Idiopathic Pulmonary Fibrosis: An overview of the disease, current and developing treatmentsIdiopathic Pulmonary Fibrosis (IPF) is a progressive fibrotic Interstitial Lung Disease (ILD) of unknown cause. Extensive scarring of lung tissue impairs function and as there is no curative treatment prognosis is very poor [1]. It is difficult to diagnose and is not well understood as there are very limited resources dedicated to research when comparing to cancer research [2]. After a brief introduction on IPF, this paper will discuss current therapies, recent clinical developments, and future developments through pre-clinical evaluations.

PathophysiologyThe epithelium becomes activated in the lungs in IPF which can trigger epithelial to mesenchymal transition (whereby epithelial cells behave like mesenchymal cells) and the release of TGF-β [3]. The differentiation and migration of fibroblasts and myofibroblasts is triggered by:• clotting factors• mediators released by activated epithelial cells (e.g. TGF-β, PDGF, Wnt)• cells which have undertaken epithelial to mesenchymal transitionFibroblasts and myofibroblasts may even differentiate directly from epithelial cells and are altered by mutations, often involving epigenetic changes [4]; they evade apoptosis, proliferate and generate extracellular matrix at a rapid rate.Myofibroblasts are more fibrotic than fibroblasts. These secrete large amounts of extracellular matrix rich in type I collagen. As the excess extracellular matrix is deposited this may act as a positive feedback mechanism for further deposition of collagen. Myofibroblasts also disrupt the basement membrane and the lung architecture through matrix metalloproteinases [5]. The result of fibroblast activation is the development of dispersed areas of fibrosis known as fibroblastic foci.Underlying this is an inability of the lung to remove the damaged tissue as it develops. Abnormalities in the immune system may enhance fibrosis and this is currently under investigation.DiagnosisThe American Journal of Respiratory and Critical Care Medicine has defined evidence based guidelines for diagnosing IPF [6]. As is the case

with many idiopathic conditions, diagnosing IPF is not straightforward and usually requires a multidisciplinary team (MDT) involving a chest physician, a radiologist, a histopathologist, a lung surgeon and a specialist nurse [7]. The difficulty lies in the fact that this is essentially a differential diagnosis. The MDT must establish that the patient suffers from ILD that is not caused by any known cause such as environmental exposure to irritants, connective tissue disease or drug toxicity. Further investigations should include High-Resolution Computed Tomography (HRCT) and, in some cases, a surgical lung biopsy; showing a pattern of Usual Interstitial Pneumonia (UIP), a subtype of ILD that includes IPF and is characterised by fibrosis in specific patterns [6].Clinical FeaturesThe main features found in IPF patients are (in decreasing order of relevance): age > 45, persistent breathlessness on exertion, persistent cough, bilateral inspiratory crackles and finger clubbing. History taking and examination, lung function testing and blood testing are essential in diagnosing ILD and finding known causes of ILD hence ruling out IPF [7].Typical features of clinical presentation for IPF patientsName of study authors Lin Pan et. al. [8]

Age

Sex (M/F)

Smoker

PRESENTING SYMPTOMS

Cough

Sputum

Dyspnoea

Fatigue

Haemoptysis

Chest pain

Recurrent unexplained fever

Leg/foot swelling

Clinical findings were recorded on clinical presentation for 75 patients diagnosed with IPF [8].ImagingThe next step in investigation should be referring the patient for a chest X-ray and an HRCT scan. X-rays are often done first and provide a basis for referring to an HRCT scan, looking for evidence of honeycombing, a common feature in most IPF patients [6].                 Typical HRCT and Chest X-RAY for an IPF patient [9] Figure 1. HRCT scan(left) showing the subtle difference between honeycombing (*, posterior part of the right lung) and emphysematous lesions (arrowhead, lateral left lung). The X-RAY(right) shows signs of reticular opacity (blurring caused by an intraparenchymal process [9]) which is also indicative of UIP [5]. Reproduced with permission from [9], Copyright Massachusetts Medical Society. At this stage there can be a diagnosis. However, often the investigations are inconclusive and further testing is required [7].Lung BiopsiesBronchoscopic and transbronchial biopsies are also tools that help make alternative diagnoses, but the most accurate diagnostic tool is a surgical lung biopsy. A surgeon obtains tissue directly from the lung which is then analysed by a histopathologist who will look for UIP-specific patterns [7].                      Typical histological slides for an IPF patient [9] Figure 2. (A) shows the presence of heavily fibrosis tissue. (B) is further magnified and shows the close anastomosis of a fibroblastic focus (*) with normal alveolar tissue (arrow). These features are characteristic of IPF. Reproduced with permission from [9], Copyright Massachusetts Medical Society.The added information from performing a surgical biopsy is usually enough for the MDT to reach a conclusion. However not all patients are eligible for a surgical biopsy and diagnosis can be very challenging [7].In order to exclude other potential causes of observed ILD such as sarcoidosis, a bronchioalveolar lavage, yielding differential cell count and analysis, may be performed [7].SurvivalMedian survival from diagnosis is 3 years but is closer to 7 years if measured from the onset of symptoms. Survival is very heterogeneous with some patients surviving more than a decade while others decline much more rapidly. Mortality increases with age and cigarette consumption during illness. Not all patients with IPF die from the disease, and there are some comorbidities which can influence survival such as emphysema [1]. The proportion of patients with IPF who die from the

disease is disputed with one study suggesting it could be less than half. Other causes of death include cardiovascular disease, cancer and infection [11]. Individuals with IPF have higher prevalence of vascular disease [12].IncidenceThe standardised mortality rate in the UK has increased from 1968 to 2008 and this is not explained solely by an ageing population but may partly reflect increasing awareness of the disease. IPF accounts for more deaths in the UK than ovarian cancer, lymphoma or leukaemia [13].The reported incidence and prevalence of IPF can vary greatly between countries [11] [13] [14]. Whilst there is likely to be a degree of international variation, research is also dependant on the methodology used. Changes in classification have occurred over recent years and IPF was only formally classified as a unique disease state in 2000, discrete from the other idiopathic interstitial pneumonias [15].The incidence of IPF in the UK was predicted to be 8.0/ 100,000 person years in 2008 [13].  In some countries such as Taiwan the incidence is much lower [11], however, higher estimates of 16.3/100,000 person years have been suggested in studies in the USA. Such findings occurred when a broader diagnostic classification was used. The results of epidemiological studies depend significantly on the breadth of criteria used [14] and are rarely based on biopsy findings [16].Incidence is higher in males and increases with age; hence we can expect to see a rise in prevalence in the future due to the aging population. Very few cases are seen in individuals younger than 55 [13]. There is some geographical variation within the UK and it appears that incidence rates may be higher in rural areas [13], perhaps due to environmental and occupational risk factors (see below).Risk FactorsAs IPF is a rare disease, the majority of research into epidemiology involves case control studies which often do not provide particularly strong evidence of a causal link and are especially prone to recall bias. Individuals with certain genotypes may have a predisposition to the disease. Exposure to environmental risk factors may then cause injury and further modify the genetic make-up of the epithelial cells, often through epigenetic changes.Tobacco smoking is an established risk factor of IPF. Smoking trends may help explain the discrepancy of incidence between the sexes. Abnormal acid gastro-oesophageal reflux shows higher prevalence in those with IPF (87%) than asthma and has been strongly associated with the disease [17].

A wide variety of environmental and occupational exposures have been attributed as potential risk factors for IPF. These include agriculture (from exposure to dust such as feeding grains or faecal material), livestock, wood dust and stone/sand [16].Viruses may play a role and research suggests that herpes virus can trigger endoplasmic reticulum stress, a key feature in the pathophysiology of the disease [5].Genetics also play a role in pathogenesis. Patients with IPF have significantly shorter telomeres in the alveolar epithelium than is expected which may prevent effective regeneration in the lung. Mutations in the telomerase enzyme (TERC and TERT) are present in some familial cases of IPF [18], and surfactant protein (C and A2) may also be mutated and accumulate.A mucin 5B gene promotor polymorphism in epithelial cells of the lung is associated with approximately 35% of sporadic cases [5]. This may be due to reduced clearance of toxic agents which contribute to the disease [19].Using this knowledge of IPF a variety of treatments have been developed, aiming to improve the survival rate of patients.

Traditional Pharmacological TherapyThe traditional pharmacological therapy for IPF is an anti-inflammatory agent used together with an immunomodulatory or anti-fibrotic agent. The anti-inflammatory agent is usually a corticosteroid, and the current recommended drug of choice is prednisone. The immunomodulatory agent recommended is usually either azathioprine or cyclophosphamide [20]. Azathioprine is a purine analogue converted to its active form in body tissues. It acts by inhibiting adenine deaminase, thus inhibiting de novo purine synthesis and therefore impairing cell proliferation, especially leukocytes and lymphocytes. Cyclophosphamide is an alkylating agent of the nitrogen mustard group. It is given orally and is activated in the liver to several cytotoxic compounds that suppress lymphocyte function [21].

Figure 3. Chemical Structure of Azathioprine

Figure 4. Chemical Structure of CyclophosphamideThe benefits of the traditional therapy, however, remain debatable. As reviewed in Mapel et al.’s paper, some older trials evaluating corticosteroid therapy in IPF suggested favourable corticosteroid response with improvements in factors associated with a better prognosis, but these

improvements are only seen in a small percentage of patients. Furthermore, the improvement is seen primarily in patients who have a better outlook, with less advanced pulmonary fibrosis [22]. The lack of useful, high-quality clinical data evaluating the benefits of corticosteroid therapy makes it difficult to derive from these studies a distinction between causation and correlation.Similarly, there is no consensus that a combined corticosteroid therapy with azathioprine or cyclophosphamide improves outcomes. Schwartz et al.’s paper in 1994 suggests that cyclophosphamide therapy is beneficial; however, it had a small sample size and was designed as an observational study rather than a treatment intervention trial [23]. Other trials [24, 25] show combined corticosteroid and cyclophosphamide therapy to be unhelpful in delaying disease progression, listed in Table 1. These trials also cannot be taken to be conclusive: in addition to a small sample size in Riha et al.’s paper, retrospective trials are less able to take into account all the pertinent risk factors involved as risk factors would have been recorded before-hand, and there is likely to be minimal standardisation of the recorded data for accuracy and consistency as compared to randomised prospective controlled trials [26].Study Therapy Sample Size,

Trial TypeOutcome

Schwartz et al., 1994 (4)

corticosteroids VS cyclophosphamide VS no treatment

39: 14 corticosteroids, 7 cyclophosphamide, 18 untreated.Prospective observational study

Patients on cyclophosphamide had better outcome

Riha et al., 2002 (5)

corticosteroids VS corticosteroids and cyclophosphamide VS no treatment

42: 27 corticosteroids, 7 corticosteroids and cyclophosphamide, 8 untreated.Retrospective review

No difference in survival

Collard et al., 2004 (6)

prednisone and cyclophosphamide VS no treatment

164: 82 prednisone and cyclophosphamide, 82 untreated.Retrospective review

No difference in survival

Consensus RecommendationThe current consensus recommendation is to use prednisone, azathioprine and N-acetylcysteine (NAC). NAC is a precursor of the major antioxidant glutathione and thus acts as a powerful antioxidant and cellular detoxifying agent. It was shown to restore depleted pulmonary glutathione levels and to improve lung function in patients with fibrotic lung disease [27]. It has been proposed more recently that an oxidant / antioxidant imbalance is involved in alveolar epithelial cell injury and thereby contributes to progressive fibrosis in IPF [5]; if this were true, the use of NAC in managing IPF would appear to have some scientific basis. This is backed up by a 2005 randomised parallel-group intervention trial with a sample size of 182 patients, which showed statistically significant improvement for patients on NAC compared to patients on traditional therapy: patients on NAC + traditional therapy had less deterioration in VC and single-breath DLCO at 6 and 12 months than patients only on placebo + traditional therapy [28].

Figure 5. Chemical Structure of N-acetylcysteineClinically, the use of any drug combination would have to take into account not only the benefit to the patient, but also possible adverse effects. The common adverse effects of NAC include nausea, vomiting, and other GI complaints. Rarely, rash and / or fever may occur. These adverse effects are rather common among drugs and are, in most cases, quite benign. Not only was there no significant additional toxicity seen with use of NAC, NAC seemed to protect against azathioprine-induced myelotoxicity [29].However, there are as of now no published trials evaluating the mortality and / or morbidity of patients on NAC therapy versus patients who are not on any therapy. Therefore while the use of NAC in addition to traditional therapy may be established as better than that of traditional therapy, the question of whether pharmacological agents really do help IPF patients still remains.

To Give, or Not to Give?Given that there is little reliable, quality evidence-based data to support the use of combined corticosteroid therapy, should it still be given to patients with IPF? Corticosteroids are used pharmacologically to treat patients with inflammatory disorders, but recent research evidence suggests that chronic inflammation plays only a minimal role in the progression of IPF [5]. Furthermore, corticosteroids are known to have many adverse effects, including, but not limited to, osteoporosis, hypertension, infertility, and a heightened risk of catching infections. Do the questionable benefits of pharmacological agents really justify their usage? To answer this question, we looked at and evaluated alternative approaches to the treatment of IPF.

The Surgical ApproachDue to the poor prognosis of IPF, patients with end-stage IPF are often considered for lung transplantation [30]. However, the efficacy of this procedure has long since been debated. Lung transplantation involves donation of one or two healthy lungs to an individual with IPF; these lungs will be free from fibrosis and should therefore give a better prognosis to those who receive them. Lung transplant has been found to decrease risk of death by around 75% [31], showing that statistically, it is an option worth considering at end-stage IPF. Lung transplants can be done unilaterally or bilaterally depending on severity of bronchiectasis [31]. However, evidence suggests that there is no significant survival benefit between patients receiving unilateral or bilateral transplantation [32].Out of all patients awaiting lung transplantation, IPF patients have the poorest prognosis [33]. Even when considering other conditions requiring lung transplant, IPF has the highest percentage of people dying while on the waiting list [34]. While this means they are prioritised by transplant teams over some other conditions, receiving a transplant will still depend upon organ availability, ABO blood group matching to avoid chronic rejection and cytomegalovirus status [31]. Despite this, transplant teams may well prioritise patients with other conditions such as Cystic Fibrosis because of their higher survival benefit from the transplantation [34]. Another cause of prioritisation over end-stage IPF sufferers is that IPF often develops in people age 50+ [13]. Transplant teams may be more likely to give organs to younger patients because of the associated survival benefit.Time on the waiting list has to be very short as prognosis is so poor. Waiting times for transplantation depend on the centre, availability of

organs and prioritisation, and IPF patients can find themselves waiting anytime from 2-12 months for a transplant [31, 33, 34]. Even though transplants are quickly prioritised, death on the waiting list for IPF remains shockingly high, stretching from 30-40% in some centres [31, 34]. This high death rate on quite often a short waiting time could suggest that patients are being referred for transplant too late into the disease process [33]. Earlier referral for transplantation may see better survival rates in IPF patients and could provide better outcomes for a lot of people currently suffering.

Are Transplants our Answer?Although, survival rates are a lot higher for patients who have received transplant [31, 34], the 5 year survival rate of patients is still only around 50% [30-33]. A lot of patients die from sepsis after the operation has occurred, and there is always the risk of chronic rejection in all transplantations. Some patients also died of cancer before 5 years. Although there is no established link between carcinoma and IPF, it has been suggested that whatever is causing the fibrosis in IPF could also be damaging and mutating the p53 gene, a mutation important in carcinogenesis. On top of this IPF and cancer share several risk factors such as smoking [9].As stated earlier giving transplant earlier in the disease may be a good option to combat the high death rates associated with end-stage IPF [9]. But even as death from organ rejection and sepsis decreases there will always be a limited supply of organ donors and therefore prioritisation of IPF and other diseases will still go on, and death will occur in some cases. Considering transplant for IPF earlier in the disease will also mean there will be more people on the waiting list for transplants and waiting times will increase for all diseases.As current pharmacological therapies and surgeries have limited benefit in IPF, the answer lies with identifying the underlying fibrotic process in IPF, and developing new clinical options to combat or reverse the disease process to improve prognosis for patients.

Tralokinumab Monoclonal AntibodiesN-acetylcysteine AntioxidantsWarfarin AnticoagulationAerosol Interferon-gamma (cytokine)Thalidomide (immunomodulatory drug)Losartan (angiotensin-II receptor antagonist) Other

Antifibrotics Pirfenidone NintedanibEndothelin Receptor Antagonists Macitentan BosentanCombination Therapies Azathioprine, Prednisolone & N-acetylcysteinePEX, Rituximab & SteroidsCurrent Clinical Trials for IPF Treatment

The current pharmacological treatments for IPF are relatively ineffective at improving the disease prognosis or slowing the disease progression. Current treatment options focus on anti-inflammatory mechanisms [6] however multiple drugs, many of which are anti-fibrotic, are in clinical trials.

PirfenidonePirfenidone is an anti-fibrotic and anti-inflammatory agent that is currently used as a treatment for mild-to-moderate IPF in most of the world. It was first approved in 2008 for use in Japan and has since been approved for use in India, Europe, China, Canada, and the USA with the FDA approving its use in October this year.

Figure 6. Chemical Structure of PirfenidoneThe exact mechanism of action of pirfenidone is unknown; however it has been shown to reduce fibroblast formation, inhibit TGF-β stimulated collagen production and down-regulate pro-fibrotic cytokines.After dealing with problems such as acute exacerbations from IPF leading to patient death, some early clinical trials for pirfenidone [35] showed that it preserved vital capacity, and this justified the CAPACITY phase III studies. [36] The two CAPACITY trials had the primary endpoint of change in percent predicted (FVC) at Week 72 after starting a course of pirfenidone. Only one of the studies met this with statistical significance, however. The results of this paper followed by a Cochrane review of non-steroid treatments for IPF [37] was considered sufficient evidence to approve the drug for use in Europe, however the FDA required more evidence to prove pirfenidone’s effectiveness, and this prompted the 2014 ASCEND study [38].The ASCEND study went on to meet its primary endpoint (change in FVC) and both of its key secondary endpoints (6MWD, PFS) with statistical significance. Following a pre-specified analysis of the ASCEND study [39] the FDA approved pirfenidone for treatment of mild-to-moderate IPF.The use of pirfenidone as IPF treatment is currently under scrutiny as it is

only slightly slowing down disease progression, and is not curative or proving to increase quality of life.

Nintedanib (BIBF 1120)Figure 7. Chemical Structure of NintedanibNintedanib, previously known as BIBF 1120, is a tyrosine kinase antagonist which targets pro-fibrotic growth factor receptors including those for FGF, VEGF and PDGF. Following successful mouse studies, nintedanib was advanced to a randomised, double-blind, placebo-controlled Phase II clinical trial. [40]

Figure 8: Graph shows the effect of different nintedanib doses on lung function, measured using FVC. A dose of 150mg of nintedanib twice daily was shown to reduce annual FVC decline by ⅔ compared to the placebo. Reproduced with permission from [40], Copyright Massachusetts Medical Society.The TOMORROW trial reported three main effects of nintedanib on IPF:• decrease the frequency of acute exacerbations• potentially slow decline in lung function• improve quality of life [41]However, these improvements can be mainly attributed to the treatment of some secondary effects of IPF, instead of the IPF cause itself. [40]Despite this, nintedanib has a lot of potential regarding IPF treatment and it might not be long before it is licensed for use, having been given priority review by the FDA and accelerated assessment by the European Medicine Agency.

Endothelin Receptor AntagonistsEndothelin receptor antagonists (ERAs) work by blocking endothelin receptors which are activated by endothelin (ET-1). ET-1 is a potent, endogenous vasoconstrictor and acts via proliferation of smooth muscle cells, fibrosis, inflammation, and endothelial dysfunction. In addition, ET-1 is a profibrotic molecule that can increase collagen synthesis and decrease interstitial collagenase production. It is thought that by blocking the actions of ET-1 that prognosis of patients with IPF might improve.ERAs also have anti-angiogenic effects, as increased vasculature is caused by IL-8 release [42] and this is thought to contribute to fibrosis. [43] It is therefore hypothesised that a reduction in angiogenesis would reduce fibrosis.BosentanAnother ERA being investigated for the treatment of IPF is bosentan. After

the first BUILD trials showed a trend to delayed IPF worsening or death, as well as improvements in some measures of dyspnoea and health-related quality of life, [44] further BUILD trials looked to demonstrate these trends further. Both of these trials failed to achieve their primary outcomes, however. BUILD-2 suggested its failure related to its primary outcome, 6MWD, not being appropriate to assess treatment effects in parenchymal lung disease. [45] BUILD-3 used IPF worsening for its primary outcome, [46] however this did not aid the success of the study.MacitentanMacitentan is an ERA approved for the treatment of pulmonary arterial hypertension, but is now being investigated for its potential benefit in IPF treatment. The MUSIC trial was a phase II study that compared macitentan versus placebo to assess its effect on FVC in IPF patients. [47] Neither the primary or secondary endpoint of the study was met, and this meant the follow-up study (MUSIC OL) was withdrawn prior to enrolment as it was not justified.

Other TherapiesLosartanLosartan is an angiotensin II receptor antagonist used mainly to treat hypertension, however its effectiveness in treating IPF is currently being trialed. A phase I clinical trial published in 2012 [48] showed a potential association between regular losartan intake and attenuation of disease progression in IPF patients.   The only other study pursued for use of losartan in treating IPF was by the University of Iowa in 2011, however it was withdrawn in the recruitment phase.Tralokinumab (monoclonal antibody)Monoclonal antibodies bind to specific target cells or proteins, which are then attacked by the patient’s immune system. Tralokinumab is a human IgG4 recombinant monoclonal antibody (mAb) for interleukin-13 (IL-13), a cytokine that is thought to possibly play a role in the pathogenesis of IPF through the promotion of inflammation, fibrosis, and mast cell activation. Targeting IL-13 is also believed to enhance repair processes in the lungs. [49] There is an ongoing phase II trial currently evaluating the efficacy and safety of tralokinumab in patients with mild-to-moderate (IPF) over a 72-week treatment period. Recruitment for a clinical trial to evaluate the efficacy of tralokinumab in adults with IPF is also underway.WarfarinAs an anticoagulant, warfarin was proposed as a potential treatment option

for IPF as previous studies have linked IPF to thrombosis-related clinical events, such as an increased risk of acute coronary syndrome and deep vein thrombosis. [50] The first study to look at the use of warfarin to treat IPF [51] was stopped due to a low probability of benefit based on an increase in mortality seen in the subjects randomised to warfarin (14 warfarin versus 3 placebo deaths).N-acetylcysteine (NAC)The PANTHER-IPF trials recently analysed the effectiveness of NAC as a monotherapy compared to the current three-drug regimen. [52] However these trials had to be halted due to severe adverse effects and an excess number of deaths in the combination-therapy group. Further research is therefore required for a conclusion on the effectiveness of NAC as a monotherapy to be drawn.Aerosol interferon gamma (IFN-γ)Interferon gamma (IFN-γ) is a Th1 cytokine which inhibits collagen production. In fibrosis, the balance of cytokines is shifted towards promoting collagen, thus inhibiting collagen production should decrease the levels of fibrosis. The effectiveness of IFN-γ in IPF treatment remains unclear however. A 1999 study found that IFN-γ and prednisolone significantly improved pulmonary function tests in patients who had previously been unresponsive to treatment. [53] However, another study in 2004 found no significant difference when IFN-γ was administered compared to a placebo. [54] Further studies are therefore required to investigate the therapeutic potential of IFN-γ.ThalidomideThalidomide is an immunomodulator with anti-inflammatory effects and anti-angiogenic properties. It has been shown to suppress TNF-α levels, [42] which may slow the deterioration of lung function in IPF patients as TNF-α plays a crucial role in fibrosis. The anti-angiogenic properties of thalidomide are of interest as they may cause a reduction in fibrosis, however further studies are required to determine whether thalidomide might be a viable treatment for IPF.

Combination therapiesFuture pharmacological treatments of IPF may lie in the use of combinations of drugs instead of one single drug, following the successful results of some trials.Azathioprine, Prednisone and NACThis combination is currently recommended for IPF treatment, having

been shown to stabilise the condition of IPF patients, slow their disease progression and decrease the deterioration rate of VC and DLCO. [28] In 2011, the American Thoracic Society recommended against this three-drug regimen as a routine treatment for IPF, [5] however it may be appropriate in some cases.PEX, Rituximab and SteroidsTwo clinical studies are currently in progress investigating the effects of combined PEX, rituximab (mAb) and corticosteroids in the United States. [55, 56] Figure 9. Table comparing two clinical studies investigating the combination therapy of PEX, rituximab and steroids.This page gives an overview of some of the drugs currently in clinical trials, however more potential options are being explored – many of which are through pre-clinical trials.

TRIAL NAME PHASE ESTIMATED COMPLETION DATE

AIM EXPERIMENTAL ARM A

EXPERIMENTAL ARM B

A Multicenter Study of Combined PEX, Rituximab and Steroids in Acute Idiopathic Pulmonary Fibrosis

2 July 2015 Effects of this combined treatment

Standard Steroid Treatment:

Intravenous Methylprednisolone

Standard Steroid Treatment + Rituximab + PEX

Combined PEX, Rituximab and Steroids in Acute IPF Exacerbations

1/2 December 2014 Feasibility, safety and efficacy in hospitalised patients with acute IPF exacerbations

Standard Steroid Treatment:

Intravenous Methylprednisolone

Standard Steroid Treatment + Rituximab + PEX

Preclinical Studies for IPFThe only drug currently approved for IPF treatment, pirfenidone, received a ‘weak no’ recommendation from four well-respected medical groups.[57] Pirfenidone can produce photosensitivity side-effects in more than 50% of patients, with little clinical benefit.[58] Therefore a new treatment for IPF is needed to improve the poor disease prognosis. Preclinical trials are underway to try and find a new alternative. Most commonly used is the bleomycin model in mice. Figure 10.  Masson’s stain of mice lungs showing the fibrotic effects of Bleomycin. Reproduced with permission from [68], Copyright Massachusetts Medical Society.Bleomycin refers to a family of structurally related compounds, used in

anti-cancer chemotherapy via DNA breakdown.[59] It causes single and double-strand breaks in tumour cells, interrupting the cell cycle. However this produces DNA-cleaving superoxide and hydrogen-free radicals which lead to an inflammatory response causing pulmonary toxicity, activation of fibroblasts and subsequently, fibrosis. Bleomycin was chosen for animal use because of the major adverse effect that results: fibrosis. Since its first use modelling IPF, numerous agents have been shown to inhibit its fibrosis. However, very few have been used clinically in IPF management and their efficacy is significantly reduced in humans compared to animal models.[60]Bleomycin causes rapid inflammatory and fibrotic reactions giving elevation of inflammatory cytokines and pro-fibrotic markers, peaking at around day 14. Certain histological hallmarks, such as mural incorporation of collagen, intra-alveolar buds and obliteration of alveolar space, leads to the assumption that it is very similar to the human disease. The bleomycin model is advantageous due to its ease of performance, making it accessible and reproducible. Instillation is normally through intratracheal administration resulting in fast inflammatory reactions causing bronchiocentric accentuated fibrosis. Techniques such as intravenous or intrapleural administration cause subpleural scarring, which is more similar to the human disease. All methods of instillation have a control group subjected to the same procedure but using saline.[59]Bleomycin has contributed largely toward understanding of IPF, elucidating roles of cytokines, growth factors and signalling pathways. Now we can identify multiple limitations with the model. To start, single bleomycin injection into a mouse causes pathogenesis resembling acute lung disease, because the injury sustained comes at one point in time, rather than the continuous and progressive deterioration in IPF; a chronic disease. A recurring problem is that all these trials test the drug’s effect over a short time – less than 31 days – therefore results obtained are for minimizing acute injury and repair.[61] Human IPF lasts much longer and therefore this has minimal relevance.[62] Additionally, fibrotic development induced by Bleomycin is partially reversible, independent from intervention, which isn’t the case in humans. This is demonstrated in the graph below from a 2009 paper[63]; at 28days Lactate Dehydrogenase (LDH) – a fibrosis level marker – levels in the lungs were similar in the control and bleomycin-exposed mice. This shows mice lungs can somewhat recover from bleomycin-induced lung fibrosis even without treatment.

Figure 11.  This graph shows the ability of mice lungs to partially recover from the fibrosis caused by bleomycin, void of any intervention. Reproduced with permission from [63], Copyright Massachusetts Medical Society.Studies also often start treatment immediately after injury is sustained, whereas in humans significant damage will have developed before symptoms and subsequent treatment are initiated. Therefore, even with promising results in mice in preclinical trials, success may not replicate in humans.Further studies have been conducted recently, refining the bleomycin mouse model. For instance, bleomycin injected biweekly models the chronic disease nature more closely as their lungs receive continuous injury for 16 weeks.[64] Ten weeks following this, mice lungs still display significant fibrosis. However, issues occur such as gaining ethical consent and a very high mortality rate.Although bleomycin is the most ubiquitous current model, there are alternatives. One study used Silicon Dioxide to induce pulmonary fibrosis in mice. Silicon Dioxide, also known as Silica, is the second commonest element in the Earth’s crust, found in many rocks. Breaking it down produces silica dust which, if deposited in the lungs is phagocytosed by macrophages which are unable to differentiate between pathogens and silica particles. This initiates an inflammatory response, releasing multiple cytokines and subsequently fibrosis as fibroblasts proliferate, producing collagen around silica particles.[65]The 2009 paper used bleomycin to induce pulmonary fibrosis to investigate the effectiveness of vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor SU5416 as a treatment. It was considered a potential therapy because of VEGF’s role in angiogenesis, which is important in fibrotic development. This study aimed to see if VEGF pathway disruption might significantly reduce pulmonary fibrosis in the mice. The results, although showing a strong correlation between increased VEGF expression and heightened angiogenesis and therefore higher pulmonary fibrosis, did not show a decrease of lung fibrosis on treatment of SU5416.Group Microvessels number (vessels/HPF)

Day 7 Day 14 Day 28

Control

BLM

E-SU5416

L-SU5416

Table 1: SU5416 inhibits angiogenesis response in bleomycin-induced pulmonary fibrosis, measured by microvessel density.[63]A more recent study[57], from Japan, looked into using carbon monoxide-bound haemoglobin vesicles. The use of carbon monoxide (CO) was based on its potent anti-inflammatory, anti-oxidant and anti-proliferative effects. Two obstacles of using CO are its extremely short half-life (1-21min) and the high risk of toxicity when administered via inhalation. However, using haemoglobin-vesicles solves both issues, and it is itself promptly metabolized to ensure there is no accumulation in the body. Finally, it is believed to carry CO to the lungs efficiently, making it a potential therapy for IPF.Some studies have used Transforming Growth Factor (TGF)-B1 as an alternative chemical to induce lung fibrosis in mice. This is a natural protein which appears to be critical in healing due to its role in angiogenesis, its anti-inflammatory and anti-fibrotic effects. However, on closer examination this is partially contradicted as TGF-B1 induces tissue injury, stimulates apoptosis of cells and decreases epithelisation; all of which inhibit healing.[66]Even still, if TGF-B1 levels are elevated chronically, there is increased collagen deposition and consequently excessive fibrosis results.[67] This has been demonstrated by increased collagen staining in Ad-TGFB lungs compared to the control (Ad-con) by a study from 2012; see image below.[68]

Figure 12. Increased collagen staining in Ad-TGFB lungs compared to Ad-con, the control. Reproduced with permission from [68], Copyright Massachusetts Medical Society.Although initially earlier studies struggled with using TGF-B1 due to the critical role it plays in airway development, causing problems with fetal-lethality.[57] However, having created live lung-specific TGF-B1 mice, an exciting opportunity has arisen to study adult lung fibrosis. In the paper previously mentioned, from 2012, TGF-B1 was instilled intratracheally into anaesthetised mice.[66]Another method of inducing pulmonary fibrosis in an animal model is the use of a skin sensitising hapten fluorescein isothiocyanate (FITC) Single

intratracheal instillation of FITC leads to reproducible lung injury, quantifiable increases in the lung collagen content and evidence of specific immunity to fluorescein hapten.[69] It has several advantages over the bleomycin model; it produces chronic persistent inflammation that leads to an altered fibroblast phenotype resulting in increased collagen deposition rather than transient fibrosis, which occurs in the bleomycin model.[69, 70] FITC also enables identification of injured areas by fluorescence, which enables tracking of an area of initial injury at a time remote from the injury. It is also less expensive than the bleomycin model.[69]

The aetiology of IPF is not well understood, meaning that any pharmacological developments do not address the primary cause of IPF, instead merely treating the associated pathology. Considering the extent of our research, we have realised the very limited number of potential treatments and the relatively unpromising results that clinical and pre-clinical trials have yielded. Therefore in order to advance treatment of this disease and improve the prognosis for patients, the underlying cause needs to first be identified.In our first meeting, it became apparent that none of our group had much knowledge about IPF. As the project progressed and consequently our understanding of IPF developed, we realised the debilitating consequences of the disease on a sufferer’s life. Simple daily tasks which are normally carried out with ease are complicated by the significant lung destruction in IPF and this leads to a severely reduced quality of life. The lack of awareness of IPF also shocked us, particularly when we learnt of the poor prognosis and survival time that accompanies a diagnosis. However, our group were reassured that most of the papers we referenced were very recent, demonstrating that there are many ongoing attempts to find a successful treatment. Hopefully a significant advancement will be made in the near future.