Oral Sub Mucous Fibrosis: Exploring Therapeutic Strategies Using -anti TGF β Drugs

Fibrosis includes a wide range of diseases including systemic sclerosis (SSc), idiopathic pulmonary fibrosis (IPF), liver and renal fibrosis, and oral submucous fibrosis (OSMF) [1-11]. It is a chronic disease, which affects a large number of individuals posing challenge to the healthcare professionals and economic burden to the patients. It has been estimated that the fibrotic diseases are responsible for approximately 45% of mortalities in the Western countries. However, developing countries have higher mortality rate [10, 12]. In renal fibrosis, differentiation of myofibroblasts and collagen synthesis is stimulated by cytokines and growth factors, which are secreted from the adjacent tubular epithelial cells, endothelial cells, or from fibroblasts. Abstract


Introduction
Fibrosis includes a wide range of diseases including systemic sclerosis (SSc), idiopathic pulmonary fibrosis (IPF), liver and renal fibrosis, and oral submucous fibrosis (OSMF) [1][2][3][4][5][6][7][8][9][10][11].It is a chronic disease, which affects a large number of individuals posing challenge to the healthcare professionals and economic burden to the patients.It has been estimated that the fibrotic diseases are responsible for approximately 45% of mortalities in the Western countries.However, developing countries have higher mortality rate [10,12].
In renal fibrosis, differentiation of myofibroblasts and collagen synthesis is stimulated by cytokines and growth factors, which are secreted from the adjacent tubular epithelial cells, endothelial cells, or from fibroblasts.

Oral Sub Mucous Fibrosis: Exploring Therapeutic Strategies Using -anti TGF β Drugs
Sheshaprasad R 1 , Anuradha Pai 2   The most crucial role is played by transforming growth factor beta-1 (TGF-β1) [13].IPF is a progressive lung disease, which is characterized by an injury of the alveolar epithelial cell, activated fibroblasts and myofibroblasts proliferation, and extracellular matrix (ECM) accumulation.TGF-β1 is associated with fibroblast proliferation and accumulation in IPF [14].Systemic sclerosis is characterized by fibrosis of various organs including the skin, kidney, heart, lungs, and digestive system.Damage of the endothelial cells takes place and fibroblasts are activated by an autoantibody-mediated mechanism.The TGF-β1 pathway is a critical signaling pathway in the development of systemic sclerosis [15] In liver fibrosis, hepatic stellate cells get activated and are transformed into myofibroblasts, which produce ECM leading to fibrosis.In OSMF, oral epithelial cells secrete TGF-β1 [16], which stimulates cytokine expression by the connective tissue leading to fibroelastic changes in lamina propria, connective tissue, blood vessels, and degenerative changes in the muscles.
Although fibrotic disorders arise in different tissues and various risk factors Table 1 are associated with the disease development and progression, there are certain common features, which are observed among the affected tissues in fibrosis.It has been reported that transforming growth factor pathway, vascular endothelial growth factor pathway, PDGF pathway, and fibroblast growth factor pathway are involved in the pathogenesis of fibrosis [17].In the fibrotic tissue, trans-differentiation of epithelial cells to myofibroblasts occurs by a process known as the epithelial-mesenchymal transition (EMT) [18,19].Additionally, there is also an increase in production of collagens, secretion of α-smooth muscle actin (α-SMA), reduction of extracellular degradative enzyme, excessive accumulation of ECM with components such as hyaluronic acid, fibronectin, proteoglycans, and collagens that lead to the formation of permanent fibrotic scar [10,[19][20][21][22][23].The excessive accumulation of ECM proteins is caused due to high production of tissue inhibitors of metalloproteinases (TIMP) by myofibroblasts, which inhibit the action of various matrix metalloproteinases (MMPs) [24,25].
Up regulation of TGF-β1, down-regulation of bone morphogenic protein (BMP) and remodeling of ECM are characteristic features of OSMF, which are characterized by rigidity of the mucous along with fibroelastic changes of the lamina propria [37][38][39][40][41].The disease affects the oral cavity, pharynx, and the upper one-third part of the esophagus and causes great difficulty in opening the mouth and eating [41][42][43].Areca nut chewing is considered as the most probable etiological factor in OSMF [40,44,45].The alkaloid and polyphenol components of areca nut were found to be the inducer and activator of TGF-β1 in epithelial cells [40].Exposure to areca nut and stimulation of TGF-β1 pathway are responsible for overproduction of collagen and decrease in degradation of collagen in OSMF [46,47].TGF-β1 induces transcription of COL1A1 procollagen gene [16] [48-50], increases activities of procollagen proteinases [46] and promotes the expression of lysyl oxidase (LOX), an enzyme essential for transforming collagen fibers into stable mature fibrillar form [51,52].It has been shown in immunohistochemical experiments that there is intense TGF-β1 staining of the epithelium, fibroblast, macrophages, and inflammatory cells in early OSMF [16,47].Furthermore, activated TGF-β1 induces myofibroblast transdifferentiation in OSMF [47,53].Therefore, TGF-β1 function is indispensable for the pathogenesis of oral submucosal fibrosis.
Submucous fibrosis is predominantly found in Indian population and occasionally in the Taiwanese population [54].There has been a tremendous rise in the incidence of submucous fibrosis in the recent years.In 2002, in Indian subcontinent alone, 5 million people were affected by the disease [54,55].It has been reported that in the Indian states of Bihar, Madhya Pradesh, Gujarat, and Maharashtra, the younger generation is getting addicted to areca nut products [46], [54].
The percentage of OSMF among patients attending orthodontics and pediatric dentistry (OPD) in Jaipur, Rajasthan, India in 2012 was found to be 3.39% [1] [54] and in a 17-year, long-term, follow-up study, the annual malignant transformation rate was found to be approximately 0.5-7.6%[54,56].Although, there are several drug therapies, surgical therapy, and physiotherapy to manage the symptoms of the disease and to alleviate the symptoms of OSMF, there is no effective treatment for this disorder [57,58].In this review paper, we aim to offer suggestions to perform clinical trials on drugs based on molecular pathways of OSMF, mainly TGF-β1 pathway.Since TGF-β1 is a common pathway involved in fibrotic diseases, the central idea of this review article is to suggest antifibrotic drugs, which act on TGF-β1 pathway that have already been clinically tested in other fibrotic diseases.

Materials and Methods
We reviewed the records of the https://clinicaltrials.gov/, the registry of clinical trials that have been conducted internationally and in the United States.We used the advanced search option of the registry, and wrote "FIBROSIS" in the text box provided for "SEARCH TERMS", selected "CLOSED STUDIES" in the drop down provided for "RECRUITMENT", STUDIES WITH RESULT" in the dropdown provided for "STUDY RESULT", and selected "INTERVENTIONAL STUDIES" for the dropdown provided for "STUDY TYPE" and checked "PHASE 3" in the "PHASE" category.Remaining text boxes were kept in the default mode.
By applying the set criteria in searching the clinical trial registry database, we obtained 89 hits.Out of these 89 hits, we screened the downloaded XML file for entries on pulmonary fibrosis.In the process, we excluded 75 entries, as the studies were not related with pulmonary fibrosis.In the next step, we removed the entries, which did not have associated publications and those entries that were redundant in nature.There were two entries, which were redundant in nature and three entries that did not have publications associated with them.Nine entries were considered for further analysis.
After performing the search in the United States government website on clinical trials data, we performed another search in the PubMed database.We chose the successfully tested drugs from the result of our previous search and used the keywords "Name of the selected drug" Gadekar [64] studied several pyridone derivatives and reported that 5-methyl-1-phenyl-2-(1H)-pyridone (which was later named as pirfenidone) had analgesic, antipyretic, and anti-inflammatory activities.Margolin [65] reported that the drug can act as an antifibrotic agent.Later in 2007, patent rights of the drug were purchased by InterMune Inc. for the United States and Europe from Marnac.In Japan, pirfenidone is sold by Shionogi as Pirespa® and in India, Cipla began the sale of the drug in October 2010.

Results
In the list of studies that we obtained, Table 2 pirfenidone and nintedanib were the two drugs, which were found to improve conditions associated with IPF; and cyclophosphamide was found to be effective in the treatment of scleroderma interstitial lung disease.On the other hand, there were some drugs in our list, which showed no promise in improving the diseased condition in idiopathic pulmonary disease.Such drugs included sildenafil, which has shown no benefit in the treatment of idiopathic pulmonary disease; warfarin that was associated with increased risk of mortality in idiopathic pulmonary disease population; bosentan that showed no effect when compared to placebo; and ambrisentan, acetylcysteine, combination of prednisone, azathioprine, and N-acetylcysteine that were associated with increased risk of idiopathic pulmonary disease progression and hospitalization.
By a study of the articles we retrieved through our literature search in PubMed, we found out that cyclophosphamide did not inhibit TGF-β1-induced signaling, as assessed by luciferase reporter gene expression in lung fibroblast model [59], nintedanib reversed TGF-β1-induced EMT in non-small cell lung cancer cells [60], pirfenidone treatment inhibited TGF-β1-induced upregulation of phosphorylation of ERK1/2, p38 and Jun amino-terminal kinases (JNK) in a renal fibrosis rat model [61].

Discussion
OSMF is a debilitating disease of the oral cavity.It is one of the most poorly understood oral diseases and no single drug has provided complete relief to the patient [62] .Corticosteroids are immunosuppressive agents, which are commonly used to treat the disease but are not effective in reversing the abnormal deposition of fibrotic tissues.Proteolytic enzymes such as hyaluronidase, collagenase, and chymotrypsin are also used to treat the disease.Hyaluronidase breaks down hyaluronic acid and reduces collagen formation.Vitamins, antioxidants, minerals, and lycopene have shown improvement in the disease condition.Antifibrotic drugs such as interferon gamma; however, improved mouth opening and reduced burning sensation; it has definite adverse effects.However, surgical treatment treats severe trismus by incising the fibrous bands; it may lead to further fibrosis [62].Lack of good quality clinical trials of drugs used in the treatment of OSMF and lack of evidence for the use of specific intervention have made the management of OSMF difficult [63].Therefore, there is a need for alternative therapies that can be used to effectively manage the disease and prevent its malignant transformation.
By performing a systematic search of the clinical trial database and PubMed literature, we obtained only two drugs namely pirfenidone and nintedanib, which have shown promise in treating fibrotic disorders of the lung.In human myometrial and leiomyoma cells, pirfenidone inhibits cell proliferation, by reducing the rate of deoxyribonucleic acid (DNA) synthesis, and also decreases levels of messenger RNAs (mRNAs) encoding collagen I and collagen III [66].In human retinal pigment epithelial cells, a TGF-β1-induced increase in fibronectin synthesis is inhibited by Pirfenidone [67].In the patent application, Margolin reported that pirfenidone inhibited, TGF-β1-induced collagen production from fibroblasts [65]and in a hamster model, pirfenidone decreased collagen gene expression [68].Moreover, pirfenidone has been shown to attenuate liver [69] and cardiac fibrosis [70].Its effect in treating OSMF has not yet been tested in preclinical and human clinical trials.In this review, we suggest pirfenidone as a potential candidate drug in the treatment of OSMF.
Pirfenidone may reduce OSMF by the following mechanisms: 1) By decreasing the levels of mRNA encoding type I and III collagen and also inhibiting TGF-β1-induced collagen production from fibroblasts [71].
4) By down regulating fibroblast growth factor (b-FGF), which interacts synergistically with other growth factors enhancing the extracellular matrix deposition in OSMF [71,76].5) By reducing the level of plasminogen activator inhibitor-1 (PAI-1), which is upregulated by TGF-β1 in OSMF [77] Originally, nintedanib was developed as a cancer drug and was used as a second-line therapy for lung cancer of adenocarcinoma subtype.Nintedanib, also known as BIBF 1120, is an orally available tyrosine kinase inhibitor, which inhibits PDGF receptor-α and -β, vascular endothelial growth factor receptor-1, -2 as well as fibroblast growth factor receptor-1, -2, and -3 [78] Most importantly, nintedanib has shown inhibition of TGF-β1 profibrotic effect in human lung fibroblast [79] and also improvement in bleomycin-induced pulmonary fibrosis in rodents [80].Based on its mode of action, we suggest the human clinical trial of this drug in the treatment of OSMF.
Moreover, we suggest that nintedanib may reduce OSMF through the following mechanism: 1) By directly preventing phosphorylation of TGF-β1 receptor and reducing excessive ECM production which is a hallmark of OSMF [81].
2) By targeting PDGF receptor-α and-β and thus reducing the level of PDGF, which is up regulated in OSMF [38].
3) By targeting fibroblast growth factor receptor-1, -2, and -3 and thereby reducing the level of fibroblast growth factor, which may be a potential biomarker for malignant transformation of OSMF [82] .
In two clinical trial programs namely TOMORROW trial which was an international phase II trial and two international phase III trials called as INPULSIS trial study conducted for Idiopathic pulmonary fibrosis (IPF) were treated with 150 mg of nintedanib 150 mg twice daily.It was noted that the reduction in mortality all-cause and those related to respiratory causes were decreased by 30% against the placebo [83].
According to another study the dosage recommended for pirfenidone is 801 mg three times per day with a two week titration period.The study recommends that the dose modifications are flexible and keeping the safety of the patient can be optimized for patients based on the observable Adverse Events (AE) [84].
In conclusion, considering the beneficial effect of pirfenidone and nintedanib in pulmonary fibrosis treatment, their role in reducing TGF-β1-induced Tubular epithelial cells, endothelial cells, fibroblasts Yes Systemic sclerosis Exposure to environmental toxins and genetic factor Multiple organs including the lungs, skin kidney, heart and digestive system Yes Oral submucous fibrosis Areca nut chewing, gutkha chewing, smokeless tobacco use Oral epithelial cells, Exposure to environmental toxins and genetic factor Multiple organs including the lungs, skin kidney, heart and digestive system Yes Acetylcysteine, Combination of prednisone, azathioprine, and N-acetylcysteine (NAC) (94, 95)

Table 1 .
Fibrosis in Different Types of Tissues with Risk Factors apjcc.waocp.orgSheshaprasad R, et al: Oral Sub Mucous Fibrosis: Exploring Therapeutic Strategies Using -anti TGF β Drugs

Table 2 .
Selected Entries From National Centre for Biotechnology Information Clinical Trial Registry apjcc.waocp.orgSheshaprasad R, et al: Oral Sub Mucous Fibrosis: Exploring Therapeutic Strategies Using -anti TGF β Drugs profibrotic effects and down regulating various receptors, cytokines and proinflammatory molecules involved in the pathogenesis of OSMF, designing preclinical and clinical trials to test the effectiveness of these drugs in treating OSMF is suggested.