Use of a novel one-nostril mask-spacer device to evaluate airway hyperresponsiveness (AHR) in horses after chronic administration of albuterol

Abstract

Inflammatory airway disease (IAD) is very common in stabled horses. Short-acting beta agonist (SABA) drugs are often used to relieve clinical signs, although long-term exposure to these drugs may result in rebound bronchoconstriction. The purpose of this study was twofold: i) to describe the deposition of radiolabeled drugs using a novel one-nostril design mask-spacer combination with a breath-activated inhaler (BAI), and ii) to determine whether treatment for 10 d with inhaled albuterol using this device would impair the ability of albuterol to prevent bronchospasm during a histamine challenge test. The percentage of radio-aerosol deposited in the total lung was 12.39% ± 5.05%. All study horses demonstrated airway hyperresponsiveness (AHR) before enrollment in the study [mean provocative concentration eliciting 35% increase in delta flow (PC35) < 6 mg/mL histamine]. There was no significant difference in airway hyperresponsiveness to post-albuterol histamine challenge before or after treatment with albuterol. A 10-d treatment with placebo, however, caused a significant increase in airway hyperresponsiveness in all horses (P < 0.001). The results of this study show that the novel mask-spacer device was effective in delivering radiolabeled aerosolized drug to the lung and that delivery of a SABA for 10 d using this device did not result in increased airway hyperresponsiveness.

Introduction

Inflammatory airway disease (IAD) is a common equine disease of domestication, with an estimated 30% to 50% of stabled horses being affected (1,2). Horses with IAD frequently demonstrate airway hyperresponsiveness (AHR) to stimuli such as inhaled histamine. Short-acting beta agonist (SABA) drugs such as albuterol are often used to relieve clinical signs associated with AHR, including cough and poor performance, by eliciting bronchodilation.

Although albuterol use is common, the safety of long-term exposure to SABA drugs is questionable, as there is evidence in rodents and humans that long-term use may result in decreased efficacy due to receptor desensitization, which may in turn exacerbate AHR (3–5). Oral administration of clenbuterol, a beta-2 adrenergic drug, over 21 d has recently been shown to result in tachyphylaxis, which causes increased airway reactivity in horses with IAD (6). The many warnings about the use of SABA drugs for maintenance therapy are often ignored, however, both in human and veterinary medicine. Recent in-vitro work in horses has indicated that, as in humans, the (S)-enantiomer found in albuterol potentiates the contractile effect of spasmogens such as histamine on isolated equine bronchial tissue (7).

Inhaled delivery of aerosolized drugs is preferable because it results in fewer side effects attributable to systemic absorption of the drug. Questions remain, however, about the efficacy of delivery of aerosolized drugs to horses. The use of a valved holding chamber (AeroChamber; Trudell Medical) and a metered dose inhaler (fenoterol) to elicit bronchodilation in horses with heaves was first described by Tesarowski et al (8). The delivery device (Equine Aeromask; Trudell Medical) is commonly used, but its large volume decreases the effective delivered dose and it is bulky and expensive. Other single-nostril devices have emerged, including the EquineHaler (Equine Healthcare) and the AeroHippus (Trudell Medical). Radiolabeling studies as well as bronchodilator trials have shown varying levels of efficacy among these devices (9–11). The purpose of the study reported here was twofold: i) to describe the deposition of radiolabeled inhaled drug using a novel one-nostril design mask-spacer combination with a breath-activated inhaler (BAI) and ii) to determine whether treatment for 10 d with inhaled racemic albuterol using the mask-spacer would impair the ability of albuterol to prevent bronchospasm during a histamine challenge test.

Materials and methods

Animals used in phase I

This study was carried out in 2 phases. In phase I, the radioaerosol study, 5 healthy mares (4 Standardbreds, 1 Thoroughbred, 7- to 20-years-old) were used. All testing was carried out under the approval and auspices of the Institutional Animal Care and Use Committee (IACUC number G422-01) of Tufts University and the Radiation Safety and Health Hazards Committee in accordance with state and federal regulations for the use of radiopharmaceuticals.

Animals used in phase II

In phase II, the albuterol study, 24 horses underwent histamine bronchoprovocation. Eight horses (2 Warmblood, 4 Thoroughbred, 2 cross breed, 8- to 20-years-old) that demonstrated airway hyperresponsiveness [mean provocative concentration eliciting 35% increase in delta flow (PC35) < 6 mg/mL histamine, mean 3.86 ± 0.67 mg/mL histamine] (Sigma-Aldrich, St. Louis, Missouri, USA) were chosen to participate. These horses were housed in box stalls with 6 to 10 h of turnout daily, bedded on shavings, and fed hay and grain. All testing for this phase of the study was carried out under the approval and auspices of the IACUC of Tufts University (G524-03).

Experimental design — phase I

A total of 7 measurements was made using 5 horses. The delivery device consisted of a small mask (total volume approximately 300 mL), which also served as a spacer device, fitting snugly over a single nostril. A breath-activated inhaler (BAI) was attached to the mask for delivery of radioaerosol (Figure 1). No bronchodilator or anti-inflammatory drugs were given during the study period.

Figure 1.

The mask-spacer device being used to deliver radioaerosol to a representative horse.

Experimental design — phase II

Before any treatment, all horses were given 10 puffs (900 μg) of albuterol using the new mask and a commercial BAI containing albuterol with hydrofluoroalkane (HFA)-propellant (Ivax, Miami, Florida, USA) (90 μg/puff) and airway reactivity to histamine was measured 15 min later to establish the ability of albuterol to protect the airways from bronchospasm. After a 1-week washout period, horses were randomly assigned to receive aerosolized albuterol/HFA-propellant (albuterol, A) or aerosolized vehicle with HFA-propellant as a control (placebo, P), (10 puffs, twice daily for 10 d). Horses were given the opposite treatment after a washout period of 7 d. Airway reactivity was again measured before each new treatment period 15 min after a single dose of aerosolized albuterol (900 μg).

Radioaerosol labeling procedure

All labeling was carried out at the Massachusetts General Hospital (MGH) Nuclear Pharmacy under the supervision of the MGH Radiation Safety Board. Radiolabel [99mTechnetium (Tc) diethylene triamine pentaacetic acid (DPTA)] and beclomethasone diproprionate (BDP)/HFA-propellant were added to an empty commercial BAI canister (Ivax) and crimped within a glove bag at MGH. The canister was then sealed with a metering valve using a custom collet and crimper (JG Machinery, Sanford, North Carolina, USA) and placed in an ultrasonic bath for 5 min for warming and pressurization before being transported to Cummings School of Veterinary Medicine at Tufts University (CSVM).

Delivery of radioaerosol

A background count was done in the scan room before measurements were made and horses were then sedated with detomidine hydrochloride [0.005 to 0.01 mg/kg bodyweight (BW) IV] (Pfizer, New York, New York, USA) and walked to the scan room. After 5 min, a total of 12 puffs of medication was given with the new mask/spacer. The following regions were scanned for 60 s each: left cranial lung, left caudal lung, right caudal lung, right cranial lung, nose, larynx/pharynx and trachea, and the mask itself. Background counts were repeated after the final scan and the pre-scan and post-scan background counts were averaged and used for correction purposes. Primary radioactive counts were corrected for background radiation and radioactive decay (12,13).

Imaging equipment

Scintigraphy was carried out using a large field-of-view planar scintillation camera (IS2 Research, Ottawa, Ontario) that was equipped with a high-resolution collimator. All static images were acquired by use of a 512 × 512-pixel matrix and stored on a hard drive until analysis. Post-acquisition nuclear medicine software was used for image display and analysis (Mirage processing application; Segami, Columbia, Maryland, USA).

Airway deposition analysis

Canister radioactivity was measured using a dose calibrator before and after delivery to determine delivered dose. Regions of interest were drawn around the left cranial, left caudal, right caudal, and right cranial lung, as well as the larynx/trachea area, the nose, and the mask. Background and decay-corrected counts were summed and the percent of total lung deposition was calculated in the following manner:

$$\begin{array}{l}\text{Total\hspace{0.17em}}\%\hspace{0.17em}\text{lung\hspace{0.17em}deposition}=100\times (\text{total\hspace{0.17em}counts\hspace{0.17em}from\hspace{0.17em}all}\hspace{0.17em}\text{lung}\\ \text{scans})/(\text{total\hspace{0.17em}lung}\hspace{0.17em}\text{scans}+\text{larynx}-\text{pharynx}+\text{trachea}+\text{nose}+\text{mask})\end{array}$$

This percentage was taken to represent the entire dose delivered in millicuries from the canister.

Attenuation correction

As a single scan detects approximately 1/4 of the total radiation deposited in the lung, both sides of the thorax were scanned to account for approximately 1/2 the radiation in the thorax and a correction factor of 2 was used (14).

Correction for clearance of DTPA

Reported half-life (t 1/2) for DTPA in the equine lung ranges from 41 to 71 min; we used the middle value of 55.7 min (15–17). Only values for lung were corrected for DTPA clearance.

Airway reactivity testing

Pulmonary function testing was carried out under light sedation (0.4 mg/kg BW xylazine hydrochloride IV) (Pfizer) using flowmetric plethysmography and histamine bronchoprovocation, as described in a previous study (18). Delta flow values were averaged for each 3-min measurement period. Doubling concentrations of histamine (histamine diphosphate monohydrate; Sigma-Aldrich, St. Louis, Missouri, USA) were delivered by nebulizer (LC Plus; PARI Respiratory Equipment, Midlothian, Virginia, USA) and compressor (Compare; Omron Healthcare, Lake Forest, Illinois, USA) from 4 mg/mL to a maximum of 32 mg/mL (2 min each) until delta flow increased by 35% (PC35). Horses were tested before being given the initial dose of albuterol on the first day of each treatment period and on day 10. Values for PC35 were then calculated by linear interpolation for each horse and timepoint using Microsoft Office Excel (Microsoft, Redmond, Washington, USA).

Statistical analysis

In phase I, percent deposition of radioaerosol was calculated and expressed as mean ± standard deviation. In phase II, data were analyzed using a mixed model to control for the multiple measurements made on each horse as part of the crossover study design. As this was a crossover design, all 8 horses were tested in both periods 1 and 2. Half the horses received first albuterol, then placebo (AP sequence) and the other half received first placebo, then albuterol (PA sequence). The model included fixed effects for the treatment, the period, and the sequence and a random effect for horse nested within the sequence. We therefore tested the hypothesis that there would be no difference in the ability of albuterol to protect the airways from bronchoconstriction induced by histamine after treatment with albuterol or placebo over a 10-day period regardless of sequence (no crossover effect). As the variances between groups were high, a nonparametric signed rank test was also done.

Results

Deposition of radioaerosol in the lung

Mean activity released from the canister after a total of 12 actuations (1 dose) was 8.56 ± 3.01 mCi. After correction for radioactive decay for all sites, as well as attenuation and DTPA clearance for the lung, percentage of deposition was highest for the nose (65.32% ± 3.89%), followed by the mask (20.84% ± 5.42%), total lung (12.39% ± 5.05%), left cranial lung (3.39% ± 1.41%), right cranial lung (3.31% ± 1.51%), right caudal lung (3.08% ± 1.42%), left caudal lung (2.61% ± 1.68%), and larynx/trachea (1.45% ± 1.01%). The deposition of radioaerosols in the lungs only is shown in Figure 2. Four out of 7 attempts resulted in a delivery of over 11% to the lower lung, and 3 out of 7 attempts resulted in a delivery of 2% to 10.5% to the lower lung, with a range of 2% to 38%, and a mean of 12.38% ± 5.04%. Deposition appeared to be diffuse rather than concentrated in the central airways and appeared most dense in the lung periphery along the costophrenic angle (Figure 3).

Figure 2.

After correction for radioactive decay for all sites, as well as attenuation and DTPA clearance for the lung, percentage of deposition was highest for the nose (65.32% ± 3.89%), followed by the mask (20.84% ± 5.42%), total lung (12.39% ± 5.05%), left cranial lung (3.39% ± 1.41%), right cranial lung (3.31% ± 1.51%), right caudal lung (3.08% ± 1.42%), left caudal lung (2.61% ± 1.68%), and larynx/trachea (1.45% ± 1.01%). Only lung counts are shown here.

Figure 3.

Deposition of radioaerosol appears to be diffuse rather than concentrated in central airways and appeared most dense in the lung periphery.

Airway hyperresponsiveness testing

All horses demonstrated airway hyperresponsiveness (AHR) before enrollment in the study (mean PC35 = 3.86 mg/mL, range = 2.85 to 4.53 mg/mL) (Figure 4). All horses experienced a significant decrease in response to histamine following a single dose (10 puffs, 900 μg) of albuterol before entering either the AP (albuterol then placebo) sequence or PA (placebo then albuterol) sequence treatment phase of the study, with a mean increase in PC35 of 4.53 mg/mL (range 2.1 to 6.7 mg/mL, P = 0.017) (Figure 4). There was no significant difference between AHR to post-albuterol histamine challenge before or after 10-day treatment with albuterol (Figure 5). In contrast, after 10-day treatment with placebo, there was a significant increase in AHR in all horses (mean decrease in PC35 of 4.3 mg/mL, range 1 to 8.1 mg/mL of histamine) despite treatment with inhaled albuterol (P < 0.01) (Figure 6). There was no effect of period, sequence, or random effect for horse nested within the sequence.

Figure 4.

Histamine bronchoprovocation was carried out on all horses before the study and they all demonstrated airway hyperresponsiveness (AHR) (mean PC35 = 3.86 mg/mL, range = 2.85 to 4.53 mg/mL). Histamine bronchoprovocation was repeated after administration of a single dose of albuterol/HFA propellant (10 actuations, 900 μg), using the new mask and breath-activated inhaler (BAI). All horses experienced a significant decrease in airway reactivity, with a resultant mean PC35 of 8.39 ± 1.69 mg/mL, range 5.65 to 10.00 mg/mL, which resulted in an increase of 4.53 mg/mL, range 2.1 to 6.7 mg/mL. * P = 0.017

Figure 5.

Horses were given a single dose of albuterol/HFA propellant (900 μg) and histamine bronchoprovocation was carried out. Horses were then treated with albuterol/HFA propellant (900 μg twice daily) for 10 d. At the end of the treatment period, a single dose of albuterol/ HFA propellant was given again and histamine bronchoprovocation was also repeated. There was no significant difference between airway hyperresponsiveness to post-albuterol histamine challenge before or after treatment with albuterol (P < 0.001), although 4 horses did experience an increase in airway hyperresponsiveness (decrease in PC35).

Figure 6.

Horses were given a single dose of albuterol/HFA propellant (900 μg) and histamine bronchoprovocation was carried out. Horses were then treated with vehicle/HFA propellant (900 μg twice daily) for 10 d. At the end of the treatment period, a single dose of albuterol/HFA propellant was given again and histamine bronchoprovocation was also repeated. The average PC35 before treatment was 8.96 ± 3.13 mg/mL histamine and after treatment it was 4.84 ± 1.44 mg/mL histamine. There was a significant increase in airway hyperresponsiveness (decrease in PC35) in all horses (mean decrease in PC35 of 4.3 mg/mL, range 1 to 8.1 mg/mL of histamine) after a single dose (900 μg) of aerosolized albuterol. * P < 0.01

Discussion

The results of the first phase of this study show that the one-nostril mask-spacer device used with a breath-activated inhaler was effective in delivering radiolabeled aerosolized drug to the lung of horses. Four out of 7 attempts resulted in a delivery of over 11% to the lower lung, with a range of 2% to 38% and a mean of 12.38% ± 5.04%. This compares well with other devices that are available for delivery of inhaled drugs to horses.

The configuration of the delivery device, type of propellant, and the patient’s breathing pattern all contribute to the total amount of drug delivered (19,20). Both small (~ 150 mL) and large (~ 750 mL) spacers have been shown to deposit significantly more of the delivered dose of inhaled drug to the lung than a jet nebulizer or metered-dose inhaler (MDI) alone in humans (21). The combination of spacer/HFA-propellant results in approximately 53% delivery to the lung in humans, whereas spacer/CFC-propellant results in a much lower (13%) delivery, albeit for drugs in solution such as beclomethasone, not those in suspension, such as fluticasone (20,22). Delivery of drug to the equine lung, even while using an HFA propellant, is even smaller. Several radiolabeling studies have been carried out in horses. The Aeromask (Trudell Medical) delivered approximately 6% of the chlorofluorocarbon (CFC)-propellant drug and 14% of HFA-propellant drug to the lower respiratory tract (10). A large, single-nostril spacer/mask device (EquineHaler; Equine Healthcare) delivered approximately 8.2% ± 5.2% of HFA-propellant drug to the lower respiratory tract (9). A third device, using a small, one-nostril mask and separate spacer (AeroHippus; Trudell Medical, London, Ontario) delivered 18.2% ± 9.3% of HFA-propellant drug to the lower airways (23). The device used in this study, a small, onenostril mask that also served as a spacer, compared well, delivering an average of approximately 12% of drug to the lung.

Despite correction for attenuation, physical decay, and DTPA clearance, it is likely that our methods failed to fully account for the deposited radioactive aerosol in the chest. One scan picks up gamma radiation in a conical region (pointed towards the horse). The greatest activity arises from radioaerosol deposited peripherally in the ipsilateral lung, where there is the least attenuation. We corrected our lung deposition data accordingly, but this may still be an underestimation since much of the dose that settles in the large (central) airways may be too deep to detect and the cranial lung is largely behind the shoulders and triceps, which further attenuates the signal.

The question of attenuation correction is important, particularly because gamma scintigraphic images are inherently 2-dimensional (24). Various methods of attenuation correction have been attempted in humans and smaller animals (25). Perhaps the most useful is the study done by Marlin et al (26), which used a point source in a live horse to demonstrate that attenuation of 99mTc was approximately 90% at a distance that was halfway across the chest. While this attests to the considerable attenuation of signal by the horse’s large chest, it indicates that the contralateral lung does not contribute significantly to measures of lung deposition (26).

It is critical to correct for DTPA clearance in the lung, as inhaled 99mTc-DTPA particles rapidly pass from the lungs to the blood. Scans conducted at the end of a study will therefore have lower counts than those done at the beginning, beyond that accounted for by physical radioactive decay. A wide range of clearance values for humans is published in the literature, with the t_1/2 ranging from 52 to 110 min. Multiple factors may influence these values, including blood flow, alveolar surface area, lung volumes, and peripheral deposition of drug (27). The lung volumes at which clearance values were obtained in the horse is not known, nor was there further measurement of blood flow or alveolar surface area for each lung area measured (15–17). Therefore, there is still some uncertainty about the actual percentage of deposition of the drug. Despite these shortcomings, our radiolabeling studies demonstrate that the mask-spacer effectively allowed drug to reach the lung in a way that compares reasonably with other devices and provided validation to continue with the second purpose of our study, which was to examine the effects of repeated (10-day) administration of inhaled albuterol on airway reactivity.

Inhaled albuterol is often used to treat inflammatory airway disease (IAD) and asthma, which is a similar disease in humans. Although it is well known that beta-2 adrenergic receptor agonist (β2-AR) drugs do not prevent or treat the inflammation associated with either disease, they are often the only therapy used for both asthma and IAD. There is longstanding and strong concern in the human medical community that long-term use of short-acting beta agonist (SABA) drugs such as albuterol may lead to failure during acute severe asthma (28). Moreover, a recent study in horses has demonstrated that use of the systemic β2-AR, clenbuterol, for 21 d leads to tachyphylaxis, which is manifested as increased airway hyperresponsiveness (6). Both HFA-albuterol and CFC-albuterol given long-term (12 mo) were shown to decrease bronchodilator ability in humans (29), and guinea pigs treated with R, S, or racemic-albuterol for 10 d had significant increase in AHR (30). Similarly, regular inhalation of both S and RS-albuterol, but not R-albuterol, induced airway inflammation in both healthy and asthmatic cats (31). On the other hand, regular treatment with R, S, and RS albuterol resulted in airway hyperreactivity in mice (4), which suggests that there may be a distinct difference in responses of species to these drugs.

Varying mechanisms appear to mediate tolerance to short-acting and long-acting β2-ARs, including receptor internalization (32), sustained release of pro-inflammatory cytokines (33), decreased expression of β2-AR gene (34) resulting in cholinergic signal amplification and resultant increased hyperresponsiveness, as well as goblet cell hyperplasia (4). Genetic polymorphisms that govern some of these mechanisms are recognized in humans (3), but this has not been studied in horses. In humans, tolerance of small airways to albuterol in vitro was prevented by concurrent treatment with corticosteroid, which underscores the importance of addressing the inflammatory nature of these diseases (32).

In contrast to findings in mice, cats, and humans, our study did not reveal any adverse effects after 10 d of twice daily treatment with inhaled albuterol. It is unclear, however, how this translates to clinical medicine. There have been 2 randomized trials of albuterol given on a regular schedule versus on an as-needed basis in asthmatics; neither study showed any evidence of tolerance to the acute bronchodilatory properties of albuterol (35,36). Thus, it may be that both horses and humans with mild-to-moderate asthma are able to tolerate use of SABA drugs (28). In addition, horses have large airways that may help to protect them from extreme airway closure. Horses may also have innate bronchoprotective mechanisms that are still not known.

Unexpectedly, AHR increased in all horses treated with HFA-vehicle alone and 4 horses developed a marked increase in AHR despite bronchodilation with albuterol 15 min before the challenge. Increased AHR has been reported in response to the oleic acid used as a vehicle for CFC-propellant drugs, but there are no reports of AHR with the ethanol used in the Ivax albuterol-HFA formulation (37,38). A comparison of HFA-BDP and HFA-vehicle in human asthmatics, however, showed that adverse effects in the respiratory system were more commonly found in the HFA-placebo group (36%) as opposed to HFA-BDP (25%), which suggests that the vehicle itself may have contributed to the adverse effects (39). In contrast, in a clinical trial in horses, no adverse effects of HFA-vehicle were seen, although vehicle administration resulted in bronchodilation, which the authors attributed to an effect of the nasal delivery device (40).

Although there were no changes made to housing or management of horses during the study period in the current study, natural fluctuations in particulates, endotoxin, and ammonia levels may have led to increased sensitivity in some horses. There was no effect of period or sequence in the model used, however, including the comparison of AHR on day 0 for horses in both the AP and the PA treatment sequences, making this less likely. It is possible that albuterol treatment protected horses to a certain extent from subsequent AHR.