ISRCTN - ISRCTN60434145: TGF-βs in the eye of patients with neovascular age-related macular degeneration

Plain English Summary

Background and study aim
Wet age-related macular degeneration (AMD) is the commonest cause of blindness in developed countries. Many proteins (called cytokines) are thought to be responsible for this disease. One of these (VEGF-A) is the target of the current therapy. With this study, we want to clarify the role of the cytokines called TGF-beta in the disease to better understand the causes and to develop a more effective therapy.

Who can participate?
Patients aged 50 and over with AMD and cataract patients as controls.

What does the study involve?
Participants affected by AMD receive, as standard treatment, a loading dose of three injections over a three month period. Before each treatment a small sample of aqueous humor (the eyeball is filled with this liquid) is collected by a syringe from the anterior chamber of the eye. Cataract controls are subjected to sample collection just before the planned cataract surgery.

What are the possible benefits and risks of participating?
Study participation may not directly help patients but the information obtained from the study may help to improve the knowledge of the disease and consequently the treatment of people with wet AMD in the future. This is a study embedded in the routine clinical procedures without any additional treatment (except the sample collection), therefore there should be no risks or disadvantages to patients taking part. All participants receive the same treatment as prescribed by their physicians.

Where is the study run from?
Ophthalmology Unit of the Department of Medicine, Surgery and Neuroscience, Siena University Hospital (Italy)

When is the study starting and how long is it expected to run for?
June 2016 to December 2016

Who is funding the study?
University of Siena

Who is the main contact?
Prof. Gian Marco Tosi
gmtosi18@gmail.com

Trial website

Contact information

Type

Scientific

Primary contact

Prof Gian Marco Tosi

ORCID ID

Contact details

Ophthalmology Unit
Department of Medicine
Surgery and Neuroscience
University of Siena
Viale Bracci
1
Siena
53100
Italy
+39 0577 586162
gmtosi18@gmail.com

Additional identifiers

EudraCT number

ClinicalTrials.gov number

Protocol/serial number

AMD001.30

Study information

Scientific title

TGF-β concentrations and activity in the aqueous humor of patients with neovascular age-related macular degeneration before and after ranibizumab treatment

Acronym

Study hypothesis

The aim of this study is to evaluate the protein concentration of active TGF-β1, TGF-β2, and TGF-β3 in the aqueous humor of patients affected by naïve nAMD, at baseline and after intravitreal anti-VEGF-A injection by performing a luciferase-based reporter assay to test the TGF-β pathway activation by aqueous humor samples of the same patients, with the aim to clarify the role of the TGF-β family members in CNV.

Ethics approval

The Ethics Committee Regione Toscana Area Great South East (CEAVSE), 20/04/2015, ref: HTRA1_GDF6_2015

Study design

Observational single-centre case-control study

Primary study design

Observational

Secondary study design

Case-control study

Trial setting

Hospitals

Trial type

Other

Patient information sheet

Not available in web format, please use the contact details below to request a patient information sheet

Condition

Age-related macular degeneration (AMD)

Intervention

We measure the concentrations of active TGF-β1, TGF-β2, and TGF-β3 by ELISA in the aqueous humor of patients affected by nAMD, who received 3 consecutive monthly intravitreal injections of ranibizumab according to the routine clinical practice management. Age-matched cataract patients serve as controls. Anterior chamber taps are performed in the operating room prior to each intravitreal injection (patients) and before cataract surgery (controls). A 30-gauge needle is inserted into the anterior chamber and 0.16-0.2 mL of aqueous is collected, centrifuged at 1500 g for 20 minutes to remove cells and debris, aliquoted and frozen at -80°C until analysis. Samples are collected at baseline (before the first injection), before the second injection, and before the third injection. The same samples are used in a luciferase-based reporter assay to test the TGF-β pathway activation. To perform the latter, Lenti-X 293T cells are transfected with a plasmid carrying the NanoLuc® gene under the transcriptional control of three copies of the SMAD binding element. The plasmid pGL4.54[luc2/TK] (Promega Corp., Madison, WI), carrying the reporter luc2 gene coding firefly luciferase under the control of the costitutive HSV-TK promoter, is co-transfected as a normalizer for transfection efficiency. Cells are then treated with 10 𝜇l of aqueous humor of the nAMD patients, naïve or treated, or of the control samples. After 3 hours Firefly and NanoLuc luciferase activities are measured for each sample.

Intervention type

Drug

Phase

Not Applicable

Drug names

Ranibizumab

Primary outcome measure

1. TGF-beta concentration is measured using ELISA kits for human TGF-β1, TGF-β2 (Quantikine ELISA kits #DB100B and #DB250, respectively; R&D Systems, Minneapolis, USA) and TGF-β3 (#SEB949Hu; Cloud-Clone Corp., Houston, TX) at baseline, 1 month and 2 months (Only baseline for controls)
2. TGF-β pathway activation is measured using the transfection of Lenti-X 293T cells and the Nano-Glo® Dual-Luciferase® Reporter Assay System (#N1610; Promega Corp., Madison, WI) at baseline, 1 month and 2 months (only baseline for controls)

Secondary outcome measures

There were no secondary outcomes of interest.

Overall trial start date

16/03/2015

Overall trial end date

21/06/2017

Reason abandoned (if study stopped)

Eligibility

Participant inclusion criteria

Case inclusion criteria:
1. Both males and females
2. Patients aged 50 and over
3. Able to provide written, informed consent to the study 
4. Active neovascular AMD in study eye with evidence of leakage from CNV on fluorescein angiography.

Control inclusion criteria:
1. Both males and females
2. Patients aged 50 and over, the age of the controls is chosen not to have statistical differences with the cases
3. Able to provide written, informed consent to the study
4. Patients in the waiting list for cataract surgery

Participant type

Patient

Age group

Senior

Gender

Both

Target number of participants

20 cases and 20 controls

Participant exclusion criteria

CASE exclusion criteria:
1. Prior treatment for neovascular age-related macular degeneration
2. Age less than 50 years old
3. Inability to comply with the study or follow up
4. Inability to perform fluorescein angiography
5. Axial length greater than 26 mm
6. Any previous ophthalmic surgery except cataract removal ( cataract surgery had to have been performed at least 9 months prior to inclusion)
7. Any other ocular disease other than neovascular age-related macular degeneration
8. Diabetes mellitus, use of immunosuppressive drugs and a malignant tumor in any location

Control exclusion criteria:
1. Any other ocular disease other than cataract
2. Any previous ophthalmic surgery
3. Age less than 50 years old 
4. Axial length greater than 26 mm
5. Diabetes mellitus, use of immunosuppressive drugs and a malignant tumor in any location

Recruitment start date

01/06/2016

Recruitment end date

21/12/2016

Locations

Countries of recruitment

Italy

Trial participating centre

Siena University Hospital
Ophthalmology Unit of the Department of Medicine Surgery and Neuroscience Viale Bracci, 1
Siena
53100
Italy

Sponsor information

Organisation

University of Siena

Sponsor details

Via Banchi di Sotto
55
Siena
53100
Italy
+39 0577 235555
urp@unisi.it

Sponsor type

University/education

Website

https://en.unisi.it

Funders

Funder type