WO2010128486A2

WO2010128486A2 - Ophthalmic formulation for the treatment and prevention of pterygium

Info

Publication number: WO2010128486A2
Application number: PCT/IB2010/052025
Authority: WO
Inventors: Stefano Sebastiani; Roberta Di Pietro; Marco Ciancaglini
Original assignee: Stefano Sebastiani; Roberta Di Pietro; Marco Ciancaglini
Priority date: 2009-05-07
Filing date: 2010-05-07
Publication date: 2010-11-11
Also published as: IT1395457B1; WO2010128486A3; ITMI20090772A1

Abstract

The present invention concerns an ophthalmic formulation for the prevention of ocular pterygium growth and development containing an extract of Curcuma longa also in the presence of other ingredients such as ascorbic acid and visco-elastic substances.

Description

OPHTHALMIC FORMULATION FOR THE TREATMENT AND PREVENTION OF PTERYGIUM

Field of invention

The present invention regards the formulation of an ophthalmic preparation to prevent the growth and development of pterygium containing an extract of Curcuma longa and other optional ingredients like ascorbic acid and visco-elastic substances. Prior art

Ocular pterygium is usually a bilateral pathology of bulbar conjunctiva, generally located at the nasal side and, occasionally, even at the temporal side of conjunctiva. It is characterized by an invasive growth of vascularized connective tissue and of conjunctival epithelium that results in tissue remodelling, cell and fibrovascular proliferation and inflammation. In spite of its benign nature, due to its growth the pterygium can invade cornea so that reducing visual function and, eventually, leading to blindness. The current therapy is generally surgical and requests, in the advanced cases, a complicated procedure with an elevated risk of relapse. Indications to pterygium surgical removal are the following: sight reduction due to the growth in proximity to the visual axis; visual axis alteration; sight reduction due to astigmatism resulting from ocular surface deformation; ocular movements limitation; progressive growth observed by the patient or the oculist; irritation symptoms and ocular discomfort; esthetic reasons.

Generally, pterygium has a major prevalence in tropical and subtropical geographical areas and in laboratories with a greater exposition to sunlight; in epidemiological studies performed on the entire population has emerged a frequency of 17% in Indonesia, of 30% in Peru, and of 7.3% in the adult population of Australia. The incidence in temperate climates is estimated between 2-7%.

Numerous epidemiological studies have given evidence to the correlation between pterygium and sunlight exposition. This correlation is compatible both with the hypothesis that the highest risk factor is the prolonged absorbance of UV rays, both with the development of this lesion that is tipically located in the ocular area more exposed to UV light. In addition, other environmental agents have been identified as potential inducers of pterygium growth and formation including the exposition to dust, low humidity, microtrauma, smoke, sand and human papilloma virus infections. The mechanism of pterygium formation has not been clarified yet. Recent observations have proved the implication of genetic factors, anti-apoptotic mechanisms, cytokines, growth factors, tissue remodelling induced by metallo- proteinases, immunological mechanisms and viral infections. The most established model recognizes an important role to oxidative stress that appears increased in pterygium epithelium compared to normal conjunctival epithelium, inducing the production of proteins favouring cell growth.

Upon these stimuli, pterygium conjunctival epithelium, characterized by squamous metaplasia and hyperplasia, invades cornea with a centripetal direction, supported by the underlying fibrovascular tissue. The overabundant stromal proliferation of fibroblasts and blood vessels is in turn accompanied by inflammatory infiltration and abundant deposition of extracellular matrix rich in elastin and collagen. Curcuma longa, a plant whose ryzome is utilized as a spice, is rich of numerous substances of the curcuminoid family, among which curcumin is considered the most active ingredient. Curcumin exerts different biological activities, including antioxidant, anticancer, anti-inflammatory, antimicrobial, anti-angiogenic, neuro-protective, immuno-modulatory and healing activities. It is devoid of side effects at an oral dosage above 10 g/die in humans and exerts a potent antioxidant activity by counteracting a number of oxygen-derived reactive substances, like superoxide anion, oxydril and hydroxyl radicals, and by inhibiting lipid peroxidation.

WO 2006/003383 describes a preparation proposed as a lubricant and for the "Dry eye syndrome" and including Curcuma longa together with other herbs expressed as %/vol: Ocinum Sanctum (5-20%), Curcuma longa (5-18%), Carum copticum (0.5- 5%), Terminala belerica (2-7.5%), Embilica officinalis (3-10%), Terminala chebula (2- 7.5%), Camphora officinarum (1-5%), Menta sylvestries (0.5-5%), Eletteria Cardamomun (2-7.5%), Santalum album (2-7.5%), Santalum rubrum (2-7.5%), Azadirachia indica (2-7.5%), Vitex negando (2-7.5%), Meringa pterygosperma (2- 7.5%), Eclipta alba (2-7.5%), Boerhaavia diffusa (5-10%), Rosa damascena (5-10%). In addition, is commercially available an ophthalmic preparation of eye drops containing herbal extracts including Curcuma longa: "Ophtacare" eye drops by Himalaya drug Company of India containing a combination of the following 8 components (%/vol): Carum copticum (6.0%), Terminalia belerica (6.5%), Embilica officinalis (13.0%), Curcuma longa (13.0%), Ocinum Sanctum (13.0%), Rosa Damascena (11.0%), Cinnamomum camphora (0.50%), Meldespumatum (37.0%). This preparation has been demonstrated to have antimicrobial, anti-infective antiinflammatory properties with possible applications in some ocular inflammatory processes as reported in the literature. Summary of the invention

The present invention provides an ophthalmic formulation for treatment pterygium containing an extract of Curcuma longa as the only active ingredient. In fact, it has been demonstrated that Curcuma longa has a double effect on pterygium explants: on the one hand it reduces cell proliferation effects induced by growth factors active on conjunctival tissue during the development of this pathology and, on the other hand, it induces apoptosis in pterygium explant-derived cells mainly represented by keratinocytes. This invention containing Curcuma longa as the only active ingredient regards any preparations for conjunctival topical use to be applied to conjunctival mucosa as liquid preparation (aqueous or oily solutions or suspensions), but also as solid preparations (ointments, powders) for the prevention or reduction of pterygium. Other substances can be present as excipients in the formulation including anti-oxidant and visco-elastic compounds or vehicles, preservatives, buffer solutions, osmolar and emulsifying substances (or tensioactives).

In the formulation of the present invention excipients such as polyethylene glycol or vaseline and non ionic emulsifying substances (or tensioactives) (such as polysorbate) could be used for a better tolerability. Ophthalmic formulations for topical use are preferably prepared with a tolerable pH, generally in the range of 6.4-7.8, sterile and devoid of exogenous particles and with a tear-isotonic osmotic pressure (around 300 mOsm/L) or anyway between 200 and 350 mOsm/L.

Brief description of the drawings Fig. 1. Phase contrast images of primary cells derived from human pterygium explants and cultured in vitro for 24 days (a). The cells appear confluent (c) and stratified in multiple layers (b, d). Magnification: 4OX. Fig. 2. Immunofluorescence analysis of CAM.5 expression (cheratinocyte specific marker) in primary cells derived from human pterygium explants and cultured in vitro for 22 days, a, c: phase contrast images; b, d: cytoplasm fluorescent labelling. Magnification: 4OX. Fig. 3. Immunofluorescence analysis of CD104A (human fibroblasts specific marker) in primary cells derived from human pterygium explants and cultured in vitro for 22 days, a, c: phase contrast images; b, d: absence of labelling. Magnification: 4OX. Fig. 4. Human pterygium explants-derived primary cultures treatment with benzalkonium chloride plus or minus Curcuma Ionga for 3, 6 and 24 hours. Representative fields in phase contrast microscopy. Magnification: 4OX. Detailed description of the invention

The present invention provides an ophthalmic formulation for pterygium treatment containing an extract of Curcuma Ionga as the only active principle. In fact, it has been demonstrated that Curcuma Ionga has a double effect on pterygium explants in that, on one hand, it reduces cell proliferation effects induced by growth factors active on conjunctival tissue in the development of this pathology and, on the other hand, it induces apoptosis in pterygium explants-derived cells mainly represented by keratinocytes. The occurrence of apoptosis or necrosis in pterygium explants treated with benzalkonium chloride (a preservative) or Curcuma Ionga in benzalkonium chloride can be revealed through a commercial kit (human Annexin V-FlTC Kit, Bender MedSystem, Vienna, Austria) for the specific detection of apoptotic and/or necrotic markers. In synthesis, whereas the solution containing Curcuma Ionga exerts pro- apoptotic and anti-proliferation effects, the solution containing benzalkonium chloride appears to induce necrotic effects. Nevertheless, the necrotic effect induced by benzalkonium chloride administered as a single compound is reduced when it is administered in combination with Curcuma Ionga.

The observed pro-apoptotic and anti-proliferation effects are able to lead to pterygium reduction or disappearance. So, the present invention containing Curcuma Ionga as the only active principle can be extended to any preparations for conjunctival topical use to apply to conjunctival mucosa as liquid compounds (aqueous or oily solutions or suspensions), but also as solid compounds (ointments, powders) for the prevention or reduction of pterygium. Other substances can be present as excipients in the formulation including anti-oxidant and visco-elastic compounds, vehicles, preservatives, buffer solutions, osmolar and emulsifying substances (or tensioactives). In the formulation of the present invention excipients like polyethylene glycol or vaseline and anionic emulsifying substances (or tensioactives) (such as polysorbate) could be used for a better tolerability. Ample choice is anyway possible to the technician of competence provided that in the formulation the following essential requisites are observed: a well tolerable pH, generally between 6.4-7.8; sterility and absence of exogenous particles; tear-isotonic osmotic pressure (around 300 mOsm/L) or anyway between 200 and 350 mOsm/L The ophthalmic compound can be formulated as eye drops, gel, cream or ointment and contains as the only active principle an extract of Curcuma longa (0.1-20% v/v), preferably 1-5% or rather 1.2-3% in aqueous or hydro-soluble solvents (i.e. alcohol). These aqueous solvents are preferably chosen among phosphate or citrate phosphate or TRIS buffer, or buffers containing histidine, trycine, lysine, glycine, serine, corrected, if necessary, to the right physiological pH with an acid or basic component.

In addition, agents increasing solubility (as the cyclodexstrines), preservatives (benzalkonium chloride), visco-elastic substances (preferably in the range 0.1-10% v/v) (such as hyaluronic acid, polyethylene glycol, mixtures of polyethylene glycol with fatty acids), or celluloses (like hydroxyl-propyl-m ethyl cellulose) can be present. Potentially, also anti-oxidant substances, like ascorbic acid in the range 1-15% v/v and chelants like EDTA, can be contained in the formulation. Results obtained with these ocular compounds are extremely interesting as indicative of an alternative strategy to the common treatment of pterygium that is, currently, only surgical and rather complex: actually, the surgical operation consists in the detachment and removal of pterygium head, followed by conjunctival sutura leaving an ample portion of bare sclera or attaching the tissue up to the corneoscleral limbus; then, it could be necessary to make a conjunctival reconstruction through the sliding of the tissue or even the autologous transplant of conjunctiva. After this type of excision procedure it is easy that the most common post-surgery complications can occur, including infections, conjunctival cysts or adherent scars limiting ocular movements.

In addition, after surgery treatment it remains possible to develop relapse of more aggressive forms with a higher proliferation index, with a prevalence that ranges between 10-80% of cases in dependence of the techniques that have been used. Due to the absence of contraindications, the utilization of eye drops according to the invention seems to be particularly advantageous in that it prevents or delays pterygium growth and reduces the necessity of surgery interventions as well as of the relative post-surgery complications. To the inventors' knowledge, the activity observed for the first time, besides representing an alternative to surgery treatment, is due to active principles present in the Curcuma longa itself and not to the combination of natural active principles present in other eye drops formulations already commercially available. Based on another aspect of the invention, the inventors can vindicate also a method for the reduction or treatment of pterygium in a patient affected with this pathology, or in a post-surgery patient to avoid relapse, by applying a compound whose only active principle is an extract of Curcuma longa, in the concentration range of 0.1 -20% v/v, preferably 1-5% or rather 1.2-3% in aqueous or hydro-soluble solvents (i.e. alcohol) for a number of days sufficient for the reduction or disappearance of the conjunctival thickening. EXAMPLES

The invention is described in the following examples and figures that however are give for illustrative and non-limiting purposes. Example 1. Cell cultures and treatments Human pterygium explants were cultured with Eagle's MEM additioned with 10% FBS, 100 U/ml penicillin, 100 Dg/ml streptomycin and 2.5 Dg/ml fungizone at 37⁰C in the presence of 5% CO₂. Primary cells were subcultured twice a week and used for experiments at 4^th, 10^th and 25^th cell culture passage. Cells were detached with 0.025% Trypsin-EDTA 1 X through 2-5 incubations of 5-10 min. Pterygium explants showed epithelial cells (keratinocytes) able to proliferate in culture conditions (Fig. 1). In particular, immuno-cytochemical characterization with a FITC-conjugated anti-cytokeratin 7 and 8 monoclonal antibodies (CAM.5) (Becton Dickinson, San Jose, CA, USA, Catalogue No. 347653) demonstrated the presence of specific human keratinocytes markers (Fig. 2) and the absence of fibroblast contamination in the cell culture (negativity for the surface marker CD104A, recognized through the R-PE conjugated anti-PDGF receptor D monoclonal antibody) (BD Pharmingen, Catalogue No. 556002) (Fig. 3).

Cells were cultured in vitro for at least 24 days and, as shown in Fig. 1 , they appeared confluent (c) and growing in multiple layers (b, d). For treatments 10 explants were sub-cultivated in 6-well plates and incubated for different experimental time-intervals (3, 6 and 24 hours) with 0.001 % v/v benzalkonium chloride aqueous solution (vehicle) or with 0.001 % v/v benzalkonium chloride and aqueous extract of 2% v/v Curcuma longa derived from a dry extract of 95% Curcuma (Curcuma longa L. e.f., cas No.: 84775-52, FARMALABOR, Milan). Five explants were not treated and used as controls. Example 2. Immuno-phenotype characterization in flow cytometry After 22 days of culture, cells were harvested through trypsinization, counted, centrifuged at 400 g for 5 min, washed in 1X PBS (phosphate-buffered saline) and incubated with 1X FACS ™ Lysing Solution for 10 min at room temperature. Cells were then centrifuged at 400 g for 5 min and permeabilized with 1X FACS Permeabilizing Solution for 10 min at room temperature in the dark. After washing with PBS plus 0.5% BSA (bovine serum albumin) and 0.1 % NaN₃, cells were incubated 30 minutes at room temperature with 5 ml (20 ml/10⁶ cells) of a FITC- conjugated anti-cytokeratin 7 and 8 monoclonal antibody (MoAb) (CD140a) (CAM.5) (Becton Dickinson, San Jose, CA, USA) or with an R-PE conjugated anti-PDGF receptor D MoAb (BD Pharmingen). Cells were then washed and fixed in 1 % paraformaldehyde for 30 min at room temperature before flow cytometry analysis with an EPICS Coulter cytometer and Expo 32 analysis software. For each sample 10,000-20,000 events were analysed. Example 3. Immunofluorescence characterization For immuno-cytochemical analysis, cells were cultured on coverslips in 24-well plates (Nunc) and fixed in 100% methanol for 5 min or Carnovsky solution. After blocking of not specific antigenic sites through incubation with 10% normal donkey serum for 20 min, samples were incubated with a FITC-conjugated anti-cytokeratin 7 and 8 MoAb (CD140a) (CAM.5) (Becton Dickinson, San Jose, CA, USA) to identify keratinocytes or with an R-PE conjugated anti-PDGF receptor D MoAb (BD Pharmingen) to identify a possible contamination with fibroblasts. Coverslips were than mounted on slides and observed with a light microscope ZEISS Axioskop. Computerized image acquisition was done through a Coolsnap Videocamera and 6.1 MetaMorph® Analysis Software (Universal Imaging Corp, Downingtown, PA). Example 4. Apoptosis and necrosis analysis with Annexin V/PI staining in flow cytometry The evaluation of the incidence of both apoptosis and necrosis after treatment with Curcuma longa and benzalkonium chloride or benzalkonium chloride alone was done with a commercial kit according to manufacturer instructions (human Annexin V-FITC Kit, Bender MesdSystem, Vienna, Austria). In brief, the cells were gently suspended with binding buffer and incubated with Annexin V-FITC for 10 minutes at room temperature in the dark. Samples were then washed and supra-vitally stained with Propidium Iodide (Pl, 50 Dg/ml) that allows the discrimination of apoptotic cells (dim fluorescence) from necrotic cells with altered membranes (bright fluorescence). The analysis was carried out with an EPICS Coulter flow cytometer equipped with Expo 32 analysis software. For each sample 10,000-20,000 events were analysed. Viable cells were Annexin V^neg/Pl^πeg, early apoptotic cells were Annexin v^pos/Pl^ne9, late apoptotic cells were Annexin v^pos/Pl^pos and necrotic cells were Annexin V^neg/Pl^pos. Statistics

Data were analysed through the two-tailed T-test (Minitab, statistical analysis software, State College, PA). Values of p<0.05 were considered significant. Table 1. Apoptotic and necrotic cells mean percentage after treatment with benzalkonium chloride and Curcuma longa for 24 hours.

Viable cells: Ann V^neg/Pl^neg; early apoptotic cells: Ann V^pos/Pl^πeg; late apoptotic cells: Ann V^pos/Pl^pos; necrotic cells: Ann V^neg/Pl^pos.

*p<0.001 compared with controls and samples treated with Curcuma longa + Benzalkonium chloride *^*p<0.001 compared with controls and samples treated with Benzalkonium chloride ^***p<0.001 compared with controls and samples treated with Curcuma longa + Benzalkonium chloride Results After treatment with Curcuma longa an increased incidence of early and late apoptosis was found in comparison with untreated controls or samples treated with the vehicle (benzalkonium chloride) for corresponding time-intervals (3, 6 and 24 hours) (Table 1 , Fig. 4). In particular, the treatment with benzalkonium chloride alone was toxic to the cells, as evidenced by an increased and early detection of necrotic features demonstrated by the high Pl staining positivity (Ann v^ne9/Pl^pos markers, see Table 1 ).

Fig. 4 displays the morphological analysis of pterygium cultures (39 days) treated with benzalkonium chloride and Curcuma longa for 3, 6 and 24 hours. The figure shows necrotic cells, characterized by cytoplasm and nuclear disruption resulting in the appearance of amorphous debris and large areas devoid of cells in the culture dish, as well as apoptotic cells, characterized by a typical shrinkage and volume decrease resulting in the disappearance of cell junctions and in the culture rarefaction.

The results obtained demonstrate the capability of Curcuma longa of inducing cell apoptosis in cells derived from human pterygium, useful effect to control the pathologic growth of the tissue. This effect is specific and quite different from the necrotic effect induced by the preservative benzalkonium chloride, which is in any case inhibited when used in combination with Curcuma longa.

Claims

1. Eye drops comprising Curcuma longa as the only active ingredient for use in the treatment of pterygium.

2. Eye drops according to claim 1 , wherein the Curcuma longa is in a concentration from 0.1 to 20 % v/v, preferably from 1 to 5% and even more preferably from 1.2 to 3%.

3. Eye drops according to claims 1-2, wherein the Curcuma is dissolved in an aqueous or water-soluble buffer solution having a pH from 6.4 to 7.8 selected in the group consisting of: phosphate, phosphate-citrate or TRIS buffer, or a buffer containing histidine, tricine, lysine, glycine, serine.

4. Eye drops according to claims 1-3 having an anti-proliferation and pro-apoptotic effect on human keratinocytes useful for the reduction or elimination of pterygium.

5. Eye drops according to claims 1-4, in liquid (aqueous or oily solutions, or suspensions) or solid (creams, ointments, powders) form.

6. Eye drops according to claims 1-5, also comprising viscoelastic and/or emulsifying and/or isotonicizing substances and/or excipients and/or preservatives and/or chelating substances.

7. Eye drops according to claims 1-6, where these isotonicizing agents are selected from: sodium chloride, citric acid, glycerol, sorbitol, mannitol, ethylene glycol, propylene glycol, dextrose.

8. Eye drops according to claims 1-7, where these viscoelastic substances are in a quantity from 0.1 to 10% and are selected in the group consisting of: hyaluronic acid, polyethylene glycol, polypropylene glycol, polyvinylpirrolidone, mixtures of polyethylene glycol with fatty acids, or cellulose.

9. Eye drops according to claims 1-8, where these excipients are selected in the group consisting of: vaseline, olive oil, optionally in combination with lanolin.

10. Eye drops according to claims 1-9, having an osmotic pressure from 200 to 350 mOsm or isotonic with tears.

11. Extract of Curcuma longa for use in the treatment of pterygium.