Sage Journals: Discover world-class research

Abstract

Objectives

Feline allergic asthma is a common chronic lower airway disease characterized by clinical signs attributed to eosinophilic inflammation, airway hyper-responsiveness (AHR) and airway remodeling. Tachykinins released from sensory nerves and immune cells bind neurokinin-1 (NK-1) receptors in the lung. The resultant neurogenic airway inflammation has been implicated in asthma pathogenesis. In mouse models and spontaneous human asthma, NK receptor antagonists reduce bronchospasm and inflammation. We hypothesized that chronic administration of maropitant, an NK-1 receptor antagonist, would decrease clinical signs of asthma, AHR and eosinophilic inflammation in experimentally asthmatic cats.

Methods

Cats (n = 6) induced to have asthma using Bermuda grass allergen (BGA) were enrolled in a randomized, prospective, placebo-controlled crossover design study. Cats received either oral maropitant (2 mg/kg) or placebo q48h for 4 weeks; following a 2 week washout, cats were crossed-over to the alternate treatment. Study endpoints included subjective clinical scoring systems after BGA challenge, ventilator-acquired pulmonary mechanics to assess AHR after bronchoprovocation with methacholine, and collection of bronchoalveolar lavage fluid to quantify airway eosinophilia. Statistical analysis was performed using a Mann–Whitney rank sum test with P <0.05 considered significant.

Results

Administration of maropitant for 1 month in experimentally asthmatic cats produced no significant difference in clinical scoring scheme (P = 0.589 and P = 1.0), AHR (P = 0.818) or airway eosinophilia (P = 0.669) compared with placebo.

Conclusions and relevance

Chronic administration of maropitant was ineffective at blunting clinical signs, AHR and airway eosinophilia in experimental feline asthma and thus cannot be recommended as a novel treatment for this disorder.

Introduction

Feline asthma is a serious chronic respiratory condition resulting in clinical signs of cough, wheeze and episodic respiratory distress attributed to airway hyper-responsiveness (AHR), eosinophilic airway inflammation, and airway remodeling.¹ The immunologic driving force behind allergic asthma is aeroallergen-induced activated T helper 2 cells, which result in chronic inflammation and subsequently orchestrate a variety of pathologic changes.² Homeostasis in the lung is maintained, in part, by the neuroimmune axis. Crosstalk between these two systems is mediated via cytokines, neuropeptides and their receptors.^3–5 In asthmatic individuals allergens and other airborne irritants induce neuropeptide release locally. Tachykinins are released both from sensory nerves and immune cells, and there is evidence that these neurogenic mediators play an important role in asthma pathogenesis both by facilitating acute bronchospasm and maintaining inflammation during periods of relative clinical quiescence.^2–4

Tachykinin release from sensitized neurons sustains and amplifies the ongoing inflammatory response in rodent models of asthma and in humans with spontaneous asthma.^6–10 This is accomplished by enhanced lymphocyte proliferation, immunoglobulin production, cytokine secretion and alterations in vascular permeability.^{3–5,11–15} Substance P (SP) is a neuropeptide released from sensory neurons and immune cells within the respiratory tract and exerts its effects by binding neurokinin-1 (NK-1) receptors.³ SP and NK-1 activity have been found to be particularly important in the development of neurogenic inflammation. SP immunoreactive nerves stimulated by local irritants and nerve growth factors are increased in humans severely affected by asthma.³ NK-1 receptors have been localized to respiratory leukocytes, the vascular endothelium and goblet cells, and are believed to contribute to cellular influx, edema and mucous production.^16,17 Further, SP acts as a mitogen for smooth muscle cells, endothelial cells and fibroblasts that may contribute to the architectural changes seen the airways of asthmatic patients.³

Corticosteroids represent the gold standard of therapy for asthma. The efficacy of corticosteroids may include attenuation of SP-induced inflammation because the NK-1 receptor gene has been found to have glucocorticoid responsive elements.^2,18 However, comorbidities including diabetes mellitus, cardiac disease and recurrent upper respiratory infections make corticosteroid therapy relatively or absolutely contraindicated in asthmatic cats. Studies examining rodent models of asthma have shown the NK-1 receptor antagonism may have promise as a means of reducing inflammatory cell influx and AHR.^7,17,19 Studies in human asthmatics utilizing mono-antagonism of the NK-1 receptor are lacking; however, examination of these studies in addition to those examining combined NK-1 and NK-2 antagonists have shown mild effects on AHR.^10,20,21 Maropitant citrate (Cerenia) is an NK-1 receptor antagonist marketed as an antiemetic in cats with an excellent safety profile.²² It has been used anecdotally for the treatment of naturally occurring feline asthma, without the benefit of objective scientific evaluation. However, if effective in modulating the SP-mediated neuroimmune axis to disrupt airway inflammation and/or AHR, maropitant may represent an effective treatment for asthmatic cats. This study hypothesized that in experimentally asthmatic cats, chronic every-other-day dosing of maropitant would reduce clinical signs after allergen challenge, AHR measured by ventilator-acquired pulmonary mechanics and airway eosinophilia.

Materials and Methods

Cats

Six cats were enrolled in a prospective, placebo-controlled, crossover study using an established experimental model of feline asthma. Asthma was induced using Bermuda grass allergen (BGA) as previously described.²³ The asthmatic phenotype was confirmed with positive skin reactivity to BGA and bronchoalveolar lavage fluid (BALF) eosinophil percentage of >17%. This study was approved by the University of Missouri Care and Use Committee (Animal Care and Use Committee protocol #7891). Cats were cared for in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Treatment

Each cat was randomly assigned to either the maropitant group (2 mg/kg PO q48h in a food treat for 28 days) or the placebo group (food treat alone). The cats received weekly aerosol challenges, including an aerosol challenge 24 h prior to bronchoprovocation testing and BALF collection. After a 2 week washout period the cats were crossed over to the alternate treatment.

Subjective clinical scoring system

A visual analog scale (VAS) was used to score clinical signs 10 mins after airway challenge with BGA. The VAS used a 10 cm scale to assess signs observed ranging from no clinical signs (0 cm) to extreme respiratory distress (10 cm). The distance from 0 to the patient score was then measured to provide a quantitative VAS score. The VAS was performed by a single investigator (MG). A clinical score was also assigned to each cat 10 mins after BGA challenge using a nine-point system (Table 1).

Table 1

A composite clinical scoring system was used to evaluate the cats following aerosol challenge with Bermuda grass allergen. The clinical visual analog scores were assessed by the same individual (MG). For the clinical score, cats were given a score between 0 and 9²⁴

Respiratory scoring system (0–9 points)

Respiratory rate changes

0 points: <50% increase in baseline RR

0.5 points: 50% increase in baseline RR

1 point: >100% increase in baseline RR

Auscultation (prechallenge)

0 points: no wheezes

1 point: wheezes

Auscultation (postchallenge)

0 points: no wheezes

1 point: wheezes

Inducible cough (prechallenge)

0 points: no

1 point: yes

Inducible cough (postchallenge: 10 mins later)

0 points: no

1 point: yes

Clinical signs (postchallenge)

0 points: no signs

0.5 points: mild-to-moderate increase in respiratory effort but not enough to affect activity or mentation; spontaneous cough

1 point: moderate signs of respiratory difficulty (expiratory push or rapid and shallow breathing)

1.5 points: moderate to severe signs of respiratory difficulty (nostrils flaring, impaired activity, obvious distress)

2 points: severe signs (open-mouth breathing, lateral collapse, obtunded)

Time in chamber (until development of clinical signs)

0 points: 10 mins

0.5 points: 5 to <10 mins

1 point: 2 to <5 mins

1.5 points: 1 to <2 mins

2 points: <1 min

RR = respiratory rate

Measurement of AHR

Ventilator-acquired pulmonary mechanics was performed on anesthetized and mechanically ventilated cats as previously described, with minor modification.²³ Briefly, ketamine (30 mg IV) was used as a premedicant, propofol was used for induction (6 mg/kg) and maintenance (0.3 mg/kg/min), and cisatracurium (0.1 mg/kg IV of with additional doses of 0.01–0.02mg/kg titrated to effect) was administered for neuromuscular blockade. Neuromuscular blockade was monitored with a peripheral nerve stimulator. Edrophonium (0.5 mg/kg IV) was administered to reverse neuromuscular blockade after completion of data collection when indicated. Pulse oximetry was used to monitor oxygen saturation (SpO₂). Initial ventilator settings were as follows: tidal volume was 10 ml/kg, respiratory rate was 10 breaths/min, inspired oxygen fraction was 0.4, inspiratory to expiratory ratio was 1:3, and the positive end expiratory pressure was 2 cm H₂O. Parameters recorded on a breath-by-breath basis on the ventilator included airway resistance, compliance and peak pressure; after inspiratory and expiratory breath hold plateau pressure and endogenous positive end expiratory pressure were measured. Methacholine (MCh) challenge was terminated when airway resistance reached 200% of baseline (EC200raw). Prior to data collection, cats were ventilated for 5 mins to allow for steady-state conditions.

Saline was nebulized for 30 s followed by 4 mins of data collection to determine baseline data. MCh was aerosolized for 30 s with 4 mins of data collection between each dose. Doubling doses of MCh from 0.0625 −32.0000 mg/ml were administered serially until the intended study endpoints were reached. The intended endpoint of the study was the effective concentration of MCh to increase airway resistance by 200% over baseline (EC200Raw) or oxygen desaturation <75%. The EC200Raw was calculated by linear interpolation of the log plot of the dose–response curve; the maximum airway resistance obtained over each 4 min period of data collection for each dose of MCh was used for this calculation.

Collection and analysis of BALF

Using a 20 ml aliquot of sterile saline, BALF was collected in a blind fashion through the endotracheal tube after completion of ventilator-acquired pulmonary mechanics.²⁵ A differential count was performed on a Wright’s stained cytospin to evaluate the percentage of eosinophils.

Statistical analysis

Mann–Whitney rank sum tests were used to evaluate the differences in VAS score, EC200Raw and BALF eosinophil percentage between the maropitant and placebo groups. A P value <0.05 was considered significant.

Results

Chronically administered maropitant failed to decrease the VAS score or the composite clinical score after allergen challenge compared with placebo (P = 0.589 and P = 1.00, respectively; Figure 1a,b). Maropitant did not significantly dampen AHR, as indicated by EC200Raw in response to MCh challenge vs placebo (P = 0.818; Figure 2). No biologically significant alteration in any other measured pulmonary mechanics parameter was noted (Table 2). Oxygen desaturation <75% after MCh challenge did not occur in any cat with any treatment; the mean SpO₂ in all cats over all time points was 98 ± 1%. Blockade of the NK-1 receptor by maropitant did not diminish airway eosinophilia as there was no significant difference in this parameter between maropitant and placebo treatment groups (P = 0.699; Figure 3).

Figure 1

(a,b) Six experimentally induced asthmatic cats receiving maropitant and placebo in random order were evaluated for clinical signs after allergen challenge using (a) a visual analog scale (VAS) score and (b) a subjective clinical composite score. The boxes represent the 25th and 75th quartiles, with a horizontal line at the median. The whiskers represent the range of the data. The black squares represent the mean. No significant difference was observed between the maropitant or placebo groups based on subjective VAS or clinical composite score (P = 0.589 and P = 1.00, respectively)

Figure 2

Six experimentally induced asthmatic cats receiving placebo and maropitant as part of a randomized, placebo-controlled, prospective, crossover study were evaluated for airway hyper-responsiveness (AHR) following methacholine (MCh) challenge. The boxes represent the 25th and 75th quartiles, with a horizontal line at the median. The whiskers represent the range of the data. The black squares represent the mean. Maropitant did not significantly dampen AHR as measured by the EC200Raw in response to MCh vs placebo (P = 0.818). Baseline airway resistance was not significantly different between treatments (P = 0.535); data not shown

Table 2

A variety of other parameters relevant to lung function were determined on experimentally asthmatic cats undergoing methacholine bronchoprovocation, including peak pressure – plateau pressure; plateau pressure – positive end expiratory pressure; and change in plateau pressure. Increases in peak pressure – plateau pressure are proportional to airway resistance. Increased plateau pressure – positive end expiratory pressure values indicate decreased respiratory compliance. Increases in plateau pressure (change in plateau pressure) imply enhanced airflow limitation. No significant changes in any parameter were observed between the maropitant and placebo groups

Group	Plateau pressure – positive end expiratory pressure	Peak pressure – plateau pressure	Δ Plateau pressure
Placebo	8.27 ± 3.37	3.32 ± 0.76	5.07 ± 2.25
Maropitant	2.08± 5.71	2.5 ± 3.45	8.42 ± 5.48
P value	0.937	0.132	0.937

Values are given as mean ± SD

Figure 3

Six experimentally induced asthmatic cats receiving placebo and maropitant in random order were evaluated for airway eosinophilia. Bronchoalveolar lavage samples were collected in a blind fashion. A differential count was performed on a Wright’s stained cytospin to evaluate the percentage of eosinophils. The boxes represent the 25th and 75th quartiles, with a horizontal line at the median. The whiskers represent the range of the data. The black squares represent the mean. In our cats, maropitant did not dampen airway eosinophilia compared with placebo (P = 0.699)

Discussion

To our knowledge, this is the first study, using an experimental feline asthma model, to evaluate the utility of chronic, 1 month use of maropitant as a treatment for asthma. In contrast to our hypothesis, chronically administered maropitant did not diminish clinical signs after allergen challenge (indicated by the lack of change in VAS score or composite clinical score), AHR or airway eosinophilia vs placebo. This is in contrast to rodent studies, which showed a more favorable response to NK-1-receptor blockade by demonstrating decreased eosinophil influx and AHR, although the effects on AHR were limited to the late phase.^17,19 Human studies evaluating NK-1 receptor blockade have shown mild effects on AHR, although studies are limited.⁷

Maropitant has been shown to be effective as an antiemetic, with excellent affinity for the NK-1 receptor. The dose of maropitant selected in this study exceeded the recommended dose by 200%, making insufficient dosing and poor receptor affinity unlikely as the cause of the lack of a statistically significant response.²² Chronic administration allowed us to reach steady-state concentrations, also making inadequate dosing unlikely. Maropitant was administered in a food treat to minimize the stress associated with oral administration of medication. The same food treat and timing was used in the placebo group to reduce introduction of variables between groups.

Neurogenic inflammation is complex, involving multiple receptors (NK-1, NK-2 and NK-3) and mediators (SP, neurokinin A [NKA], neurkokinin B). The NK-1 receptor is located predominantly on the bronchial vessels, epithelial cells, submucosal glands and vascular endothelium.³ The tachykinin receptor localized to airway smooth muscle is predominantly NK-2.³ While SP is capable of binding to NK-2 receptors, the major mediator for this receptor type is NKA.^3,26,27 Human asthmatic patients are hypersensitive to both SP and NKA, with increased NKA and SP concentrations within BALF.^12,13,28 As maropitant is highly specific for the NK-1 receptor, this may provide an explanation for the lack of response in clinical signs and AHR in the cats of this study.

There are several additional reasons why maropitant was not effective in this study. First, tachyphylaxis, the diminished response to a drug/compound after multiple doses, secondary to chronic inflammation and multiple allergen challenges may result in a subsequent minimal response to NK-1 receptor antagonism. The release of SP and its binding to the NK-1 receptor results in rapid internalization of the NK-1 receptor, leading to desensitization of cells to SP signaling.²⁹ Second, the presence of chronic inflammation may result in depletion of SP (as has been shown for vasoactive intestinal polypeptide in asthmatic compared with healthy humans,^17,30–32) making the effect of NK-1 receptor blockade less effective in the management of chronic asthma. Finally, the perpetuation of airway inflammation is dependent on multiple redundant pathways and it has been speculated that following the establishment of a persistent cell-mediated inflammatory response, blockade of tissue NK-1 receptors is insufficient to prevent further eosinophil influx and airway remodeling.^3,33,34

Conclusions

Maropitant did not reduce airway AHR or airway eosinophilia in experimentally induced feline asthma. Thus, this study did not support the use of maropitant in feline asthma. It is possible that dual receptor blockade (NK-1 and NK-2) may be more efficacious at blunting the hallmark features of allergic asthma. Further study is needed to determine if chronic airway allergic inflammation depletes neurogenic inflammatory mediators and therefore limits the utility of downstream tachykinin receptor antagonism to treat allergic airway disease.³⁵

Footnotes

Acknowledgements

We would like to thank Dr Hans Rindt for his technical assistance.

Conflict of interest

The authors do not have any potential conflicts of interest to declare.

Funding

Funding for this study was provided by the Winn Feline Foundation. Maropitant was generously donated by Zoetis.

Accepted: 16 March 2015

References

Padrid

. Feline asthma. CVT Update. In: Bonagura

(ed). Kirk’s current veterinary therapy xiii: small animal practice. Philadelphia, PA: Saunders, 2000, pp 805–810.

Reinero

. Advances in the understanding of pathogenesis, and diagnostics and therapeutics for feline allergic asthma. Vet J 2011; 190: 28–33.

O’Connor

O’Connell

O’Brien

, et al. The role of substance P in inflammatory disease. J Cell Physiol 2004; 201: 167–180.

Barnes

Baraniuk

Belvisi

. Neuropeptides in the respiratory tract. Part I. Am Rev Respir Dis 1991; 144: 1187–1198.

Barnes

Baraniuk

Belvisi

. Neuropeptides in the respiratory tract. Part II. Am Rev Respir Dis 1991; 144: 1391–1399.

Nieber

Baumgarten

Rathsack

, et al. Substance P and beta-endorphin-like immunoreactivity in lavage fluids of subjects with and without allergic asthma. J Allergy Clin Immunol 1992; 90: 646–652.

Veres

Rochlitzer

Braun

. The role of neuro-immune cross-talk in the regulation of inflammation and remodelling in asthma. Pharmacol Ther 2009; 122: 203–214.

Ramalho

Almeida

Beltrao

, et al. Substance P antagonist improves both obesity and asthma in a mouse model. Allergy 2013; 68: 48–54.

De Swert

Tournoy

Joos

, et al. The role of the tachykinin NK1 receptor in airway changes in a mouse model of allergic asthma. J Allergy Clin Immunol 2004; 113: 1093–1099.

10.

Joos

Van Schoor

Kips

. The effect of inhaled FK224, a tachykinin NK-1 and NK-2 receptor antagonist, on neurokinin A-induced bronchoconstriction in asthmatics. Am J Respir Crit Care Med 1996; 153: 1781–1784.

11.

Schelfhout

Van De Velde

Maggi

, et al. The effect of the tachykinin NK(2) receptor antagonist MEN11420 (nepadutant) on neurokinin A-induced bronchoconstriction in asthmatics. Ther Adv Respir Dis 2009; 3: 219–226.

12.

Joos

De Swert

Pauwels

. Airway inflammation and tachykinins: prospects for the development of tachykinin receptor antagonists. Eur J Pharmacol 2001; 429: 239–250.

13.

Joos

Germonpre

Pauwels

. Role of tachykinins in asthma. Allergy 2000; 55: 321–337.

14.

Nadel

. Neutral endopeptidase modulates neurogenic inflammation. Eur Resp J 1991; 4: 745–754.

15.

Adcock

Peters

Gelder

, et al. Increased tachykinin receptor gene expression in asthmatic lung and its modulation by steroids. J Mol Endocrinol 1993; 11: 1–7.

16.

Goetzl

Sreedharan

. Mediators of communication and adaptation in the neuroendocrine and immune systems. FASEB J 1992; 6: 2646–2652.

17.

Tiberio

Turco

Leick-Maldonado

, et al. Effects of neurokinin depletion on airway inflammation induced by chronic antigen exposure. Am J Respir Crit Care Med 1997; 155: 1739–1747.

18.

Ihara

Nakanishi

. Selective inhibition of expression of the substance P receptor mRNA in pancreatic acinar AR42J cells by glucocorticoids. J Biol Chem 1990; 265: 22441–22445.

19.

Maghni

Taha

Afif

, et al. Dichotomy between neurokinin receptor actions in modulating allergic airway responses in an animal model of helper T cell type 2 cytokine-associated inflammation. Am J Respir Crit Care Med 2000; 162: 1068–1074.

20.

Joos

Pauwels

. Tachykinin receptor antagonists: potential in airways diseases. Curr Opin Pharmacol 2001; 1: 235–241.

21.

Ichinose

Nakajima

Takahashi

, et al. Protection against bradykinin-induced bronchoconstriction in asthmatic patients by neurokinin receptor antagonist. Lancet 1992; 340: 1248–1251.

22.

Hickman

Cox

Mahabir

, et al. Safety, pharmacokinetics and use of the novel NK-1 receptor antagonist maropitant (Cerenia) for the prevention of emesis and motion sickness in cats. J Vet Pharmacol Ther 2008; 31: 220–229.

23.

Chang

Dodam

Cohn

. Comparison of direct and indirect bronchoprovocation testing using ventilator-acquired pulmonary mechanics in healthy cats and cats with experimental allergic asthma. Vet J 2013; 198: 444–449.

24.

Eberhardt

DeClue

Reinero

. Chronic use of the immunomodulating tripeptide feG-COOH in experimental feline asthma. Vet Immunol Immunopathol 2009; 132: 175–180.

25.

Nafe

Guntur

Dodam

, et al. Nebulized lidocaine blunts airway hyper-responsiveness in experimental feline asthma. J Feline Med Surg 2013; 15: 712–716.

26.

Boichot

Biyah

Germain

, et al. Involvement of tachykinin NK1 and NK2 receptors in substance P-induced microvascular leakage hypersensitivity and airway hyperresponsiveness in guinea-pigs. Eur Respir J 1996; 9: 1445–1450.

27.

Boot

de Haas

Tarasevych

, et al. Effect of an NK1/NK2 receptor antagonist on airway responses and inflammation to allergen in asthma. Am J Respir Crit Care Med 2007; 175: 450–457.

28.

Cheung

van der Veen

den Hartigh

, et al. Effects of inhaled substance P on airway responsiveness to methacholine in asthmatic subjects in vivo. J Appl Physiol 1994; 77: 1325–1332.

29.

Leeman

Slack

, et al. A substance P (neurokinin-1) receptor mutant carboxyl-terminally truncated to resemble a naturally occurring receptor isoform displays enhanced responsiveness and resistance to desensitization. Proc Natl Acad Sci U S A 1997; 94: 9475–9480.

30.

Ollerenshaw

Jarvis

Woolcock

, et al. Absence of immunoreactive vasoactive intestinal polypeptide in tissue from the lungs of patients with asthma. N Engl J Med 1989; 320: 1244–1248.

31.

Dey

Hoffpauir

Said

. Co-localization of vasoactive intestinal peptide- and substance P-containing nerves in cat bronchi. Neuroscience 1988; 24: 275–281.

32.

Dey

Altemus

Michalkiewicz

. Distribution of vasoactive intestinal peptide- and substance P-containing nerves originating from neurons of airway ganglia in cat bronchi. J Comp Neurol 1991; 304: 330–340.

33.

Schratzberger

Reinisch

Prodinger

, et al. Differential chemotactic activities of sensory neuropeptides for human peripheral blood mononuclear cells. J Immunol 1997; 158: 3895–3901.

34.

Numao

Agrawal

. Neuropeptides modulate human eosinophil chemotaxis. J Immunol 1992; 149: 3309–3315.

35.

Kraneveld

Nijkamp

. Tachykinins and neuro-immune interactions in asthma. Int Immunopharmacol 2001; 1: 1629–1650.

Chronic neurokinin-1 receptor antagonism fails to ameliorate clinical signs,airway hyper-responsiveness or airway eosinophilia in an experimental model of feline asthma

Abstract

Objectives

Methods

Results

Conclusions and relevance

Introduction

Materials and Methods

Cats

Treatment

Subjective clinical scoring system

Measurement of AHR

Collection and analysis of BALF

Statistical analysis

Results

Discussion

Conclusions

Footnotes

Acknowledgements

Conflict of interest

Funding

References