AMP challenges have been utilized in both adults and children. The test is highly specific and therefore can be used to discriminate between asthma and COPD.
AMP is ubiquitous in the human body, and inhalation challenges are not associated with side effects. However, doses administered to subjects are 50-100-fold higher compared to methacholine, thus reaching a dose plateau where subjects cannot be pushed further. Whether or not AMP inhalation induces airway eosinophilia is debatable (van den Berge et al. 2004).
Outcomes and Interpretation of Results
AMP challenges have been used for clinical research to assess the effects of asthma medications on airway responsiveness (Figure 10.5). Nonsteroidal
FIGURE 10.5 Percent change in forced expiratory volume in 1 s (FEVj) at doubling concentrations of adenosine monophosphate (AMP) (mg/mL) in patients with asthma without treatment (left) and following treatment with inhaled corticosteroids (ICSs) (right). The provocative concentration of AMP causing a 20% fall in FEV1 (PC20) shifts approximately 4 doubling concentrations higher with ICS treatment.
therapeutic agents, including the anti-immunoglobulin E (IgE) antibody, omali- zumab, have been shown to shift the PC20 to AMP (Currie et al. 2003; Prieto et al. 2006). Studies of this nature demonstrate the usefulness of AMP to assess asthma therapies but also provide evidence of IgE and thus mast cell/basophil involvement in the pathophysiology of airway constriction induced by AMP. Lee et al. evaluated the effectiveness of single and short-term dosing regime of a histamine Hj receptor antagonist on AMP PC20. Treatment with levocetirizine significantly increased AMP PC20 values compared to the placebo, without effecting prechallenge FEVJ (Lee et al. 2004). The leukotriene receptor antagonist, montelukast, has also been shown to be a valuable add-on therapy for asthmatics not well controlled by ICSs due to genetic susceptibility to p2-receptor genotype (Sims et al. 2003). The effectiveness of montelukast in this subject group was assessed using AMP PC20, given that it is thought to be a more sensitive method to detect ICS inflammatory effects as opposed to methacholine(O’Connor et al. 1992; van den Berge et al. 2001).
Interestingly, other intervention studies with ICSs verify that changes in airway hyperresponsiveness to indirect stimuli such as AMP provide more information on airway inflammatory control than symptoms and lung function or airway reactivity to direct stimuli like methacholine or histamine. For example, O’Connor et al. demonstrated that inhaled glucocorticosteroids decreased airway hyperresponsiveness to AMP to a significantly greater extent than it reduces airway hyperresponsiveness to methacholine in subjects with mild asthma. They proposed that this might represent a reduction in mast cell numbers and activity (O’Connor et al. 1992). Ketchell et al. expanded on these findings to demonstrate that following inhalation of fluticasone propionate (100-1000 pg) in mild stable asthma, there was a reduction in airway hyperresponsiveness to AMP, whereas a single inhalation of fluticasone propionate 1000 pg did not affect airway hyperresponsiveness to histamine (Ketchell et al. 2002). Wilson et al. reported that 4 weeks of treatment with 160 mcg ciclesonide once daily in subjects with mild persistent asthma significantly increased the AMP PC20 from 13 to 140 mg/mL, compared to only 17 mg/mL with placebo (Wilson et al. 2006). This eightfold increase in mean PC20 to AMP was associated with a decrease in sputum eosinophils and significant improvements in exhaled nitric oxide levels. Similarly, Taylor and colleagues reported that ICSs decreased airway hyperresponsiveness to AMP in a dose-dependent manner, which was in line with a decrease in sputum eosinophils (Taylor et al. 1999). On the other hand, Green et al. showed that after 4 weeks of treatment with twice the equivalent dose of budesonide (800 pg/day), there was only a 0.4 doubling dose reduction of airway hyperresponsiveness to methacholine from baseline, which was associated with a 1.6-fold reduction in sputum eosinophils (Green et al. 2006). Young children have also been included in studies comparing responses to both methacholine and AMP. These studies have reflected that AMP responsiveness is more likely to be associated with atopy, IgE, and inflammatory markers including blood eosinophils compared with methacholine (Bakirtas and Turktas 2006; Choi et al. 2007). These findings suggest that airway responsiveness to AMP appears to provide better reflection of changes in inflammatory markers than responsiveness to methacholine.
Bronchoprovocation Tests for the Evaluation of Drug Efficacy in Asthma 211 Allergen Inhalation Challenge
Allergens are natural stimuli that when inhaled by sensitized individuals can induce three hallmarks of allergic asthma: bronchoconstriction, airway inflammation, and AHR. The mechanism by which allergens induce asthmatic responses is an active area of study with established animal and human allergen challenge models. Inhaled allergens can be administered to subjects with allergic asthma to provoke controlled asthma exacerbations for the evaluation of the immunobiology and pathophysiology of allergic asthma, and also to test the efficacy of novel therapeutics for the treatment of asthma (Diamant et al. 2013).
The pathogenesis of allergic asthma is driven by allergen exposure and subsequent activation of the innate and adaptive immune responses. The inhaled allergen is a pro-inflammatory stimulus that activates immune cells in the airway, resulting in asthmatic symptoms of wheezing, shortness of breath, chest tightness, and cough. More specifically, the innate immune system detects inhaled allergens through pattern recognition receptors, such as toll-like receptors (TLR4, TLR9), which leads to the release of pro-inflammatory cytokines (IL-1, IL-6, IL-25, IL-33) and thymic stromal lymphopoietin (TSLP) (Willart et al. 2012; Chu et al. 2013). IgE bound on the surface of mast cells and basophils are able to bind and cross-link allergen, which results in the induction of their activation and the release of inflammatory mediators (histamines, leukotrienes, prostaglandins) (Gould and Sutton 2008; Stone et al. 2010; Dullaers et al. 2012).
The adaptive immune response mediated by antigen-presenting cells (APCs), specifically dendritic cells, T cells, and B cells, which drives an adaptive immune response against allergens. Dendritic cells (professional APCs) process and present antigens to Th2 cells (CD4+ T cells), thus initiating the Th2 response, a characteristic of allergic disease (Mempel et al. 2004; Trivedi and Lloyd 2007; Dua et al. 2010). Furthermore, a myriad of cells (T cells, basophils, ILC2s) are able to produce IL-4 and IL-13 (Mandler et al. 1993; Ogata et al. 1998; Smith et al. 2015), which acts as a maturation factor for B cells that allow them to isotype-switch into IgE- producing plasma cells (Kasaian et al. 1995; Zuidscherwoude and van Spriel 2012). Specifically, Th2 cells present allergen peptides to B cells via T cell receptor and B cell receptor interactions (Robinson 2010), which initiates IgE isotype-switching of B cells, allowing B cells to mature into IgE-secreting plasma cells (Luger et al. 2009). Like T cells, B cells are also able to secrete cytokines, such as IL-4 and IL-13, which further propagates the Th2 response.
Allergic asthmatics typically respond to allergens in two phases, the EAR and the late asthmatic response (LAR), which are measured by a decline in the FEVj. The EAR is characterized by an acute onset of bronchoconstriction, largely due to cross-linking of allergen-specific IgE bound to high-affinity IgE receptors (FceRI) on mast cells and basophils (Figure 10.2), and subsequent activation and release of the bronchoconstrictor mediators including histamine, leukotrienes, and proinflammatory cytokines. The LAR is more a delayed bronchoconstriction occurring approximately 4-8 h postchallenge and is associated with cellular infiltration of the airways, which is predominantly eosinophilic in nature (Gauvreau et al. 2000; Gauvreau and Evans 2007).
FIGURE 10.6 Percent change in forced expiratory volume in 1 s (FEVj) at doubling titrations of sensitizing allergen extract in subjects with mild allergic asthma without treatment (left) and following treatment with inhaled corticosteroids (ICSs) (right). The provocative concentration of allergen causing a 15% fall in FEV1 (PC15) shifts approximately 1.5 doubling dilutions with higher ICS treatment.
During an allergen inhalation challenge, asthmatic subjects inhale an aeroal- lergen to which they are sensitized, as determined by skin prick testing. Common aeroallergens include house dust mite, cat dander, and pollens. Allergen extracts are diluted in saline, nebulized, and delivered by inhalation in increasing (often doubling) dilutions. The FEV1 is measured 10 min after each inhalation, and the challenge is stopped when a 20% fall in FEV1 is reached (Gauvreau and Evans 2007; Diamant et al. 2013). In the PC15 model of allergen challenge, the acute response to allergen is expressed as a PC15, that is, the concentration of allergen causing a 15% decline in FEVj (Figure 10.6). In the EAR/LAR model of allergen challenge, a predefined amount of allergen is inhaled and spirometry is measured regularly for 7-10 h to capture both the EAR and LAR. In such cases, responses to allergen can be expressed as the maximum percent fall in FEV1 or as the area under the curve (AUC) of the EAR and LAR. A positive EAR is defined as a >20% fall in FEV1 and a positive LAR is defined as a >15% fall in FEV (Figure 10.7).