Pharmaceutical compositions for treatment of dry eye disease and associated disorders

Abstract

The present invention relates to pharmaceutical compositions containing Acetylcholine agonist like Cinitapride, Prucalopride, Citicoline, Epibatidine, Lobeline, Cytosine, Cytisinicline for treatment of dry eye syndrome and its related disorders in suitable dosage form. The inventive concept provided herein are methods and pharmaceutical compositions comprising acetylcholine receptor agonist or pharmaceutically acceptable salt thereof by preparing oral dosage forms like Nasal drops, ophthalmic drops, oral mouth liquid spray or sublingual or dispersible dosage form for the management of dry eye syndrome and its associated disorders in suitable dosage form accordingly performing preclinical study to prove the efficacy of acetylcholine receptor agonist.

Description

[0001] PHARMACEUTICAL COMPOSITIONS FOR TREATMENT OF DRY EYE DISEASE AND ASSOCIATED DISORDERS

[0002] FIELD OF THE INVENTION:

[0003] The present invention relates to pharmaceutical compositions containing Acetylcholine agonist like Cinitapride, Prucalopride, Citicoline, Epibatidine, Lobeline, Cytosine, Cytisinicline for treatment of dry eye syndrome and its related disorders in suitable dosage form.

[0004] BACKGROUND OF THE INVENTION:

[0005] Dry eye disease (DED) is a multifactorial, age-related, chronic, progressive disease of the ocular surface resulting in pain, visual impairment, tear fdm hyperosmolarity and instability and inflammation. Patients with DED are more susceptible than others to eye infections and damage to the surface of the eye (cornea).

[0006] Dry eye disease is characterized by a reduction in tear volume, rapid breakup of the tear film, or an increase in the evaporative properties of the tear film layer. This disease affects daily life activities including reading, driving, and the ability to tolerate contact lenses.

[0007] While the prevalence of DED is difficult to report because of varying definitions and diagnostic criteria, it is estimated that approximately 16 million adults in the United States have been diagnosed with DED with a higher prevalence among women than men and increasing prevalence with age.

[0008] Dry eye syndrome is the most prevalent ocular disease globally. It is characterized by eye discomfort, functional visual disturbances, tear film instability, and ocular surface alterations.

[0009] The tear film consists of three layers: the lipid layer on the outside, the aqueous layer in the middle, and the inner mucus layer. Dry eye syndrome can be classified into the aqueous- deficient and evaporative etiological subtypes.

[0010] Aqueous deficient dry eye syndrome results from the reduction in aqueous tear production, and evaporative dry eye syndrome is characterized by excessive evaporation of the tear film resulting from the lack of a protective lipid layer.

[0011] The lacrimal functional unit, which includes the cornea, conjunctiva, meibomian glands, and the main and accessory lacrimal glands, is useful in understanding dry eye and tear dynamics. Lacrimal glands, which secrete electrolytes, water, proteins, and mucins known as lacrimal gland fluid into the tear film, are innervated by parasympathetic and sympathetic nerves.

[0012] Table 1 : Symptoms and current treatment options for Dry Eye Syndrome

[0013] Table 2: Drugs used for Treatment of Dry eye syndrome:

[0014] Mechanism of Action of Tear drops production:

[0015] The parasympathetic system predominantly regulates the secretions of lacrimal glands. The parasympathetic nervous system releases Acetyl choline, which acts on muscarinic and nicotinic receptors.

[0016] Muscarinic receptors are important mediators of secretions by the lacrimal and salivary glands. Muscarinic receptors have also been reported to be present in the cornea, conjunctiva, and meibomian glands, suggesting that muscarinic receptors may affect secretions by these tissues.

[0017] Choline is a precursor of Ach, which is a neurotransmitter of the parasympathetic nervous system. Choline deficiency may contribute to the pathophysiology of dry eye syndrome by the reducing parasympathetic tone.

[0018] Another important pathological mechanism of dry eye syndrome is inflammation. Conjunctival inflammation manifests as T-cell infiltrates and the upregulation of cluster of differentiation (CD) 3, CD4, and CD8, as well as lymphocyte activation markers such as CD I la and human leukocyte antigen (HLA)-DR. Pro-inflammatory cytokines, such as interleukin (IL)- 1, IL-6, IL-8, and tumor necrosis factor have been reported to be elevated in the ocular surface.

[0019] Choline deficiency has been reported to cause oxidative damage in various organs. Thus, choline deficiency may enhance inflammation in dry eye syndrome and delay wound healing on the ocular surface. Nicotine receptors stimulated with ACh in inflammatory cells inhibit the release of pro-inflammatory cytokines in a concentration-dependent manner.

[0020] It has also been reported that choline is involved in cell damage and metabolism, protects against hypoxia-induced injuries of vessels and vascular endothelial cells, and is essential for tissue repair after injury Choline modulates the function of inflammatory cells and elevates the mRNA levels of enzymes and receptors involved in metabolism.

[0021] Impaired choline uptake or phosphorylation reduces mitochondrial adenosine triphosphate (ATP) synthesis, which leads to AMP-activated protein kinase (AMPK) activation and induces mitophagy causing inflammation.

[0022] Ocular pain and discomfort are the characteristic symptoms of dry eye syndrome. The main symptoms of dry eye syndrome include dryness, itchiness, burning, stinging, grittiness, foreign body sensation, tearing, tired eyes, redness, and blurred vision. Pain is a result of the network activity of multiple areas related to sensory, cognitive, and emotional functions. Pain pathways in the central nervous system are modulated by the cholinergic system. There are two types of ACh: muscarinic and nicotinic receptors.

[0023] Muscarinic acetylcholine receptors are ubiquitously found in the cerebral cortex. The perception of pain is modified by cholinergic transmission. Choline has an analgesic effect by activating 7 nicotinic receptors.

[0024] The antinociceptive effects of cholinergic agonists are related to gamma-aminobutyric acid- ergic (GABAergic) signaling, as well as nicotinic and muscarinic modulation of nociceptive transmission. Choline deficiency may contribute to the development of ocular symptoms associated with dry eye syndrome.

[0025] Choline is a precursor of phosphatidylcholine and a component of the tear film. Choline alfoscerate is a surfactant that can stabilize the aqueous and lipid layers of the tear film. The effect of oral choline alfoscerate has been reported in patients with dry eye syndrome. Choline alfoscerate may affect dry eye syndrome mainly through antinociception in the central nervous system and an improvement in tear stability as an emulsifier in tears. Choline alfoscerate, in combination with a cholinesterase inhibitor, may have a sufficient effect on tear secretion.

[0026] Choline acetyltransferase and acetylcholinesterase have been found in the corneal epithelium. Cholinergic signaling stimulation of the corneal epithelium promotes the directional migration of corneal epithelial cells and wound healing through muscarinic acetylcholine receptors (mAChRs) and nicotinic acetylcholine receptors (nAChRs).

[0027] Choline acetyltransferase is completely regenerated 28 days after corneal abrasion. Citicoline eye drops were applied to the recovery of comeal sensitivity after laser in situ keratomileusis by enhancing comeal innervation. Choline salicylate has been developed to increase the viscosity of eye drops.

[0028] Nitrogen mustard, which is a vesicant responsible for irreversible comeal injury, damages the comeal epithelium and stroma through the sphingomyelin-ceramide pathway. Detergent chemicals, widely used in household products, are toxic, and they induce inflammation via the alteration of phospholipid and sphingolipid metabolism.

[0029] Choline is involved in the development and differentiation of the retina and the eye. Choline and its metabolites perform various functions in the visual pathway, which includes the ocular surface, retina, optic nerve, and visual cortex of the brain.

[0030] Choline deficiency is associated with retinal hemorrhage, glaucoma, and dry eye syndrome. Choline and its metabolite supplementation may be effective in treating these diseases.

[0031] Betaine, which is an intermediate of choline, has been reported to reduce comeal staining and protect the ocular surface integrity against the environmentally desiccating stress as an Osmo protectant and an anti-inflammatory agent. Betaine decreases tear osmolarity and inflammatory cytokines, including tumor necrosis factor-, IL-1, IL-8, C-C motif chemokine ligand 2 (CCL2), and C-C motif chemokine ligand- 20 (CCL20).

[0032] Tear hyperosmolarity is an essential mechanism underlying dry eye syndrome as a potent inflammatory stress that impairs normal cell functions of comeal and conjunctival epithelial cells. Citicoline, it is dietary supplements in the US, but was originally a prescription drug in Japan. Citicoline is a phospholipid composed of ribose, pyrophosphate, cytosine, and choline. It is water soluble and highly bioavailable. Supplementation with citicoline increases choline stores available for other biosynthetic pathways. Citicoline appears to decrease glutamate levels in the brain and increase adenosine triphosphate, which in turn offers protection against ischemic neurotoxicity. Increased glucose metabolism in the brain and cerebral blood flow has also been demonstrated, as well as increased availability of the neurotransmitter’s acetylcholine, norepinephrine, and dopamine. Antioxidant effects of citicoline have been demonstrated in several models, including brain, renal, and hepatic injury. Citicoline also enhances antioxidant mechanisms in the body, while suppressing the damaging effects of free radicals on neural tissue.

[0033] It also promotes anti-inflammatory activities and optimizes patterns associated with the release of neurotransmitters. Citicoline is considered as a comprehensive therapeutic agent for supporting brain health.

[0034] Citicoline - legal status in EU 1 Citicoline (more specifically citicoline inner salt) is a food ingredient designed to supplement natural intake of Choline and cytidine in the body. It is a highly purified substance which upon intake is readily hydrolyzed, breaking down to choline and cytidine which are normal body constituents, and which then undergo further metabolism and incorporation into normal pathways of metabolism.

[0035] Citicoline is intended to be used in food supplements aimed at a target population of middle- aged to elderly adults, at a maximum level of 500 mg / day, and in foods for particular nutritional uses, specifically foods for special medical purposes, at a maximum level of 250 mg / serving, and with a maximum daily intake from these types of foods of 1000 mg / day. Citicoline is not intended to be consumed by children.

[0036] Possibly Effective for Decline in memory and thinking skills that occurs normally with age. Taking citicoline by mouth or by IV seems to help with memory loss in people over 50 years old. A group of eye disorders that can lead to vision loss (glaucoma). Taking citicoline by mouth, as a shot, or as eye drops might improve vision in some people with glaucoma.

[0037] CDP choline - also known as citicoline or cytidine-5-diphosphocholine - is a naturally occurring phospholipid. It’s a popular nootropic, a nutrient that passes the blood-brain barrier to support brain health and enhance cognitive function. CDP choline is made up of compounds choline and cytidine. Choline is an essential nutrient, which we need for making the building blocks for our cell membranes and nervous system.

[0038] Another benefit of citicoline is it increases cerebral metabolism impacting neurotransmitters. Citicoline helps to boost the function of the neurotransmitter acetylcholine, one of the most abundant neurotransmitters in the body. Found both in the central nervous system and the peripheral nervous system, it’s responsible for muscle contraction, regulates endocrine functions, and plays a vital role in memory storage and cognition. Citicoline’s role in neurotransmitter function helps to keep many of the body’s processes running smoothly.

[0039] Choline, a component of citicoline, is an essential nutrient we only receive from our diet. It’s found naturally in foods such as organ meats, eggs, chicken, fish, and to a lesser extent, in some plant-based foods such as cauliflower and broccoli.

[0040] Although choline exists in many foods, it’s richest sources tend to be organ-based meats, which are not common in the American diet. If adequate consumption of choline can’t be established via diet alone, supplemental citicoline may be used to increase its levels.

[0041] Citicoline is marketed as tablets and injection has been approved for the Treatment of depression, schizophrenia, stroke, Parkinson disease, brain injury, and cognitive deficits.

[0042] Citicoline is chemically known as 5'-O-[hydroxy({hydroxy[2-(trimethyl ammonia) ethoxy] phosphoryl}oxy)phosphoryl]cytidine molecular formula is C14H26N4O11P2, its molecular weight is 488.32. Citicoline is soluble in water and methanol; solubility in water decreases as the pH increases.

[0043] Citicoline is the generic name of the pharmaceutical substance that chemically is cytidine-5'- diphosphocholine (CDP-choline), which is identical to the natural intracellular precursor of phospholipid phosphatidylcholine .

[0044] Citicoline, also known as cytidine-5 -diphosphocholine or CDP-choline, is hydrolyzed into cytidine and choline in the intestine. CDP-choline, Citicoline, Cytidine 5 '-diphosphocholine, Cytidine diphosphate-choline and it is indicated to treat Neuroprotection, reduced oxidative stress, Reduced Neuro inflammation, Reduced edema, Treat Glaucoma and Prevention of diabetes induced retinal neurodegeneration.

[0045] Its mechanism of action Citicoline has beneficial effects on neurological functions; it acts by increasing the synthesis of phosphatidylcholine, the primary neuronal phospholipid and enhancing the production of acetylcholine. Brain phospholipid synthesis is impaired following stroke and ischemic events.

[0046] Citicoline enhances glucose metabolism and increases blood flow to the brain. Poor circulation to the brain can cause neurological problems, including memory loss, fatigue, vertigo, and stroke.

[0047] Oral dosages of 250 to 2,000 mg daily have been evaluated in adolescents and adults in clinical trials. Citicoline is water-soluble, with more than 90% oral bioavailability. Citicoline is well absorbed following intramuscular administration. After intramuscular doses of citicoline 1,000 mg, peak increases in plasma choline levels were seen in 0.4 hours, with levels increasing from 11 micromol / L (baseline) to 25 micromol / L.

[0048] No interactions found with food. Choline derived from citicoline crosses the blood-brain barrier, presumably serving as a source of acetylcholine and phosphatidylcholine (lecithin) synthesis. A major portion of a dose of citicoline appears to be incorporated into tissues and / or used in biosynthetic / biodegradation pathways, including lecithin / lipid membrane synthesis. Citicoline is metabolized in the liver to free choline. The liver is capable of synthesizing lecithin from choline. The half-life of free choline is 2 hours after intramuscular administration.

[0049] Only small amounts of dose are recovered in the urine and feces (less than 3% each). Approximately 12% of a dose is eliminated through the lungs as carbon dioxide.

[0050] Cinitapride

[0051] Cinitapride is a gastroprokinetic agent and antiulcer benzamide with agonist activity at 5-HT1 and 5-HT4 receptors and antagonist activity at 5-HT2 receptors.

[0052] It is a benzamide with gastroprokinetic and antiemetic properties typically used for the treatment of gastrointestinal motility disorders such as gastroesophageal reflux disease (GERD), non-ulcer dyspepsia, and delayed gastric emptying.

[0053] It is indicated to treat gastrointestinal disorders associated with motility disturbances like gastroesophageal reflux disease (GERD), non-ulcer dyspepsia and delayed gastric emptying.

[0054] Cinitapride is a substituted benzamide with 5-HT receptor antagonist and agonist activity. The mechanism of action of cinitapride involves increasing the release of acetylcholine in the gastrointestinal tract, which in turn stimulates the contraction of smooth muscle in the gastrointestinal wall. Cinitapride also acts as a 5-HT4 receptor agonist, which further enhances the motility of the gastrointestinal tract.

[0055] In addition, cinitapride inhibits the activity of dopamine receptors in the chemoreceptor trigger zone (CTZ) of the brain. The CTZ is involved in the regulation of nausea and vomiting, and by inhibiting dopamine receptors in this area, cinitapride can reduce feelings of nausea and prevent vomiting. Overall, cinitapride acts on multiple pathways to improve gastrointestinal motility and reduce symptoms of nausea and vomiting.

[0056] Prucalopride

[0057] Prucalopride is a dihydrobenzofurane carboxamide derivative from the benzofurane family that selectively stimulates 5-HT4 receptors and thus, it presents enterokinetic properties. MOTEGRITY ® is indicated for the treatment of chronic idiopathic constipation (CIC) in adults. MOTEGRITY can be taken with or without food.

[0058] The high selectivity of prucalopride allowed further development as it prevented the cardiac adverse reactions observed due to non-target effects of precedent therapies.

[0059] Prucalopride, a selective serotonin type 4 (5-HT4) receptor agonist, is a gastrointestinal (GI) prokinetic agent that stimulates colonic peristalsis (high-amplitude propagating contractions [HAPCs]), which increases bowel motility.

[0060] Prucalopride was devoid of effects mediated via 5-HT2A, 5-HT2B, 5-HT3, motilin or CCK-A receptors in vitro at concentrations exceeding 5-HT4 receptor affinity by 150-fold or greater. In isolated GI tissues from various animal species, prucalopride facilitated acetylcholine release to enhance the amplitude of contractions and stimulate peristalsis. In rats and dogs, prucalopride stimulated gastrointestinal motility with contractions starting from the proximal colon to the anal sphincter.

[0061] The mechanism of action of prucalopride involves binding to and activating 5-HT4 receptors in the gastrointestinal tract, which stimulates the release of acetylcholine and increases the contraction of smooth muscle in the gastrointestinal wall. This leads to an increase in intestinal motility and acceleration of colonic transit time, which helps to relieve constipation.

[0062] In addition, prucalopride also modulates the sensitivity of sensory nerves in the gut, leading to an increase in colonic sensory threshold and a decrease in visceral hypersensitivity. This helps to reduce symptoms such as abdominal pain and discomfort, which are often associated with chronic constipation. Overall, prucalopride acts on multiple pathways to improve intestinal motility and reduce symptoms of chronic constipation.

[0063] OBJECTIVE OF THE INVENTION:

[0064] The inventive concept provided herein are methods and pharmaceutical compositions comprising acetylcholine receptor agonist or pharmaceutically acceptable salt thereof by preparing oral dosage forms like Nasal drops, ophthalmic drops, oral mouth liquid spray or sublingual or dispersible dosage form for the management of dry eye syndrome and its associated disorders in suitable dosage form accordingly performing preclinical study to prove the efficacy of acetylcholine receptor agonist.

[0065] SUMMARY OF THE INVENTION:

[0066] The invention is explained in more detail with reference to the following Examples, which are to be considered as illustrative and not to be construed to limit the scope of the invention as set forth in the appended claims.

[0067] The liquid spray formulations of the present invention are for intranasal administration. In one aspect, the invention is directed to stable liquid spray formulations comprising an effective amount of Acetylcholine agonist like Cinitapride, Prucalopride, Citicoline, Epibatidine, Lobeline, Cytosine, Cytisinicline, a pharmaceutically acceptable salt thereof, or a derivative thereof, water, a cosolvent and a permeation enhancer.

[0068] In another aspect, the invention is directed to stable liquid spray formulations comprising an effective amount of Cinitapride, Prucalopride, Citicoline, Epibatidine, Lobeline, Cytosine, Cytisinicline, a pharmaceutically acceptable salt or a derivative thereof, water and a permeation enhancer that do not contain alcohol.

[0069] Further provided herein, in some embodiments, is a method of improving ocular discomfort, comprising the local administration of a therapeutically effective amount of an acetylcholine receptor agonist into the nasal cavity of an individual in need thereof, wherein the agonist selectively binds to the peripheral acetylcholine receptor.

[0070] In some embodiments is a method of improving ocular discomfort, comprising the local administration of a therapeutically effective amount of an acetylcholine receptor agonist into the nasal cavity of an individual in need thereof, wherein the agonist selectively binds to the peripheral acetylcholine receptor and is administered in an amount that is not systemically bioavailable.

[0071] Applicants have created new liquid formulations for nasal administration that are stable and comfortable for the user. The formulations that do not contain alcohol are especially suitable for administration to children. Further, the alcohol-free formulations may be suitable for patients in recovery from alcohol addiction, liquid formulations for nasal administration is in a simple solution form. As used herein, the term “simple solution” refers to a solution in which the solute(s) has fully dissolved in the solvent.

[0072] In another embodiment, the present invention relates to liquid compositions of an acetylcholine receptor agonist for administration via metered dispensing system for increased patient compliance wherein, the said metered dispensing system is a spray pump and / or an actuator system with simple, user-friendly operation to deliver precise dose over nasal mucosa.

[0073] In another embodiment, the liquid spray formulations of the present invention have a pH from about 3.0 to about 6.0, preferably about 4.5. In another embodiment, the liquid spray formulation containing citicoline is studied further by using Schirmer Tear Test for tear formation by using New Zealand rabbit.

[0074] The details of the examples of test samples and animal experiment study are given below.

[0075] TABLE 3: Example of compositions containing Citicoline. TABLE 4: Example of compositions containing Cinitapride.

[0076] TABLE 5: Example of compositions containing Prucalopride.

[0077] Schirmer Tear Test procedure and results:

[0078] Schirmer Tear Test Experimental procedures:

[0079] In vitro wetting of the STT strip: The wetting of the STT strip will be investigated in vitro to determine the kinetics of fluid movement along the strip. This data is used to convert wetting measured in the in vivo studies (in mm), to volume of tears (in pL). By doing so, an estimate of reflex tear-flow volumes in New Zealand white rabbits can be made. Saline volumes of 2, 3, 4, 5, 6, 7, 8 and 10 pL were added to the strip, and the wetted length of the strip was determined at 10s intervail.

[0080] In vivo tests: Each rabbit is manually restrained in a standard commercial polycarbonate rabbit restrainer and tested by using the STT, which does not involve topical anesthesia. Performance of the test involved insertion of the notched end of a commercial tear-test paper over the lower lid margin at the juncture of the temporal and middle third of the lid. The eyelids should be held shut for the period of the test. The length of tear strip wetted shall be recorded (in mm), every 10 s for a total of 3 min. Test strips should be held in place throughout the period of recording. The eyes were tested sequentially, with recording for the second eye commencing immediately after completion of testing in the first. All test strips should be from the same lot and manufacturer. The test is noninvasive and causes minimal distress to the rabbits. Tests were performed indoors in an environment of constant thermostatically controlled temperature and humidity.

[0081] From the efficacy model performed in rabbits, as per the raw data compilation of parameters in Schirmer tear test experiment was performed by keeping varenicline nasal spray as control and citicoline nasal spray as test sample. Test sample having citicoline nasal spray has shown significant tear production effect in comparison to control sample.

[0082] The data shows promising tear production effects of Citicoline nasal spray at given strength and is more efficacious compared to varenicline nasal spray at given strength of formulation by nasal route as nasal spray in New Zealand rabbits.

Claims

CLAIMS:

1. The liquid spray formulations of the present invention are for intranasal administration. In one aspect, the invention is directed to stable liquid spray formulations comprising an effective amount of Acetylcholine agonist like Cinitapride, Prucalopride, Citicoline, Epibatidine, Lobeline, Cytosine, Cytisinicline, a pharmaceutically acceptable salt thereof, or a derivative thereof, water, a cosolvent and a permeation enhancer.

2. In another aspect, the invention is directed to stable liquid spray formulations comprising an effective amount of Cinitapride, Prucalopride, Citicoline, Epibatidine, Lobeline, Cytosine, Cytisinicline, a pharmaceutically acceptable salt or a derivative thereof, water and a permeation enhancer that do not contain alcohol.

3. A method of improving ocular discomfort, comprising the local administration of a therapeutically effective amount of an acetylcholine receptor agonist into the nasal cavity of an individual in need thereof, wherein the agonist selectively binds to the peripheral acetylcholine receptor.

4. A method of improving ocular discomfort, comprising the local administration of a therapeutically effective amount of an acetylcholine receptor agonist into the nasal cavity of an individual in need thereof, wherein the agonist selectively binds to the peripheral acetylcholine receptor and is administered in an amount that is not systemically bioavailable.

5. A new liquid formulation for nasal administration that are stable and comfortable for the user. The formulations that do not contain alcohol are especially suitable for administration to children. Further, the alcohol-free formulations may be suitable for patients in recovery from alcohol addiction, liquid formulations for nasal administration is in a simple solution form. As used herein, the term “simple solution” refers to a solution in which the solute(s) has fully dissolved in the solvent.

6. The present invention relates to liquid compositions of an acetylcholine receptor agonist for administration via metered dispensing system for increased patient compliance wherein, the said metered dispensing system is a spray pump and / or an actuator system with simple, user-friendly operation to deliver precise dose over nasal mucosa.

7. The liquid spray formulations of the present invention have a pH from about 3.0 to about 6.0, preferably about 4.

5. In another embodiment, the liquid spray formulation containing citicoline is studied further by using Schirmer Tear Test for tear formation by using New Zealand rabbit.

8. The wetting of the STT strip will be investigated in vitro to determine the kinetics of fluid movement along the strip. This data is used to convert wetting measured in the in vivo studies (in mm), to volume of tears (in pL) . By doing so, an estimate of reflex tearflow volumes in New Zealand white rabbits can be made. Saline volumes of 2, 3, 4, 5, 6, 7, 8 and 10 pL were added to the strip, and the wetted length of the strip was determined at 10s intervail.

9. Each rabbit is manually restrained in a standard commercial polycarbonate rabbit restrainer and tested by using the STT. Performance of the test involved insertion of the notched end of a commercial tear-test paper over the lower lid margin at the juncture of the temporal and middle third of the lid. The eyelids should be held shut for the period of the test. The length of tear strip wetted shall be recorded (in mm), every 10 s for a total of 3 min. The eyes were tested sequentially, with recording for the second eye commencing immediately after completion of testing in the first. Tests were performed indoors in an environment of constant thermostatically controlled temperature and humidity.

10. From the efficacy model performed in rabbits, as per the raw data compilation of parameters in Schirmer tear test experiment was performed by keeping varenicline nasal spray as control and citicoline nasal spray as test sample. Test sample having citicoline nasal spray has shown significant tear production effect in comparison to control sample.

11. The data shows promising tear production effects of Citicoline nasal spray at given strength and is more efficacious compared to varenicline nasal spray at given strength of formulation by nasal route as nasal spray in New Zealand rabbits.

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