Fibrosis-Modulating Effects of Metformin and Pirfenidone in Oral Submucous Fibrosis

Phase 1

Recruiting

• Conditions: Oral Submucous Fibrosis
• Interventions: Drug: Pirfenidone (PFD); Drug: Metformin Hydrochloride 500Mg Tablet; Drug: Beclomaethasone and Vitamin E

• Registration Number: NCT06871904
• Lead Sponsor: Ziauddin University

Brief Summary

The goal of this clinical trial is to learn if metformin and antifibrotic drugs (pirfenidone) can modulate fibrosis and improve treatment outcomes in patients with oral submucous fibrosis (OSF). The study also aims to investigate the molecular mechanisms underlying their effects on exosome secretion and protein expression.

The main questions it aims to answer are:

Do metformin and antifibrotic drugs alter exosome secretion and biological activity in OSF cell lines? What molecular pathways are influenced by these drugs in modulating fibrosis? Does treatment with metformin and antifibrotic drugs improve clinical outcomes in OSF patients? Researchers will compare metformin and antifibrotic drug treatment groups to a control group to see if these drugs lead to significant changes in fibrosis-related exosomal protein expression and clinical improvement in OSF patients.

Participants will :

Undergo in vitro experiments on OSF cell lines to analyze drug effects using qPCR, Western Blot, and LCMS for protein profiling.

Participate in a randomized, double-blind clinical trial where they receive metformin, antifibrotic drugs, or a placebo.

Undergo clinical evaluations and laboratory tests to assess treatment efficacy. This study aims to develop an affordable and effective fibrosis-targeted therapy for OSF by repurposing metformin, potentially improving patient outcomes and reducing the risk of malignant transformation.

Detailed Description

Oral submucous fibrosis stands as a persistent inflammatory and potentially malignant condition affecting the oral cavity, marked by progressive fibrosis of the oral mucosa. Existing therapies, such as corticosteroids, are costly and merely treat the symptoms without addressing the molecular pathways that cause fibrosis, which results in limited efficacy, relapse, and side effects. Given the financial constraints of affected communities, there is a desperate need for affordable and accessible treatments. This research project aims to investigate the effects of metformin and antifibrotic drugs on the secretion and biological activity of exosomes in oral submucous fibrosis (OSF) cell lines. The study will explore how these treatments impact morphological changes, exosomal gene expression, and protein profiles in OSF cell lines compared to control and vehicle-controlled groups. Additionally, the project will identify molecular pathways influenced by metformin and antifibrotic drugs, with a focus on their modulation of exosomal protein content and functional profiles. The clinical relevance of exosomal protein profiles in OSF treatment, particularly through the repurposing of metformin, will also be evaluated. Methodologically, the study involves in vitro experiments on OSF cell lines, employing techniques such as qPCR, Western Blot, and Liquid Chromatography-Mass Spectrometry (LCMS) for protein profiling, alongside a clinical trial to assess therapeutic efficacy.

The project is structured around several key objectives:

1. Impact Assessment: Evaluate how metformin and antifibrotic drugs affect exosome secretion and activity in OSF cell lines.

2. Morphological and Gene Expression Analysis: Analyze morphological changes and exosomal gene expression profiles in OSF cell lines post-treatment compared to control groups.

3. Molecular Pathways: Identify and analyze the molecular pathways modulated by metformin and antifibrotic drugs that influence exosomal protein content and functions.

4. Exosome Characterization: Characterize exosomes isolated from different treatment groups using advanced techniques such as dynamic light scattering (DLS), scanning electron microscopy (SEM), Western Blot (WB) and Liquid Chromatography-Mass Spectrometry (LCMS).

5. Clinical Relevance: Explore the potential clinical applications of the exosomal protein profiles for OSF treatment, focusing on the repurposing of metformin.

6. Methodology: The study includes both in vitro and clinical components, with the in vitro studies involving dose-response experiments, microscopy, gene expression analysis, and exosome isolation and characterization. The clinical trial will assess the efficacy of metformin and antifibrotic drugs in patients with OSF through randomized double-blind controlled trials.

This research has the potential to uncover new therapeutic strategies for OSF, particularly through the repurposing of metformin, which could lead to better clinical outcomes for patients suffering from this precancerous condition.

Study Timeline

• Status Verified: 2025-03
• Study First Submitted: 2025-03-05
• Study First Submitted QC: 2025-03-11
• Study First Posted: 2025-03-12
• Study Start: 2025-04-07
• Last Update Submitted: 2025-04-29
• Last Update Posted: 2025-05-04
• Primary Completion: 2025-10-30
• Study Completion: 2025-12-01

Recruitment & Eligibility

• Status: RECRUITING
• Sex: All
• Target Recruitment: 45

Inclusion Criteria

1. Patients with OSF having fibrous bands
2. Patients with limited mouth opening due to OSF in Stage 2 and Stage 3
3. Patients who have not received any treatment for OSF in the previous three months
4. Patients with habits of pan, Chalia, Gutkha
5. The age group between 18 and 45 years

Exclusion Criteria

1. Patients presenting with OSCC
2. Patients with limited mouth opening due to impaction of the third molar
3. Patients with limited mouth opening due to temporomandibular joint disorder
4. Any history of Metformin intolerance or contraindications to its use.
5. Medical conditions (e.g., cardiovascular disease, renal/hepatic impairment) or drug therapy (>6 months) with immunosuppressants, corticosteroids, or antifibrotics.
6. Pregnancy or lactation.
7. Participation in other clinical trials concurrently.
8. Inability / unwilling to provide informed written consent.

Study & Design

• Study Type: INTERVENTIONAL
• Study Design: PARALLEL

Arm && Interventions

Group	Intervention	Description
Metformin + Supportive Care	Beclomaethasone and Vitamin E	Group 2 (Metformin + Supportive Care): Metformin: 500 mg twice daily + Beclomethasone mouthwash three times + Capsule vitamin E 400mg daily.
Pirfenidone + Supportive Care	Pirfenidone (PFD)	Group 3 (Pirfenidone + Supportive Care): Pirfenidone: 200 mg twice daily + Beclomethasone mouthwash three times + Capsule vitamin E 400mg daily.
Pirfenidone + Supportive Care	Beclomaethasone and Vitamin E	Group 3 (Pirfenidone + Supportive Care): Pirfenidone: 200 mg twice daily + Beclomethasone mouthwash three times + Capsule vitamin E 400mg daily.
Beclomaethasone and Vitamin E	Beclomaethasone and Vitamin E	Group 1 Control (Supportive Care): Beclomethasone mouthwash three times + Capsule vitamin E 400mg daily.
Metformin + Supportive Care	Metformin Hydrochloride 500Mg Tablet	Group 2 (Metformin + Supportive Care): Metformin: 500 mg twice daily + Beclomethasone mouthwash three times + Capsule vitamin E 400mg daily.

Primary Outcome Measures

Name	Time	Method
Change in Exosome Secretion and Characterization in OSF Cell Lines	24, 48, and 72 hours post-treatment	This outcome evaluates the effect of Metformin and Pirfenidone on exosome secretion and size distribution in OSF cell lines. Exosomes will be isolated and characterized using Scanning Electron Microscopy (SEM) and Dynamic Light Scattering (DLS) to assess morphological changes, size, and concentration differences across treatment groups.
Changes in Fibrosis-Related Gene Expression in OSF Cell Lines	24 and 48 hours post-treatment	This outcome assesses the effect of Metformin and Pirfenidone on fibrosis-related gene expression (TGF-β, Collagen 1A1, α-SMA) in OSF cell lines. Changes will be analyzed using quantitative PCR (qPCR).
Reduction in Fibrosis Severity in OSF Patients	Baseline, 4 weeks, 8 weeks, and 12 weeks, 16 weeks, 20 weeks and 24 weeks	This outcome measures clinical improvement in Oral Submucous Fibrosis (OSF) patients treated with Metformin, Pirfenidone, or Supportive Care (Beclomethasone + Vitamin E). Severity will be assessed using the Burning Mouth Index by verbal numeric rating scale (0-10) at baseline and every 4 weeks during the trial and Interincisal Mouth Opening (IMO).
Change in TGF-β and Collagen Levels in Exosomes from OSF Cell Lines	24, 48, and 72 hours post-treatment	This outcome assesses whether Metformin and Pirfenidone reduce fibrosis-related molecular markers (TGF-β and Collagen) in exosomes isolated from OSF cell lines. Exosomal protein levels will be analyzed using Western Blot and Liquid Chromatography-Mass Spectrometry (LCMS) to determine drug-induced changes in fibrosis-related pathways.

Secondary Outcome Measures

Name	Time	Method
Morphological Changes in OSF Cell Lines Post-Treatment	24, 48, and 72 hours post-treatment	This outcome evaluates the morphological alterations in OSF cell lines following treatment with Metformin and Pirfenidone. Changes in cell shape, size, and fibrosis-associated structural modifications will be assessed using Scanning Electron Microscopy (SEM).
Size and Distribution of Exosomes in OSF Cell Lines	24, 48, and 72 hours post-treatment	This outcome measures changes in exosome size, distribution, and concentration after treatment with Metformin and Pirfenidone. Characterization will be performed using Dynamic Light Scattering (DLS) and Scanning Electron Microscopy (SEM).
Functional Impact of Treated OSF cells	24, 48, 72 and 96 hours post- treatment	This outcome evaluates whether Metformin- and Pirfenidone-treated OSF cell lines, as compared to control untreated OSF cells, influence fibroblast migration and proliferation. Cell behavior will be assessed through Scratch migration and proliferation assays to determine functional effects.
Safety and Viability of OSF Cell Lines Post-Treatment	24, 48, and 72 hours post-treatment	This outcome measures the cytotoxicity and cell viability of OSF cell lines following treatment with Metformin and Pirfenidone. MTT assay will be used to assess potential toxic effects of the drugs at different concentrations.

Trial Locations

Locations (1)

Ziauddin University; 🇵🇰 Karachi, Sindh, Pakistan