SOLUBLE MELATONIN TRIPARTATE ADDUCT FOR THE PREVENTION AND TREATMENT OF RARE AND SEVERE EYE SIGHT-THREATENING CONDITIONS AND NEURO-OPHTHALMIC DISORDERS

Abstract

The invention relates to soluble melatonin tripartate adduct comprising three structurally distinct regions and optionally trehalose, its stable sterile lyophilized powder and preservative-free aqueous solution or ointment, and to related pharmaceutical forms thereof, wherein the active melatonin moiety is adducted to a nucleoside in an optimal ratio range, with or without the presence of C.sub.2-C.sub.5 short chain amino acid. The invention further relates to the pharmaceutical field, more precisely it refers to the ophthalmic use of said melatonin tripartate adduct and to the method to prevent or treat serious ophthalmic pathologies in mammals and humans, more precisely rare and severe eye sight-threatening conditions and neuro-ophthalmic disorders in human and veterinary medicine, wherein said acute and chronic pathophysiological alterations and oculopathies are mainly caused by oxidative stress and related hypoxia-inducible factors (HIFs) and inflammation affecting the eyes. The pharmaceutical composition of said melatonin tripartate adduct may optionally contain either retinol or sodium hyaluronidate, or hyaluronic acid, L-carnosine, L-acetylcarnosine or a mixture thereof. Viscosity enhancers may be optionally included in the composition. Furthermore, the pharmaceutical composition of the invention is chemically and physically stable and safe.

Claims

1. A soluble pharmaceutical composition comprising (i) a melatonin tripartate adduct (MTA) of general formula (I): ##STR00002## and (ii) trehalose in an amount of from 1% to 5% by weight, wherein MTA is adducted to the nucleoside adenosine in the presence of C.sub.2-C.sub.5 low-chain amino acid glycine, an active moiety melatonin, adenosine and glycine being at a molar range ratio varying as from 1:1:1 up to of 1:6:1, the MTA is optimally in a form of a stable sterile lyophilized powder.

2. (canceled)

3. The pharmaceutical composition according to claim 1, wherein the melatonin tripartate adduct (MTA) consists of melatonin of pharmaceutical grade with a purity content in a range of from 99.0% to 101.0%, calculated on a dry basis.

4. The pharmaceutical composition according to claim 1 the melatonin tripartate adduct (MTA) consists of the active moiety melatonin, adenosine and glycine at the molar range ratio 1:4:1.

5. The pharmaceutical composition according to claim 1, wherein a concentration of the melatonin tripartate adduct in a pharmaceutical product varies from 0.01 mg/ml to 10 mg/ml, preferably between 0.05 mg/ml and 2.5 mg/ml in sterile distilled water or saline solution.

6. The pharmaceutical composition according to claim 1 wherein pharmaceutical grade trehalose is admixed to melatonin tripartate adduct at a convenient amount so that the final concentration of the trehalose in the composition is from about 1.5% to 3% by weight.

7. The pharmaceutical composition according to claim 1, wherein the sterile lyophilized powder of the MTA is admixed to trehalose and is suitably dissolved in sterile water in aseptic conditions to yield a sterile ophthalmic solution of melatonin tripartate to elicit the desired therapeutic effects of the adduct when intended and/or addressed to the ophthalmic field.

8. The pharmaceutical composition according to claim 7, further optionally containing at least one of: retinol, sodium hyaluronidate, hyaluronic acid, L-carnosine, L-acethylcarnosine or mixtures thereof.

9. The pharmaceutical composition according to claim 7, further optionally containing at least one of: hydroxypropylmethylcellulose (HPMC) or carboxymethylcellulose (CMC) or sodium hyaluronate or hyaluronic acid, to yield an ophthalmic viscosurgical device (OVD) with a viscosity suitable for ophthalmic surgery.

10. The pharmaceutical composition according to claim 9, wherein HPMC or CMC are in a range from 0.1% to 0.5%, preferably at 0.3%, or sodium hyaluronate or hyaluronic acid at 1% by weight with respect to the total weight of the solution.

11. The pharmaceutical composition according to claim 7, wherein the pharmaceutical composition is in the form of sterile gel, emulsion and ointment, where the latter is consisting from a pharmacologically effective amount of melatonin tripartate and suitable excipients for the conventional preparation of an ointment such as liquid paraffin at a concentration between 5% and 25%, more preferably between 10% and 20%, optimally 15% by weight, anhydrous lanolin between 1% and 15%, more preferably between 5% and 10%, optimally 10% by weight, and white vaseline between 60% and 95%, more preferably between 70% and 85%, optimally 73.5% by weight.

12. The pharmaceutical composition according to claim 7, having a pH in a range between 7.0 and 7.7, preferably 7.2±0.2, and optimally pH 7.4.

13. The pharmaceutical composition according to claim 7, having an isotonicity of the solution in a range from 260 to 480 mOsm/kg, preferably between 310 and 350 mOsm/kg.

14. A process for preparing a sterile preservative-free ophthalmic solution of melatonin tripartate adduct, comprising the steps of: (i) preparing melatonin tripartate adduct (MTA) sterile lyophilized powder by adducting activated melatonin, adenosine and glycine at the optimal molar ratio of 1:4:1, by a conventional lyophilisation process; and (ii) admixing in sterile environment said sterile lyophilized powder consisting of melatonin tripartate adduct in a desired volume of distilled water and then with an appropriate amount of pharmaceutical grade trehalose; and (iii) adding optionally retinol or sodium hyaluronidate, hyaluronic acid, L-carnosine, L-acethylcarnosine each in a convenient amount or a mixture thereof, and admixing thoroughly to yield a clear and transparent solution; and (iv) adjusting the pH to about 7.4 and the osmolarity within the preferred parameters of 310 and 350 mOsm/kg; and (v) processing the bulk solution through a sterilizing membrane or cartridge filter of an appropriate efficiency to meet the sterility requirements; and (vi) apportioning a fixed volume of the preservative-free sterile ophthalmic solution into a primary unit-dose strip or multi-dose container equipped from a drop-metering system, using nitrogen as a purge and process gas.

15. A sterile preservative-free pharmaceutical ophthalmic composition according to claim 7 for the treatment of oculopathies and ophthalmic diseases of the posterior segment of the eye by intraocular or topical ocular administration, consisting essentially of a sterile isotonic aqueous solution containing melatonin tripartate adduct at a concentration varying as from 0.01 mg/ml to 10 mg/ml, preferably between 0.05 mg/ml and 2.5 mg/ml.

16. A packaged unit-dose or multi-dose container, holding from 0.2 to 20 ml of sterile preservative-free ophthalmic solution of melatonin tripartate adduct, according to claim 14 for ophthalmic use

17. The packaged unit-dose or multi-dose container according to claim 16, wherein the sterile preservative-free ophthalmic solution of melatonin tripartate adduct is dispensed at between 10 μg and 200 μg/dose of melatonin active, more preferably between 20 μg and 50 μg/dose, while the more preferred dose is 25 μg in each eye.

18. A pharmaceutical composition consisting of the sterile preservative-free ophthalmic composition according to claim 7 for use in therapy of eye sight-threatening ocular diseases and neuro-ophthalmic disorders.

19. A pharmaceutical composition consisting of sterile preservative-free ophthalmic solution according to claim 7, suitable for the production of a medicinal product for the prevention and treatment of oculopathies or ophthalmic diseases in humans, mammals and animals.

20. The pharmaceutical composition according to claim 17 for use in the prophylaxis and treatment of a human ophthalmic disease or clinical condition, comprising administering an effective amount of melatonin tripartate adduct of the invention into the periocular area in a subject in need thereof.

21. The pharmaceutical composition according to claim 17 for use in the prophylaxis and treatment of at least one condition selected from the group of retinopathy of prematurity (ROP), cataract in babies and strabismus, degenerative ophthalmic diseases as choroidal neovascularization (CNV), retinal vein occlusion (RVE), retinitis and optic neuritis (ON), uveitis, glaucoma, retinal glaucomatous, cataract, age-related macular degeneration (AMD), retinitis pigmentosa (RP), pathological myopia (PM), photocheratitis, amblyopia, chorioretinopathy, diabetic macular degeneration (DMD), diabetic macular edema (DME), proliferative diabetic retinopathy and retinal detachment comprising the administration of an effective amount of melatonin tripartate adduct in a subject in need thereof.

22. The pharmaceutical composition according to claim 17 for use in the prophylaxis and treatment of at least one condition selected from the group of diseases with high inflammatory expression as dry eye syndrome (DES), computer vision syndrome or display device syndrome (CVS), ocular changes during and after space travel comprising the administration of an effective amount of melatonin tripartate adduct in a subject in need thereof.

23. The pharmaceutical composition according to claim 17 for use in the prophylaxis and treatment of at least one condition selected from the group of ophthalmic diseases or conditions from bacterial or viral origin such as trachoma, ocular toxoplasmosis, ocular malaria, virus-related posterior uveitis infections that include human herpes virus, measles, rubella, and arboviruses such as Ebola, Marburg, Dengue, West Nile, Chikungunya, SARS-CoV-2 and Zika comprising the administration of an effective adjuvant amount of melatonin tripartate adduct in a subject in need thereof.

24. The pharmaceutical composition according to claim 17 for use in the prophylaxis and treatment of the ophthalmic conditions, the injuries and the inflammation induced by peri—and post—ophthalmic surgery and oxidative stress in affected subject comprising the administration of an effective amount of melatonin tripartate adduct.

Description

Example 1

[0084] Production of Melatonin Tripartate Adduct and Trehalose

[0085] Melatonin (0.1 mole; 23.2 g), adenosine (0.4 moles; 106.8 g) and glycine (0.1 mole; 7.54 g), at a molar ratio 1:4:1 respectively, were dissolved in about 1 litre of a solvent mixture (water/ethanol 70%-30%), To each sample was added trehalose (1.5%) by weight as cryoprotectant agent. The solution was filtered and lyophilized to yield melatonin tripartate adduct with 1.5% trehalose (about 152.54 g, molecule melatonin 23.28 g=15.26%). The lyophilized powder was accurately recovered and dissolved in ethanol at about a concentration of 5 mg/ml. During the precipitation process the flow rate of melatonin solution was adjusted to 2 ml/min and the flow rate of water was adjusted to 10 ml/min. A gas pressure of 0.2 bar was used to ensure the production of homogenous lyophilized powder. Microjet reactor temperature was adjusted to 25-40° C. throughout the precipitation process. The lyophilized samples contained added trehalose (1.5%) by weight as cryoprotectant agent. The obtained sterile lyophilized powders were dispersed in sterile water to obtain a concentration of melatonin tripartate adduct+trehalose of about 6 mg/ml for all preparations. The concentrations of the obtained mixtures of melatonin tripartate adduct and of trehalose have been reported here below in Table 1.

TABLE-US-00002 TABLE 1 Melatonin tripartate + Melatonin Trehalose Sample Trehalose concentration concentration concentration mixture n. mg/ml mg/ml mg/ml and (%) 1 2.69 0.41 1.5 (0.15%) 2 2.75 0.42 1.5 (0.15%) 3 2.62 0.40 1.5 (0.15%)

[0086] All formulations proved to be soluble and to yield a transparent solution of the desired nominal concentration of melatonin tripartate.

Example 2

[0087] Production of Sterile Melatonin Tripartate Ophthalmic Solution and Package in Preservative-Free Multidose Containers [0.4 mg MLT/ml=20 μg MLT/50 μl=drop 1/20 ml=2.62 mg/ml MLT-TRP]

[0088] The sterile lyophilized powder (13.35 g) resulting from sample mixture 1 of Example 1 have been further dissolved in sterile bidistilled water to yield 5.0 litres solution, then sterilized by filtration in aseptic conditions using 0.22 μm sterilizing membrane filter. An aliquot of about 2.0 litres of the collected sterile solution is then apportioned into sterile containers (multidose strips of eye drops each of 0.5 ml volume resulting 3570 strips) and 2.5 litres remnant volume is used to fill in aseptic conditions 460 special preservative-free S ml multidose container equipped with a volumetric precision drop dispenser. All batches tested for pH (result=7.4), melatonin potency (result=99.1% of declared nominal dose), clarity (result=limpid & transparent), osmolarity (result=335 mOsm/kg), sterility (result=conform) and eye tolerance test (results=complies with the test).

Example 3

[0089] Production of Melatonin Tripartate Sterile Ophthalmic Ointment [1.5 mg MLT/g=3.93 mg/g MLT-TRP]Sterile lyophilized powder of melatonin tripartate adduct (7.86 g) resulting from sample mixture 1 of Example 1, has been thoroughly admixed in aseptic conditions with a suitable mixer with 2000 g of a sterile mixture of pharmaceutical grade excipients consisting of liquid paraffin 15%, anhydrous lanolin 10% and white vaseline 73.5% to yield 1830 g of a pale whitish semisolid ointment. The bulk ointment has bee then apportioned aseptically in 3 grams tubes of melatonin tripartate each, resulting 520 tubes of sterile ophthalmic ointment with a production yield of 85%.

[0090] The batch was tested for melatonin potency (result=98.7% of declared nominal value), pH (result=7.2) and sterility (result=conform).

Example 4

[0091] Treatment of Uveitis with Ophthalmic Melatonin Tripartate Sterile Solution

[0092] Experiment was conducted in 12 male chinchilla rabbits weighing 2 kg each. In 8 rabbits, acute immunogenic uveitis was induced in both eyes. Melatonin tripartate adduct eye drop solution and trehalose of Example 2, at the active concentration of melatonin 0.5 mg/ml, and the placebo solution consisting of 0.9% saline solution and 3% of trehalose, both packed in the special preservative-free multidose container equipped with a 25 μl drop-volume special dispensing system, have been used for test Two groups of 4 animals received the drop instillation according to the scheme reported hereby: the first group received 25 μl of melatonin (as tripartate adduct) eye drops solution; the second group received placebo solution in the same ratio. The solutions were dropped 3 times daily into both eyes for 8 consecutive days.

TABLE-US-00003 Rabbit Group n. Rabbit n. Treatment 1-Healthy (control) 4 no treatment 2-Placebo 4 saline solution (25 μl, 3 times/day for 8 consecutive days) 3-Melatonin tripartate 4 melatonin tripartate eye drops solution (25 μl melatonin, 3 times/day for 8 consecutive days)

[0093] In animals treated with melatonin tripartate eye drops solution, a significant reduction of conjunctival oedema, corneal oedema, and iridial oedema has been recorded.

[0094] All animals with uveitis exhibited reduction of antioxidant activity (AOA) of tears, increasing of SOD (superoxide dismutase) and α.sub.2-macroglobulin demonstrating pronounced oxidative stress. Table 1 reports the biochemical parameters determined into the rabbit aqueous humour on day 10 of uveitis after treatment with melatonin tripartate eye drops solution and placebo.

TABLE-US-00004 TABLE 1 Antioxidant SOD α.sub.2-Macroglobulin Group Activity (μM) (mg/ml) (nmol/min .Math. ml) 1 856.05 ± 54.16  0.79 ± 0.06  3.41 ± 1.65 2 342.01 ± 59.98*  22.1 ± 0.91* 18.64 ± 2.63* 3 504.91 ± 17.01* ** 11.31 ± 0.72* **  8.2 ± 1.42* ** *p < 0.01 compared to healthy animals **p < 0.01 compared to animals that did not receive treatment

[0095] The results showed that melatonin tripartate eye drops solution when administered in eye drops to rabbits affected by uveitis showed a remarkable anti-inflammatory effect due to the antioxidant activity of melatonin.

Example 5

[0096] Protective Effect of Melatonin Tripartate in a Rat Pups Model of Induced Cataract

[0097] 60 Sprague-Dawley albino rat pups having an average weight of 10 g±1 g, were randomized into 3 groups: [0098] Group A received subcutaneous injection of normal saline. [0099] Group B injected with 40 μmole sodium selenite/kg subcutaneously and treated with one drop of 0.9% sodium chloride ophthalmic solution twice daily during 30 consecutive days and considered as positive control. [0100] Group C injected with 60 μmole/kg sodium selenite subcutaneously and treated at the same time with one drop of 10 μg melatonin (as tripartate) ophthalmic solution, instilled twice daily during 30 consecutive days.

[0101] The obtained results, reported in the Table 2 hereby, indicate that the treatment of rats with eye drops solution of melatonin (as tripartate) 10 μg eye drop twice daily, starting on the day of sodium selenite injection during 39 days, decreased the cataract formation after 15 and 21 days of treatment;

TABLE-US-00005 TABLE 2 Incidence of cataract % Group 15 days 21 days A 0 0 B 27 53 C 32.53 14.78

Example 6

[0102] Protection with Melatonin Tripartate from Oxygen-Induced Retinopathy (OIR) in Pups Mice Model

[0103] Pups mice model were exposed to 75% oxygen for 5 days and then returned to room air (21% oxygen). The mice were randomly divided into 3 groups:

[0104] Control group: kept in room air (21% oxygen);

[0105] OIR (oxygen-induced retinopathy) group: exposing to 75% oxygen (without treatment);

[0106] OIR (oxygen-induced retinopathy)+melatonin tripartate group: exposing to 75% oxygen+10 μg melatonin (as tripartate) eye drops solution one in each eye twice day during the period of exposure to 75% oxygen.

[0107] The effects of melatonin on the expression of inflammatory factors in the retina of OIR mice have been investigated and tabulated hereby. The results of Table 3 show significant overexpression of the protein levels of TNF-α and IL-1β in retinas of OIR mice, which were downregulated following to melatonin adduct treatment

TABLE-US-00006 TABLE 3 Group TNF-α (%) IL-1β (%) Control 0 0 OIR (non treated) 43.6 59.8 OIR + Mel Trip 21.7 18.43 OIR = mice with oxygen-induced retinopathy Mel Trip = melatonin tripartate

[0108] Reported results of the above Table show that melatonin tripartate significantly reduced inflammatory cytokines expression in the retina of OIR model.

Example 7

[0109] Melatonin Tripartate Effects on the Intraocular Pressure (IOP) in Glaucomatous Mice

[0110] The study was carried out on 15 normal adult mice (Group A-control) and 15 glaucomatous mice (Group B). Mice were housed in temperature—and light—controlled rooms maintained according to a 12-hour light/dark cycle; all animals were fed ad libitum and studied at age 3, 6, 9, and 12 months.

[0111] Melatonin tripartate eye drops solution was administered to the cornea at a concentration of 25 μg of melatonin (as tripartate) in both eyes. Control group received the same volume of 0.9% saline solution. The intraocular pressure (IOP) was measured each hour for 5 hours being the main risk factors for the development of glaucoma producing changes in the retina and optic nerve.

[0112] The obtained results have been reported in Table 4 and evidenced that melatonin tripartate decreased IOP in glaucomatous mice, respectively, with a maximum effect at 4 hours in both groups of animals.

TABLE-US-00007 TABLE 4 IOP (% of control) Group 0 h 1 h 2 h 3 h 4 h 5 h A 162 160 163 159 157 158 B 153 160 153 140 115 141

[0113] The administration of melatonin tripartate eye drops produced an acute hypotensive effect, reducing the progressive increase of IOP in a recognized model of glaucoma, protecting the retina from further progression of this pathology.

Example 8

[0114] Protective Effect of Melatonin Tripartate in Diabetic Rat Retina

[0115] The fluorescein leakage test (FLT) provides information on the effects of xenobiotics on the impermeability (gate function) of epithelial cell monolayers, and their recovery after exposure. The study was carried out on 21 male Wistar rats (weighing 200-250 g), maintained at a 12-h light/dark cycle (lights on from 07:00 to 19:00). Food and water were provided ad libitum, and the room temperature was maintained.

[0116] Rats were randomly divided into three groups: (1) control group; (2) diabetic group (without treatment); (3) diabetic+melatonin tripartate group.

[0117] The above last group was treated with 20 μg melatonin tripartate eye drop solution for each eye twice a day, 1 week after induction of diabetes and continued for 7 consecutive weeks. At the end of the experiment (8 weeks), the retinas were examined by fluorescein angiography to evidence blood vessel abnormalities in retina, after administration of 10% fluorescein sodium by intraperitoneal injection of 0.1 ml. Leakage (blood vessel abnormalities) was characterized by the presence of a hyperfluorescent lesion graded as follows: 0, no leakage; 1, slight leakage; 2, moderate leakage; and 3, prominent leakage. 8 weeks after induction of diabetes, the experiment results were tabulated and revealed the development of retinal alterations in diabetic rats. More specifically, the leakages in the diabetic group were significantly higher than in the control group, while treatment with melatonin tripartate eye drops solution during 7 consecutive weeks significantly decreased this score compared to the diabetic control group, as reported here below in Table 5.

TABLE-US-00008 TABLE 5 Fluorescein leakage Group (FLT) score Control 0.3 Diabetic (non treated) 2.7 Diabetic + Mel Trip 1.2 Met Trip = melatonin tripartate

[0118] The results are justified in view that melatonin tripartate is a direct and powerful free-radical scavenger, directly neutralizing ROS and NO as well as other toxic reactants. Melatonin tripartate eye drop solution might be beneficial in preventing retinopathy also in diabetic patients.

Example 9

[0119] Melatonin Tripartate in the Prevention and Treatment of Various Retinal Diseases Associated with Increase of VEGF, Vascular Leakage and Angiogenesis.

[0120] The experimental study was carried out evaluating the effects produced by melatonin (as tripartate) and hypoxia-inducible factor-1α(HIF-1α) protein levels in retinal pigment of epithelial cells under hypoxia.

[0121] Retinal pigment epithelial cells (2×10.sup.6 cells/well) were cultured with CoCl.sub.2 (cobalt chloride) at 150 μM with or without melatonin (as tripartate) (10.sup.−5 to 10.sup.−11 M) for 24 h. Protein was extracted from cultured cells. HIF-1α, ELISA kit was used for the measurement of intracellular HIF-1α, protein, and the results were expressed as percentages of the control (cells cultured under normoxia and without melatonin tripartate adduct) (mean±SD, n=3). HIF-1α, protein level in the controls was 89.1 pg/mg protein. The results are reported in following Table 6:

TABLE-US-00009 TABLE 6 VEGF Group (% Control) Normoxia 100 CoCl.sub.2 (150 μM) 260 Melatonin (as tripartate) (10.sup.−11M) 255 Melatonin (as tripartate) (10.sup.−8M) 230 Melatonin (as tripartate) (10.sup.−8M) 70 *P < 0.05, compared with the cells cultured under hypoxia and without melatonin.

[0122] Melatonin tripartate adduct significantly inhibited hypoxia-induced accumulation of HIF-1α, protein suggesting that it may have potential value in the prevention and treatment of various retinal diseases associated with increase of VEGF, vascular leakage and angiogenesis.

Example 10

[0123] Melatonin Tripartate for the Relief of Symptoms from Ophthalmic Conjunctivitis by SAR-CoV-2

[0124] On days 8 and 9 of hospitalization (disease day 12 and 13), patients reported redness, foreign body sensation, and tearing in both eyes, without blurred vision. They excluded that they had touched their eyes with their hands. Slit-lamp examination bilaterally showed moderate infection of the conjunctiva, conjunctival follicles of the lower eyelid and preauricular and palpable lymph nodes. No sub-conjunctival haemorrhage or corneal lesions or anterior chamber inflammation were observed. Slit lamp scans showed clear signs of acute viral conjunctivitis. The examination of the disease on days 12 and 13 showed a moderate conjunctival infection and follicles of the conjunctiva of the lower eyelid. Clinical examinations on day 17 of the disease and on day 19 showed that treatment with the adduct of melatonin ophthalmic drops (20 μg of melatonin/drop of solution three times a day for 12 days) in each eye gradually attenuated the onset of symptoms. The clinical presentation of the cases met the criteria for acute viral conjunctivitis. It is likely that the ophthalmic drops of the melatonin adduct of the invention have significantly contributed to the treatment of symptoms. Conjunctival swabs tested positive for SARS-CoV-2 RNA for at least 5 days before Ct values gradually decreased. Conjunctival swab values remained positive (Cycle Cutoff: 38.47) but showed a trend of decreasing values and signs of clinical improvement. The conjunctival swabs remained positive for SARS-CoV-2 until days 14 and 17 after the onset of the disease. On day 19 RT-PRC tested negative for SARS-CoV-2. Conjunctival specimens were negative for SARS-CoV-2 on day 19 and day 20 of illness, respectively. In conclusion SARS-CoV-2 is capable of causing ophthalmic complications such as viral conjunctivitis in the intermediate phase of the disease. The aim of this study is to report the ocular characteristics and the presence of viral RNA of acute respiratory syndrome of coronavirus 2 (SARS-CoV-2) in conjunctival swabs of two patients suffering from confirmed new coronavirus disease 2019 (COVID-19). Written informed consent was obtained from the respective patients. Participants aged 38 and 62 with confirmed COVID-19 and bilateral acute conjunctivitis appeared 12 and 13 days after the onset of the disease. Based on the detailed ophthalmological examination, reverse transcription PRC (RT-PRC) was performed to confirm the presence of the SARS-CoV-2 virus. Acute viral conjunctivitis improved clinically significantly following administration of 20 μg melatonin/drop of solution three times a day for 12 consecutive days. However, conjunctival sampling may not be useful for early diagnosis as the virus may not initially be present in the conjunctiva.

[0125] Therefore, the present invention is well adapted to carry out the objects and attain the ends and advantages mentioned as well as those that are inherent therein. While numerous changes may be made by those skilled in the art, such changes are encompassed within the spirit of this invention as defined by the appended claims.