Patent Description:
Corneal or conjunctival epithelial cell damage occurs in many corneal diseases such as dry eye disease; occupational dry eye caused by insufficient rate of blinking; lack of tear production such as is found, for example, in Sjögren's syndrome; Meibomian oil deficiency; drug or preservative induced cell damage; mechanical cell damage induced by such factors as contact lens wearing; and ocular surface disease.

Treatments for corneal or conjunctival epithelial cell damage known in the art tend to be strictly palliative and are generally aimed at lessening the severity of the symptoms of the condition rather than at treating the condition by healing the damaged cells. For example, Sjögren's Syndrome (henceforth "SS") is normally treated by use of moisture replacement therapies such as by application of artificial tears, which have only limited effectiveness, or by drugs that affect the immune system, which during chronic treatment lead to side effects in up to <NUM>% of cases. See, for example, <NPL>.

Aragona et al. (<NPL>) have disclosed a treatment for SS that involves application of a hypotonic solution containing a high concentration of hyaluronate, and hypothesized that the effectiveness of the formulation is likely to be due to its role in controlling the expression of the CD44 hyaluronate receptor in the corneal and conjunctival cells, thereby reducing inflammation in the eyes. It has been additionally reported that when artificial tears are used to treat SS, more mucoadhesive artificial tears are more effective (<NPL>), hyaluronate being well-known as a mucoadhesive polymer; see, for example, <NPL>.

Other disclosures that teach that hypotonic solutions are preferred treatments for dry eye syndrome include <NPL>, which reports that hypotonic hyaluronate was more effective against Dry Eye Syndrome than was isotonic hyaluronate, and <NPL> which likewise teaches that hypotonic solutions are particularly beneficial in the treatment of dry eyes, and suggests that their effectiveness arises because they counter the hyperosmolarity typical of dry eye syndrome. A similar conclusion is reached by <NPL>, which teaches that application of autologous serum is a preferred treatment for SS, but then cites the teaching of Aragona et al. that topical application of hyaluronic acid, preferably hypotonic hyaluronic acid, has also shown to be effective.

<CIT> discloses humectant eye drops that are useful for treatment of dry eye syndrome. The eye drops have non-Newtonian rheological properties that mimic the behavior of natural tears, and comprise an aqueous solution of a low molecular weight humectant polyol at approximately isotonic concentration, an anionic polymer having a molecular weight between <NUM>,<NUM> and <NUM>,<NUM>,<NUM>, and less than <NUM> inorganic salt. Eye drops of this formulation containing <NUM>% (w/v) glycerol, hyaluronate, and carbomer have been shown to be effective in treating dry eye syndrome; see, for example, <NPL>, and <NPL>. This composition was also shown to be effective in treating irritation of mucous or skin cells; see <CIT>.

<CIT> (henceforth '<NUM>) discloses an ophthalmic preparation and method for treating conjunctivochalasis, a disease of the conjunctival folds. The preparation comprises an aqueous solution of glycerol, a normal component of human blood. In contrast to artificial tear solutions known in the art, the preparation disclosed in '<NUM> provides a statistically significant reduction in the severity of the condition as measured by the Lid Parallel Conjunctival Folds (LIPCOF) scale. See, for example, <NPL>.

A glycerol-containing humectant eye drop composition is disclosed in <CIT>. The composition disclosed therein comprises an isotonic solution comprising <NUM> - <NUM>% polyvinylpyrrolidone, <NUM> - <NUM>% hydroxypropyl methycellulose, <NUM> - <NUM>% glycerol, and a preservative. No assertion of non-Newtonian rheological properties is made. A similar eye drop composition containing glycerol and propylene glycol is disclosed in <CIT>.

A glycerol-containing humectant eye drop composition comprising <NUM>% glycerol and poly(l-lysine)-graft-poly(ethylene glycol) as an excipient was shown to extend the tear film break-up time relative to a commercially available artificial tear formulation comprising propylene glycol and polyethylene glycol (<NPL>).

Despite these advances, compositions and methods for treating corneal irritation, in particular that arising from SS, that heal the damaged cells rather than merely ease the symptoms remain a long-felt, but as yet unmet need.

The present invention is directed to an ophthalmic preparation for use in the treatment of irritation or damage to conjunctival or corneal epithelial cells resulting from Sjögren's syndrome, according to claim <NUM>.

Furter developments are according to dependent claims <NUM>-<NUM>.

In the following description, various aspects of the invention will be described. For the purposes of explanation, specific details are set forth in order to provide a thorough understanding of the invention. It will be apparent to one skilled in the art that there are other embodiments of the invention that differ in details without affecting the essential nature thereof.

The inventor has discovered that surprisingly, glycerol is an effective substance for treatment of irritation of corneal epithelial cells, irritation of conjunctival epithelial cells, and hence, damage to corneal or conjunctival cells when these conditions are caused by factors other than infection. Topical application of glycerol (e.g. in an aqueous solution) to the affected eye reduces or even eliminates entirely corneal or conjunctival irritation.

The invention herein disclosed is an ophthalmic preparation for treatment or prevention of irritation to corneal or conjunctival epithelial cells, the preparation comprising an aqueous glycerol solution. The solution comprises <NUM>% glycerol (w/v). In preferred embodiments of the invention, the solution is isotonic.

In preferred embodiments of the invention, the composition comprises an aqueous glycerol solution in which the concentration of inorganic salts is less than <NUM>. In preferred embodiments of the invention, the viscosity of the solution is controlled by addition of a quantity of high molecular weight polymer (MW > <NUM><NUM> Dalton) such as hyaluronate, carbomer or a mixture thereof, sufficient to bring the solution to the desired viscosity. All ingredients are of purity sufficient for use in eye drops.

The solutions may then be transferred to a container appropriate for dispensing it as eye drops.

While in some embodiments of the invention, the only active ingredient present in the composition is glycerol, the composition may comprise in addition a pharmaceutically effective concentration at least one pharmacologically active agent. If necessary, any stabilizer, preservative, antioxidant, buffer or combination thereof appropriate for use with the pharmacologically active agent may be added to the solution in any concentration suitable for use in eye drops.

It is within the scope of the invention to disclose the use of the eye drops in the non-surgical treatment of, or prevention of, irritation of and irritation to the corneal or conjunctival epithelial tissue, particularly damage due to causes other than infection, and a method of non-surgical treatment or prevention of irritation of and hence, damage to the corneal or conjunctival epithelial tissue, particularly damage due to causes other than infection. A non-limiting example of a condition that can be treated by the eye drop composition disclosed herein is SS.

A typical protocol for use of the eye drops disclosed herein to treat or to alleviate corneal and/or conjunctival epithelial cell damage is to place drops in the affected eye three to eight times daily until the severity of condition is reduced to an acceptable level. In particularly severe cases, more frequent applications may be necessary, and in less severe cases, one or two daily treatments may be sufficient. The progress of the treatment can be measured by the use of techniques such as Lissamine Green staining or Rose Bengal staining to track the condition of the epithelial cells. In some preferred embodiments of the treatment, application of the composition disclosed herein is performed for no more than three months, by which time statistically significant improvement of the condition of the corneal or conjunctival epithelial cells is observed. In some preferred embodiments of the treatment, application of the composition disclosed herein is performed for no more than one month, by which time statistically significant improvement of the condition of the corneal or conjunctival epithelial cells is observed.

It is within the scope of the invention to include within the method prophylactic application of the composition disclosed herein in order to prevent recurrence of the condition. After the course of therapeutic treatment, which typically lasts no more than three months, a maintenance regimen comprising prophylactic application of the eye drops is begun. Application of the eye drops one to three times daily is usually sufficient to prevent recurrence of the irritation.

In contrast to methods known in the art, in particular, those that use drugs that affect the immune system, no side effects were observed in any of the treatment protocols in which the invention disclosed herein was tested. In particular, no side effects were observed with long-term use of eye drops containing as much as <NUM>% (w/v) glycerol.

The following examples of the preparation and use of the ophthalmological composition herein disclosed are intended to assist a person having ordinary skill in the art to make and use the invention, and are not to be construed as being in any way limiting. Examples <NUM> and <NUM> are according to the invention whereas examples <NUM>, <NUM> and <NUM> are not according to the invention.

A suitable concentration of preservative may optionally be added.

A composition was prepared as described in Example <NUM> above and was tested on <NUM> patients suffering from Sjögren's syndrome. Results of the study are summarized in Table <NUM>; the value in each column is the mean score with the standard error of the mean given in parentheses. Lissamine Green staining evaluated by Oxford Grade is a measure of the severity of dry eye syndrome, while OSDI (Ocular Surface Disease Index) is a measure of patient satisfaction. At the conclusion of the treatment, the patients' eyes were free of measurable damage.

These results are surprising and unexpected, since the Schirmer's test, which measures the level of tear formation was very low at the start of the study (<NUM> ± <NUM>) and did not change significantly after <NUM> months of treatment (<NUM> ± <NUM>). That is, in the patients treated according to the method herein disclosed, using the composition herein disclosed, objective measures of the level of eye irritation symptomatic of Sjögren's Syndrome due to the severely reduced tear production characteristic of the condition showed a significant decrease, even though tear production did not increase. This observation cannot be explained by the known physicochemical moisturizing effect of glycerol.

An experiment was performed to investigate the effect in vitro of a solution containing glycerol on human corneal epithelial cells, in particular, on the expression of barrier genes Involucrin, Occludin, Filaggrin, and Cadherin-<NUM>.

Immortalized human corneal epithelial cells (HCEC cell line) cultured in DMEM/F12 with <NUM>% FBS and <NUM> ng/ml human epidermal growth factor (Invitrogen - Gibco). The cells were treated for three hours with one of the following three compositions: (a) an aqueous solution of glycerol (<NUM>% w/v); (b) <NUM>µg/ml Polyinosinic:polycytidylic acid (p(I:C), an activator of TLR3 to induce inflammation; and (c) a combination of the two previous compositions.

The expression of the barrier genes was determined at the mRNA level by use of quantitative "real-time" PCR (Q-PCR). Q-PCR was performed on an ABI Prism <NUM> sequence detection system (Applied Biosystems, Foster City, CA) using the <NUM>' nuclease assay. Total RNA was isolated using TRIzol (Invitrogen) and <NUM>µg of total RNA were reverse-transcribed into cDNA by using <NUM> U of AMV reverse transcriptase (Promega, Madison, WI, USA) and <NUM>µg/µl random primers (Promega). PCR amplification was performed by using the TaqMan primers and probes. As internal controls, transcripts of cyclophilin A (PPIA) were determined.

The pro-inflammatory challenge p(I:C) (<NUM>µg/ml) markedly decreased the expressions of Involucrin, Occludin, Filaggrin, and Cadherin-<NUM>. However, of greatest importance, co-incubation of the human corneal epithelial cells with glycerol (<NUM>%) during the p(I:C)challenge significantly prevented the barrier-impairing actions of the TLR3 agonist.

Claim 1:
An ophthalmic preparation for use in the treatment of irritation or damage to conjunctival or corneal epithelial cells resulting from Sjögren's syndrome, said ophthalmic preparation comprising an aqueous solution of glycerol, characterized in that said ophthalmic preparation comprises an aqueous solution containing <NUM>% (w/v) glycerol, <NUM>% (w/v) sodium hyaluronate, and <NUM>% Carbomer <NUM>, said solution buffered to a pH of approximately <NUM>.