NGF eye-drops topical administration in patients with retinitis pigmentosa, a pilot study

The present pilot study (EudraCT n. 2008-004561-26) followed the tenets of the Declaration
of Helsinki and was approved by the ethics committee of the institution. All the enrolled
patients were fully informed as to the nature and goals of the study. Written informed
consent was obtained from all patients.

Patient recruitment and Inclusion criteria

Sixteen eyes of 8 patients (6 males, 2 females; average age 49.7 ± 14.3 years) affected
by RP were included in the study (Table 1). All patients had progressive forms of RP based on history, clinical findings and
ERG abnormalities. Furthermore, patients met the following inclusion criteria: (1)
typical RP with a rod-cone pattern of retinal dysfunction, as determined by standard
Ganzfeld electroretinography, dark-adapted Tuebinger perimetry, and classic fundus
appearance. (2) Advances stage of the disease (at baseline: central portion of visual
field with Goldman V/4e 15 deg; fERG 1 uV). (3) Known inheritance pattern and/or
genotype under study. (4) At least 1 years of fERG and clinical examination follow-up,
with a minimum of three visits. (5) No or minimal ocular media opacities. (6) No concomitant
ocular (e.g. glaucoma, amblyopia) or systemic diseases. Patients with non- Usher syndromic
sub-types of RP, Leber’s congenital amaurosis or early onset RP with atypical functional
patterns were excluded.

Table 1. Patient details

Measures of ocular function and electroretinography

A full general and ophthalmologic examination (including detailed family history,
anterior segment biomicroscopy, BCVA, direct and indirect ophthalmoscopy, intraocular
pressure measurement) was performed on each patient at baseline.

Best-corrected visual acuities were obtained with a projected Snellen chart. Kinetic
visual fields were measured to the V4e white test light of the Goldmann perimeter
against the standard background of 31.5 apostilbs. Goldmann visual fields were digitized
and total visual field areas were calculated.

Cone focal ERGs (fERG) were recorded from the central 18° region using a uniform red
field superimposed on an equiluminant steady adapting background, used to minimize
stray-light modulation 19], 20]. The stimulus was generated by a circular array of eight red LEDs (? maximum, 660 nm;
mean luminance, 93 cd/m
²
) presented on the rear of a Ganzfeld bowl (white-adapting background). A diffusing
filter in front of the LED array made it appear as a circle of uniform red light.
fERGs were recorded in response to the sinusoidal 95 % luminance modulation of the
central red field. Flickering frequency was 41 Hz. Patients fixated monocularly at
a 0.25° central fixation mark, under the constant monitoring of an external observer.
Pupils were pharmacologically (1 % tropicamide and 2.5 % phenylephrine hydrochloride)
dilated to a diameter ?8 mm, and all subjects underwent a pre-adaptation period of
20 min to the stimulus mean luminance. fERGs were recorded by an Ag–AgCl electrode
taped on the skin over the lower eyelid. A similar electrode, placed over the eyelid
of the contralateral patched eye, was used as reference (inter-ocular recording).
fERG signals were amplified (10
⁶
-fold), bandpass filtered between 1 and 100 Hz (6 dB/oct), and averaged (12-bit resolution,
2-kHz sampling rate, 200–600 repetitions in 2–6 blocks). Off-line discrete Fourier
analysis quantified the amplitude and phase lag of the response fundamental harmonic
(1st harmonic) at 41 Hz.

Ocular and systemic complications potentially related to ngf administration

During the entire period of assessment (40 days; see below) particular attention was
paid to detect ocular and/or systemic side effects. Potential ocular complications
included inflammation (external or uveitis), pain, development of lens opacities,
and increased intraocular pressure. Systemic complications previously reported in
the literature include allergic reactions, systemic pain as well as weight loss 21].

A comprehensive medical evaluation was carried out by a general physician at day zero,
and at the end of the NGF treatment. All patients received oral and written information
about the experiment procedures before signing the informed consent.

Nerve growth factor isolation

NGF (2.5S) was purified from male mouse submandibular glands as already described
15], 22]. Briefly, the extract of submandibular glands of adult male mice was passed through
subsequent cellulose columns, to separate NGF by adsorption. NGF-containing fractions
were analyzed by spectrophotometry and Western blot analysis. NGF purity (95 %) was
estimated by high-performance liquid chromatography, while its biological activity
was evaluated by neurite outgrowth stimulation in rat PC12 cells. Purified NGF was
dialyzed, lyophilized under sterile conditions, and stored at ?20 °C until used. At
the time of use, purified NGF was dissolved in 0.9 % sterile saline solution in concentrations
of 200 µg/mL. The concentration of NGF in this solution was stable over the 10 day
treatment time.

NGF administration schedule

A total of 1 mg of NGF diluted in 5 mL of saline solution was administered in the
form of eye drops onto the conjunctiva of both eyes for 10 consecutive days 3 times
a day. This amount is considered sufficient to reach and stimulate NGF receptors in
most cerebral cholinergic areas of the brain and optic pathways, as previously reported
13]. We preferred to use murine NGF, instead of human-recombinant NGF, because contrasting
results have been reported on the efficacy of the latter, mainly due to a lack of
in vivo studies 16].

Testing schedule

fERG examinations were performed at baseline, at the end of the 10 days period of
NGF administration and 30 days later. BCVA measurement and Goldmann visual field examination
were performed at baseline and 30 days after the end of NGF administration.

Data analysis

Changes in BCVA and fERG amplitude obtained after treatment were evaluated as individual
changes, as well as in the contest of test–retest variability data obtained from a
large cohort of RP patients followed clinically at the Visual Electrophysiology Service
of the Institute of Ophthalmology at Universita’ Cattolica del S. Cuore, which have
been subject of a long term follow-up study 23].

In each patient, the pre and post treatment total areas of Goldmann visual fields
were compared. A percentage difference 20 % was considered clinically significant
according to previous studies on test–retest variability in patients with RP 24].