroid onto coated charcoal, followed by centrifugation at 1500 x g for 15 min at 4 C. An aliquot of the supernatant was thereafter removed for liquid scintillation counting (PerkinElmer, Freiburg, Germany). The concentration of unlabeled cortisol in the sample was then determined from standard curves assayed in triplicate. The amounts of cortisol in the unknown samples were quantified by reference to the appropriate standard curve and calculated as nanomoles per liter using Multicalc software (Wallac, Turku, Finland). The coefficients of variability for analytical imprecision were 8.1% (intraassay) and 10.2% (interassay; n = 6).
The assay was based on the competition between unlabeled cortisol (antibody) and a fixed quantity of the tritium-labeled compound for binding to a protein with a high specificity and affinity for cortisol. The amount of labeled protein-cortisol complex formed was inversely related to the amount of unlabeled cortisol present in the assay sample. Measurement of protein-bound radioactivity enabled the amount of unlabeled cortisol in the sample to be calculated.
Analysis of serum activities of liver enzymes and hematological profiles
Serum activities of liver enzymes, ALP, ALT, AST, GGT, and lactate dehydrogenase were determined before, during, and after treatment with dexamethasone-ear drops according to standard procedures. Briefly, catalytic activities were determined using standard methods recommended by the German Society of Clinical Chemistry (22) with an automated analyzer (model 704, Hitachi, Mannheim, Germany) using commercially available reagents (Roche, Mannheim, Germany). All enzyme activities were determined at 37 C, and assay procedures were optimized according to the manufacturer’s instructions. Moreover, serum was analyzed for creatinine kinase (CK) after adding acetylcysteine using a CK kit (Roche) according to the manufacturer’s instructions, as recommended by the International Federation of Clinical Chemistry (23). Additionally, serum levels of protein, albumin, bilirubin, urea, electrolytes, and glucose were determined by routine clinical analyses.
Furthermore, EDTA-treated blood was analyzed for hematological constituents using a semiautomated analyzer (Technicon H1, Bayer Diagnostics, Muenchen, Germany). Blood hemoglobin concentrations were determined by the cyanomethemoglobin procedure. The red blood cell count, thrombocyte count, white blood cell count, and mean cell volume were measured directly. The values of red blood cell indices (packed cell volume, mean cell hemoglobin, and mean cell hemoglobin concentrations) were calculated.
Statistical analysis
Data presented in figures and tables are the mean ± SEM. To determine the effect of ototopic administration of dexamethasone-containing ointment on HPA function (by measuring resting and ACTH-stimulated plasma cortisol concentrations) and on liver enzyme activities, data before treatment (d 0), during placebo treatment (d 5), during dexamethasone as well as placebo application (d 11, 12, and 19), and 1 wk after drug withdrawal (d 33) were compared using repeated measures ANOVA. To examine the differences between baseline values vs. placebo and treatment values, Dunnett’s multiple comparison test was employed. P < 0.05 was considered significant.
Results
General effects
Physical and otoscopic examinations of the experimental animals showed, no apparent clinical abnormalities of the external auditory canal, the skin, or cutaneous pigmentation during the treatment period. Interestingly, at the ototopic dexamethasone therapeutic doses used for 3 wk, we were able to observe in all dogs enormous polydypsia and polyuria during the entire treatment period, without appreci-able changes in food intake. However, as a recovery sign after dexamethasone withdrawal, polydypsia and polyuria ceased after 7 d.
Plasma cortisol levels
One specific goal of the present study was to evaluate the responsiveness of the HPA after otic administration of topical glucocorticoids in therapeutic doses for 21 d. During placebo challenge, cortisol concentrations were slightly higher than baseline cortisol levels, but did not statistically differ from baseline values. Differences from placebo were significant at all time points of otic dexamethasone application for HPA responses and adrenocortical cortisol secretion (Fig. 1). Plasma cortisol concentrations decreased significantly in all animals during the treatment periods, as measured on d 11 and 19 [F(4,36) = 16.04; P < 0.0001]. As illustrated in Fig, 1, the average plasma cortisol level was suppressed to 1.24 ± 0.27 nmol/liter on d 11, and suppression was more pronounced (<1 nmol/liter) on d 19. In terms of mean percent dexamethasone induced at therapeutic doses twice daily for 21 d, there was an approximately 92–95% suppression of 0800 h plasma cortisol (baseline cortisol, 14.75 ± 4.76 nmol/liter). All transotic-treated dogs showed a similar trend in cortisol reduction. One week after cessation of ototopical dexamethasone treatment, there was a recovery of the cortisol response (Fig. 1); however, the variance for cortisol concentration was significantly different between values before and after treatment [F(9,9) = 366.4; P < 0.0001]. Cortisol levels increased within above 100% to values obtained during the treatment periods, but were 50% lower than pretreatment values (d 0; 14.75 ± 4.76 vs. 8.83 ± 0.90 nmol/liter; P < 0.05).
FIG. 1. Plasma cortisol values before treatment, during administration of placebo, during administration of dexamethasone-containing preparation and placebo, and after cessation of treatment. All 10 dogs were examined for cortisol response on each occasion. The differences in baseline, placebo, and dexamethasone and placebo were statistically significant. ***, P < 0.001, control vs. d 11 and 19 dexamethasone administration; *, P < 0.05, control vs. treatment withdrawal.
An ACTH challenge was administered before, during, and after pretreatment with placebo or otic glucocorticoid preparation to determine whether dexamethasone exerted an inhibitory effect on cortisol secretion at the level of the adrenal glands. ANOVA indicated that synthetic ACTH was able to stimulate significantly endogenous cortisol release before treatment to approximately 700% above basal values (18.63 ± 2.61 vs. 152.83 ± 23.94 nmol/liter). As depicted in Fig. 2, the interaction between dexamethasone treatment and ACTH challenge represented a significant difference in plasma cortisol concentrations between baseline (d 0) and otic treatment (d 12; 152.83 ± 23.94 vs. 40.93 ± 6.75 nmol/liter; P = 0.0004). During the treatment period, approximately 3.7-fold lower ACTH-stimulated cortisol concentrations were found, but these were significantly higher than cortisol values measured on d 0 before ACTH challenge (40.93 ± 6.75 vs. 18.63 ± 2.61 nmol/liter; P = 0.0008). Thus, otic dexamethasone did show an ability to block the cortisol response to an ACTH challenge. In a similar pattern as cortisol levels after treatment cessation, an ACTH stimulation test failed to fully up-regulate endogenous cortisol release 7 d after discontinuation of treatment (Fig. 2). At this time after ACTH stimulation, cortisol levels increased, but did not reach the values seen before dexamethasone challenge (107.80 ± 12.30 vs. 152.83 ± 23.94 nmol/liter).
FIG. 2. Effects of topical dexamethasone pretreatment on cortisol response 60 min after ACTH challenge. Before, during, or after administration of a dexamethasone-containing preparation and/or placebo in the external auditory canal twice daily, dogs were challenged with ACTH (250 μg). ***a, P < 0.001, cortisol levels on d 0 (control), before vs. after ACTH stimulation test; ***b, P < 0.001, cortisol values during placebo, before vs. after ACTH stimulation test; ***c, P < 0.001, cortisol values after ACTH response on d 0 (control) vs. ACTH response on d 12 during dexamethasone application (by Student’s t test).
Effects on liver enzymes
As a marker of possible glucocorticoid-induced hepatic dysfunction, serum levels of specific enzymes were analyzed. Marked significant alterations in various liver enzymes were seen during treatment with otic dexamethasone compared with enzyme activities on d 0 (control) and during placebo treatment. As shown in Fig. 3A, 21-d otical administration of a dexamethasone-containing preparation increased progressively and significantly the mean total serum activity of ALP. On d 11, however, a less enhanced, nonsignificant activity (134.50 ± 16.30 U/liter) was seen compared with mean basal and placebo activities (99.00 ± 14.85 and 94.90 ± 14.87 U/liter, respectively), and a 4-fold significant activity increase was determined by d 19 (control vs. d 19, 99.00 ± 14.85 vs. 426.20 ± 75.20 U/liter; P < 0.001). Serum ALP activity peaked 1 wk after discontinuation of the treatment, with a mean value approximately 5 times greater than that at baseline (512.20 ± 90.42 vs. 99.00 ± 14.85 U/liter; P < 0.001 vs. d 0). Likewise, in dexamethasone-treated dogs, serum AST, ALT, and GGT activities increased significantly by d 11 and 19 (Fig. 3, B–D). Except for AST, the progressive increase in the activities of ALT and GGT did not return to baseline 7 d after drug withdrawal and were still significantly elevated. The remaining biochemical parameters (albumin, bilirubin, urea, electrolytes, glucose, CK, and lactate dehydrogenase) were within the normal physiological range and were not altered by ototopical glucocorticoid treatment in any of the animals examined (summarized in Table 1).
FIG. 3. Dexamethasone-induced increase in liver enzyme activity: ALP (A), AST (B), ALT (C), and GGT (D). Asterisks indicate significant differences in serum enzyme levels between baseline and dexamethasone treatment. Values are the mean ± SEM. *, P < 0.05; **, P < 0.01; ***. P < 0.001.
TABLE 1. Effects of ototopic dexamethasone on serum biochemical parameters compared with baseline and placebo
Hematological parameters
After 21 d of topical dexamethasone administration, significant differences were not observed in total leukocyte counts between experimental animals before and after treatment. There was a significant increase in the mean number of segmented neutrophils on d 11 and 19 during dexamethasone application (P < 0.05 vs. d 0), whereas eosinophils decreased during these treatment periods and remained at this reduced level even 7 d after drug withdrawal, but lymphocytes showed only a tendency to decrease without statistical significance. All other hematological parameters did not demonstrate such relevant changes during treatment; data are summarized in Table 2.
TABLE 2. Effects of ototopic dexamethasone on hematological parameters compared with baseline and placebo
Discussion
To determine the importance of ototopical dexamethasone-containing preparation on HPA function, hepatic metabolic activity, and the immune system, we investigated changes in endogenous cortisol production by the adrenal gland, several liver enzyme activities, and other additional biochemical and immunological parameters in clinically healthy beagle dogs. Although treatment with topical corticosteroids may be of benefit at the expense of systemic side effects, the large number of studies have shown that, for instance, asthmatic patients exposed to oral, parenteral, or inhaled glucocorticoids have a marked suppression of endogenous cortisol production (24), indicating chronic adrenal suppression, a surrogate marker for possible adverse effects in other tissues (2, 25, 26). Indeed, these studies have focused only on adrenal insufficiency, not on the possible role of exogenous glucocorticoids in liver function, i.e. liver enzyme activities. Conceivably, it has been argued from the findings of in vitro studies of cell cultures (hepatocytes and HeLa cells) that exogenous glucocorticoids might induce enzyme activities by de novo protein synthesis (27, 28). Even though ototopical corticosteroids are frequently used in children with inflammatory disorders in the outer and middle auditory canal, studies showing a direct correlation between these exogenous glucocorticoids and eventual insufficiency in adrenal reserve as well as hepatic function have not been conducted to date in humans or experimental animals.
This study produced several interesting findings that merit discussion and that together provide an explanation for the differences between previous studies comparing the systemic activities of dermal, inhaled, and oral glucocorticoids. The main findings of the present study were that ototopical dexamethasone treatment twice daily for 21 d in therapeutic doses caused a marked influence on HPA responses and liver function: 1) basal and ACTH-stimulated plasma cortisol levels dramatically decreased during the treatment periods; 2) exogenous dexamethasone produced enhanced activities of several liver enzymes; and 3) there was a significant increase in differential counts of segmented neutrophils, whereas eosinophils and lymphocytes decreased, as in dogs and other experimental animals receiving oral or parenteral glucocorticoids.
In the present experiment after ototopical administration, dexamethasone itself as a mixture component with antimicrobial agent (neomycin) in relatively low dose (0.6 mg/ear, twice daily; 60 μg/kg body weight once) strongly decreased the resting plasma cortisol level from 18.63 to 0.79 nmol/liter, which peaked on d 19 of treatment. Even though in this study the resting plasma ACTH level was not determined, if it had been decreased, this would indicate a preferential and major suppression of CRH, which, in turn, would stimulate the pituitary-adrenocortical axis to produce ACTH (29). Thus, it seemed logical to assume that dexamethasone might have greatly hampered the feedback sensitivity of the HPA. In contrast, previous studies have argued, in line with our findings, that at the pituitary level, the discriminative ability of the dexamethasone suppression test can be enhanced by reducing the dose of dexamethasone (30, 31). Hence, because dexamethasone at the chosen dose in our study totally abolished the significant increase in endogenous cortisol release from the adrenal cortex, it can be anticipated that dexamethasone might also abolish the basal increase in plasma ACTH concentrations, but this remains speculative at present.
The earlier time to peak cortisol response in dexamethasone-treated compared with placebo-treated animals indicates an accelerated decrease in HPA and, hence, adrenal sensitivity to dexamethasone. This acute adrenal insufficiency, reflected by the marked suppression of cortisol, might be attributed to the occurrence of acute adrenal crisis. These conditions agree with previous studies of the effects of topical inhaled corticosteroids applied in asthma therapy (24, 32, 33). However, these studies have addressed the issue of HPA suppression and adrenal crisis in association with high-dose, long-term, inhaled glucocorticoid treatment (34).
Similarly, in confirming our findings, it has been shown that locally administered dermal glucocorticoid-containing ointments also potentially suppress the endogenous cortisol release (4, 35); however, increased sensitivity to systemic adverse effects has been reported in patients with pathological skin changes, such as in atopic dermatititis after treatment with potent topical glucocorticoids (3, 36, 37). In this study, it has been argued that in diseased skin the degree of prompt endogenous cortisol suppression should be encouraged by increased percutaneous absorption (38, 39). Indeed, this effect is assumed to be enhanced by various factors: extent and severity of disease, greater surface area of treated skin, and dose and duration of topical glucocorticoid treatment (13, 40). In humans (children), otitis externa or interna might induce damage in ear canal stratum corneum and thus might enhance systemic absorption of dexamethasone and, as a result, produce marked adrenal suppression, as described by Levin and Maibach (13), of hydrocortisone with increased serum hydrocortisone levels in children with atopic dermatitis.
Although the present study was undertaken in healthy experimental animals without changes in ear canal skin integrity, the percutaneous drug absorption and the dexamethasone-induced influence on endogenous cortisol production might agree with the results obtained for skin, described above. However, it can strongly be assumed that in the ear, the percutaneous penetration and cutaneous metabolism of synthetic glucocorticoids might involve different pharmacokinetics than obviously oral or parenteral administration, as well as those of dermally administered corticosteroids. In normal human skin, it has been shown that the doses absorbed after 24-h skin application of, for example, hydrocortisone, is up to 5% (41). However, as reported by Feldmann and Maibach (42), the extent of penetration of this compound through human skin varies depending on the anatomical site to which the compound is applied. A maximum percutaneous penetration of hydrocortisone was observed on the scrotum (40%). A similar percentage of absorption was observed after application of the hydrophobic pesticide parathion in the human ear canal, whereas in the scrotum, absorption was as twice as much as that in the ear canal (43). Although at present there are no data available for ear canal corticosteroid flux, it can be assumed that percutaneous penetration could be highly facilitated by the thin stratum corneum, occlusive condition, high blood circulation, and temperature in a given dog ear canal. Thus, percutaneous absorption of dexamethasone at low therapeutic dose in the ointment should be even more pronounced in the ear than in the skin and should have highly suppressed, even to undetectable, resting plasma cortisol levels during the treatment period.
Furthermore, in the present study the standard Synacthen (Novartis, Basel, Switzerland) test was conducted to directly test the adrenal reserve and indirectly assess HPA function, because chronic ACTH deficiency leads to a quiescent adrenal gland and, hence, to an inadequate cortisol response to exogenous ACTH at pharmacological doses (250 μg). This test is supposed to be preferable because it should present a more physiological stimulus to the adrenal gland and thus be more sensitive in detecting adrenal impairment (26). Accordingly, during ototopical dexamethasone treatment, the RIA procedure yielded 73% lower cortisol levels measured 1 h after the administration of exogenous ACTH than baseline values (152.83 ± 23.94 vs. 40.93 ± 6.75 nmol/liter), suggesting insufficiency of the adrenal glands. This is in good agreement with data from asthmatic patients treated with inhaled glucocorticoids, in whom standard ACTH-stimulated cortisol production was markedly blunted (44, 45), but in these subjects, supraphysiological glucocorticoid doses were used. As evidenced by a subnormal Synacthen stimulation test (250 μg) result, the data from our experimental animals indicate progressive suppression of the HPA to the most advanced phase and probable functional adrenal gland atrophy. With a pronounced or prolonged ACTH decrease, in keeping with our results, the adrenal glands may not be able to produce cortisol spontaneously, even after maximum stimulation with ACTH analogs (46). However, despite the controversy in the widespread published data for normal responses after either standard-dose (250 μg) or low-dose (1 μg) ACTH test (47, 48), the latter not only measures adrenal gland responsiveness, but may detect subtle degrees of adrenal atrophy (49); we believe that the standard test in the present study might have detected central adrenal axis insufficiency. Moreover, not all studies concur that the low-dose ACTH test is more sensitive than the high-dose ACTH test (50, 51). We did not use the low-dose ACTH test, because our test ototopical preparation contained dexamethasone, which may blunt the cortisol response to low-dose ACTH and may limit the value of the test method (26).
It was also worth assessing the duration of HPA recovery after drug withdrawal. Even if among the commonly and topically used glucocorticoids, dexamethasone, prednis-olone, and methylprednisolone, dexamethasone, with the longer half-life (36–54 h), suppresses ACTH for the longest period (46), resting plasma cortisol concentrations increased rapidly by d 7 after treatment cessation, but did not reach baseline. In almost all animals examined with suppressed adrenal response, the response to 250 μg ACTH, reflected by mean plasma cortisol values, returned gradually and steadily to baseline within 7 d after discontinuation of the treatment. In marked contrast to these short-term data, there is evidence that the recovery of suppressed adrenal response after long-term, high-dose glucocorticoid treatment may take more than a year (52), but other factors might be involved, leading to the delayed recovery of higher centers (53). Although the HPA can be suppressed by a very brief treatment, clinically evident adrenal insufficiency is perhaps rare in patients treated for less than 1 wk (54). Thus, it can be suggested that if short-term treatment does suppress the HPA, this suppression would last for only a few days (55). In our study, complete HPA recovery was evident weeks after cessation of daily dexamethasone administration.
Because glucocorticoids have an immense effect on the scenario of carbohydrate and protein metabolism in the liver as well as on total leukocyte constitution (56), we studied, in an additional series of experiments, whether ototopical dexamethasone treatment may have influenced serum biochemical and hematological parameters. Despite the antiinflammatory and immune-suppressive effects mediated by glucocorticoids, their prolonged and high-dose usage can cause hepatic injury with different individual and species susceptibilities (19, 57). The inference that the balance of enzyme activities may be altered during glucocorticoid treatment, amplifying the glucocorticoid action, comes from the influence of glucocorticoid on liver metabolic function. This is an accepted index of whole tissue enzyme activities, particularly in the liver. When the present studies were initiated, no corresponding data were available after ototopical glucocorticoid, particularly dexamethasone application.
One important effect of a 21-d administration of dexamethasone in our animals was the significant progressive increase in serum enzyme activities, in particular, ALP, ALT, AST, and GGT, compared with placebo values. These enzymes remained elevated 1 wk after cessation of the treatment. Long-term and high-dose treatment with oral and/or parenteral glucocorticoids produced a definite induction of these liver enzyme activities (58, 59). This result is also in accordance with the significant increase in levels of serum enzymes in vitro in cultured hepatocytes (27). It is unknown to us at present whether this effect is achieved by directly modulating the production of the enzymes in hepatocytes or by damaging these cells, with enzymes being released into the blood. Because we found no significant changes in glucose and protein levels in the serum of treated animals compared with baseline values, it may indicate that the increased enzyme activities result from increased biosynthesis or de novo synthesis of enzyme proteins or are due to decreased degradation of these enzymes in the liver, even after treatment has been discontinued (60). The increased enzyme activity that may result from glucocorticoid-induced hepatocellular damage may not support our data (27). In fact, few studies have shown that oral or parenteral glucocorticoid administration can result in morphological liver changes with a disruption of the hepatocyte microfilament network, thus leading to altered tight junction permeability of the blood-bile barrier of the hepatocytes (61). However, in this study, ototopical dexamethasone treatment may not alter the subcellular organelles and plasma membranes of hepatocytes; thus, the possible mechanisms of enzyme induction remain unclear at present. Otherwise, the increase in enzyme activities was not accompanied by any change in serum contents of electrolytes, urea, bilirubin, and albumin during dexamethasone treatment.
Daily glucocorticoid administration did not significantly alter total leukocyte count. Leukocytosis, with increased number of neutrophils, is considered partly to be the modulatory effect of glucocorticoids in generating blood cells. There was a dexamethasone-induced increase in the number of neutrophils in this study, which might be an effect of the glucocorticoid to increase the liberation of matured neutrophils from bone marrow (62). From the differential numbers, eosinophils and lymphocytes were decreased in the presence of exogenous glucocorticoid, which may be interpreted as an immunosuppressive effect of the drug. This decreasing effect might not be attributable to the toxic effect of the drug, but, rather, may result from redistribution of mature circulating cells (63).
In summary, we have provided evidence for the effects of ototopical dexamethasone-containing preparations on the HPA and hepatic function in healthy experimental animals. Treated animals exhibited consistently decreased resting and ACTH-stimulated plasma cortisol levels. Although we did not determine the plasma ACTH level, the rapid and marked decrease in basal cortisol levels may have resulted from acute adrenal insufficiency, suggesting an impaired hypothalamic-pituitary-mediated negative feedback mechanism. Concomitantly, dexamethasone altered serum activities in several liver enzymes and white blood cell constitution, which may indicate potential adverse glucocorticoid consequences. Thus, the complications for HPA and liver function have to be considered during treatment of inflammatory processes in the ear with glucocorticoid-containing ointments. In this regard, additional studies should be performed to assess and clarify the role of this neuroendocrine system in the pathophysiology of otitis externa and media.
Acknowledgments
We gratefully acknowledge the help and skilful technical assistance of Giesela Lochmann and Ina Hochheim. We are thankful to Prof. M. Kietzmann (Hanover, Germany) and Dr. Y. Endawoke (Leipzig University, Leipzig, Germany) for thoughtful comments and critical reading of the manuscript.
References
Geddes DM 1992 Inhaled corticosteroids: benefits and risks. Thorax 47:404–407
Lipworth BJ 1999 Systemic adverse effects of inhaled corticosteroid therapy: a systematic review and meta-analysis. Arch Intern Med 59:941–955
Patel L, Clayton PE, Addison GM, Price DA, David TJ 1995 Adrenal function following topical steroid treatment in children with atopic dermatitis. Br J Dermatol 132:950–955
Garden JM, Freinkel RK 1986 Systemic absorption of topical steroids. Metabolic effects as an index of mild hypercortisolism. Arch Dermatol 122:1007–1010
Wihl JA, Andersson KE, Johansson SA 1997 Systemic effects of two nasally administered glucocorticosteroids. Allergy 52:620–626
Hanania NA, Chapman KR, Kesten S 1995 Adverse effects of inhaled corticosteroids. Am J Med 98:196–208
Livanou T, Ferriman D, James VH 1967 Recovery of hypothalamo-pituitary-adrenal function after corticosteroid therapy. Lancet 2:856–859
Goldstein DE, Konig P 1983 Effect of inhaled beclomethasone dipropionate on hypothalamic-pituitary-adrenal axis function in children with asthma. Pediatrics 72:60–64
Gustafsson P, Tsanakas J, Gold M, Primhak R, Radford M, Gillies E 1993 Comparison of the efficacy and safety of inhaled fluticasone propionate 200 micrograms/day with inhaled beclomethasone dipropionate 400 micrograms/day in mild and moderate asthma. Arch Dis Child 69:206–211
Brown PH, Blundell G, Greening AP, Crompton GK 1991 Screening for hypothalamo-pituitary-adrenal axis suppression in asthmatics taking high dose inhaled corticosteroids. Respir Med 85:511–516
Keller-Wood ME, Dallman MF 1984 Corticosteroid inhibition of ACTH secretion. Endocr Rev 5:1–24
Clark DJ, Lipworth BJ 1997 Evaluation of corticotropin releasing factor stimulation and basal markers of hypothalamic-pituitary-adrenal axis suppression in asthmatic patients. Chest 112:1248–1252
Levin C, Maibach HI 2002 Topical corticosteroid-induced adrenocortical insufficiency: clinical implications. Am J Clin Dermatol 3:141–147
Thiers BH 1989 Topical steroid therapy of atopic skin diseases. Allergy Proc 10:413–416
Rupprecht M, Hornstein OP, Schluter D, Schafers HJ, Koch HU, Beck G, Rupprecht R 1995 Cortisol, corticotropin, and ?-endorphin responses to corticotropin-releasing hormone in patients with atopic eczema. Psychoneuroendocrinology 20:543–551
Rupprecht M, Rupprecht R, Koch HU, Haack D, Muller OA, Hornstein OP 1991 Multihormonal response to dexamethasone. A study in atopic dermatitis and normal controls. Acta Derm Venereol 71:214–218
Glaze MB, Crawford MA, Nachreiner RF, Casey HW, Nafe LA, Kearney MT 1988 Ophthalmic corticosteroid therapy: systemic effects in the dog. J Am Vet Med Assoc 192:73–75
Solter PF, Hoffmann WE, Chambers MD, Schaeffer DJ, Kuhlenschmidt MS 1994 Hepatic total 3-hydroxy bile acids concentration and enzyme activities in prednisone-treated dogs. Am J Vet Res 55:1086–1092
Rutgers HC, Batt RM, Vaillant C, Riley JE 1995 Subcellular pathologic features of glucocorticoid-induced hepatopathy in dogs. Am J Vet Res 56:898–907
Center SA, Slater MR, Manwarren T, Prymak K 1992 Diagnostic efficacy of serum alkaline phosphatase and -glutamyltransferase in dogs with histologically confirmed hepatobiliary disease: 270 cases (1980–1990). J Am Vet Med Assoc 201:1258–1264
Abraham GE, Bustler JE, Teller RC 1972 Radioimmunoassay for plasma cortisol. Anal Lett 5:757
German Society of Clinical Chemistry 1972 Recommendation for measuring enzyme activity in human serum. Z Klin Chem Klin Biochem 10:91
International Federation of Clinical Chemistry 1991 Method for creatine kinase. Eur J Clin Chem Clin Biochem 29:435–456
Todd GR, Acerini CL, Buck JJ, Murphy NP, Ross-Russell R, Warner JT, McCance DR 2002 Acute adrenal crisis in asthmatics treated with high-dose fluticasone propionate. Eur Respir J 19:1207–1209
Pedersen S, O’Byrne P 1997 A comparison of the efficacy and safety of inhaled corticosteroids in asthma. Allergy 52:1–34
Crowley S, Hindmarsh PC, Holownia P, Honour JW, Brook CG 1991 The use of low doses of ACTH in the investigation of adrenal function in man. J Endocrinol 130:475–479
Hadley SP, Hoffmann WE, Kuhlenschmidt MS, Sanecki RK, Dorner JL 1990 Effect of glucocorticoids on alkaline phosphatase, alanine aminotransferase, and -glutamyltransferase in cultured dog hepatocytes. Enzyme 43:89–98
Hanford WC, Kottel RH, Fishman WH 1981 Alkaline phosphatase protein increases in response to prednisolone in HeLa cells. Biochem J 200:461–464
Bugajski J, Gadek-Michalska A, Bugajski AJ 2001 A single corticosterone pre-treatment inhibits the hypothalamic-pituitary-adrenal responses to adrenergic and cholinergic stimulation. J Physiol Pharmacol 52:313–324
Yehuda R, Boisoneau D, Lowy MT, Giller EL 1995 Dose-response changes in plasma cortisol and lymphocyte glucocorticoid receptors following dexamethasone administration in combat veterans with and without posttraumatic stress disorder. Arch Gen Psychiatry 52:583–593
Yehuda R, Southwick SM, Krystal JH, Bremner D, Charney DS, Mason JW 1993 Enhanced suppression of cortisol following dexamethasone administration in posttraumatic stress disorder. Am J Psychiatry 150:83–86
Wong J, Black P 1992 Acute adrenal insufficiency associated with high dose inhaled steroids. Br Med J 304:1415
Zwaan CM, Odink RJ, Delemarre-van de Waal HA, Dankert-Roelse JE, Bokma JA 1992 Acute adrenal insufficiency after discontinuation of inhaled corticosteroid therapy. Lancet 340:1289–1290
Gallant C, Kenny P 1986 Oral glucocorticoids and their complications. J Am Acad Dermatol 14:161–177
Turpeinen M 1988 Influence of age and severity of dermatitis on the percutaneous absorption of hydrocortisone in children. Br J Dermatol 118:517–522
Turpeinen M 1989 Adrenocortical response to adrenocorticotropic hormone in relation to duration of topical therapy and percutaneous absorption of hydrocortisone in children with dermatitis. Eur J Pediatr 148:729–731
Bartorelli A, Rimondini A 1984 Severe hypertension in childhood due to prolonged skin application of a mineralocorticoid ointment. Hypertension 6:586–588
Turpeinen M, Salo OP, Leisti S 1986 Effect of percutaneous absorption of hydrocortisone on adrenocortical responsiveness in infants with severe skin disease. Br J Dermatol 115:475–484
Wester RC, Maibach HI 1992 Percutaneous absorption in diseased skin. In: Maibach HI, Surber C, eds. Topical corticosteroids. Basel: Karger; 128–141
Feldmann RJ, Maibach HI 1967 Regional variation in percutaneous penetration of 14C cortisol in man. J Invest Dermatol 48:181–183
Maibach HI, Feldmann RJ, Milby TH, Serat WF 1971 Regional variation in percutaneous penetration in man. Arch Environ Health 23:208–211
Matsuda K, Katsunuma T, Iikura Y, Kato H, Saito H, Akasawa A 2000 Adrenocortical function in patients with severe atopic dermatitis. Ann Allergy Asthma Immunol 85:35–39
A Shohat M, Mimouni M, Shuper A, Varsano I 1986 Adrenocortical suppression by topical application of glucocorticosteroids in infants with seborrheic dermatitis. Clin Pediatr 25:209–212
Brus R 1999 Effects of high-dose inhaled corticosteroids on plasma cortisol concentrations in healthy adults. Arch Intern Med 159:1903–1908
Sorkness CA 1998 Establishing a therapeutic index for the inhaled corticosteroids. II. Comparisons of systemic activity and safety among different inhaled corticosteroids. J Allergy Clin Immunol 102:S52–S64
Krasner AS 1999 Glucocorticoid-induced adrenal insufficiency. J Am Med Assoc 282:671–676
Crowley S, Hindmarsh PC, Honour JW, Brook CG 1993 Reproducibility of the cortisol response to stimulation with a low dose of ACTH (1–24): the effect of basal cortisol levels and comparison of low-dose with high-dose secretory dynamics. J Endocrinol 136:167–172
Dickstein G, Shechner C, Nicholson WE, Rosner I, Shen-Orr Z, Adawi F, Lahav M 1991 Adrenocorticotropin stimulation test: effects of basal cortisol level, time of day, and suggested new sensitive low dose test. J Clin Endocrinol Metab 72:773–778
Dickstein G, Spigel D, Arad E, Shechner C 1997 One microgram is the lowest ACTH dose to cause a maximal cortisol response. There is no diurnal variation of cortisol response to submaximal ACTH stimulation. Eur J Endocrinol 137:172–175
Mayenknecht J, Diederich S, Bahr V, Plockinger U, Oelkers W 1998 Comparison of low and high dose corticotropin stimulation tests in patients with pituitary disease. J Clin Endocrinol Metab 83:1558–1562
Weintrob N, Sprecher E, Josefsberg Z, Weininger C, Aurbach-Klipper Y, Lazard D, Karp M, Pertzelan A 1998 Standard and low-dose short adrenocorticotropin test compared with insulin-induced hypoglycemia for assessment of the hypothalamic-pituitary-adrenal axis in children with idiopathic multiple pituitary hormone deficiencies. J Clin Endocrinol Metab 83:88–92
Henzen C, Suter A, Lerch E, Urbinelli R, Schorno XH, Briner VA 2000 Suppression and recovery of adrenal response after short-term, high-dose glucocorticoid treatment. Lancet 355:542–545
Ford LR, Willi SM, Hollis BW, Wright NM 1997 Suppression and recovery of the neonatal hypothalamic-pituitary-adrenal axis after prolonged dexamethasone therapy. J Pediatr 131:722–726
Carella MJ, Srivastava LS, Gossain VV, Rovner DR 1993 Hypothalamic-pituitary-adrenal function one week after a short burst of steroid therapy. J Clin Endocrinol Metab 76:1188–1191
Streck WF, Lockwood DH 1979 Pituitary adrenal recovery following short-term suppression with corticosteroids. Am J Med 66:910–914
Casadevall M, Saperas E, Panes J, Salas A, Anderson DC, Malagelada JR, Pique JM 1999 Mechanisms underlying the anti-inflammatory actions of central corticotropin-releasing factor. Am J Physiol 276:G1016–G1026
Bhagwat AG, Ross RC 1971 Prednisolone-induced hepatic injury. Ultrastructural and biochemical changes in rabbits. Arch Pathol 91:483–492
Sanecki RK, Hoffmann WE, Gelberg HB, Dorner JL 1987 Subcellular location of corticosteroid-induced alkaline phosphatase in canine hepatocytes. Vet Pathol 24:296–301
DeNovo RC, Prasse KW 1983 Comparison of serum biochemical and hepatic functional alterations in dogs treated with corticosteroids and hepatic duct ligation. Am J Vet Res 44:1703–1709
Seetharam S, Sussman NL, Komoda T, Alpers DH 1986 The mechanism of elevated alkaline phosphatase activity after bile duct ligation in the rat. Hepatology 6:374–380
Van Noorden CJ, Frederiks WM, Aronson DC, Marx F, Bosch K, Jonges GN, Vogels IM, James J 1987 Changes in the acinar distribution of some enzymes involved in carbohydrate metabolism in rat liver parenchyma after experimentally induced cholestasis. Virchows Arch B Cell Pathol 52:501–511
Dale DC, Fauci AS, Guerry IV D, Wolff SM 1975 Comparison of agents producing a neutrophilic leukocytosis in man. Hydrocortisone, prednisone, endotoxin, and etiocholanolone. J Clin Invest 56:808–813
Bloemena E, Weinreich S, Schellekens PT 1990 The influence of prednisolone on the recirculation of peripheral blood lymphocytes in vivo. Clin Exp Immunol 80:460–466(Getu Abraham, Jutta Gotts)
Address all correspondence and requests for reprints to: Dr. Getu Abraham, Leipzig University, Institute of Pharmacology, Pharmacy, and Toxicology, An den Tierkliniken 15, D-04103 Leipzig, Germany. E-mail: gabraham@rz.uni-leipzig.de.
Abstract
To clarify whether ototopical glucocorticoid treatment is associated with impaired hypothalamic-pituitary-adrenal axis (HPA) activity and altered hepatic metabolism, one commercially available dexamethasone-containing ointment was tested. At present, very little is known about the effects of ototopical glucocorticoid treatment on HPA and liver function. Ten beagle dogs received two daily therapeutic doses of dexamethasone (0.6 mg/ear) in the outer auditory canal for 21 d in a single-blind, placebo-controlled study. Resting cortisol concentrations were assessed before, during, and after treatment using an RIA system. Adrenal function and HPA feedback sensitivity were measured by a standard dose (250 μg) ACTH stimulation test. Serum biochemical and hematological parameters were measured, whether ototopical glucocorticoids affect hepatic function was studied, and blood cell counts were made. Ototopical dexamethasone treatment induced a marked suppression (to about 100%) of resting plasma cortisol concentrations below the placebo effect (P < 0.0001) within the first 11 d, and these remained reduced during the entire treatment period up to d 19. As well, an ACTH stimulation test found a markedly reduced rise in plasma cortisol concentrations (P = 0.0004). Concomitantly, significant increases in serum activities of alkaline phosphatase, -glutamyl transferase, alanine transaminase, and aspartate transaminase were detected. Moreover, we found a significant reduction in differential leukocyte counts of eosinophils and lymphocytes, whereas neutrophils increased. Although cortisol levels and hematological parameters returned to baseline 7 d after treatment cessation, liver enzyme activities remained elevated. In conclusion, these findings suggest that after ototopical application, dexamethasone is sufficiently absorbed from the auditory canal to suppress HPA function as well as to alter metabolic and hemopoietic profiles. Thus, in long-term treatment of otitis externa or media, the systemic adverse suppression of HPA has to be considered in relation to stress exposure, whereas changes in serum enzyme activities may not be interpreted as hepathopathy.
Introduction
OTOTOPICAL GLUCOCORTICOID formulations, with or without an antimicrobial or antimycotic agent, have commonly been used for the treatment of acute or chronic otitis externa or acute otitis media with otorrhea commonly in humans (children). Although topical corticosteroid therapy seemed to be more beneficial by minimizing the degree of the well-known systemic bioactivity than oral or parenteral glucocorticoids, it is now becoming increasingly accepted that such forms of application of glucocorticoid-containing drugs are associated with systemic adverse effects at the clinically recommended doses. Despite this widespread argument, however, even ototopical therapy with topical glucocorticoids such as dexamethasone might lead to transcutaneous systemic absorption and, hence, to any total systemic adversative burden, but this remains to be evaluated.
Experimental findings have emphasized over the past years the role of topical glucocorticoids as relevant triggering factors for many systemic alterations, such as suppression of the hypothalamic-pituitary-adrenal axis (HPA), depression of leukocyte numbers, alteration in hepatic enzyme activities, and changes in bone metabolism and growth (1). For example, when using inhaled glucocorticoids for the treatment of asthma (for review, see Ref. 2) or topical glucocorticoids for atopic dermatitis (3, 4) and nasal rhinosinusitis (5), a detectable suppression of the HPA could be demonstrated. A decreased responsiveness of the adrenal gland is recognized, most likely as sensitive and reproducible indices of the systemic adverse effects of glucocorticoids (6, 7). For this effect on the HPA, most studies have focused on assessing endogenous cortisol production level in plasma or serum, which is suppressed by exogenous glucocorticoids, as a reliable and suitable method of measurement (8, 9). Moreover, an integrated cortisol determination in plasma after an ACTH stimulation test at high dose (250 μg) is regarded to also be sensitive for detecting adrenocortical suppression, for example, in patients taking long-term inhaled corticosteroids (10). However, the mode and magnitude of such iatrogenic adrenal suppression, measured as depressed levels of cortisol after exogenous glucocorticoid treatment, are supposed to be associated with excessive high dose and long-term administration of glucocorticoids, which might even result in reversible manifestations of Cushing’s syndrome (11). In asthmatic patients, it has been shown that prolonged treatment with excessive glucocorticoids caused chronic suppression of the HPA and secondary adrenocortical insufficiency, which persisted for months or more than a year after discontinuation of treatment (7, 12), but information is lacking regarding the duration or the effects of suppression after ototopical administration of antiinflammatory doses. In contrast, considerable debate continues about the clinical significance of basal nonstimulated cortisol concentrations, because although these offer an indication of the relative systemic activity of inhaled corticosteroids at the recommended therapeutic doses, they presumably do not indicate the degree of adrenal reserve in response to various stress factors. Hence, an ACTH stimulation test might offer an assessment of impaired HPA negative feedback based on the measurement of cortisol levels.
Supporting this, a few studies have examined the biosensitivity of HPA to dermal topical glucocorticoids, which could be mediated by transcutaneous absorption in humans and experimental animals (3, 13, 14), in part with conflicting results. Patients with atopic dermatitis may have decreased plasma cortisol levels without therapeutic intervention (15). Rupprecht et al. (16) found that subjects with atopic dermatitis display impaired suppression of the HPA by glucocorticoids, which seemed to be a phenomenon of glucocorticoid resistance. In contrast, in healthy subjects and experimental animals, topical glucocorticoids suppressed endogenous cortisol release; however, this was found to be dose related and dependent on the route and duration of treatment, usually applied for a long period of time at higher doses (17). Such conditions have, however, failed to be considered during the use of ear drops containing antiinflammatory components such as dexamethasone.
Although the results of previous studies have indicated that oral or parenteral glucocorticoids alter hepatic functions, little information exists concerning the induction of serum liver enzyme activities during topical treatment with glucocorticoids in general, and even less is known about this process specifically during therapy with ototopical glucocorticoids. In this context, a unique spectrum of morphological and biochemical changes to the liver has been reported that is markedly reflected in serum by high activities of several liver enzymes, such as alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and -glutamyl transferase (GGT) (18). These enzymes are often used as markers to test the state of integrity of liver cell membranes, perhaps referred as liver dysfunction, either as a result of drug application or any disease-related hepatic disorders. Accordingly, a more pronounced progressive increase has been described for hepatic ALP and GGT activities after oral or parenteral glucocorticoids and in hepatic diseases (19, 20). However, the results of studies evaluating the extent to which glucocorticoids, after ototopical (or dermal) administration, affect these enzymes and how long these effects persist after cessation of treatment have been rarely presented.
Because previous studies have shown that exogenous topical (inhaled or dermal) glucocorticoids, usually at high doses, induce complete suppression of cortisol secretion, which is controlled by HPA, partly through a negative feedback mechanism, we investigated in this study the use of an ototopical dexamethasone-containing preparation at recommended doses to detect changes in HPA function (responsiveness and recovery), by measuring resting cortisol levels compared with ACTH-stimulated cortisol release. Moreover, we assessed serum hepatic enzyme activities and hematological parameters during a twice-daily application of dexamethasone-containing ear drops for 21 d. We compared both basal and post-ACTH cortisol levels after administration of a placebo and a pharmacologically active agent, dexamethasone, in the same animal.
Materials and Methods
Animals
Animal experiments were approved by the local ethical committee for animal welfare and were carried out in accordance with the guidelines of the German law relating to animal welfare.
In these studies a total of 10 adult beagle dogs of mixed gender (four males and six females) between 3 and 4 yr of age, weighing 11–16 kg, were enrolled. The animals were housed in solid-bottom cages with an outlet, three and four dogs per cage, respectively; they were fed commercial diet food and received water ad libitum. Animals were clinically healthy and received no steroid therapy for at least 3 months before this experiment, except for routine vaccination and deworming. Otoscopic examination showed no symptoms of inflammatory disorders of the external auditory canal. Each dog served as its own control.
Experimental design and blood sampling
A single-blind, nonrandomized, placebo-controlled, repeated measure design was used with washout periods of 1 wk. Three days before any treatment (d 0), a complete physical and special otoscopic examination, the baseline complete blood cell count and serum biochemical analysis, as well as determination of basal plasma cortisol levels before and after ACTH stimulation test were performed in all dogs. Moreover, in this pretreatment period, both ears (left and right) were cleaned with dry soft tissues. Dogs had an initial placebo run, a 5-d period during which 20 drops of placebo (0.60 g placebo ointment) were administered in the left ear twice daily at 0800 and 1800 h. Thereafter, dogs received an ototopic drug combination in recommended therapeutic doses containing dexamethasone-21-acetate (1 mg/g), neomycin sulfate (5 mg/g), and clotrimazole (10 mg/g), two doses for 21 d each: 20 drops (0.60 g ointment, 60 μg/kg body weight dexamethasone) of dexa-methasone-containing ear gel twice daily (total daily dose, 1200 μg) in the right ear canal at 0800 and 1800 h. Correspondingly, dogs were given daily vs. twice daily 20 drops (0.60 g) of a nonactive agent containing placebo formulation while receiving dexamethasone-containing ear drops. With a 1-wk washout period the whole study was designed to take about 40 d. Clinical and otoscopic examinations were carried out every morning before drug application during the treatment phase. Blood samples for the analysis of total blood cell count, serum biochemical profile (including urea, electrolytes, and liver function tests), and plasma cortisol levels were collected from cephalic venipuncture in EDTA- or lithium-heparin-displaced tubes (Sarstedt, Hamburg, Germany) at different time points: on d 0 (baseline), d 5 (placebo phase), d 11 and 19 (during treatment with dexamethasone and placebo in parallel for 21 d), and d 33 (7 d after last application of pharmacologically active substances). For determination of endogenous cortisol concentrations, all blood samples from each animal were collected frequently at 0800 h during these five periods. On similar days, a response challenge test to a standard dose (250 μg) synthetic ACTH was performed. Serum and plasma samples were obtained after centrifugation of whole blood (4000 x g, 10 min, 4 C), and the specimens were then aliquoted in 1.5-ml polypropylene tubes and stored at –20 C until analysis.
ACTH stimulation test
To assess the feedback sensitivity of the HPA, adrenocortical suppression and responsiveness after application of ototopical dexamethasone-containing formulations in the external auditory canal was studied by a serial determination of baseline plasma cortisol levels and the cortisol concentrations after ACTH challenge test. All samples for cortisol determination were obtained at 0800 h and immediately before and 1 h after iv administration of an ACTH bolus (250 μg). Adrenocortical function was assessed on d 0 (before drug treatment), 5 (after placebo), 12 (during topical glucocorticoid application), and 33 (after drug withdrawal). ACTH was administered in the morning 1 h before the usual time of treatment.
Plasma cortisol determination
Plasma cortisol was measured with a slight modification of the methods described by Abraham et al. (21) using an RIA kit with commercially available [3H]cortisol (specific activity, 50–90 Ci/mmol; Amersham Biosciences, Freiburg, Germany). All assays were performed in duplicate in a blind fashion by a separate technician. In brief, perturbing components were separated by precipitating the blood plasma with absolute alcohol. For this purpose, 100 μl thawed blood plasma were mixed with 900 μl ethanol (99.98%) and centrifuged for 15 min at 4 C. Thereafter, 100 μl supernatant were transferred to new incubation tubes, and samples were dried overnight at 37 C in a drying closet. After drying the samples, 100 μl phosphate buffer were added to each sample. Samples were incubated after the addition of aliquots of 100 μl [3H]cortisol at a final concentration of 1.4–2.5 nM (125 nCi/ml) and 100 μl polyclonal antibody (raised in rabbits against cortisol) at final volumes of 300 μl for 4 h at 4 C. Nonspecific binding was determined in the presence of phosphate buffer instead of the antibody. Separation of protein-bound [3H]cortisol from unbound steroid was achieved by adsorption of the free steroid onto coated charcoal, followed by centrifugation at 1500 x g for 15 min at 4 C. An aliquot of the supernatant was thereafter removed for liquid scintillation counting (PerkinElmer, Freiburg, Germany). The concentration of unlabeled cortisol in the sample was then determined from standard curves assayed in triplicate. The amounts of cortisol in the unknown samples were quantified by reference to the appropriate standard curve and calculated as nanomoles per liter using Multicalc software (Wallac, Turku, Finland). The coefficients of variability for analytical imprecision were 8.1% (intraassay) and 10.2% (interassay; n = 6).
The assay was based on the competition between unlabeled cortisol (antibody) and a fixed quantity of the tritium-labeled compound for binding to a protein with a high specificity and affinity for cortisol. The amount of labeled protein-cortisol complex formed was inversely related to the amount of unlabeled cortisol present in the assay sample. Measurement of protein-bound radioactivity enabled the amount of unlabeled cortisol in the sample to be calculated.
Analysis of serum activities of liver enzymes and hematological profiles
Serum activities of liver enzymes, ALP, ALT, AST, GGT, and lactate dehydrogenase were determined before, during, and after treatment with dexamethasone-ear drops according to standard procedures. Briefly, catalytic activities were determined using standard methods recommended by the German Society of Clinical Chemistry (22) with an automated analyzer (model 704, Hitachi, Mannheim, Germany) using commercially available reagents (Roche, Mannheim, Germany). All enzyme activities were determined at 37 C, and assay procedures were optimized according to the manufacturer’s instructions. Moreover, serum was analyzed for creatinine kinase (CK) after adding acetylcysteine using a CK kit (Roche) according to the manufacturer’s instructions, as recommended by the International Federation of Clinical Chemistry (23). Additionally, serum levels of protein, albumin, bilirubin, urea, electrolytes, and glucose were determined by routine clinical analyses.
Furthermore, EDTA-treated blood was analyzed for hematological constituents using a semiautomated analyzer (Technicon H1, Bayer Diagnostics, Muenchen, Germany). Blood hemoglobin concentrations were determined by the cyanomethemoglobin procedure. The red blood cell count, thrombocyte count, white blood cell count, and mean cell volume were measured directly. The values of red blood cell indices (packed cell volume, mean cell hemoglobin, and mean cell hemoglobin concentrations) were calculated.
Statistical analysis
Data presented in figures and tables are the mean ± SEM. To determine the effect of ototopic administration of dexamethasone-containing ointment on HPA function (by measuring resting and ACTH-stimulated plasma cortisol concentrations) and on liver enzyme activities, data before treatment (d 0), during placebo treatment (d 5), during dexamethasone as well as placebo application (d 11, 12, and 19), and 1 wk after drug withdrawal (d 33) were compared using repeated measures ANOVA. To examine the differences between baseline values vs. placebo and treatment values, Dunnett’s multiple comparison test was employed. P < 0.05 was considered significant.
Results
General effects
Physical and otoscopic examinations of the experimental animals showed, no apparent clinical abnormalities of the external auditory canal, the skin, or cutaneous pigmentation during the treatment period. Interestingly, at the ototopic dexamethasone therapeutic doses used for 3 wk, we were able to observe in all dogs enormous polydypsia and polyuria during the entire treatment period, without appreci-able changes in food intake. However, as a recovery sign after dexamethasone withdrawal, polydypsia and polyuria ceased after 7 d.
Plasma cortisol levels
One specific goal of the present study was to evaluate the responsiveness of the HPA after otic administration of topical glucocorticoids in therapeutic doses for 21 d. During placebo challenge, cortisol concentrations were slightly higher than baseline cortisol levels, but did not statistically differ from baseline values. Differences from placebo were significant at all time points of otic dexamethasone application for HPA responses and adrenocortical cortisol secretion (Fig. 1). Plasma cortisol concentrations decreased significantly in all animals during the treatment periods, as measured on d 11 and 19 [F(4,36) = 16.04; P < 0.0001]. As illustrated in Fig, 1, the average plasma cortisol level was suppressed to 1.24 ± 0.27 nmol/liter on d 11, and suppression was more pronounced (<1 nmol/liter) on d 19. In terms of mean percent dexamethasone induced at therapeutic doses twice daily for 21 d, there was an approximately 92–95% suppression of 0800 h plasma cortisol (baseline cortisol, 14.75 ± 4.76 nmol/liter). All transotic-treated dogs showed a similar trend in cortisol reduction. One week after cessation of ototopical dexamethasone treatment, there was a recovery of the cortisol response (Fig. 1); however, the variance for cortisol concentration was significantly different between values before and after treatment [F(9,9) = 366.4; P < 0.0001]. Cortisol levels increased within above 100% to values obtained during the treatment periods, but were 50% lower than pretreatment values (d 0; 14.75 ± 4.76 vs. 8.83 ± 0.90 nmol/liter; P < 0.05).
FIG. 1. Plasma cortisol values before treatment, during administration of placebo, during administration of dexamethasone-containing preparation and placebo, and after cessation of treatment. All 10 dogs were examined for cortisol response on each occasion. The differences in baseline, placebo, and dexamethasone and placebo were statistically significant. ***, P < 0.001, control vs. d 11 and 19 dexamethasone administration; *, P < 0.05, control vs. treatment withdrawal.
An ACTH challenge was administered before, during, and after pretreatment with placebo or otic glucocorticoid preparation to determine whether dexamethasone exerted an inhibitory effect on cortisol secretion at the level of the adrenal glands. ANOVA indicated that synthetic ACTH was able to stimulate significantly endogenous cortisol release before treatment to approximately 700% above basal values (18.63 ± 2.61 vs. 152.83 ± 23.94 nmol/liter). As depicted in Fig. 2, the interaction between dexamethasone treatment and ACTH challenge represented a significant difference in plasma cortisol concentrations between baseline (d 0) and otic treatment (d 12; 152.83 ± 23.94 vs. 40.93 ± 6.75 nmol/liter; P = 0.0004). During the treatment period, approximately 3.7-fold lower ACTH-stimulated cortisol concentrations were found, but these were significantly higher than cortisol values measured on d 0 before ACTH challenge (40.93 ± 6.75 vs. 18.63 ± 2.61 nmol/liter; P = 0.0008). Thus, otic dexamethasone did show an ability to block the cortisol response to an ACTH challenge. In a similar pattern as cortisol levels after treatment cessation, an ACTH stimulation test failed to fully up-regulate endogenous cortisol release 7 d after discontinuation of treatment (Fig. 2). At this time after ACTH stimulation, cortisol levels increased, but did not reach the values seen before dexamethasone challenge (107.80 ± 12.30 vs. 152.83 ± 23.94 nmol/liter).
FIG. 2. Effects of topical dexamethasone pretreatment on cortisol response 60 min after ACTH challenge. Before, during, or after administration of a dexamethasone-containing preparation and/or placebo in the external auditory canal twice daily, dogs were challenged with ACTH (250 μg). ***a, P < 0.001, cortisol levels on d 0 (control), before vs. after ACTH stimulation test; ***b, P < 0.001, cortisol values during placebo, before vs. after ACTH stimulation test; ***c, P < 0.001, cortisol values after ACTH response on d 0 (control) vs. ACTH response on d 12 during dexamethasone application (by Student’s t test).
Effects on liver enzymes
As a marker of possible glucocorticoid-induced hepatic dysfunction, serum levels of specific enzymes were analyzed. Marked significant alterations in various liver enzymes were seen during treatment with otic dexamethasone compared with enzyme activities on d 0 (control) and during placebo treatment. As shown in Fig. 3A, 21-d otical administration of a dexamethasone-containing preparation increased progressively and significantly the mean total serum activity of ALP. On d 11, however, a less enhanced, nonsignificant activity (134.50 ± 16.30 U/liter) was seen compared with mean basal and placebo activities (99.00 ± 14.85 and 94.90 ± 14.87 U/liter, respectively), and a 4-fold significant activity increase was determined by d 19 (control vs. d 19, 99.00 ± 14.85 vs. 426.20 ± 75.20 U/liter; P < 0.001). Serum ALP activity peaked 1 wk after discontinuation of the treatment, with a mean value approximately 5 times greater than that at baseline (512.20 ± 90.42 vs. 99.00 ± 14.85 U/liter; P < 0.001 vs. d 0). Likewise, in dexamethasone-treated dogs, serum AST, ALT, and GGT activities increased significantly by d 11 and 19 (Fig. 3, B–D). Except for AST, the progressive increase in the activities of ALT and GGT did not return to baseline 7 d after drug withdrawal and were still significantly elevated. The remaining biochemical parameters (albumin, bilirubin, urea, electrolytes, glucose, CK, and lactate dehydrogenase) were within the normal physiological range and were not altered by ototopical glucocorticoid treatment in any of the animals examined (summarized in Table 1).
FIG. 3. Dexamethasone-induced increase in liver enzyme activity: ALP (A), AST (B), ALT (C), and GGT (D). Asterisks indicate significant differences in serum enzyme levels between baseline and dexamethasone treatment. Values are the mean ± SEM. *, P < 0.05; **, P < 0.01; ***. P < 0.001.
TABLE 1. Effects of ototopic dexamethasone on serum biochemical parameters compared with baseline and placebo
Hematological parameters
After 21 d of topical dexamethasone administration, significant differences were not observed in total leukocyte counts between experimental animals before and after treatment. There was a significant increase in the mean number of segmented neutrophils on d 11 and 19 during dexamethasone application (P < 0.05 vs. d 0), whereas eosinophils decreased during these treatment periods and remained at this reduced level even 7 d after drug withdrawal, but lymphocytes showed only a tendency to decrease without statistical significance. All other hematological parameters did not demonstrate such relevant changes during treatment; data are summarized in Table 2.
TABLE 2. Effects of ototopic dexamethasone on hematological parameters compared with baseline and placebo
Discussion
To determine the importance of ototopical dexamethasone-containing preparation on HPA function, hepatic metabolic activity, and the immune system, we investigated changes in endogenous cortisol production by the adrenal gland, several liver enzyme activities, and other additional biochemical and immunological parameters in clinically healthy beagle dogs. Although treatment with topical corticosteroids may be of benefit at the expense of systemic side effects, the large number of studies have shown that, for instance, asthmatic patients exposed to oral, parenteral, or inhaled glucocorticoids have a marked suppression of endogenous cortisol production (24), indicating chronic adrenal suppression, a surrogate marker for possible adverse effects in other tissues (2, 25, 26). Indeed, these studies have focused only on adrenal insufficiency, not on the possible role of exogenous glucocorticoids in liver function, i.e. liver enzyme activities. Conceivably, it has been argued from the findings of in vitro studies of cell cultures (hepatocytes and HeLa cells) that exogenous glucocorticoids might induce enzyme activities by de novo protein synthesis (27, 28). Even though ototopical corticosteroids are frequently used in children with inflammatory disorders in the outer and middle auditory canal, studies showing a direct correlation between these exogenous glucocorticoids and eventual insufficiency in adrenal reserve as well as hepatic function have not been conducted to date in humans or experimental animals.
This study produced several interesting findings that merit discussion and that together provide an explanation for the differences between previous studies comparing the systemic activities of dermal, inhaled, and oral glucocorticoids. The main findings of the present study were that ototopical dexamethasone treatment twice daily for 21 d in therapeutic doses caused a marked influence on HPA responses and liver function: 1) basal and ACTH-stimulated plasma cortisol levels dramatically decreased during the treatment periods; 2) exogenous dexamethasone produced enhanced activities of several liver enzymes; and 3) there was a significant increase in differential counts of segmented neutrophils, whereas eosinophils and lymphocytes decreased, as in dogs and other experimental animals receiving oral or parenteral glucocorticoids.
In the present experiment after ototopical administration, dexamethasone itself as a mixture component with antimicrobial agent (neomycin) in relatively low dose (0.6 mg/ear, twice daily; 60 μg/kg body weight once) strongly decreased the resting plasma cortisol level from 18.63 to 0.79 nmol/liter, which peaked on d 19 of treatment. Even though in this study the resting plasma ACTH level was not determined, if it had been decreased, this would indicate a preferential and major suppression of CRH, which, in turn, would stimulate the pituitary-adrenocortical axis to produce ACTH (29). Thus, it seemed logical to assume that dexamethasone might have greatly hampered the feedback sensitivity of the HPA. In contrast, previous studies have argued, in line with our findings, that at the pituitary level, the discriminative ability of the dexamethasone suppression test can be enhanced by reducing the dose of dexamethasone (30, 31). Hence, because dexamethasone at the chosen dose in our study totally abolished the significant increase in endogenous cortisol release from the adrenal cortex, it can be anticipated that dexamethasone might also abolish the basal increase in plasma ACTH concentrations, but this remains speculative at present.
The earlier time to peak cortisol response in dexamethasone-treated compared with placebo-treated animals indicates an accelerated decrease in HPA and, hence, adrenal sensitivity to dexamethasone. This acute adrenal insufficiency, reflected by the marked suppression of cortisol, might be attributed to the occurrence of acute adrenal crisis. These conditions agree with previous studies of the effects of topical inhaled corticosteroids applied in asthma therapy (24, 32, 33). However, these studies have addressed the issue of HPA suppression and adrenal crisis in association with high-dose, long-term, inhaled glucocorticoid treatment (34).
Similarly, in confirming our findings, it has been shown that locally administered dermal glucocorticoid-containing ointments also potentially suppress the endogenous cortisol release (4, 35); however, increased sensitivity to systemic adverse effects has been reported in patients with pathological skin changes, such as in atopic dermatititis after treatment with potent topical glucocorticoids (3, 36, 37). In this study, it has been argued that in diseased skin the degree of prompt endogenous cortisol suppression should be encouraged by increased percutaneous absorption (38, 39). Indeed, this effect is assumed to be enhanced by various factors: extent and severity of disease, greater surface area of treated skin, and dose and duration of topical glucocorticoid treatment (13, 40). In humans (children), otitis externa or interna might induce damage in ear canal stratum corneum and thus might enhance systemic absorption of dexamethasone and, as a result, produce marked adrenal suppression, as described by Levin and Maibach (13), of hydrocortisone with increased serum hydrocortisone levels in children with atopic dermatitis.
Although the present study was undertaken in healthy experimental animals without changes in ear canal skin integrity, the percutaneous drug absorption and the dexamethasone-induced influence on endogenous cortisol production might agree with the results obtained for skin, described above. However, it can strongly be assumed that in the ear, the percutaneous penetration and cutaneous metabolism of synthetic glucocorticoids might involve different pharmacokinetics than obviously oral or parenteral administration, as well as those of dermally administered corticosteroids. In normal human skin, it has been shown that the doses absorbed after 24-h skin application of, for example, hydrocortisone, is up to 5% (41). However, as reported by Feldmann and Maibach (42), the extent of penetration of this compound through human skin varies depending on the anatomical site to which the compound is applied. A maximum percutaneous penetration of hydrocortisone was observed on the scrotum (40%). A similar percentage of absorption was observed after application of the hydrophobic pesticide parathion in the human ear canal, whereas in the scrotum, absorption was as twice as much as that in the ear canal (43). Although at present there are no data available for ear canal corticosteroid flux, it can be assumed that percutaneous penetration could be highly facilitated by the thin stratum corneum, occlusive condition, high blood circulation, and temperature in a given dog ear canal. Thus, percutaneous absorption of dexamethasone at low therapeutic dose in the ointment should be even more pronounced in the ear than in the skin and should have highly suppressed, even to undetectable, resting plasma cortisol levels during the treatment period.
Furthermore, in the present study the standard Synacthen (Novartis, Basel, Switzerland) test was conducted to directly test the adrenal reserve and indirectly assess HPA function, because chronic ACTH deficiency leads to a quiescent adrenal gland and, hence, to an inadequate cortisol response to exogenous ACTH at pharmacological doses (250 μg). This test is supposed to be preferable because it should present a more physiological stimulus to the adrenal gland and thus be more sensitive in detecting adrenal impairment (26). Accordingly, during ototopical dexamethasone treatment, the RIA procedure yielded 73% lower cortisol levels measured 1 h after the administration of exogenous ACTH than baseline values (152.83 ± 23.94 vs. 40.93 ± 6.75 nmol/liter), suggesting insufficiency of the adrenal glands. This is in good agreement with data from asthmatic patients treated with inhaled glucocorticoids, in whom standard ACTH-stimulated cortisol production was markedly blunted (44, 45), but in these subjects, supraphysiological glucocorticoid doses were used. As evidenced by a subnormal Synacthen stimulation test (250 μg) result, the data from our experimental animals indicate progressive suppression of the HPA to the most advanced phase and probable functional adrenal gland atrophy. With a pronounced or prolonged ACTH decrease, in keeping with our results, the adrenal glands may not be able to produce cortisol spontaneously, even after maximum stimulation with ACTH analogs (46). However, despite the controversy in the widespread published data for normal responses after either standard-dose (250 μg) or low-dose (1 μg) ACTH test (47, 48), the latter not only measures adrenal gland responsiveness, but may detect subtle degrees of adrenal atrophy (49); we believe that the standard test in the present study might have detected central adrenal axis insufficiency. Moreover, not all studies concur that the low-dose ACTH test is more sensitive than the high-dose ACTH test (50, 51). We did not use the low-dose ACTH test, because our test ototopical preparation contained dexamethasone, which may blunt the cortisol response to low-dose ACTH and may limit the value of the test method (26).
It was also worth assessing the duration of HPA recovery after drug withdrawal. Even if among the commonly and topically used glucocorticoids, dexamethasone, prednis-olone, and methylprednisolone, dexamethasone, with the longer half-life (36–54 h), suppresses ACTH for the longest period (46), resting plasma cortisol concentrations increased rapidly by d 7 after treatment cessation, but did not reach baseline. In almost all animals examined with suppressed adrenal response, the response to 250 μg ACTH, reflected by mean plasma cortisol values, returned gradually and steadily to baseline within 7 d after discontinuation of the treatment. In marked contrast to these short-term data, there is evidence that the recovery of suppressed adrenal response after long-term, high-dose glucocorticoid treatment may take more than a year (52), but other factors might be involved, leading to the delayed recovery of higher centers (53). Although the HPA can be suppressed by a very brief treatment, clinically evident adrenal insufficiency is perhaps rare in patients treated for less than 1 wk (54). Thus, it can be suggested that if short-term treatment does suppress the HPA, this suppression would last for only a few days (55). In our study, complete HPA recovery was evident weeks after cessation of daily dexamethasone administration.
Because glucocorticoids have an immense effect on the scenario of carbohydrate and protein metabolism in the liver as well as on total leukocyte constitution (56), we studied, in an additional series of experiments, whether ototopical dexamethasone treatment may have influenced serum biochemical and hematological parameters. Despite the antiinflammatory and immune-suppressive effects mediated by glucocorticoids, their prolonged and high-dose usage can cause hepatic injury with different individual and species susceptibilities (19, 57). The inference that the balance of enzyme activities may be altered during glucocorticoid treatment, amplifying the glucocorticoid action, comes from the influence of glucocorticoid on liver metabolic function. This is an accepted index of whole tissue enzyme activities, particularly in the liver. When the present studies were initiated, no corresponding data were available after ototopical glucocorticoid, particularly dexamethasone application.
One important effect of a 21-d administration of dexamethasone in our animals was the significant progressive increase in serum enzyme activities, in particular, ALP, ALT, AST, and GGT, compared with placebo values. These enzymes remained elevated 1 wk after cessation of the treatment. Long-term and high-dose treatment with oral and/or parenteral glucocorticoids produced a definite induction of these liver enzyme activities (58, 59). This result is also in accordance with the significant increase in levels of serum enzymes in vitro in cultured hepatocytes (27). It is unknown to us at present whether this effect is achieved by directly modulating the production of the enzymes in hepatocytes or by damaging these cells, with enzymes being released into the blood. Because we found no significant changes in glucose and protein levels in the serum of treated animals compared with baseline values, it may indicate that the increased enzyme activities result from increased biosynthesis or de novo synthesis of enzyme proteins or are due to decreased degradation of these enzymes in the liver, even after treatment has been discontinued (60). The increased enzyme activity that may result from glucocorticoid-induced hepatocellular damage may not support our data (27). In fact, few studies have shown that oral or parenteral glucocorticoid administration can result in morphological liver changes with a disruption of the hepatocyte microfilament network, thus leading to altered tight junction permeability of the blood-bile barrier of the hepatocytes (61). However, in this study, ototopical dexamethasone treatment may not alter the subcellular organelles and plasma membranes of hepatocytes; thus, the possible mechanisms of enzyme induction remain unclear at present. Otherwise, the increase in enzyme activities was not accompanied by any change in serum contents of electrolytes, urea, bilirubin, and albumin during dexamethasone treatment.
Daily glucocorticoid administration did not significantly alter total leukocyte count. Leukocytosis, with increased number of neutrophils, is considered partly to be the modulatory effect of glucocorticoids in generating blood cells. There was a dexamethasone-induced increase in the number of neutrophils in this study, which might be an effect of the glucocorticoid to increase the liberation of matured neutrophils from bone marrow (62). From the differential numbers, eosinophils and lymphocytes were decreased in the presence of exogenous glucocorticoid, which may be interpreted as an immunosuppressive effect of the drug. This decreasing effect might not be attributable to the toxic effect of the drug, but, rather, may result from redistribution of mature circulating cells (63).
In summary, we have provided evidence for the effects of ototopical dexamethasone-containing preparations on the HPA and hepatic function in healthy experimental animals. Treated animals exhibited consistently decreased resting and ACTH-stimulated plasma cortisol levels. Although we did not determine the plasma ACTH level, the rapid and marked decrease in basal cortisol levels may have resulted from acute adrenal insufficiency, suggesting an impaired hypothalamic-pituitary-mediated negative feedback mechanism. Concomitantly, dexamethasone altered serum activities in several liver enzymes and white blood cell constitution, which may indicate potential adverse glucocorticoid consequences. Thus, the complications for HPA and liver function have to be considered during treatment of inflammatory processes in the ear with glucocorticoid-containing ointments. In this regard, additional studies should be performed to assess and clarify the role of this neuroendocrine system in the pathophysiology of otitis externa and media.
Acknowledgments
We gratefully acknowledge the help and skilful technical assistance of Giesela Lochmann and Ina Hochheim. We are thankful to Prof. M. Kietzmann (Hanover, Germany) and Dr. Y. Endawoke (Leipzig University, Leipzig, Germany) for thoughtful comments and critical reading of the manuscript.
References
Geddes DM 1992 Inhaled corticosteroids: benefits and risks. Thorax 47:404–407
Lipworth BJ 1999 Systemic adverse effects of inhaled corticosteroid therapy: a systematic review and meta-analysis. Arch Intern Med 59:941–955
Patel L, Clayton PE, Addison GM, Price DA, David TJ 1995 Adrenal function following topical steroid treatment in children with atopic dermatitis. Br J Dermatol 132:950–955
Garden JM, Freinkel RK 1986 Systemic absorption of topical steroids. Metabolic effects as an index of mild hypercortisolism. Arch Dermatol 122:1007–1010
Wihl JA, Andersson KE, Johansson SA 1997 Systemic effects of two nasally administered glucocorticosteroids. Allergy 52:620–626
Hanania NA, Chapman KR, Kesten S 1995 Adverse effects of inhaled corticosteroids. Am J Med 98:196–208
Livanou T, Ferriman D, James VH 1967 Recovery of hypothalamo-pituitary-adrenal function after corticosteroid therapy. Lancet 2:856–859
Goldstein DE, Konig P 1983 Effect of inhaled beclomethasone dipropionate on hypothalamic-pituitary-adrenal axis function in children with asthma. Pediatrics 72:60–64
Gustafsson P, Tsanakas J, Gold M, Primhak R, Radford M, Gillies E 1993 Comparison of the efficacy and safety of inhaled fluticasone propionate 200 micrograms/day with inhaled beclomethasone dipropionate 400 micrograms/day in mild and moderate asthma. Arch Dis Child 69:206–211
Brown PH, Blundell G, Greening AP, Crompton GK 1991 Screening for hypothalamo-pituitary-adrenal axis suppression in asthmatics taking high dose inhaled corticosteroids. Respir Med 85:511–516
Keller-Wood ME, Dallman MF 1984 Corticosteroid inhibition of ACTH secretion. Endocr Rev 5:1–24
Clark DJ, Lipworth BJ 1997 Evaluation of corticotropin releasing factor stimulation and basal markers of hypothalamic-pituitary-adrenal axis suppression in asthmatic patients. Chest 112:1248–1252
Levin C, Maibach HI 2002 Topical corticosteroid-induced adrenocortical insufficiency: clinical implications. Am J Clin Dermatol 3:141–147
Thiers BH 1989 Topical steroid therapy of atopic skin diseases. Allergy Proc 10:413–416
Rupprecht M, Hornstein OP, Schluter D, Schafers HJ, Koch HU, Beck G, Rupprecht R 1995 Cortisol, corticotropin, and ?-endorphin responses to corticotropin-releasing hormone in patients with atopic eczema. Psychoneuroendocrinology 20:543–551
Rupprecht M, Rupprecht R, Koch HU, Haack D, Muller OA, Hornstein OP 1991 Multihormonal response to dexamethasone. A study in atopic dermatitis and normal controls. Acta Derm Venereol 71:214–218
Glaze MB, Crawford MA, Nachreiner RF, Casey HW, Nafe LA, Kearney MT 1988 Ophthalmic corticosteroid therapy: systemic effects in the dog. J Am Vet Med Assoc 192:73–75
Solter PF, Hoffmann WE, Chambers MD, Schaeffer DJ, Kuhlenschmidt MS 1994 Hepatic total 3-hydroxy bile acids concentration and enzyme activities in prednisone-treated dogs. Am J Vet Res 55:1086–1092
Rutgers HC, Batt RM, Vaillant C, Riley JE 1995 Subcellular pathologic features of glucocorticoid-induced hepatopathy in dogs. Am J Vet Res 56:898–907
Center SA, Slater MR, Manwarren T, Prymak K 1992 Diagnostic efficacy of serum alkaline phosphatase and -glutamyltransferase in dogs with histologically confirmed hepatobiliary disease: 270 cases (1980–1990). J Am Vet Med Assoc 201:1258–1264
Abraham GE, Bustler JE, Teller RC 1972 Radioimmunoassay for plasma cortisol. Anal Lett 5:757
German Society of Clinical Chemistry 1972 Recommendation for measuring enzyme activity in human serum. Z Klin Chem Klin Biochem 10:91
International Federation of Clinical Chemistry 1991 Method for creatine kinase. Eur J Clin Chem Clin Biochem 29:435–456
Todd GR, Acerini CL, Buck JJ, Murphy NP, Ross-Russell R, Warner JT, McCance DR 2002 Acute adrenal crisis in asthmatics treated with high-dose fluticasone propionate. Eur Respir J 19:1207–1209
Pedersen S, O’Byrne P 1997 A comparison of the efficacy and safety of inhaled corticosteroids in asthma. Allergy 52:1–34
Crowley S, Hindmarsh PC, Holownia P, Honour JW, Brook CG 1991 The use of low doses of ACTH in the investigation of adrenal function in man. J Endocrinol 130:475–479
Hadley SP, Hoffmann WE, Kuhlenschmidt MS, Sanecki RK, Dorner JL 1990 Effect of glucocorticoids on alkaline phosphatase, alanine aminotransferase, and -glutamyltransferase in cultured dog hepatocytes. Enzyme 43:89–98
Hanford WC, Kottel RH, Fishman WH 1981 Alkaline phosphatase protein increases in response to prednisolone in HeLa cells. Biochem J 200:461–464
Bugajski J, Gadek-Michalska A, Bugajski AJ 2001 A single corticosterone pre-treatment inhibits the hypothalamic-pituitary-adrenal responses to adrenergic and cholinergic stimulation. J Physiol Pharmacol 52:313–324
Yehuda R, Boisoneau D, Lowy MT, Giller EL 1995 Dose-response changes in plasma cortisol and lymphocyte glucocorticoid receptors following dexamethasone administration in combat veterans with and without posttraumatic stress disorder. Arch Gen Psychiatry 52:583–593
Yehuda R, Southwick SM, Krystal JH, Bremner D, Charney DS, Mason JW 1993 Enhanced suppression of cortisol following dexamethasone administration in posttraumatic stress disorder. Am J Psychiatry 150:83–86
Wong J, Black P 1992 Acute adrenal insufficiency associated with high dose inhaled steroids. Br Med J 304:1415
Zwaan CM, Odink RJ, Delemarre-van de Waal HA, Dankert-Roelse JE, Bokma JA 1992 Acute adrenal insufficiency after discontinuation of inhaled corticosteroid therapy. Lancet 340:1289–1290
Gallant C, Kenny P 1986 Oral glucocorticoids and their complications. J Am Acad Dermatol 14:161–177
Turpeinen M 1988 Influence of age and severity of dermatitis on the percutaneous absorption of hydrocortisone in children. Br J Dermatol 118:517–522
Turpeinen M 1989 Adrenocortical response to adrenocorticotropic hormone in relation to duration of topical therapy and percutaneous absorption of hydrocortisone in children with dermatitis. Eur J Pediatr 148:729–731
Bartorelli A, Rimondini A 1984 Severe hypertension in childhood due to prolonged skin application of a mineralocorticoid ointment. Hypertension 6:586–588
Turpeinen M, Salo OP, Leisti S 1986 Effect of percutaneous absorption of hydrocortisone on adrenocortical responsiveness in infants with severe skin disease. Br J Dermatol 115:475–484
Wester RC, Maibach HI 1992 Percutaneous absorption in diseased skin. In: Maibach HI, Surber C, eds. Topical corticosteroids. Basel: Karger; 128–141
Feldmann RJ, Maibach HI 1967 Regional variation in percutaneous penetration of 14C cortisol in man. J Invest Dermatol 48:181–183
Maibach HI, Feldmann RJ, Milby TH, Serat WF 1971 Regional variation in percutaneous penetration in man. Arch Environ Health 23:208–211
Matsuda K, Katsunuma T, Iikura Y, Kato H, Saito H, Akasawa A 2000 Adrenocortical function in patients with severe atopic dermatitis. Ann Allergy Asthma Immunol 85:35–39
A Shohat M, Mimouni M, Shuper A, Varsano I 1986 Adrenocortical suppression by topical application of glucocorticosteroids in infants with seborrheic dermatitis. Clin Pediatr 25:209–212
Brus R 1999 Effects of high-dose inhaled corticosteroids on plasma cortisol concentrations in healthy adults. Arch Intern Med 159:1903–1908
Sorkness CA 1998 Establishing a therapeutic index for the inhaled corticosteroids. II. Comparisons of systemic activity and safety among different inhaled corticosteroids. J Allergy Clin Immunol 102:S52–S64
Krasner AS 1999 Glucocorticoid-induced adrenal insufficiency. J Am Med Assoc 282:671–676
Crowley S, Hindmarsh PC, Honour JW, Brook CG 1993 Reproducibility of the cortisol response to stimulation with a low dose of ACTH (1–24): the effect of basal cortisol levels and comparison of low-dose with high-dose secretory dynamics. J Endocrinol 136:167–172
Dickstein G, Shechner C, Nicholson WE, Rosner I, Shen-Orr Z, Adawi F, Lahav M 1991 Adrenocorticotropin stimulation test: effects of basal cortisol level, time of day, and suggested new sensitive low dose test. J Clin Endocrinol Metab 72:773–778
Dickstein G, Spigel D, Arad E, Shechner C 1997 One microgram is the lowest ACTH dose to cause a maximal cortisol response. There is no diurnal variation of cortisol response to submaximal ACTH stimulation. Eur J Endocrinol 137:172–175
Mayenknecht J, Diederich S, Bahr V, Plockinger U, Oelkers W 1998 Comparison of low and high dose corticotropin stimulation tests in patients with pituitary disease. J Clin Endocrinol Metab 83:1558–1562
Weintrob N, Sprecher E, Josefsberg Z, Weininger C, Aurbach-Klipper Y, Lazard D, Karp M, Pertzelan A 1998 Standard and low-dose short adrenocorticotropin test compared with insulin-induced hypoglycemia for assessment of the hypothalamic-pituitary-adrenal axis in children with idiopathic multiple pituitary hormone deficiencies. J Clin Endocrinol Metab 83:88–92
Henzen C, Suter A, Lerch E, Urbinelli R, Schorno XH, Briner VA 2000 Suppression and recovery of adrenal response after short-term, high-dose glucocorticoid treatment. Lancet 355:542–545
Ford LR, Willi SM, Hollis BW, Wright NM 1997 Suppression and recovery of the neonatal hypothalamic-pituitary-adrenal axis after prolonged dexamethasone therapy. J Pediatr 131:722–726
Carella MJ, Srivastava LS, Gossain VV, Rovner DR 1993 Hypothalamic-pituitary-adrenal function one week after a short burst of steroid therapy. J Clin Endocrinol Metab 76:1188–1191
Streck WF, Lockwood DH 1979 Pituitary adrenal recovery following short-term suppression with corticosteroids. Am J Med 66:910–914
Casadevall M, Saperas E, Panes J, Salas A, Anderson DC, Malagelada JR, Pique JM 1999 Mechanisms underlying the anti-inflammatory actions of central corticotropin-releasing factor. Am J Physiol 276:G1016–G1026
Bhagwat AG, Ross RC 1971 Prednisolone-induced hepatic injury. Ultrastructural and biochemical changes in rabbits. Arch Pathol 91:483–492
Sanecki RK, Hoffmann WE, Gelberg HB, Dorner JL 1987 Subcellular location of corticosteroid-induced alkaline phosphatase in canine hepatocytes. Vet Pathol 24:296–301
DeNovo RC, Prasse KW 1983 Comparison of serum biochemical and hepatic functional alterations in dogs treated with corticosteroids and hepatic duct ligation. Am J Vet Res 44:1703–1709
Seetharam S, Sussman NL, Komoda T, Alpers DH 1986 The mechanism of elevated alkaline phosphatase activity after bile duct ligation in the rat. Hepatology 6:374–380
Van Noorden CJ, Frederiks WM, Aronson DC, Marx F, Bosch K, Jonges GN, Vogels IM, James J 1987 Changes in the acinar distribution of some enzymes involved in carbohydrate metabolism in rat liver parenchyma after experimentally induced cholestasis. Virchows Arch B Cell Pathol 52:501–511
Dale DC, Fauci AS, Guerry IV D, Wolff SM 1975 Comparison of agents producing a neutrophilic leukocytosis in man. Hydrocortisone, prednisone, endotoxin, and etiocholanolone. J Clin Invest 56:808–813
Bloemena E, Weinreich S, Schellekens PT 1990 The influence of prednisolone on the recirculation of peripheral blood lymphocytes in vivo. Clin Exp Immunol 80:460–466(Getu Abraham, Jutta Gotts)