5FU vs 5FU With Viscoelastic Formulation for the Prevention of Scarring Post-trabeculectomy
Condition: Glaucoma; Trabeculectomy; Wound Healing
MeSH terms: Cicatrix
Study type: Interventional
Phase: Phase 4

Summary: 
      Trabeculectomy is the most effective method of lowering intraocular pressure in glaucoma that
      is not well controlled with drop medication. The post-operative wound healing response
      remains the major barrier in surgical success. Scar tissue formation results in occlusion of
      the surgical drainage site created. As a consequence, the intraocular pressure rises once
      again. The current use of anti-scarring agents such as 5 Fluorouracil (5FU), is a well
      established method employed by ophthalmologists to clinically reduce the postoperative
      scarring response. However, in patients with a high risk of bleb failure, repeated
      subconjunctival injections of 5FU following bleb needling is often required resulting in
      increased clinical load and patient inconvenience. A slow release formulation of 5FU would be
      of benefit by releasing the 5FU into the subconjunctival space over an extended period
      thereby providing a prolonged therapeutic effect. In addition, hyaluronic acid is known to
      possess antifibrotic properties and from its gel-like physical state, would potentially act
      as a physical tissue spacer that may further limit active subconjunctival scarring at the
      site where it is injected. We hypothesise that injecting a mixture of 5FU with hyaluronic
      acid would give better outcomes than injecting 5FU alone.

      The aim of this study is to determine whether bleb needling with subconjunctival injection of
      Viscoelastic/5FU formulation is an effective method for prolonged drug delivery in preventing
      the post-operative scarring response. This will be a prospective case-controlled study
      involving patients who have already undergone trabeculectomy and who require bleb needling
      and subconjunctival 5FU injections.
    

Detailed description: 
      Specific Aims

      The aim of this study is to determine whether needling of the conjunctival bleb after
      trabeculectomy with subconjunctival injection of a 5 Fluorouracil (5FU)/ viscoelastic
      formulation is an effective method for prolonged drug delivery in preventing post-operative
      scarring in the conjunctiva.

      Study Design

      Cell Culture Experiments

      Cell culture experiments were carried out to determine the effect of the hyaluronic acid, at
      varying commercially available concentrations, on human Tenon's fibroblasts, the target cell
      type of our proposed HA/ 5FU mixture. We also perform tests to examine the pharmacokinetic
      drug release profiles of the different 5FU/HA formulations. This allowed us to determine the
      most effective formulation to use for patients in the clinical arm of the study.

      Briefly, human Tenon's fibroblasts attained from tissue explants from cadaver eyes were grown
      until confluent in fetal calf serum containing the standard antibiotics for tissue culture,
      gentamicin and amphotericin, and placed in an incubator at 37°C under 95% humidity with 5%
      CO2. The cells were passaged when confluent and used for experiments between passages 3-6.

      Alpha smooth muscle actin expression Tenon's fibroblasts were seeded on 24 well plates and
      incubated in fetal calf serum (FCS) containing the standard antibiotics for tissue culture,
      gentamicin and amphotericin, and placed in an incubator at 37°C under 95% humidity with 5%
      CO2. The culture medium was removed after 24 hours and replaced with fresh medium described
      below.

      Collagen I production Standard 3-D collagen I gels seeded with fibroblasts for gel
      contraction assays were employed to quantify collagen production by fibroblasts treated with
      different HA concentrations. Gels treated with TGF beta (a known stimulator for collagen I
      synthesis and inducer of alpha smooth muscle actin expression ie myofibroblast phenotype)
      acted as positive control. Type I collagen derived from rat tail were mixed with culture
      medium and following confluency, trypsinised cells were resuspended in this mixture and cast
      in a 24 well plate and allowed to polymerise in an incubator at 37°C. After polymerisation,
      the gels were gently detached from the plates in which they are cast and suspended in fresh
      medium described below

      All experiments were conducted in triplicate and repeated 3 times.

      Human Tenon's fibroblasts were cultured in fresh media containing HA alone, 5-FU/HA (HA
      concentrations10, 14, 23 mg/ml) or cultured without HA or 5FU (controls). Fibrosis and
      scarring response of the myofibroblasts was assessed through Collagen I and alpha smooth
      muscle actin (ASMA) expression which were quantified by Real Time -Polymerase Chain Reaction
      (RT-PCR). WST-1 assay determined cell viability. 5-FU solution or powder was dispersed in HA
      and mechanically stirred and then vortex mixed.

      Our results showed that HA markedly reduced ASMA and collagen I mRNA expression by
      myofibroblasts compared to controls without HA. Treatment with HA following 5FU (10mg/ml)
      pretreatment resulted in a greater reduction in ASMA and collagen I expression compared to HA
      treatment alone, and statistical significance reached with 23mg/ml HA (p<0.05). Cell
      proliferation and viability was not affected by HA.

      Our team of investigators has completed the experiments examining the release of 5FU from the
      5FU/ HA mixture. 5FU solution or powder was mechanically stirred and vortex mixed in HA and
      the release of 5FU was quantified using high performance liquid chromatography (HPLC). The
      concentration of 5FU used was 10mg/ml and the concentration of HA used was 10, 14 and 23
      mg/ml.

      The 5FU/HA mixture delayed drug release to 48 hours, with HA of 14mg/ml and 23mg/ml showing a
      greater delay in drug release than 10mg/ml, (p<0.05). There was no significant difference in
      rate of drug release between powder and solution form of 5FU although the pH was notably
      different ie 6.6 and 10 respectively.

      The results of our in-vitro studies and drug release assays demonstrate that this 5FU/HA
      mixture has the desired therapeutic effect on Human Tenon's myofibroblasts. The optimum
      concentration for HA is 23mg/ml and it would be combined with 5FU 10mg/ml. We expect that 5FU
      would be released from the mixture over 48 hours; both HA and 5FU will have anti-scarring
      effects in the subconjunctival space.

      Patient Recruitment:

      Patients will be recruited and randomized at the first postoperative visit where a 5-FU
      injection is indicated according to clinical parameters indicating active scar formation.
      (usually after the first post-operative month when suturelysis has been completed). All post
      operative care apart from the 5FU/viscoelastic mixture injection in the control group will be
      unchanged from current treatment standards.

      At least 50 patients who have under gone trabeculectomy at the Singapore National Eye Centre
      will be recruited in the study from glaucoma clinics at Singapore National Eye Centre. This
      will consist of different types of glaucoma. 25 patients will receive subconjunctival
      injection of 5FU after bleb needling while 25 will receive injections of a 5FU/ viscoelastic
      formulation.

      Patient assessment and examination at baseline

      All patients (cases and controls) will have the following examination: Snellen visual acuity,
      slit-lamp examination of the anterior segment, bleb and intraocular pressure measurement with
      a Goldmann tonometer. Tonometry will also be performed pre injection using a pneumatonometer
      (Reichert) according to manufacturer's guidelines.

      Colour photographs of the bleb will be captured and stored digitally for masked bleb grading.
      The Visante OCT will be used to image the bleb morphology in dark conditions. It is a new
      non-contact instrument that rapidly obtains high-resolution images of the angle. The image
      capture scan takes less than 10 seconds and is akin to taking a photograph. The device allows
      qualitative and quantitative angle imaging, which is objective and reproducible. This will
      take place prior to the needling procedure.

      Bleb needling & subconjunctival injection

      Having taken informed consent from the patient, the patient will be assigned into either the
      case or control group. In brief, needling of the conjunctival bleb will be carried out at the
      slit lamp using an aseptic technique. The doctor performing the procedure will use sterile
      gloves and clean the patient's eyelids and peri-orbital skin with povidone iodine 10%. The
      eye will be anaesthetised with Guttae Tetracaine 0.5% and a wire speculum inserted to
      separate the eyelids. Povidone iodine is the inserted into the conjunctival sac. 0.1ml of
      lignocaine 2% is injected into the subconjunctival space, temporal or nasal to the bleb
      before needling of the bleb is performed with a 27 guage needle attached to a 1ml syringe.
      5FU solution (10mg/ml) (control arm) or 5FU/viscoelastic formulation (10mg/ml of 5FU in
      23mg/ml HA, (50/50) volume/volume) (experimental arm) is injected posterior to the bleb after
      needling is complete. Injections are given within the same quadrant as the bleb. Post
      injection the conjunctival sac is thoroughly rinsed and re-examined on the slit-lamp. The IOP
      is checked at the end of the procedure using the Goldmann tonometer and the pneumotonometer.

      Patients will receive current standard of care with respect to intraoperative antifibrosis
      therapy and the timing and frequency of postoperative 5-FU. Further administration of
      subconjunctival 5-FU solution or 5FU/HA formulation, (depending on the treatment arm the
      patient was initially assigned to) at subsequent office visits will be administered when
      required according to current clinical practice for patients in both treatment arms. This
      will be recorded in the study.

      Follow-up examinations

      Bleb imaging with the Visante anterior segment OCT and IOP measurements will be recorded at
      every visit ie week 1, and 12. The need for further needling or other intervention eg
      massage/suture removal/lysis (if relevant) will be clinically decided by the ophthalmologist
      at each visit and bleb imaging will be performed before any additional procedures perfomed if
      it falls on a research visit ie week 1 or 12. Bleb grading from slit lamp photographs will be
      performed at baseline pre-injection and at week 12.
    

Intervention name(s): 5 Fluorouracil with hyaluronic acid; 5 Fluorouracil
Intervention type(s): Drug; Drug
Intervention description(s): Participants will receive a subconjunctival injection of 0.1ml of a mixure of 5 Fluorouracil (50mg/ml) with hyaluronic acid (23mg/ml) once at enrollment into the study.; Pariticpants will receive a subconjunctival injection of 0.1ml of 5 Fluorouracil 50mg/ml once at enrollment
Other intervention name(s): Healon 5 - hyaluronic acid 23mg/ml
Intervention MeSH terms: Fluorouracil; Hyaluronic Acid
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: None (Open Label)
Primary purpose: Treatment
Keywords: Trabeculectomy; Wound healing; 5 Fluorouracil; Hyaluronic acid; post-operative scarring