Animal models for anti-emphysema drug discovery.

Review

Animal models for antiemphysema drug discovery

Expert Opin. Drug Discov. Downloaded from informahealthcare.com by Fudan University on 05/07/15 For personal use only.

Concetta Gardi†, Blerta Stringa & Piero A Martorana 1.

Animal models of emphysema

2.

CS model and drug intervention

3.

Conclusion

4.

Expert opinion

†

University of Siena, Department of Molecular and Developmental Medicine, Siena, Italy

Introduction: Emphysema is characterized by an abnormal and permanent enlargement of airspaces accompanied by destruction of their walls. Up to now, there is no cure for emphysema, and animal models may be important for new drug discovery. Areas covered: Herein, the authors review animal models of emphysema since the protease-antiprotease hypothesis as well as the results obtained with compounds tested in these models. Of particular importance are animal models of cigarette smoke exposure since it is the most important risk factor of emphysema. The authors also analyze two approaches to drug testing, that is, the approach aimed at preventing emphysema and the one aimed at reversing it. Expert opinion: It has been suggested that early and late interventions do not have the same protective effect and that late interventions are much more likely to reveal treatments beneficial in humans. However, this is not always the case, and a compound that prevents emphysema when administered as an early intervention can also have the same protective effect when given as a late intervention. Furthermore, the fact that a compound detected by means of early intervention is now in clinical practice shows that early intervention studies can be predictive for efficacy in humans. Keywords: a-1-antitrypsin, animal models of emphysema, anti-inflammatory agents, antioxidants, chronic obstructive pulmonary disease, cigarette smoke exposure, emphysema, mouse strains, protease inhibitors, reversal of emphysema Expert Opin. Drug Discov. (2015) 10(4):399-410

1.

Animal models of emphysema

Introduction Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality worldwide and is predicted to become the third most common cause of death by 2020 [1]. The pathogenesis of COPD is characterized by an abnormal inflammatory response both in the conducting airways and in parenchyma. Although various environmental pollutants are important causes for the development of this disease, cigarette smoke (CS) is the most important risk factor [2,3]. The CS-induced oxidative stress results in lung inflammation, protease/antiprotease imbalance, accelerated aging and apoptosis [4]. COPD includes emphysema, chronic bronchitis and small airway disease. Emphysema is defined in anatomical terms as ‘a condition of the lung characterized by abnormal, permanent enlargement of air spaces distal to the terminal bronchioles, accompanied by the destruction of their walls and without obvious fibrosis’ [5]. The therapy of COPD is mainly symptomatic relying on the administration of bronchodilators such as b2-agonists or muscarinic receptor antagonists. Also, inhaled corticosteroids are administered in an attempt to inhibit the underlying inflammation. However, the effectiveness of these agents in patients with COPD remains controversial [6]. Inhaled corticosteroids remain the treatment of choice in 1.1

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Since the formulation of the protease-antiprotease theory of emphysema in the early 1960s, a variety of animal models of emphysema have been developed. In an attempt to find a therapy for the human condition, starting > 40 years ago, pharmacological agents have been tested in vivo in models of emphysema. Cigarette smoke (CS) has long been recognized as the major risk factor for the development of emphysema in humans. However, only in 1990, after many unsuccessful attempts, the first animal model of CS-induced emphysema was developed. The knowledge of the advantages and limitations of the animal models of CS-induced emphysema is imperative for the interpretation of the results obtained. In recent years, studies have been published that claim the possibility of not only preventing but also reversing the structural emphysematous changes by means of pharmacological agents.

This box summarizes key points contained in the article.

patients with a high risk of exacerbations. Evidence about other therapies, such as antibiotics or mucolytics, is emerging in selected patients [7]. Recently, roflumilast, a novel selective PDE4 inhibitor, has been approved by both the European Medicines Agency and the US FDA for patients with COPD with severe flow limitations and with symptoms of chronic bronchitis, and it may be useful in reducing exacerbations [8]. However, up to now, there is no cure for COPD. Thus, there is still a medical need for new potential therapeutic targets that may lead to the discovery and development of novel and effective agents for this devastating disease. A bit of history The animal models used for the investigation of either biological or natural products or synthetic compounds with the potential of preventing the development and progression of emphysema obviously reflect the knowledge of the time about the pathogenesis of this disease. The modern era of the knowledge of the development of emphysema started with two publications in the early 1960s. A clinical investigation showed that patients with a genetic deficiency in a-1antitrypsin (A1AT), the major serum protease inhibitor, developed severe, early onset emphysema [9]. Almost at the same time, an experimental study reported that intratracheal administration of papain (a protease with elastolytic activity) to rats resulted in emphysema [10]. Thanks to these two works, the protease/antiprotease hypothesis for the development of emphysema as well as a novel animal model of this disease was born. Thus, starting from the early 1970s agents were tested in animal models of emphysema induced either with an intratracheal administration or with an aerosol of papain [11-13]. This protease digests the elastic fibers of the 1.2

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lung and induces a severe form of emphysema of the panlobular type within 2 -- 3 weeks, offering a very good base for the investigation of inhibitors of the emphysematous lesion. Subsequently, papain was replaced in the 1980s by porcine pancreatic elastase (PPE), a protease with specific elastolytic activity, which when given intratracheally also induces severe panlobular emphysema [14]. Purified human leukocyte elastase (HLE) was then introduced since it was considered to be biologically more relevant for the human disease. Like papain and PPE, HLE was found to cause decreased elastin content and increased airspace size. However, unexpectedly, this protease was found to induce greater pulmonary hemorrhagic lesions but less emphysema than PPE. Increasing the dose of HLE in order to induce more severe emphysema resulted in death of the animals from pulmonary hemorrhage. Different susceptibilities of lung tissue to these enzymes and differing accessibility to alveolar elastin have been considered as possible explanations for the range of severity of the emphysema [15]. Thus, starting in the early 1980s, the investigation of compounds with the potential of preventing emphysema was done in animal models in which the emphysema was induced either with PPE or with HLE. During those years, the most widely tested compounds were the oligopeptides chloromethyl ketones synthesized by the group of powers in Atlanta (US) [16-18]. Eglin-c, a polypeptide purified from the medicinal leech Hirudo medicinalis [19], and furoyl saccharin, a nonpeptidic acylating agent [20,21], were also investigated in these models. The models of papain and PPE-induced emphysema are used sometimes even now since these are models of severe panlobular emphysema, however, mainly for the investigation of some specific pathogenetic, mechanistic or environmental questions. Even though CS had been recognized for a long time to be the main risk factor for COPD in humans, up to the 1980s, exposure of laboratory animals to CS had not succeeded in inducing emphysema. In 1986, Snider et al. [22] wrote that smoke-exposed rats and hamsters developed focal alveolar epithelial hyperplasia and mild fibrosis, but not emphysema. The first unequivocal animal model of emphysema induced by CS exposure was reported in 1990 [23]. Guinea pigs exposed to the smoke of 10 cigarettes/day, 5 days/week for up to 12 months developed progressive emphysema and alterations in pulmonary function tests similar to those seen in patients with COPD. Since then, there has been a large number of studies investigating the development of CS-induced emphysema in various species of laboratory animals. Consequently, investigation in an animal model of CS-induced emphysema should today be the reference preclinical study in drug discovery since this model offers the greatest similarity to the human condition. However, there are various points that should be considered before embarking on a long and costly experimental investigation.

Expert Opin. Drug Discov. (2015) 10(4)

Animal models for anti-emphysema drug discovery

A.

these animal species lack the anatomical basis for the development of centrilobular emphysema. It is always surprising to read in publications the finding of ‘centrilobular emphysema’ in mice exposed to CS.

B.

Strains of mice Different strains of inbred mice respond differently to the exposure of CS, that is, there are susceptible and resistant strains [30-32]. The strain C57Bl/6J has been found to be susceptible and is presently the most widely used in studies of CS exposure. This strain is sensitive to oxidants [30,32], which is probably related to the low activity levels of the nuclear factor erythroid-derived 2, like 2 (Nrf2)-mediated antioxidant pathways [33,34]. Additionally, these mice have a moderate deficiency in serum A1AT (-24%) [35], which makes them more susceptible to the effects of neutrophil elastase (NE). Also, alveolar macrophages isolated from C57Bl/6J mice when exposed to CS extract release more proinflammatory cytokines and MMP than macrophages from a resistant strain such as Institute of Cancer Research and have a higher baseline production of reactive oxygen species [34]. In these mice, a modest emphysema, which is reflected by a slight increase of the mean linear intercept (Lm) (+10.5%) but no decrease in the internal surface area (ISA; a calculated parameter of lung destruction), starts to develop after 3 months of CS exposure [32]. At this time interval, a positive reaction for mouse NE is immunohistochemically seen on the alveolar septa. Similarly, a mild apoptosis can be observed in the lungs of these mice. Also, at 3 months, 75% of C57Bl/6J mice develop goblet cell metaplasia in their large and middle size bronchi. Six to 7 months of smoke exposure results in overt emphysema in C57Bl/6J mice. The lesion is characterized by disseminated foci of severe emphysema interspersed by apparently normal parenchyma. The increase of the Lm ranges from +14 to +17.5% to 21.3% [30,32,36]. In other laboratories, the increase of the Lm ranges from 13 to 38% [31,37]. The magnitude of the decrease of the ISA is usually lower. However, much more sensitive as a parameter of lung parenchyma destruction is the destructive index that increases by +213% [30]. Additionally, the lungs of CS exposed C57Bl/6J mice have significantly lower lung elastin values than those of control animals [30]. Thus, chronic exposure of mice to CS closely mimics the human condition. However, there are some limitations, which we should be aware of. Mainly, the model is suitable for the investigation of potential drugs on emphysema and small airways remodeling [38,39] but not on large bronchi changes. Additionally, the severity of the parenchymal lesion is usually moderate.


1.3.2

Figure 1. Emphysema in rodents. An anatomical characteristic of the rodents is that they lack the respiratory bronchioles. Thus, the terminal bronchioles lead directly into alveolar ducts (left micrograph). Since centrilobular emphysema is a lesion of the respiratory bronchioles, these animal species lack the anatomical basis for the development of centrilobular emphysema. Administration of an emphysema-inducing agent (in this case an aerosol of papain) induces a form of panlobular emphysema characterized by enlargement of the air spaces distal to the terminal bronchioles, accompanied by destruction of the alveolar walls (right micrograph). Reproduced from [29] with permission of NRC Research Press.

1.3

How to choose the animal model Animal species

1.3.1

The first point is the choice of the animal species. As reported in a recent review on animal models of smoke exposure [24], in a list of 155 analyzed studies a large portion (115 studies) was carried out in mice. The advantages and/or disadvantages of the mouse as animal of choice have been previously listed [25,26]. Advantages are the relative low cost, the rapid reproductive cycle and large litter sizes. Also, mouse biology has been extensively studied, and inbred strains and their mutants are available. Another additional advantage of the mouse is the possibility of gene manipulation with the induction of gain or loss function. A potential disadvantage is that mice are obligatory nose breathers. This means they have a different pattern of particle filtration from that of mouth breathers such as humans [27]. Submucosal glands are absent in the mouse, and goblet cells are found mainly in the trachea. Thus, the appearance of goblet cells in their bronchi should be considered as metaplasia. Due to these anatomical features, the mouse is not a good model of the bronchial changes of COPD. On the other hand, in guinea pigs submucosal glands are irregularly distributed in their bronchial tree and goblet cells are present [28]. Another anatomical characteristic of the mouse lung is the lack of respiratory bronchioles, a feature that is shared by other rodents (Figure 1) [29]. Since centrilobular emphysema is a lesion of the respiratory bronchioles [5],

Age and gender Although the reason for the lack of a more severe lesion is not known, one can speculate that while very young and healthy animals with all their defensive and reparative processes intact are exposed to CS, smokers with COPD are usually elderly persons either underweight or obese and possibly with 1.3.3


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diabetes and a heart condition. This is a general problem involving most animal models of human diseases. The logical comment about these points would be: let’s use older mice. In a recent study, investigating the effect of age on the outcome of the pulmonary lesions, C57Bl/6 mice were exposed to CS for 6 months starting at age 3 months or age 12 months with air-exposed controls. There was no difference in airspace size between the two control groups, and CS induced exactly the same amount of emphysema in young and old mice. The severity of smoke-induced small airway remodeling was also identical in both groups [39]. Thus, the question of the age appeared to be resolved; however, with a mean life span of 24 months, 12-month-old mice may be a bit young. To our knowledge, there are no CS studies that have been carried out on mice with either metabolic or cardiovascular diseases. Gender may also be of importance in the development of emphysema. Males and females from CS- or papain-induced animal models of emphysema [40,41] as well as men and women smokers [42] seem to respond differently in type and localization of lung damage, suggesting that sex-related differences should be taken into account. 1.3.4

Cigarettes and exposure systems

Other important points to consider in planning an experiment on CS exposure are the type of cigarette and the exposure method (either nose only or whole body) to be used. Since the late 1960s, the Kentucky University has developed a program for the production of reference cigarettes to be used in experimental studies. There are a few types of these cigarettes such as 1R1, 1R3F (F is for filter), 2R4F (low nicotine), 1R5F (ultra low nicotine) and 3R4F. A welldocumented list of these cigarettes can be found under reference 24. The obvious goal for the development of these reference cigarettes was an attempt to standardize the noxa of smoking among the various laboratories. However, in a recent publication on animal models of COPD, Wright et al. wrote, ‘It is our impression that currently available cigarettes (such as Kentucky 2R4F or 2R5F) cause considerably less emphysema than older ones (such as Kentucky 1R1 or 2R1), and this is a serious problem, both in terms of producing disease and particularly in terms of examining interventions since one never knows whether interventions that ameliorates small increases in air space size would really be effective with more severe emphysema’ [28]. In our laboratory, we chose to use commercial cigarettes such as Marlboro red (Virginia filter cigarettes: 12 mg of tar and 0.9 mg of nicotine) mainly for two reasons: their use probably better mimics the human situation and their greater availability. Mice are exposed to CS delivered by a smoking machine by ‘nose only’ [43] or ‘whole body’ [32] methodology. Both methods have been extensively used. One would expect that the ‘nose only’ method with controlled delivery smoke concentration should result in more homogeneous and severe lung changes; however, both methods produce a relatively similar pathology [24]. This is consistent with the notion that mice 402

are obligatory nose breathers; thus, the way in which (by ‘nose only’ or by ‘whole body’) the animals are exposed to CS does not make too much difference because both methodologies model ‘second hand smoking’ [27]. It is also worth noting that the ‘nose only’ methodology requires the daily immobilization of the animals for the smoke inhalation period, and this has been found in C57Bl/6 mice to result in hypothermia and stress (reduced body weight gain and increased adrenal weight) [44]. The duration of the smoke exposure varies with the susceptibility of the strain of mice used. The length of the exposure for the C57Bl/6J mice is usually 6 -- 7 months. At the end of the exposure period, the assessment of the lung lesion must include some morphometrical parameters since emphysema is defined in anatomical terms. Lm is widely used as a measure of air space enlargement [45] and so are the ISA of the lung [46] and the destructive index modified for the use on laboratory animals [47] as indices of lung destruction. 2.

CS model and drug intervention

CS-induced emphysema is presently the animal model of choice for testing compounds with the potential of ameliorating this disease. This report presents a selected review of the literature of these studies according to their pharmacological approach as we have done in a previous work [48]. The first approach consists of pharmacological interventions designed at slowing down the rate at which alveolar wall is lost in emphysema, that is, substances that interrupt the chain of events leading to destruction of the extracellular matrix of the lung and loss of respiratory units. The second approach is an attempt to reverse the process of alveolar loss by inducing alveolar growth and regeneration. First approach: prevention The studies listed here are also classified either as ‘early intervention,’ if the administration of the compound starts on day 1 of the smoking period, or as ‘late intervention,’ if the administration starts at a later time, usually no earlier than 3 months in a 6-month exposure [49]. The studies mentioned here are also provided in a summarized form in Table 1. 2.1

Protease inhibitors Various protease inhibitors were investigated. Human A1AT was injected intraperitoneally every 48 h at one dose level to transgenic mice that expressed extremely low levels of human A1AT but were tolerant to exogenous human A1AT. The mice were exposed to CS for 6 months. Treatment with A1AT produced an approximate twofold increase in serum A1AT levels and elastase inhibitory capacity and abolished smoke-induced elevations in lavage neutrophils and matrix breakdown products. A1AT oxidized to remove antiproteolytic activity did not increase serum elastase inhibitory capacity but did prevent neutrophil influx. Treatment with A1AT for 6 months provided 63% protection against increased 2.1.1



Table 1. Effect of various substances on cigarette smoke-induced emphysema.


Substance

A1AT ZD0892 AZD9668 Ilomastat MnTE-2-PyP AZ1 Curcumin NAC, MPG Infliximab Roflumilast GPD-1116 Celecoxib Clarithromycin Simvastatin BAY compounds Hyaluronan

Type of compound

Protection

Elastase inhibitor Elastase inhibitor Elastase inhibitor MMP inhibitor SOD mimetic MPO inhibitor Polyphenol Nonenzymatic antioxidants Anti-TNF-a antibody PDE4 inhibitor PDE4 inhibitor COX2 inhibitor Macrolide antibiotic HMGCoA reductase inhibitor sGC stimulators Glycosaminoglycan

Ref.

Early intervention

Late intervention

+ + + + + + + + n.i. + + + + + + +

n.i. + n.i. n.i. + n.i. n.i. + + n.i. n.i. + + n.i. +

[49,50] [51] [52] [53] [55] [56] [57] [59] [61] [35,78] [63] [64] [65] [66,67] [68] [69,70]

(+): Protection; (-): No protection; A1AT: a-1-antitrypsin; HMGCoA: 3-hydroxyl-3methyl-glutaril-coenzyme A; MPG: N-(2-mercaptopropionyl)-glycine; MPO: Myeloperoxidase; NAC: N-acetylcysteine; n.i.: Not investigated; sGC: Soluble guanylate cyclase; SOD: Superoxide dismutase.

airspace size (emphysema) and abolished smoke-mediated increases in plasma TNF-a. The authors concluded that A1AT therapy ameliorates smoke-induced inflammation and matrix breakdown, possibly via an anti-inflammatory mechanism related to TNF-a suppression, and provides partial protection against emphysema [50]. Similarly, human A1AT administered by inhalation directly into the lungs protected mice from the development of emphysema induced by a 6-month CS exposure [51]. The activity of the orally active inhibitor of serine elastase ZD0892 was investigated in a model of CS-induced emphysema in the guinea pig by using the early as well as the late intervention protocol. A 6-month smoke exposure produced emphysema and increases in lavage neutrophils, desmosine, hydroxyproline and plasma TNF-a. With the early intervention protocol ZD0892 prevented the increases in lavage neutrophils, desmosine and hydroxyproline as well as emphysema (by 45%) and plasma TNF-a (by 30%). Animals exposed to smoke for 4 months and to smoke plus ZD0892 for 2 months (late intervention protocol) were not protected against emphysema [52]. Also, the oral administration of AZD9668 (an NE inhibitor) reduced the inflammatory response to CS in mice and rats, as indicated by a reduction in bronchoalveolar lavage (BAL) neutrophils and IL-1b, and prevented airspace enlargement and small airway wall remodeling in guinea pigs after chronic CS exposure, whether given as early or late intervention [53]. Among proteases, another interesting target is represented by MMP. Ilomastat, a broad-spectrum MMP inhibitor, was administered by inhalation in mice, and the animals were exposed to daily CS for 6 months. CS significantly increased the recruitment of macrophages into the lungs of these animals, leading to concomitant alveolar airspace enlargement and emphysema. In animals treated daily with nebulized

ilomastat for 6 months, lung macrophage levels were greatly reduced and neutrophil accumulation was also inhibited. Corresponding reductions in airspace enlargement of up to 96% were observed [54]. Thus, both serine protease and MMP inhibitors were found to protect against CS-induced emphysema and also to reduce the influx of inflammatory cells into the lungs. Antioxidant compounds A certain number of studies investigated the potential protective effect of antioxidants against CS-induced lung damage [55]. The role of extracellular superoxide dismutase (SOD3) in the development/progression of emphysema was investigated in mice. SOD3-knockout (KO), SOD3-transgenic and wild type (WT) C57Bl/6J mice were exposed to CS for 3 days -- 6 months. In addition, groups of SOD3-KO and WT mice received daily subcutaneous injections of a SOD mimetic compound (MnTE-2-PyP). CS exposure caused airspace enlargement as well as impaired lung function and exercise capacity in SOD3-null mice, which were improved in mice either overexpressing SOD3 or treated with the SOD mimetic agent. These phenomena were associated with SOD3-mediated protection against oxidative fragmentation of extracellular matrix, such as heparin sulfate and elastin, thereby attenuating lung inflammatory response. The authors concluded that SOD3 attenuates emphysema and reduces oxidative fragmentation of extracellular matrix in mouse lung [56]. Myeloperoxidase, a neutrophil and macrophage product, is not only important in bacterial killing but also drives inflammatory reactions and tissue oxidation. The effects of AZ1, a 2-thioxantine myeloperoxidase inhibitor, were investigated in guinea pigs exposed to CS for 6 months. One group of animals received AZ1 from smoking on day 1 and another 2.1.2


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group after 3 months of smoke exposure. At 6 months, both treatments abolished smoke-induced increases in lavage inflammatory cells and prevented or stopped progression of emphysema. Protection appeared to be related to inhibition of oxidative damage and down-regulation of the smokeinduced inflammatory response [57]. Among natural compounds, dietary polyphenols have been reported to attenuate the development of emphysema due to their anti-inflammatory and antioxidant properties. In C57BL/6J mice, oral curcumin administration attenuated CS-induced pulmonary inflammation and emphysema. In fact, curcumin treatment (1 h before each CS exposure) significantly inhibited CS-induced increase of neutrophils and macrophages in BAL fluid after 10 days of CS exposure and significantly attenuated CS-induced airspace enlargement after 12 weeks of CS exposure [58]. The effect of nonenzymatic antioxidant drugs, such as N-acetylcysteine (NAC) and N-(2-mercaptopropionyl)glycine (MPG), on emphysema were also investigated. NAC is a potential drug in the treatment of COPD, and it acts as an antioxidant to prevent lung injury [59]. In a CS model in C57Bl/6J mice, both NAC and MPG were found to reduce parenchyma destruction [60]. All these results taken together indicate that antioxidant interventions have some anti-inflammatory effects and could prevent the development of CS-induced emphysema. Anti-inflammatory drugs Since chronic inflammation plays a pivotal role in the pathogenesis of COPD and emphysema [4], it was to be expected that substances with anti-inflammatory activity would be investigated in laboratory animals chronically exposed to CS. Cytokines play a pivotal role in many processes that lead to the development of COPD [61]. For example, TNF-a is believed to play an important role in the induction of lung inflammation involved in the pathogenesis of emphysema. The effect of infliximab, an antibody against TNF-a, on CS-induced emphysema was investigated in rats exposed to CS for 74 days. After 30 days of smoke exposure, the rats were injected subcutaneously every 10 days with infliximab. Infliximab partially prevented CS-induced emphysema, and this effect was accompanied by a reduction in lung inflammation and alveolar septa cell apoptosis [62]. PDEs are a large family of intracellular enzymes that degrade cyclic nucleotides. The PDE4 subtype specifically targets cAMP, a second messenger that exerts inhibitory effects on many inflammatory cells. Thus, substances that prevent the degradation of cAMP by inhibiting the activity of PDE4 will potentiate the anti-inflammatory action of this second messenger. Therefore, it was postulated that these substances may also limit inflammation in COPD and ameliorate emphysema. The specific PDE4 inhibitor roflumilast was given either at 1 mg/kg (low dose) p.o. or at 5 mg/kg (high dose) p.o. to C57Bl/6J mice, and the animals were exposed to CS for 7 months. Chronic smoke exposure caused a significant 2.1.3

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recruitment into the lungs of inflammatory cells of both the innate and adaptive immune system, emphysema and a drop in lung desmosine content. Low dose prevented the recruitment of dendritic cells and T-lymphocytes but did not prevent emphysema. High dose prevented the increase of all these types of cells and fully prevented the development of emphysema and the loss of lung desmosine content [36,63]. The effect of GDP-1116, another PDE4 inhibitor, was investigated in the senescence-accelerated mouse (SAM)P1. GDP-1116 was given orally and the mice exposed to either fresh air or CS for 8 weeks. GDP-1116 attenuated smoke-induced emphysema by inhibiting the increase of smoke-induced MMP-12 activity and protecting lung cells from apoptosis [64]. Celecoxib, a specific COX2 inhibitor, is widely used to treat inflammation. In a recent study [65], celecoxib was given via intragastric feeding daily for 20 weeks to rats exposed to CS. In this model, celecoxib prevented the increase of Lm, attenuated lung inflammation, inhibited serum NO production, iNOS, COX2 expression and PGE2 production induced by the CS exposure. Furthermore, celecoxib attenuated the activation of phospho-IkBa and NF-8B in CS-exposed rats. The authors concluded that the protective effect of celecoxib was mediated by its effects on NF-8B-regulated gene expression, which ultimately reduced the development of CS-induced pulmonary emphysema [65]. Clarithromycin is a macrolide antibiotic that has been reported to ameliorate chronic inflammation in various animal models, via mechanisms independent of its antibacterial activity. The effect of clarithromycin against CS-induced emphysema was investigated as early intervention in mice exposed to CS daily for 6 months and treated with clarithromycin (25 -- 100 mg/kg p.o. b.i.d.). Also, clarithromycin (50 or 100 mg/kg) was administered during the second half of a 6-month exposure period as late intervention. Drug treatment for 6 months prevented airspace enlargement and the destruction of the alveolar walls and impaired the accumulation of macrophages in BAL fluid. The administration of clarithromycin as late intervention reduced emphysema compared with the smoke-exposed group without treatment. In vitro, clarithromycin suppressed the activation of macrophages stimulated with TNF-a. It was concluded that clarithromycin prevented CS-induced emphysema in both study protocols by modulating lung inflammation [66]. Statins are 3-hydroxy-3-methyl-glutaryl-coenzyme-A reductase inhibitors that have pleiotropic pharmacologic properties, including anti-inflammatory and antioxidant actions. The effect of simvastatin given orally was investigated as an early intervention in a rat model of emphysema induced by exposure to CS for 16 weeks. Simvastatin inhibited lung parenchymal destruction and development of pulmonary hypertension (PH) and also inhibited peribronchial and perivascular infiltration of inflammatory cells and induction of MMP-9 activity in lung tissue. Simvastatin additionally prevented pulmonary vascular remodeling and the changes in endothelial iNOS expression induced by smoking [67]. It was also tested in guinea




Table 2. Reversal of cigarette smoke-induced emphysema in mouse models. Substance

Strain

Study protocol

Effect

Ref.

L-NIL

C57Bl/6J

Reversal

[73]

Vitamins C and E

C57Bl/6J

Reversal

[74]

Vitamin C

SMP30-KO

Reversal

[75]

Ciglitazone

C57BL/6J

Reversal

[76]

LGF

AKR/J

Smoke exposure: 8 months Treatment: 3 months Smoke exposure: 60 days Treatment: 60 days Smoke exposure: 2 months Treatment: 2 months Smoke exposure: 5 months Treatment: 2 months Smoke exposure: 6 months Treatment: 2 weeks

Reversal

[77]

LGF: Liver growth factor; L-NIL: N6-(1-iminoethyl)-L-lysine.

pigs exposed to CS for 6 months. The drug was given orally at one dose level starting 3 months after the beginning of the exposure. It prevented the smoke-induced emphysema and reversed the smoke-induced PH [68]. The potential involvement of the soluble guanylate cyclase (sGC) in the development of CS-induced emphysema and PH was investigated in the mouse and in the guinea pig. Mice exposed to CS for 6 months were treated with BAY 63-2521 (methyl N-[4,6-diamino-2-[1-[(2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-pyrimidinyl]-N-methyl-carbaminate), while guinea pigs were exposed to CS from 6 cigarettes/day for 3 months and treated with BAY 41-2272 (pyrazolopyridine 5-cyclopropyl-2-[1-(2fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-4ylamine). Both BAY compounds are sGC stimulators. Both sGC stimulators prevented the development of PH and emphysema in the two different CS-exposed animal models. sGC stimulation prevented peroxynitrite-induced apoptosis of alveolar and endothelial cells, reduced CS-induced inflammatory cell infiltrate in lung parenchyma and inhibited adhesion of CS-stimulated neutrophils. The authors concluded that treatment of COPD animal models with sGC stimulators can prevent CS-induced PH and emphysema [69]. Thus, in the animal model, the inhibition of the inflammatory process by agents acting on different pathogenic pathways results in a protective effect against the development of emphysema. Other interventions A different and interesting approach has also been reported. Hyaluronan (HA) belongs to a family of structurally similar polysaccharides called glycosaminoglycans. It is a matrix substance in which fibrous constituents of the matrix such as elastin and collagen as well as cells are embedded. A study was designed to determine if aerosolized HA could prevent emphysema development and elastic fiber injury in mice exposed to CS for 6 months. HA treated mice were protected against emphysema and elastic fiber breakdown products (desmosine and isodesmosine) in BAL fluid. The authors

concluded that protective effect of HA may have been related to its ability to bind to lung elastic fibers, thereby, preventing their breakdown by elastases [70]. In addition, when the administration of HA was delayed and started 1 month after the beginning of smoke exposure, HA also significantly reduced the severity of emphysema [71]. Second approach: reversing emphysema As mentioned earlier, the second approach is an attempt to reverse the process of alveolar loss by inducing alveolar growth and regeneration. A landmark study showed in 1997 that the adult mammalian lung is able to form new alveolar walls [72]. In that study, the authors used rats instilled intratracheally with elastase. This resulted in lung changes characteristic of human emphysema: increased lung volume, larger but fewer alveoli and diminished volume-corrected surface area due to destruction of alveolar walls. Treatment with an analogue of vitamin A, all-trans-retinoic acid, reversed these changes providing remediation of emphysema and suggesting the possibility of a similar effect in humans. This was the beginning of a novel therapeutic approach to emphysema, that is, new alveolar formation [73]. The methodology here does not differ from that used in the studies presented under first approach. Here, however, the results obtained after the administration of a compound should indicate a reversal rather than a slowing down of the development of emphysema. The studies mentioned under this heading are also entered in a summarized form in Table 2. 2.2

2.1.4

iNOS inhibition As reported for simvastatin [67] and celecoxib [65] earlier, some anti-emphysema compounds also have an inhibiting action on iNOS. In an interesting study, C57BL/6J mice were exposed to CS for 8 months. This resulted in increases in the Lm and percentage air space and decreases in the number of alveoli and septal wall thickness. Treatment with the iNOS inhibitor N(6)-(1-Iminoethyl)-L-lysine dihydrochloride in drinking water for 3 months without further smoke exposure restored lung structure and selectively reduced the number of 2.2.1


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granulocytes, macrophages, activated macrophages and T cells in the lung compartment. In addition, investigation and quantification of elastic fibers structure revealed destruction of elastic fibers during tobacco-smoke exposure and regeneration upon N(6)-(1-Iminoethyl)-L-lysine dihydrochloride treatment. The authors concluded that CS-induced emphysema occurs in an iNOS-dependent manner in mice and that selective iNOS inhibition offers the potential to reverse emphysema [74]. Vitamins C and E Vitamins C and E are potent antioxidants that can modulate the oxidant--antioxidant imbalance induced in lungs by CS. C57BL/6 mice were exposed for 60 days to CS and supplemented with vitamins C and E for a further 60 days. Supplementation of the vitamins reduced oxidative stress (evaluated as catalase and SOD activity) and inflammation (neutrophil count and TNF-a). Finally, vitamins C and E improved lung repair after emphysema, reducing Lm and stimulating regeneration of collagen and elastic fibers [75]. In a more recent work, the effect of vitamin C on CS-induced emphysema was investigated in senescence marker protein-30 knockout (SMP30-KO) mice, which cannot synthesize vitamin C. Additionally, SMP30-KO mice manifest a shorter-than-normal lifespan, during which they develop emphysema within 8 weeks of exposure to CS due to excessive oxidative stress. Exposure to CS for 2 months resulted in significant emphysema in these mice. The animals no longer underwent smoke exposure but received in drinking water either a minimal or a physiological dose of vitamin C for 2 months. Smoke-induced emphysema persisted in the low vitamin C group after smoking cessation, whereas the physiological dose of vitamin C provided pulmonary restoration (18.5% decrease of the Lm and 41.3% decrease of destructive index). This dose of vitamin C diminished oxidative stress, increased collagen synthesis and improved VEGF levels in the lungs. These results indicate that vitamin C is able to restore CS-induced emphysema [76].


2.2.2

PPARg agonists Recent studies have indicated a role for acquired immunity in the development of emphysema in humans and mice and a potential contributor to the immune dysregulation observed in emphysema is PPARg. Thus, a study was carried out to investigate if ciglitazone, a PPARg agonist could reverse CS-induced emphysema. C57Bl6 mice were exposed to 3 months of CS followed by intranasal treatment with ciglitazone twice a week for 2 months, while continuing to be exposed to smoke. After a total of 5 months of smoke exposure, mice treated with ciglitazone showed reduced emphysema, as quantified by reduction in lung volume and Lm [77]. 2.2.3

Liver growth factor Liver growth factor (LGF) is an albumin-bilirubin complex with antifibrotic, antioxidant and antihypertensive actions. The effect of LGF was investigated in AKR/J mice exposed 2.2.4

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to CS for 6 months, which developed pulmonary emphysema and inflammation. After 6 months, the animals were injected intraperitoneally twice a week for 2 weeks with purified LGF. The authors reported that LGF treatment returned the Lm to control values [78]. However, in the manuscript it is not clear if the authors used historical data for the morphometric changes obtained at 6 months (before the beginning of the treatment). If this were the case, one could question the obtained results, particularly since the delta percent change of the Lm in the smoked animals at 6 months was modest (+12.1%), and such small changes may have poor reproducibility. This study shows how important a transparent presentation of the methods and results is, particularly when the claim of the study is a revolutionary reversal of established emphysema. 3.

Conclusion

Despite the limitations, the use of animal models continues to be important for the study of the pathogenetic mechanisms underlying the development of emphysema and for the evaluation of new drugs. The choice of the animal model must take into account a number of variables, including the species, age and sex as well as the type of substance that induces emphysema and the duration of exposure. Among models currently proposed, the CS animal model remains the one closest to the human situation. Current pharmacological interventions aim at limiting pulmonary emphysema mainly through two types of approaches: the prevention of parenchymal destruction and/or the attempt to reverse the process of alveolar loss by inducing alveolar growth and regeneration. This paper provides a selected review of the results obtained in these studies (Figure 2). 4.

Expert opinion

Recently, Churg et al. published an interesting, thoughtprovoking and controversial article entitled ‘Everything prevents emphysema: are animal models of CS-induced COPD any use?’ [49]. With regard to the prevention of emphysema, the authors are of the opinion that ‘an early intervention and a late intervention are not to have the same protective effect’ (against emphysema) and that ‘animal models employing some kind of late intervention are much more likely to point to treatments that actually may be of benefit in humans’. These statements intuitively appear to be so right that arguing against may seem useless. However, it is well known that CS-induced emphysema develop very slowly and that its severity is only modest to moderate and hardly increases even after long exposure periods. In our own experience by using two strains of mice (C57Bl/6J and DBA/2) exposed to CS for 10 months, we found that after 1 month there were practically no changes in the delta percent of the Lm in both strains. After 3 months, there was a 10.5% increase of the Lm in the C57Bl/6J mice and an 18.9% increase in the DBA/2. After 6 months of CS exposure, the increase of the Lm was



Antiinflammatory treatments:

Antioxidants: • SOD mimetics (cigarette smoke, other pollutants)

• Anti-cytokines

• Polyphenols

• PDE4 inhibitors • COX2 inhibitors

• MPO inhibitors

Oxidative stress

Inflammation

• Nonenzymatic antioxidants

• Clarithromycin • Simvastatin • sGC stimulators

Proteaseantiprotease imbalance

Reversing compounds:


• Retinoic acid Antiproteases:

• Vitamins C and E

• A1AT

• iNOS inhibitors

• NE inhibitors

• PPARγ agonists

• MMP inhibitors Emphysema

• LGF

Figure 2. Potential therapeutic targets and pharmacological agents found to be effective in animal models of emphysema. A1AT: a-1-antitrypsin; LGF: Liver growth factor; MPO: Myeloperoxidase; NE: Neutrophil elastase; iNOS: Nitric oxide synthase; sGC: Soluble guanylate cyclase; SOD: Superoxide dismutase.

17.7 and 21.4% in C57Bl/6J and DBA/2 mice, respectively. At 10 months, the Lm of the C7Bl/6J did not increase further; actually, there was a small decrease of the delta percent (10.4%). This was mainly due to an increase of the Lm of the control animals, an observation that we made repeatedly. In the DBA mice, at this time period, there was a slight further increase (23.9%) of the Lm. The magnitude of the decrease of ISA was lower than that of the increase of the Lm [32]. These data, coupled with the data of the literature, suggest that due to the slow development and progression of the emphysema it does not really make any difference if the potential antiemphysema compound is applied at day 1 or at day 90 of the exposure period. In both cases, it is prevention of a modest-to-moderate developing lesion. This is reflected by the fact that, with few exceptions, compounds that are effective when administered as early intervention are also effective when administered as late intervention. Thus, it is difficult to understand why early and late interventions do not have the same protective effect and why late interventions should more likely point to treatments that may be of benefit to humans [49]. In our own experience, roflumilast was found to be effective when administered starting at day 1 (early intervention) [36] as well as when administered starting at 4 months after the beginning of smoke exposure in a 10-month

exposure study (late intervention). In another study, roflumilast, given at the same inhibitory dose as in the early intervention study, fully prevented progression of airspace enlargement during the additional 6 months of CS exposure [79]. Thus, these results indicate that a compound that prevents emphysema when administered as an early intervention can indeed have the same protective effect when given as a late intervention. Furthermore, since roflumilast is in clinical practice, these results also show that early intervention studies can provide predictive power for efficacy in humans. Also, simvastatin was reported to prevent emphysema development when given as early intervention in the rat [67]. The efficacy of this compound is presently being tested in clinical trials. The results are controversial, ranging from protection to lack of effect [80-82].

Declaration of interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.


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Affiliation

Concetta Gardi†1 MSc PhD, Blerta Stringa2 MSc & Piero A Martorana3 DVM MSc † Author for correspondence 1 Assistant Professor of General Pathology, University of Siena, Department of Molecular and Developmental Medicine, Via Aldo Moro 2 - Siena, Italy Tel: +39 0 577 234002; E-mail: [email protected] 2 Research Fellow, University of Siena, Department of Molecular and Developmental Medicine, Via Aldo Moro 2 Siena, Italy 3 Retired, Previously Pharmaforschung Hoechst AG, Frankfurt/M, Germany

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