The Veterinary Journal 194 (2012) 349–353

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The Veterinary Journal

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Endocrine effects of inhaled budesonide compared with inhaled fluticasonepropionate and oral prednisolone in healthy Beagle dogs

Marika Melamies a,⇑, Outi Vainio a, Thomas Spillmann a, Jouni Junnila b, Minna M. Rajamäki a

a Department of Equine and Small Animal Medicine, Faculty of Veterinary Medicine, Viikintie 49, 00014 University of Helsinki, Finlandb 4Pharma, Metsänneidonkuja 6, 02130 Espoo, Finland

a r t i c l e i n f o

Article history:Accepted 25 April 2012

Keywords:Adrenal suppressionInhaled corticosteroidsDogOral corticosteroidACTH stimulation test

1090-0233/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.tvjl.2012.04.029

⇑ Corresponding author. Tel.: +358 50 357 1878.E-mail address: marika.melamies@helsinki.fi (M. M

a b s t r a c t

Orally administered corticosteroids are commonly used to treat chronic respiratory disease, but adverseeffects suggest that the inhalation route may be safer. To compare the systemic effects of inhaled and oralcorticosteroids, a prospective, randomised, placebo-controlled cross-over study was conducted. Sixhealthy neutered female Beagle dogs were randomly allocated to four treatment groups: (1) budesonideinhalation (200 lg twice daily); (2) fluticasone inhalation (250 lg twice daily); (3) oral prednisolone(1 mg/kg once daily); and (4) placebo inhalation (room air twice daily). Each treatment and wash-outperiod lasted 4 weeks. The endocrine status of each dog was assessed on days 0, 28 and 35 using the adre-nocorticotropic hormone (ACTH) stimulation test. The effects of treatments were assessed using a linearmixed effects model.

After the 4 week treatment period, a significant decrease was observed in the basal serum cortisol levelof the prednisolone group (P < 0.03), and a decrease was also seen in the ACTH-stimulated peak cortisollevels of both the prednisolone and fluticasone groups (P < 0.001), compared with the budesonide groupin which no suppression was detected. The results showed that cortisol production in dogs was stronglysuppressed by oral prednisolone and by inhaled fluticasone.

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Introduction

Chronic inflammatory airway diseases, most notably chronicbronchitis and eosinophilic bronchopneumopathy, are commoncauses of coughing in dogs. Cornerstone therapies have relied onthe oral administration of corticosteroids, although this isfrequently associated with undesirable adverse effects. Inhaledcorticosteroids (ICSs) have replaced oral products as the first-lineanti-inflammatory therapy for asthma in humans, and species-specific modification of inhalation chambers and masks has ledto ICSs being more commonly used in companion animals. How-ever, to our knowledge, there is only a single report describingthe therapeutic use of fluticasone propionate (FP) and beclometh-asone dipropionate in canine chronic inflammatory airway disease(Bexfield et al., 2006). No studies comparing the clinical efficacy ofdifferent ICSs in dogs have been published.

The most common systemic adverse effects related to oralcorticosteroid treatment in dogs are polydipsia and polyuria,polyphagia, weight gain, hair loss and suppression of the hypotha-lamic–pituitary–adrenal (HPA) axis, leading to decreasedendogenous cortisol production. ICSs are also capable of causing

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elamies).

dose-related systemic adverse effects, but to a lesser extent thanorally administered preparations (Ahmet et al., 2011). ICSs aredelivered via inhaled air to the target organ and they reach thesystemic circulation either by direct absorption through therespiratory epithelium or via the gastrointestinal tract after unin-tentional ingestion (Derendorf et al., 1998; Donnelly and Seale,2001). The swallowed proportion of the drug is, however, almostcompletely inactivated by the effective first-pass hepatic metabo-lism (99% for FP and 90% for budesonide (BUD)) (Ryrfeldt et al.,1979; Harding, 1990; Lipworth and Jackson, 1999).

The systemic adverse effects of inhaled FP and oral prednisonehave already been studied in healthy dogs, with less HPA axis sup-pression when using FP compared to prednisone (Cohn et al.,2008). There do not appear to be any reports describing the sys-temic adverse effects of inhaled BUD, whereas significant suppres-sion of the HPA axis has been detected with oral BUD at a dose of3 mg/m2 for 30 days in dogs with inflammatory bowel disease(Tumulty et al., 2004). In humans, the long-term use of FP has beenfound to cause dose-related adrenal suppression more often thanBUD at equal dosages (Clark et al., 1996; Clark and Lipworth,1997; Lipworth, 1999; Kaliner, 2006). The objective of the currentstudy was to compare the endocrine effects of inhaled BUD,inhaled FP and oral prednisolone in healthy Beagle dogs atclinically relevant doses.

350 M. Melamies et al. / The Veterinary Journal 194 (2012) 349–353

Materials and methods

Dogs and verification of health

Six healthy neutered female laboratory Beagle dogs were enrolled in the study.At the start of the study, the dogs were 2.5 years old and their mean bodyweight(BW) was 17 kg (range: 11–19 kg). The health status of each dog was assessed bythorough physical examination, haematology and serum biochemistry panels (ala-nine aminotransferase, albumin, alkaline phosphatase (ALP), blood urea nitrogen,calcium, creatinine, glucose, potassium, protein, sodium and total bilirubin) as wellas by urinalysis (bacterial culture, protein-to-creatinine ratio, reagent strip (Multis-tix 10 SG, Siemens Healthcare Diagnostics), sediment and specific gravity). Latero-lateral and ventro-dorsal thoracic radiographs were taken. Additionally, faecal flo-tation and sedimentation tests were performed.

The study protocol was approved by the Committee of Experimental Animals ofWestern Finland.

Study design

This study was an open label, prospective, randomised, placebo-controlled,cross-over design. All dogs received all of the following four treatment protocolsin random order: (1) BUD inhalation (Pulmicort HFA, AstraZeneca) 200 lg twicedaily; (2) FP inhalation (Flixotide Evohaler, GlaxoSmithKline) 250 lg twice daily;(3) prednisolone orally (Prednisolon, Leiras) 1 mg/kg once daily; (4) placebo inhala-tion twice daily. Inhaled medications were administered through an aerosol cham-ber (AeroDawg, Trudell Medical International) attached to an anaesthetic mask(face mask with rubber diaphragm, Kruuse). Before each medication, the me-tered-dose inhaler (MDI) was shaken and attached tightly to the aerosol chamber.An anaesthetic mask was then fitted to cover the dog’s nose and mouth, the med-ication was sprayed into the chamber and eight breaths were counted. The placebogroup breathed room air through the chamber and mask.

All dogs had their own masks, aerosol chambers and MDIs during the 4-weekstudy period, and the inhalation devices were carefully washed after each treat-ment period. The last medication was given 24 h before blood sampling. Blood sam-ples (serum and K2EDTA samples, 6 mL each) were taken at the same time of theday (between 09:00 and 11:00 h) in the same environment where the dogs werehoused. To avoid the possible effect of circadian rhythm on cortisol excretion(Derendorf, 1997), as well as stress-induced elevation in cortisol levels, we stand-ardised the environmental factors and sample-taking protocol as far as possible,by constant sampling time and place and the same two persons collecting thesamples.

Each treatment and each washout period lasted 4 weeks. The health status wasassessed on days 0, 14, 28 and 35 by physical examination, haematology, serumbiochemistry and urinalysis, and additionally by ACTH stimulation test on days 0,28 and 35. All blood samples were taken from the cephalic vein and urine samplesby cystocentesis using ultrasonographic guidance. All haematological, serum bio-chemical and urinalysis samples were stored at room temperature and analysedwithin 2 h.

Table 1Serum biochemistry and urinalysis results (mean ± SD) in the four treatment groups(n = 6 per group).

Day 0 Day 14 Day 28 Day 35

PlaceboALP (U/L) 110 ± 43 107 ± 48 114 ± 46 109 ± 46Prot (g/L) 57 ± 3 55 ± 4.7 57 ± 5.7 55 ± 1.8U pr/cr 0.08 ± 0.02 0.11 ± 0.02 0.11 ± 0.02 0.10 ± 0.02

PrednisoloneALP (U/L) 105 ± 45 261 ± 91* 246 ± 101* 119 ± 43Prot (g/L) 58 ± 2.1 65 ± 3.4* 65 ± 2.3* 57 ± 3.0U pr/cr 0.08 ± 0.02 0.15 ± 0.04 0.47 ± 0.33* 0.15 ± 0.03

FPALP (U/L) 115 ± 47 93 ± 19 99 ± 22 103 ± 34

ACTH stimulation test

The adrenocorticotropic hormone (ACTH) stimulation test is the most reliableway to diagnose adrenal dysfunction in dogs (Herrtage, 2005; Lathan et al.,2008), and in our study the adrenocorticotropic status was assessed with thelow-dose ACTH stimulation test (Kerl et al., 1999; Frank et al., 2004) using a syn-thetic analogue of ACTH (Synacthen, Alliance; 5 lg/kg IV) administered via a plasticcatheter (Optiva 20 G, Smiths Medical). The catheter was immediately flushed with5 mL of physiological saline and a second blood sample was taken 60 min later. Ser-um samples for cortisol assays were frozen at �80 �C until analysis.

Cortisol assays were performed by a separate technician who was blinded to thetreatments. Samples were analysed in duplicate with a commercial radioimmuno-assay (RIA) kit validated for canine use (Coat-A-Count Cortisol, Siemens HealthcareDiagnostics). The intra-assay coefficient of variation (CV) for serum cortisol deter-mination was 5.6% at the mean concentration level of 42.6 nmol/L and 6.4% at thelevel of 280.2 nmol/L (both CVs were calculated from 10 duplicate determinations).The samples were analysed in one series, and no inter-assay CV% was determined.The detection limit of the serum cortisol assay was 6.9 nmol/L.

Prot (g/L) 58 ± 2.3 58 ± 2.3 58 ± 2.7 55 ± 3.1U pr/cr 0.10 ± 0.04 0.10 ± 0.03 0.16 ± 0.12 0.10 ± 0.02

BUDALP (U/L) 100 ± 25 107 ± 28 117 ± 38 107 ± 42Prot (g/L) 56 ± 3.5 57 ± 2.8 58 ± 1.6 55 ± 2.9U pr/cr 0.09 ± 0.03 0.12 ± 0.03 0.11 ± 0.02 0.10 ± 0.02

FP, fluticasone propionate; BUD, budesonide; ALP alkaline phosphatase (referencerange, 33–215 U/L); prot, protein (reference range, 58–77 g/L); U pr/cr, urineprotein-to-creatinine ratio (reference range, <0.5).* Significant difference compared to placebo, P < 0.001.

Statistical analysis

The effect of treatments on the continuous response variables was assessedusing a linear mixed effects model, where treatment, period, day of period, andinteraction terms between treatment and period, and treatment and day of periodwere used as fixed effects and dog as a random effect. With urine protein-to-creat-inine ratio and urine-specific gravity, the dog-related variance component was veryclose to zero. To get the model to converge, linear analysis was performed, exclud-ing the very small random effect of individual dogs.

Carry-over effect was excluded from all models, as the day 0 ACTH-stimulatedcortisol results proved that the 4-week wash-out was sufficiently long to normalisethe HPA axis function before the next period. Data were expressed as means ± SDand range. A P-value <0.05 was considered significant. All statistical analyses wereperformed at 4Pharma using SAS System for Windows, version 9.2 (SAS Institute).

Results

Dogs and verification of health

All of the dogs showed normal physical examination results. Nosignificant changes were detected in thoracic radiographs, and allfaecal tests to detect parasites were negative. Results on days 0,14, 28 and 35 of haematological analyses, including differential cellcounts, were within the reference range in all dogs with minorexceptions. Serum biochemistry panels and urinalysis results werewithin the reference range, except changes detected in serum ALPand protein values and urine protein-to-creatinine ratio in theprednisolone group on days 14 and 28 (Table 1).

ACTH stimulation test

Test results are presented in Table 2. On day 0, no significantdifferences were seen in baseline or ACTH-stimulated peak cortisollevels when treatment groups were compared with the placebogroup. Furthermore, no significant changes were noted in the pla-cebo or BUD groups in basal or peak cortisol levels after 4 weeks oftreatment relative to day 0 (P > 0.05 in each case). However, on day28, prednisolone had significantly suppressed the basal cortisol le-vel compared with the placebo (P = 0.016) or BUD (P = 0.029) group(Fig. 1). Additionally, the peak cortisol level after the ACTH stimu-lation test was significantly lower in the prednisolone and FPgroups than in the placebo or BUD group (Fig. 2; P < 0.001 in eachcase).

One week after discontinuation of treatment (day 35) in the FPgroup, significant elevation of the basal cortisol level was foundcompared with day 28 (P < 0.001). The level was also higher thanthat in the placebo group (Fig. 1; P = 0.035), but no significantchange was observed compared with the basal cortisol level inthe FP group on day 0 (P = 0.099). On day 35, no significant differ-ences between the treatment groups were detected in ACTH-stimulated peak cortisol concentrations (Fig. 2).

Table 2ACTH stimulation test results (mean ± SD (range)) in the four treatment groups (n = 6per group).

Cortisol(nmol/L)

Day 0 Day 28 Day 35

PlaceboBasal 36 ± 10 (20–51) 36 ± 10 (26–51) 37 ± 14 (24–64)Peak 288 ± 40 (237–335) 298 ± 25 (266–333) 297 ± 38 (251–346)

PrednisoloneBasal 39 ± 18 (23–71) 14 ± 4 (8–19) 51 ± 25 (31–93)Peak 333 ± 22 (305–372) 111 ± 15 (91–126)* 266 ± 20 (249–301)

FPBasal 42 ± 11 (26–60) 20 ± 10 (11–35) 55 ± 7 (45–65)Peak 314 ± 59 (246–417) 156 ± 67 (72–254)* 267 ± 18 (242–294)

BUDBasal 46 ± 17 (22–70) 35 ± 14 (16–50) 41 ± 15 (19–59)Peak 288 ± 38 (253–361) 274 ± 19 (249–292) 292 ± 35 (243–332)

FP, fluticasone propionate; BUD, budesonide.* Significant difference compared to placebo or BUD, P < 0.001.

Fig. 1. Basal cortisol concentrations (nmol/L) in four treatment groups (n = 6 ineach) on days 0, 28 and 35. Normal values range from 10 to 100 nmol/L (indicatedas light grey bar beside the vertical axis). The line within the box is the medianvalue and the upper and lower edges of the box represent the 75th and 25thpercentiles, respectively. Outliers are marked as open circles (s). Smallest andlargest values (not outliers) are shown by whiskers. A statistically significantdifference is designated with an asterisk. On day 28, the cortisol levels in theprednisolone group were significantly suppressed relative to the placebo (P = 0.016)or BUD (P = 0.029) groups. On day 35, the cortisol level in the FP group wassignificantly elevated relative to the placebo group (P = 0.035).

Fig. 2. ACTH stimulated peak cortisol concentrations (nmol/L) in four treatmentgroups (n = 6 in each) on days 0, 28 and 35. Normal results range from 200 to600 nmol/L (indicated as light grey bar beside the vertical axis). The line within thebox is the median value and the upper and lower edges of the box represent the75th and 25th percentiles, respectively. Outliers are marked as open circles (s).Smallest and largest values (not outliers) are shown by whiskers. A statisticallysignificant difference is designated with an asterisk. On day 28, the peak cortisollevel was significantly lower in the prednisolone and FP groups than in the placeboor BUD group (P < 0.001 in each case).

M. Melamies et al. / The Veterinary Journal 194 (2012) 349–353 351

Discussion

Both BUD and FP are potent corticosteroids used for the treat-ment of chronic respiratory diseases as inhaled formulas, althoughfew studies have been undertaken to investigate efficacy and ad-verse effects of these compounds in small animals, particularlydogs. Bexfield et al. (2006) described the use of FP and beclometh-asone dipropionate, compared with oral or parenteral corticoste-roid, in canine chronic inflammatory airway disease. Treatmentwith ICSs resulted in reduction of symptoms and side-effects,although both of these responses were owner-assessed. Inexperimental canine asthma models, inhaled BUD has been shownto significantly reduce airway eosinophilia, airway hyper-responsiveness and production of allergen-induced bone marrow

progenitors (Woolley et al., 1994a, 1994b; Inman et al., 1997).However, neither study investigated efficacy of BUD in naturally-occurring canine respiratory disease and did not consider other po-tential corticosteroids for comparison.

The use of FP has been studied in cats experimentally inducedallergic airway inflammation (Kirschvink et al., 2006; Cohn et al.,2010; Leemans et al., 2011). Cohn et al. (2010) reported that a dos-age of 44 lg twice daily for 3 weeks reduced airway eosinophilia aseffectively as higher dosages (110 or 220 lg twice daily), withoutsignificant suppression of the HPA axis at any dose. Leemanset al. (2011) treated cats only for 4 days with oral prednisoloneor FP and concluded that prednisolone was more effective. A singlestudy has investigated the effects of inhaled BUD towards allergen-specific IgE testing, but did not consider therapeutic impact or ad-verse effects (Chang et al., 2011). Further studies are required toinvestigate the efficacy of inhaled corticosteroids in cats and dogsto provide a more objective approach to treatment and to deter-mine if higher doses are required in the acute stages of airwaydisease.

Anti-inflammatory therapy with oral corticosteroids has beenthe standard therapy for chronic inflammatory airway diseases indogs for decades (McKiernan, 2000). Thus, for comparison, weplaced one treatment group on conventional medication (prednis-olone 1 mg/kg once daily) (McKiernan, 2000). The pharmacologicalproperties of orally and IV administrated BUD in dogs have beenreported (Ryrfeldt et al., 1979), but no studies describing the phar-macological properties of FP in dogs exist. The pharmacokineticcharacteristics of BUD, including elimination half-life, total clear-ance and volume of distribution, do not markedly differ betweendogs and humans (Ryrfeldt et al., 1979; Donnelly and Seale,2001). The dosages of BUD and also FP used in our study weretherefore determined according to the existing treatment recom-mendations for asthmatic children and were estimated to be clin-ically relevant (Campbell et al., 1998; Herjavecz et al., 1999; Busseet al., 2008; Levy et al., 2009; Schramm and Carroll, 2009).

After both 2 and 4 weeks of prednisolone treatment, we found asignificant increase in serum ALP and protein concentrations and

352 M. Melamies et al. / The Veterinary Journal 194 (2012) 349–353

after 4 weeks also in the urine protein-to-creatinine ratio, com-pared with the placebo group. An elevated ALP value is commonlyassociated with oral corticosteroid treatment and is mainly due tothe formation of the corticosteroid-induced ALP isoenzyme (Solteret al., 1994). Moore et al. (1992) previously reported elevated pro-tein concentrations in dogs after 5 weeks’ treatment with nearlyidentical anti-inflammatory doses of prednisone. In our dogs, theserum protein concentrations tended to increase whilst remainingwithin the reference range, thus rendering the clinical importanceof this finding as unremarkable. Mild elevations in the canine urineprotein-to-creatinine ratio reported with immunosuppressivedoses of corticosteroids for 42 days were caused by mesangial cellproliferation and glomerular changes in the kidneys (Waters et al.,1997). Although the dose of prednisolone was lower in our study,the detected elevations in the urine protein-to-creatinine ratiomight be attributed to similar changes. Five weeks of anti-inflammatory glucocorticoid treatment in dogs has previouslybeen associated with decreased numbers of eosinophils and lym-phocytes (Moore et al., 1992). In the current study, however,changes in differential cell counts were not detected, which mightbe due to the shorter duration of medication.

The most common systemic adverse effect in dogs after pro-longed corticosteroid administration is HPA axis suppression.Accordingly, marked suppression was observed after 4 weeks ofFP or prednisolone therapy in our study. This finding was in agree-ment with earlier findings in FP- and prednisone-treated dogs, andalso with human studies (Clark et al., 1996; Clark and Lipworth,1997; Kaliner, 2006; Cohn et al., 2008). However, unlike a previ-ously reported study (Cohn et al., 2008), our work alsodemonstrated that FP but not BUD significantly suppressed theHPA axis relative to placebo. One week after discontinuation oftreatment, we observed a significant increase in the basal cortisollevel in the FP group compared with the placebo group. However,the values did not exceed the reference range, and thus, the clinicalimportance of this finding is negligible.

There was a minor systemic bioactivity of BUD relative to FP atclinically relevant doses which was probably due to differences inthe pharmacological properties of these drugs. Tapering the dosageof any corticosteroid reduces systemic effects, but the equivalentdosages for equal endocrine effects or clinical responses are un-known since clinical trials comparing the efficacies of FP, BUDand prednisolone are lacking. Numerous studies of ICSs in humanshave established that the pharmacokinetics and pharmacodynam-ics of these drugs are very complex (Clark and Lipworth, 1997; Lip-worth, 1999; Adams et al., 2007). It has been suggested that FP maybe more efficient than BUD because of superior retention in thelung tissue due to its higher lipophilicity and its higher glucocorti-coid receptor binding affinity resulting in a longer duration of localanti-inflammatory action (Hogger and Rohdewald, 1994; Johnson,1996). However, BUD dissolves better in bronchial fluid due to itslower lipophilicity (Hogger et al., 1993) and is also able to formfatty acid esters in the cells of the respiratory tract, unlike FP. Theseester conjugates provide a local depot of latent, gradually regener-able, active BUD, thereby prolonging the duration of the anti-inflammatory effect in the lungs (Tunek et al., 1997; Nave et al.,2007). This may explain the good results in treating mild to mod-erate human asthma with even once daily administration of BUD(Scott et al., 2001; Selroos et al., 2004).

In the present study, dogs had a 4-week washout period be-tween treatments. Normal basal and ACTH-stimulated peak corti-sol levels were seen in all groups on days 0 and 35. Thissuggested that even after 4 weeks’ treatment with anti-inflamma-tory doses of prednisolone, the HPA axis’ recovery of suppression iscomplete within 1 week and any carry-over effect of the previoustreatment period was excluded. The HPA axis recovery was de-tected 1 week after discontinuation of medication whereas, based

on biweekly testing after 5 weeks of daily doses of prednisone(equal to ours), Moore and Hoenig (1992) concluded that recoverywas complete 2 weeks after medication ceased. Behaviouralchanges, including water consumption, urine excretion andappetite, would have been interesting additional variables to fol-low during the treatment periods since a previous study detecteda significant increase in water consumption in prednisone-treateddogs (Cohn et al., 2008).

Conclusions

The HPA axis was strongly suppressed after 4 weeks treatmentwith FP or prednisolone, but not BUD, at clinically relevant doses inhealthy dogs. These findings suggested that inhaled BUD may be asafer choice in the treatment of chronic respiratory diseases indogs sensitive to the systemic adverse effects of corticosteroidtreatment. However, further studies are required to assess thetherapeutic doses of ICSs in dogs and to compare the systemic ad-verse effects of dosages with equivalent clinical efficacy.

Conflict of interest statement

None of the authors of this paper has a financial or personalrelationship with other people or organisations that could inappro-priately influence or bias the content of the paper.

Acknowledgements

This study was carried out at the Department of Equine andSmall Animal Medicine, Faculty of Veterinary Medicine, Universityof Helsinki, and supported by a grant from the Helvi KnuuttilaFoundation. Anu Lappalainen, DVM, is thanked for assessing theradiographs. The contributions of Satu Sankari and technician Kai-sa Aaltonen to cortisol analyses are greatly appreciated.

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