Review

a-1-Antitrypsin deficiency: clinical variability, assessment, and treatment Robert A. Stockley1 and Alice M. Turner2 1

Queen Elizabeth Hospital Birmingham, Mindelsohn Way, Edgbaston, Birmingham B15 2WB, UK University of Birmingham, Queen Elizabeth Hospital Birmingham Research Laboratories, Mindelsohn Way, Edgbaston, Birmingham B15 2WB, UK

2

The recognition of a-1-antitrypsin deficiency, its function, and its role in predisposition to the development of severe emphysema was a watershed in our understanding of the pathophysiology of the condition. This led to the concept and development of intravenous replacement therapy used worldwide to protect against lung damage induced by neutrophil elastase. Nevertheless, much remained unknown about the deficiency and its impact, although in recent years the genetic and clinical variations in manifestation have provided new insights into assessing impact, efficacy of therapy, and development of new therapeutic strategies, including gene therapy, and outcome measures, such as biomarkers and computed tomography. The current article reviews this progress over the preceding 50 years. Fifty years of exploring the implications of a-1antitrypsin deficiency The original observation of a missing a1 protein based on paper electrophoresis of blood samples resulted in the first description of the clinical features of a-1-antitrypsin-deficient (AATD) subjects [1]. The subsequent 20–25 years consolidated an association of basal panacinar emphysema (see Glossary) with AATD, the identification of neutrophil elastase (NE) as its cognate proteinase with the ability to cause emphysema in animal models [2], and the logical development of a-1-antitrypsin (AAT) augmentation by regular infusions to protect against and hence stabilise the ongoing NE-mediated lung damage. This straightforward concept and approach to patient management aims has changed little over the ensuing 20–30 years. However, in this time and particularly in the past 10–15 years, emerging data have led to a better understanding of the diseases associated with AATD and hence treatment, management, and expectations. This review explores the current state of clinical, biochemical, radiological, and genetic knowledge in the management of AATD, with particular emphasis on the key clinical questions (Box 1). Corresponding author: Turner, A.M. ([email protected]). Keywords: a-1-antitrypsin deficiency; gene therapy; biomarkers. 1471-4914/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/ j.molmed.2013.11.006

a-1-Antitrypsin AAT is a 52-kDa glycoprotein predominantly synthesised in hepatocytes from two alleles located on chromosome 14, although a small amount of protein is also made by monocytes/macrophages and airway epithelial cells that may have some local implications in the lung. AATD is the result of any of a variety of abnormalities in the AAT gene, including single point mutations, insertions and deletions, and even gene deletion. The majority of these variations have been covered in other reviews (e.g., [3]); however, the original and most common defect is a point mutation at position 342, which results in an amino acid change (Glu–Lys). The point mutation causes: (i) a slight reduction in the association rate constant for NE [4]; (ii) a change in protein charge detected by isoelectric focussing; and (iii), most importantly, a destabilisation of the tertiary structure, resulting in AAT polymerisation, where the reactive loop of one molecule inserts into the opened b sheets of a second molecule and so on [5]. This leads to protein retention in the endoplasmic reticulum and failure of secretion, resulting in a critically low plasma and lung level of AAT (as the latter depends mainly on diffusion from plasma). The active site of AAT is characterised by methionine at position 358, which confers target enzyme specificity. Although its greatest association rate constant is with NE, hence the implication of NE as the major cause of the emphysematous process, it also inhibits two other serine proteinases produced by neutrophils that cause lung damage. Proteinase 3 (Pr3) is present at much higher concentrations than NE and can also induce emphysema in animal models [6]. The third neutrophil serine proteinase is cathepsin G, which also produces some of the airway features of chronic obstructive pulmonary disease (COPD) [7]. Thus, it remains uncertain which is/are the key enzyme/s that is/are uncontrolled in the lungs of AATD patients and cause tissue damage leading to various pathological changes. The situation is further complicated by the ability of NE to activate other enzymes, such as the metalloproteinases and the cysteine proteinase cathepsin B, which have also been implicated in tissue damage [8] leading to emphysema. Furthermore, AAT may have other anti-inflammatory and immunomodulatory effects, including reduction of neutrophil adherence to the endothelium, reduction of Toll-like receptor expression, and reduction of selected pulmonary proinflammatory cytokines [9,10]. Whatever the mechanism (direct or indirect), there is Trends in Molecular Medicine, February 2014, Vol. 20, No. 2

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Review Glossary a-1-Antitrypsin deficiency (AATD): low circulating levels of a-1-antitrypsin. Apical centrilobular emphysema: typical pattern of emphysema seen in COPD unrelated to AATD, where emphysema occurs in the upper zones and is most marked in the centre of pulmonary lobules. Asthma: a condition in which intermittent airflow obstruction due to bronchospasm occurs; hallmarks include bronchodilator responsiveness (an increase in FEV1 of over 400 ml, or 12% of baseline value) and variability of peak expiratory flow rate (over 20% variability between days or times of day). Augmentation therapy: intravenous infusion of a-1-antitrypsin in order to raise circulating levels closer to normal. Basal panacinar emphysema: typical pattern of emphysema seen in AATD, where emphysema occurs in the lower zone and is present throughout the lung lobules. Biomarker: a biological signal that can be used to identify clinical events, diseases, or disease subgroups; typically measured in blood or (less commonly) a secretion such as sputum. Bronchiectasis: a condition in which the bronchi (airways) become dilated; patients are typically more prone to respiratory infections and produce large volumes of sputum every day. Bronchodilator: a drug capable of dilating passages within the bronchial tree; such as b2 agonists. Chronic obstructive pulmonary disease (COPD): a condition in which airflow obstruction, diagnosed on spirometry, occurs and remains after bronchodilation. The condition typically occurs in older smokers, although wide variability is seen. DNA methylation: methylation of DNA, such as adding methyl residues to nucleic acids. Elastin: an elastic connective tissue found in the lung, among other human tissues. Emphysema: a condition in which the alveoli (air sacs) of the lungs are damaged and enlarged, causing breathlessness. Exacerbations: flare-ups of COPD, where symptoms are worse for several days. Exacerbations may be infective or non-infective and may be defined by the type of symptoms which change, or by the degree to which they affect healthcare utilisation. If they occur two or more times per year the patient is known as a ‘frequent exacerbator’. EXACTLE: acronym describing one of the major trials of augmentation therapy, short for ‘Alpha-1-Antitrypsin (AAT) To Treat Emphysema In AAT-Deficient Patients’ (Clinicaltrials.gov identifier: NCT00263887). Expression quantitative trait loci (eQTLs): genomic loci that regulate expression levels of messenger RNA. Forced expiratory volume in 1 s (FEV1): the amount of air blown out in 1 s when performing a forced expiration as part of spirometry. Fulminant hepatic failure: severe sudden onset liver failure. Gamma glutamyl transferase (GGT): an enzyme which catalyses the transfer of the gamma-glutamyl moiety of glutathione to an acceptor such as an amino acid, a peptide; commonly used in clinical practice to assess liver disease but with potential for wider application. Gas transfer: also known as transfer factor; a physiological test of lung function referring to the ability of the lung to transport gases from inspired air to the bloodstream. Gene therapy: the introduction of normal genes into cells in place of missing or defective ones in order to correct genetic disorders. Genome-wide association study (GWAS): a study that looks as SNPs throughout the human genome. Most GWASs will use over 500 000 SNPs, which are chosen to ensure that all areas of the genome are included in the study. Hepatocellular carcinoma: a primary cancer of the liver. Induced pluripotent stem cells (iPSCs): adult cells that have been genetically reprogrammed to an embryonic stem cell like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells. Intramuscular: referring to an injection given into muscle tissue. MZ: heterozygous genotype for AATD consisting of one normal ‘M’ allele and one ‘Z’ allele. Neonatal cholestasis: a condition occurring immediately after birth in which bile fails to flow from the liver to the gastrointestinal tract. Neutrophil elastase (NE): a proteolytic enzyme released from neutrophils, which is capable of breaking down elastin. Periodic acid–Schiff (PAS): a stain used to detect polysaccharides such as glycogen and mucosubstances such as glycoproteins in tissue sections. Recombinant adenovirus: a structurally altered virus without potential to cause human disease. Single nucleotide polymorphism (SNP): a change of a single nucleic acid in the genome. Stem cells: undifferentiated cells of a multicellular organism capable of giving rise to indefinitely more cells of the same type, and from which certain other types of cells arise by differentiation.

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SZ: heterozygous genotype for AATD consisting of one S allele, which is a deficiency allele resulting in a less severe deficiency than the Z allele, and one copy of the Z allele. Z allele: one of the more common variants in the AAT gene that results in severe AATD; occurs due to an SNP which results in a single amino acid change in the structure of the protein.

general acceptance that AATD is a susceptibility factor in the generation of the COPD syndrome. Clinical variation The initial patients described were young smokers with early onset basal panacinar emphysema. This has remained the ‘classical’ clinical phenotype and led to a testing policy confined to young COPD patients with a dominance of basal emphysema rather than the apical centrilobular emphysema of patients with COPD and normal AAT. Selective testing thus reinforced this as the clinical phenotype. However, significant variations in the ‘classical’ clinical phenotype have now become recognised (Figure 1). Studies with families confirmed the genetic nature of the deficiency [11] together with its archetypal clinical manifestations. However, it was noted from these family studies that those individuals identified by family screening (the non-index cases) had much milder disease, even if they smoked [12]. This suggested that factors other than genetic deficiency and smoking played a role in disease progression. Furthermore, more comprehensive studies have recently shown that airflow obstruction is not concordant between siblings [12]. However, airflow obstruction is only one indirect aspect of the physiological features of emphysema, and gas transfer tests, a more direct measure, were concordant in the same sibling test subjects. The assessment of the amount of basal emphysema by computed tomography (CT) densitometry was also discordant, whereas apical changes were concordant. This observation was in keeping with previous studies (e.g., [13]) where gas transfer was more a general feature of emphysema (including the apices), whereas spirometric measure related better to lower zone emphysema. Further, an apical predominance of emphysematous change was a feature of those AATD patients with normal spirometry and low gas transfer, whereas a basal predominance was present in those with abnormal spirometry but with normal gas transfer [14]. Subsequent studies suggested that in AATD ‘never smokers’ the initial physiological changes affect gas transfer in the late 20s, and is reflected in the development of low apical density consistent with the earliest emphysematous change [15]. It has not been possible to carry out the same exercise in smokers because most present with established disease, although longer-term follow-up of the Swedish AATD birth cohort [16] may provide this data with time. In more established disease the presence of apical emphysema has been recognised since the original pathological studies published in 1972 [17], although accompanied by a basal predominance. Nevertheless, cases have been described where the emphysema seen on a CT scan is almost entirely apical [13]. This suggests that at least in some patients, the pathological processes are more complex. Indeed, in the Exacerbations and CT scan as Lung Endpoints (EXACTLE) augmentation trial it was noted

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bronchiectasis [20]. However, CT studies have shown that bronchiectatic change is a feature of 20–30% of AATD patients [21], which is similar to that seen in usual COPD [22]. Furthermore, such a change can be identified in most AATD patients, if carefully quantified [21], and seems to colocate most with the emphysematous pathology. This suggests that a common pathophysiological process is responsible, although further studies, such as progression with and without augmentation, are needed to confirm this concept. Pure bronchiectasis, however, remains uncommon, and studies of bronchiectatic cohorts have failed to provide evidence of AATD being a significant susceptibility factor [23]. By the time of diagnosis many patients indicate that they had initially been told they had asthma. This may, in part, reflect an assumption that young patients presenting with breathlessness are at an age when COPD and emphysema are highly unlikely, and asthma is common. Nevertheless, reversibility of airflow obstruction is also a feature of COPD in AATD. The incidence of reversible airflow obstruction depends on the criteria used to confirm it and may be present in up to 80% of patients [24]. In addition, the degree of reversibility does influence subsequent decline in spirometry [25], hence reinforcing the

Box 1. Outstanding questions and future directions  Why do some patients with AATD remain healthy?  What influences different clinical phenotypes?  Why do some patients stabilise after smoking cessation and others progress?  Which patients require or benefit from proposed management strategies?  Biomarkers to predict rate of progression and monitor response to therapy are urgently needed.

that radiologically quantified emphysema showed uniform progression throughout the lung, but augmentation only provided a benefit at the bases [18]. The data suggest that a more complex or less responsive process(es) plays a role in the upper versus lower lung. Clearly, further studies are required, particularly in those AATD patients with a predominance of apical disease. These studies may require greater application of the World Health Organisation (WHO) recommendation of testing all subjects with COPD once for AATD [19] in order to identify this ‘less typical’ phenotype. The early pathological studies also identified some bronchiectasis in the lungs of AATD patients [17]. Subsequent case reports identified patients with almost pure

(A)

(B) Extrapulmonary disease

Pulmonary disease

Reversibility with short acng bronchodilator

Chronic bronchis

Liver dysfuncon incl. cirrhosis

Bronchiectasis

Emphysema Panniculis Fixed airflow obstrucon

Wegener’s granulomatosis Ulcerave colis

R TRENDS in Molecular Medicine

Figure 1. Clinical manifestations of a-1-antitrypsin deficiency (AATD). The relationship between each of the illustrated pulmonary features and their relative frequency is shown in the Venn diagram (A); for example, the size of the circle representing emphysema shows that most patients exhibit this feature. Chronic bronchitis occurs in a smaller proportion of patients than emphysema, as shown by the smaller circle; it is represented by an image of sputum because sputum production is the defining feature of this phenotype. Bronchiectasis also occurs but does so in conjunction with emphysema in most cases, as shown by the degree of overlap between the two circles; the computed tomography (CT) images here show tubular and cystic disease, and are included for illustrative purposes only. Most patients with emphysema exhibit disease in the lower zone, as shown in the reconstructed CT image; upper zone disease occurs less often, as shown by the relative size of the arrows leading to the upper image. The box with the dashed line represents the proportion of patients who respond to a short-acting bronchodilator (such as salbutamol) – they improve their forced expiratory volume in the first second of expiration (FEV1) by at least 12% of its baseline value and at least 200 ml. The box with the solid line represents those patients who have a fixed airflow obstruction – this is defined as a ratio between their FEV1 and their forced vital capacity of less than 0.7 after receiving a bronchodilator. The boxes overlap, showing that some patients will exhibit a degree of reversibility as well as residual airflow obstruction. Furthermore, some patients with lung disease, such as those with emphysema, lie outside the box of airflow obstruction, meaning that they have normal spirometry. (B) Images are shown that represent some of the extrapulmonary manifestations of AATD, which include liver dysfunction, up to and including cirrhosis, and panniculitis of the skin. Wegener’s granulomatosis and colitis are also associated.

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Review importance of standard bronchodilator therapy in AATD when airflow obstruction is present. Again, longitudinal follow-up of the Swedish birth cohort may provide further insight into whether asthma plays a role in the natural history of lung disease. Chronic bronchitis has been recognised as an important feature of usual COPD and is also a feature of AATD, affecting up to 40% of patients [26]. Chronic bronchitis is certainly associated with more evidence of neutrophilic inflammation in the airways [27], and this is greater than in usual COPD. Further, active NE [28] and Pr3 [29] are often present in the expectorated secretions of AATD patients. How this influences the decline in airflow obstruction or the progression of emphysema remains unknown. Exacerbations, as in usual COPD, are also a feature of AATD. The episodes are associated with evidence of increased inflammation, which is also greater than in usual COPD [28]. The concept of ‘frequent’ exacerbations also applies to AATD and reflects both the health status of the patient [30] and the subsequent decline in lung function [24]. As in usual COPD some episodes of exacerbation do not require intervention with new or increased medication [31]. However, episodes last longer in AATD than in usual COPD, and the severity of emphysema influences the symptoms and the duration [32]. The impact of exacerbations has received little attention in AATD, although the number is reflected in both spirometric and overall gas transfer decline [24], suggesting obstructive damage at least to the small airways. With increased inflammation and presence of free NE and Pr3, it might be expected that AAT augmentation therapy would have a beneficial effect on exacerbations. Indeed, in retrospective analysis patients reported fewer episodes while on AAT augmentation therapy than preceding it [33]. However, the recurrent and frequent healthcare exposure associated with augmentation may well have had an effect on general health and perception of exacerbation episodes. In the EXACTLE trial, exacerbations were not reduced, although in a post hoc analysis more severe exacerbations requiring hospitalisation were less in patients on augmentation therapy compared with placebo [34]. This supports the concept that AAT augmentation reduced the inflammatory response in airways (as seen in the stable clinical state [35]) during an exacerbation, and hence the severity of the episode. Other conditions The other major morbidity in AATD due to homozygosity of the common Z variant gene relates to the liver. The Z gene variant protein product polymerises in the hepatocyte and accumulates within the endoplasmic reticulum [3]. This accumulated protein produces the classic Periodic acid– Schiff (PAS) positive inclusion bodies seen histologically [5]. The protein which accumulates in the hepatocyte enters the physiological degradation pathway; however, this is overloaded, and the end result is tissue injury and cell death. This is partly offset by hepatocyte replenishment, but fibrosis also occurs owing to the injury, which leads to cirrhotic change [3]. The exact processes still require elucidation, but there must also be factors that 108

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vary the damaging and reparative processes, as few patients develop end stage liver disease in adult life. In the neonatal period a greater proportion of babies have transient jaundice if they have AATD, and a few develop fulminant hepatic failure leading to death or the urgent need for liver transplantation. Approximately 80% of those with neonatal cholestasis are healthy by the age of 18 years [36], and only 3% die before the age of 12 years from cirrhosis [37]. The clinical course and biochemical feature of liver disease can fluctuate, but in adulthood few progress to liver failure. Clinical manifestations peak in middle age, although of our current cohort of approximately 700 adults studied over 16 years far fewer have developed liver disease than the incidence of 7.9% of adults who reported liver disease and 3.4% reporting cirrhosis using a self-completed questionnaire [38]. More recently, over 60% of patients referred to a tertiary reporting service had a history or clinical evidence of liver disease [39]. This may reflect acquisition bias in a specialist centre; however, no features correlated with the histological changes, and no data were available on the clinical significance or prognosis. Thus, the clinical risk of the biochemical and histological features of liver disease in AATD remains unresolved, but the clinical impact is probably low. Factors that determine this highly variable course remain largely unknown. Studies have also indicated an increased risk of hepatocellular carcinoma, although the prevalence and odds ratio is unclear. A more extensive review of the liver in AATD has recently been published [40]. Other rare associations with AATD include panniculitis [41], Wegener’s granulomatosis [42], and ulcerative colitis [43], all of which appear to have a central neutrophilic pathophysiology and, by implication, damage as a result of uncontrolled neutrophilic proteolytic enzymes. Sporadic case reports of other associations are too infrequent to substantiate a link. Epigenetics and genetic modifiers Data suggest that although AATD predisposes to several clinical conditions the outcome is highly variable, indicating that other genetic or environmental factors may play a role (Table 1). Epigenetics refers to changes in the switching on/off of gene expression by factors other than DNA sequence; this might include environmental or lifestyle factors. Methylation is probably the best studied form of epigenetic change, in terms of predisposition to disease. DNA methylation in individuals with AATD has not been widely studied; however, it has already been demonstrated that hypomethylation of SERPINA1, the gene that codes for AAT, is associated with COPD [44]. Modification of genetic signals by environmental factors, such as cigarette smoke, is also recognised and termed gene–environment interaction. It is well known that there is a gene–environment interaction between AATD itself and lung disease, such that individuals homozygous for the PiZ variant may develop lung disease in the absence of environmental modifiers, whereas heterozygous patients (PiMZ) will only develop lung disease if exposed to smoke [45]. Genomewide association studies (GWASs) in the general population were unable to detect the effect of AATD on lung function until a smoking interaction term was added

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Table 1. Key modifiers of pulmonary phenotype in AATDa Disease modifier Cigarette smoke Environmental ozone Environmental particulate matter Occupational exposure to dust/gases/fumes IREB2 rs2568494 GC rs2070741 and rs7041 TNFA rs361525 MMP cluster (various SNPs) GSTP1 rs947894 NOS3 various SNPs Bronchodilator reversibility

Effect More severe pulmonary disease More severe emphysema, more rapid decline of gas transfer More rapid FEV1 decline More rapid gas transfer decline; effect also seen on FEV1 decline in women More severe emphysema Increased incidence of bronchiectasis and airway bacterial colonisation Increased incidence of chronic bronchitis Worse gas transfer Lower FEV1 Lower FEV1 and gas transfer More rapid FEV1 decline

Refs [116] [47,48] [47] [48,117] [52] [53] [54] [50] [118] [119] [25]

a

Abbreviations: IREB2, iron regulatory protein 2; rs, reference number from the Single Nucleotide Polymorphism Database (dbSNP) for the stated polymorphism; GC, Vitamin D binding protein; TNFA, tumour necrosis factor a; MMP, matrix metalloprotease; GSTP1, glutathione S transferase P1; NOS3, nitric oxide synthase 3; SNP, single nucleotide polymorphism; FEV1, forced expiratory volume in 1 s.

[46], further emphasising the importance of this concept. The effect of air pollution and occupation on development [47] and progression [48] of lung disease is also notable in homozygous individuals, although it remains uncertain to what degree this differs from heterozygous patients. More recently, familial patterns of phenotypic variation have been observed, even among individuals with the same AATD genotype [12], even after adjustment for environmental factors, suggesting that other genetic factors play a role. The majority of genetic modifiers studied to date in AATD are single nucleotide polymorphisms (SNPs) known to influence clinical phenotype in usual COPD. This is largely because the majority of work has focussed on candidate genes owing to the limitations of power inherent in studying a rare disease such as AATD, where cohorts are small in comparison to that required for a standard GWAS. Genes or regions thought to influence both AATD and usual COPD include the MMP1/3/12 cluster [49,50] and IREB2 [51,52]. Other genes with suggestive association in AATD that is weaker or differs in COPD include GC [53] and TNFA [54]. Differences in genetic modifiers might be expected in genes coding for proteases or inflammatory cytokines, which are known to interact with AAT in vivo, as the interaction could differ according to protein structure or genotype. Candidate gene studies have inherent limitations, in that they focus on areas known in pathogenesis; GWASs can overcome this issue but would be difficult in AATD without augmenting power [55]. Gene expression data may be used to increase the power of genetic association studies in two main ways: (i) selecting SNPs for typing [51] or (ii) calculation of expression quantitative trait loci (eQTLs) in the context of GWASs [56]. Both methods improve power by reducing multiple statistical tests. New genomic technologies, such as next generation sequencing, can examine the whole genome including non-coding regulatory elements; the clinical utility of such technology has been reviewed elsewhere [57]. Changes in the transcriptome of pulmonary epithelium [58] and alveolar macrophages [59] have been described in COPD, but few functional studies have been conducted. Non-coding RNAs regulate lung development and response to inflammation [60], both processes relevant to COPD and cell specific [61]. None of this has yet been reported in AATD, although such studies are now underway.

Therapy Augmentation therapy The susceptibility of AATD patients to the development of emphysema and the ability of neutrophil serine proteinases to produce many of the features of lung disease in experimental animals suggested cause and effect [2,6,7]. For this reason, the logic of replenishing plasma (and hence lung) AAT was both sensible and subsequently shown to be feasible [62]. The infused half-life of AAT suggested that a reasonable regimen would be weekly infusions to restore AAT protection. However, it was important to maintain the trough levels above the protective threshold, predicted to be approximately 11 mm or 80 mg/dl. This value was chosen as a value between that for the MZ and SZ heterozygotes where there appears to be little or no increased susceptibility [45,63]. The clinical evidence was subsequently supported by in vitro data that suggested an exponential relationship between the amount of neutrophilic destruction of connective tissue and the prevailing AAT level, which has an inflection point at approximately 11 mm [64]. This, together with the ability of such a regimen to normalise lung fluid AAT [65] and reduce lung inflammation [35], provides further biochemical support for a dose of 60 mg/kg/week intravenously. When augmentation was introduced, the gold standard measure for the severity of disease and its progression in AATD was the forced expired volume in 1 s (FEV1). However, the FEV1 is an effort-dependent test with more daily variability than change over several years. For this reason, combined with the general rarity of AATD, a formal trial with FEV1 as the primary outcome was deemed impractical [66]. Therefore, the FDA and regulatory bodies in several countries accepted that biochemical improvement was sufficient to grant a license for treatment. Since then, several observational studies have provided indirect evidence of efficacy. Firstly, a comparison of the decline in FEV1 in patients in Germany (where therapy was available) and Denmark (where it was not) suggested a benefit [67]. Secondly, a German study suggested that when augmentation was given to patients the subsequent decline in lung function slowed down [68]. Thirdly, a comparison of reported decline in cohorts where augmentation was available compared with those where it was not also suggested benefit [69]. 109

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Table 2. Major trials using augmentation therapy in AATDa Study design

Randomised controlled trial

Observational

Number of patients enrolled 56

Clinical findings in the augmentation group

P-value versus control

Refs

AAT 250 mg/kg i.v. every 4 weeks vs placebo for 3 years

Reduced decline in quantitative CT measures of emphysema Reduced FEV1 decline Reduced decline in quantitative CT measures of emphysema Reduced exacerbation rate Reduced FEV1 decline

0.07 0.25

[73]

0.07 0.01

[34]

0.02

[67]

Reduced mortality Reduced FEV1 decline Reduced exacerbations

0.02 0.40 0.04

[70]

FEV1 decline of 57 ml/year

N/A

[121]

77

Prolastin 60 mg/kg i.v. every week vs placebo for 2 years

295

AAT 60 mg/kg/week i.v. for 1 year vs no treatment AAT 60 mg/kg varying frequencies vs no treatment Prolastin or Trypsone 60 mg/kg, varying frequencies for 18 months, compared with 18 months pretreatment AAT 60 mg/kg/week, variable duration of treatment, no control

1048 Case series

Intervention and control groups

127

443

[120]

a

Abbreviations: AAT, a-1-antitrypsin; i.v., intravenous; CT, computed tomography; FEV1, forced expiratory volume in 1 s.

However, perhaps the most quoted data on FEV1 decline came from the National Institutes of Health (NIH) registry. Analysis of FEV1 decline for individuals receiving augmentation was significantly lower in patients whose FEV1 ranged between 35% and 60% of that predicted for age and sex [70]. These data, however, are likely to be influenced, at least, in part, by the socioeconomic modifiers that influence patient accessibility to augmentation. Importantly, this range is where the FEV1 declines fastest [25] and hence where an efficacy signal would be most likely to be detected. The data have not only been used to support the efficacy of augmentation but also to suggest lack of efficacy above and below this FEV1 range. This concept is now being challenged because the measurements of gas transfer progress even when FEV1 is stable [25], and lung density declines steadily throughout the whole of the physiological range [71]. Lung density measurements have now become accepted as a primary outcome measure in the study and management of emphysema in general, because it is the most specific measure of emphysema and the most sensitive to change [72], and hence is the best indicator of the predominant pathology of AATD. Several studies have suggested that augmentation therapy reduces the progression in group mean data using densitometry as an outcome [34,73,74]. Whether this indicates long-term healthcare benefits, or is necessary or effective in all patients remain to be determined. Some of the major augmentation studies are summarised in Table 2. Inhaled therapy The inconvenience of regular infusions of AAT and its large volume of distribution has led to the proposal and study of augmentation by inhalation [75]. Despite the convenience, there has been much debate about the potential benefit or harm. The emphysema process is accepted as one dependent on excessive connective tissue destruction by migrating neutrophils in the lung interstitium. This site is fully amenable to AAT in the plasma, and hence the local concentration would be enhanced by intravenous 110

augmentation. However, even if inhaled AAT could be delivered to the alveolar region, which provides nebulisation challenges, the epithelial barrier would be expected to restrict movement of AAT into the interstitium to provide the necessary protection at this site [76]. Nevertheless, it is possible that such a strategy could prove beneficial by two indirect means. Firstly, neutrophils have to be recruited to the lung in order to cause interstitial damage. Previous studies indicated that leukotriene B4 (LTB4) is one of the major neutrophil chemoattractants in AATD [77]. Free elastase activity in the airways can potentially stimulate airway macrophages to release LTB4 [78] and intravenous augmentation reduces both elastase activity and LTB4 [35]. In this respect, direct inhalation may be more effective at inhibiting airway elastase, and hence LTB4 production, if delivered to the site of the origin of the polymorphonuclear neutrophil (PMN) signal (Figure 2). Secondly, these baseline processes of inflammation and chemoattractant generation are enhanced during acute exacerbations of COPD, but even more so in patients with AATD [28]. These episodes are predominantly focused on the bronchial tree and hence accessible to all forms of nebulisation. Either prophylactic or acute nebulisation for individual events is thus likely to reduce the elastase-dependent inflammation and damage incurred during exacerbations. Whether this would influence the emphysema progression remains unknown; although, total decline in gas transfer is related to the number of exacerbation episodes in AATD [24], inferring potential benefit. Furthermore, recent studies in usual COPD have suggested that the occurrence of exacerbations relates to decline in lung densitometry [79], although whether this reflects cause or effect is currently unknown. A study of inhaled AAT is currently underway and may provide some insight into these unknowns (NCT01217671; www.clinicaltrials.gov). Other strategies for increasing the plasma level of AAT including preventing polymerisation of AAT in the liver, facilitating AAT secretion by chemical chaperones and the

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Inhaled AAT

(3) Macrophage

(1) Elastase

Airway lumen

Epithelial cells Intersal space

(4) (6) (2) LTB4

(5)

Endothelial cells Lumen of blood vessel Circulang neutrophil

Systemic AAT

Systemic marker of damage TRENDS in Molecular Medicine

Figure 2. Mechanisms of disease and biomarkers in a-1-antitrypsin deficiency (AATD). Uninhibited elastase activity in the airways (1) stimulates macrophages to produce the chemoattractant leukotriene B4 (LTB4), which influences neutrophil migration into the interstitium (2). Inhaled a-1-antitrypsin (AAT) (3) would inhibit elastase and block this axis. Migrating neutrophils cause local connective tissue destruction, in part, due to the action of elastase required for them to migrate through tissue (4). Augmentation therapy given intravenously raises local AAT levels in the lung when it diffuses from the blood; this restricts the area of damage (5). Local biomarkers of the proteolytic activity in the interstitial space (6) pass via lymph into the systemic circulation, such that pulmonary processes can be measured in the peripheral blood.

use of synthetic elastase inhibitors are all undergoing exploration as potential therapeutic strategies and have been covered in detail elsewhere [80]. However, the prevention of polymerisation of AAT in the lung itself may be important. Studies have shown that AAT polymers are present in the lung interstitium [81], which may be derived (at least in part) from local production [82]. Because these polymers are able to activate neutrophils they may be responsible for the colocalisation with PMNs [83], generating local proteinase damage. Preventing polymerisation locally may reduce local PMN arrest during transition and hence the degree of associated interstitial damage. Biomarkers Although lung physiology and radiological density are, by definition, biomarkers of lung damage in AATD, the term is commonly used in the context of a biochemical marker or relevant metabolite of the disease process or activity. Identification of biomarkers has enormous potential in the management of chronic lung diseases in general and, specifically, AATD. Markers specific for the presence and extent of the tissue damage leading to progressive lung and liver disease would enable clinicians to predict both the long-term outcome and efficacy of interventions without extended periods of observation. However, to date few such studies have been undertaken. Gamma glutamyl transferase (GGT) is a recognised marker of liver dysfunction, and although increased in heavy alcohol consumers and those with established liver disease [84], it was a poor predictor of future liver problems despite the association of rising levels of GGT with deteriorating liver function. However, GGT was independently associated with airflow obstruction and mortality [84], probably reflecting its role in oxidative lung injury. Indeed, GGT is also a predictor of cardiovascular mortality [85], suggesting a further role as

a non-liver biomarker. Its further use in AATD has yet to be explored. The central role of neutrophilic inflammation, NE, and damage to lung elastin leading to emphysema in AATD suggests that indicators of this process would provide ideal lung biomarkers. Early studies confirmed the presence of increased neutrophilic inflammation in the lungs of patients with AATD [86]. The inflammation in general is greater than in subjects with usual COPD, especially during exacerbations [28]. This is thought to depend on the effects of uninhibited NE in the airways on the inflammatory cascade in general, and the production of local PMN chemoattractants. Studies have confirmed the presence of free NE in airway secretions in AATD [28] and partial resolution of the inflammation with augmentation therapy [35]. This superficial evidence of the biochemical efficacy of AAT augmentation was consistent with the original argument to grant the licence to such therapy in the absence of conventional clinical trials. More specific attempts to mark the tissue destructive process have been limited, despite potentially promising results. Degradation products of tissue elastin (assumed to reflect that in the lung) relate to the severity of emphysema [87], and a preliminary study suggested some response to augmentation therapy [88]. All subsequent studies, however, have been negative, perhaps reflecting the widespread distribution and metabolism of elastin [89]. However, the predictive nature of these measures for long-term progression is currently unknown. More recently, the development of a specific NE degradation product of fibrinogen (Aa360VAL) has been used as a footprint of local inhibited activity of NE [90]. This peptide is increased in AATD, reflects the severity of airflow obstruction, and responds appropriately to augmentation therapy. Further studies are clearly required to determine 111

Review the potential and usefulness of this and other specific biomarkers in AATD. Gene and stem cell therapy Gene therapy The classical form of gene therapy inserts normal genes into cells of patients with a genetic mutation. Studies of AAT gene therapy in animals have used retroviral [91], adenoviral [92–96], adeno-associated viral [97–99], and liposomal [100,101] vectors to transfect cells. Recombinant adeno-associated viral vectors (rAAVs) containing the AAT gene have proven capable of achieving higher therapeutic levels of AAT [97,98], and were less likely to induce an inflammatory response than adenoviral vectors. They also have a specific site for incorporation into the human genome (ASV site), thus giving the genetic material carried by the vector the potential for long-term expression. Routes of administration for gene therapy include liver or airway directed therapy. Liver directed treatment [91,96], such as portal vein injection of the vector [92,98], is likely to be impractical for use in humans if it required repeated delivery at short-time intervals, but might be acceptable if only required once. Airway instillation of the vector suggested that nebulised treatment might be a viable alternative [93,101], but intramuscular injection has been the most successful method to date [97,99]. This method will not influence liver disease because the abnormal protein would still be produced by native liver cells. Phase I human trials have been completed [102,103], and an interim report of the Phase II work seems promising, although further optimisation work is still needed [104]. The dose of vector required means that patients have to undergo a high number of intramuscular injections. Thus, the dose cannot reasonably be increased to try and achieve true therapeutic levels; they remain at 3–5% of target at present. However, intrapleural administration in animals using a non-human primate adenovirus has proven more effective in sustaining levels [105]. A different approach to gene therapy is to direct small DNA fragments to replace the abnormal sequence directly in the liver. This has been optimised in vitro for PiZ-AAT [106] and proven effective [107], but the practicalities and safety of in vivo delivery and efficacy have yet to be addressed. Further developments at the preclinical stage include the use of simultaneous knockdown of Z-AAT with overexpression of M-AAT mediated by use of rAAVs containing miRNA signatures [108]. A similar approach using small hairpin RNA (shRNA) and an AAV has also been tested in mice [109]; these and other aspects of gene therapy have been reviewed in more detail elsewhere [110]. Stem cells Stem cells may be used as a delivery platform for gene therapy to many tissues because of their pluripotent potential. AAT gene transfer has been achieved by transplanting lentivirally transduced haemopoietic stem cells into mice, and demonstrated sustained levels of human AAT expression [111]. Other stem cell strategies have focused on utilising mesenchymal stem cells (MSCs). Hepatocyte-like cells, which were differentiated from human MSCs, were 112

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genetically modified to provide a potential source of transformed hepatocytes for autologous transplantation [112]. Other studies have used lentiviral vectors and rAAVs to transduce bone marrow and adipose-derived stem cells before transplanting them into mouse models with promising results [113,114]. Induced pluripotent stem cells (iPSCs) are differentiated or ‘adult’ cells that are genetically reprogrammed to an embryonic-like state; they have the potential to generate unlimited cells for autologous cell based treatments for many diseases. This technology has been used to correct the point mutation in human iPSCs derived from individuals with PiZZ AATD [115]. Furthermore, the corrected iPSCs could differentiate into hepatocyte-like cells in vitro and were functional when transplanted into the liver of a mouse. This approach could provide sustained AAT gene expression but has a risk of other mutations arising during prolonged iPSC culture. Careful screening would therefore be essential for their safe use in clinical practice. Concluding remarks Although much has been learnt about AATD over the decades, and augmentation therapy has become widely available, much remains unknown. The true natural history of the condition in smokers and never smokers has yet to be determined, and may help explain why some individuals are particularly susceptible to the development of lung disease, fulminant hepatic failure, panniculitis, and Wegener’s granulomatosis, whereas others are not. In addition, the reasons for and variations in the type and distribution of lung disease remain unknown. The assumption that NE is the direct cause of lung problems remains central. However, evidence is emerging that NE is also proinflammatory itself and may be the trigger for a more complex inflammation and proteolytic cascade that leads to pathological lung changes. This indicates the occurrence of phenomena that may not be fully responsive to AAT augmentation therapy. The next few years requires the use of new therapeutic agents, trials, and development of specific biomarkers in order to decide who needs treatment, when, what with, and by which route. The key will be deciding who is at risk of the clinical variants that AATD predisposes to and to intervene specifically to prevent the various manifestations. Disclaimer statement Professor R.A. Stockley has served on advisory boards for Grifols, Kamada, CSL Behring, and Baxter and has received fees for lecturing for Grifols and non-commercial grant funding from Grifols.

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