Technical Field of the Invention

The invention relates to the pharmaceutical composition containing the nanoparticle loaded with epigallocatechin gallate (EGCG) effective in the treatment of dry eye disease and the production method of this composition.

State of the Art (Prior Art)

Dry Eye Disease is a disease of the preocular tear film that damages the ocular surface and is associated with symptoms of ocular discomfort. Dry eye disease is also called keratitis sicca, sicca syndrome, xerophthalmia, ocular surface disease or dysfunctional tear syndrome, or only dry eyes. Keratoconjunctivitis sicca is a Latin word and its literal translation is “corneal and conjunctival drying”.

Dry eye is common and affects approximately 10-20% of the population. It is a multifactorial disorder of the tear film and ocular surface that results in eye discomfort, visual impairment, and ocular surface damage. The eye surface deteriorates due to tear hyperosmolarity and tear film imbalance. Tear film can be impaired by decreased tear production, change in tear composition, damage to the eye surface and inflammation. The dryness of the ocular surface epithelium causes apoptosis of the epithelial cells, which ultimately leads to exacerbation of the dry eye.

Dry eye disease is associated with inflammation of the ocular surface, and tear hyperosmolarity is an important mediator of this inflammation. Excessive expression of proinflammatory cytokines on the ocular surface is observed in dry eye patients. Different inflammatory mediators such as interleukin(IL)-ip, IL-6, IL-17, interferon-y, tumor necrosis factor a, chemokine (C-C motif) ligand 2 and matrix metalloproteinases play a role in inflammation associated with dry eye disease of the ocular surface.

Classification of Dry Eye Disease

Dry eye disease is divided into two as aqueous tear deficiency and evaporative dry eye as an etiological classification. Aqueous tear deficiency is caused by decreased lacrimal tear secretion. Sjogren’s syndrome and non-Sjbgren’s syndrome are divided into two subgroups.

Sjogren's syndrome is an autoimmune exocrinopathy in which activated T lymphocytes infiltrate the lacrimal and salivary glands, causing apoptosis of acinar and ductal cells and subsequent dysfunction. Dry eye, gland hyposecretion is exacerbated by the neurosecretory block, which may be caused by antibodies directed against the muscarinic receptors of the glands or by inflammatory cytokines in the tear film. Clinically, patients show both signs of dry eye and dry mouth (xerostomia). Diagnosis can be supported by laboratory tests for autoantigens described by surface epithelial cells (anti-Ro and anti -La). Sjogren’s syndrome may occur as the primary disease, but it is the more commonly known secondary autoimmune condition, and systemic lupus erythematosus, polyarteritis nodosa, poly angiomatosis granulomatosis, systemic sclerosis, primary biliary cirrhosis, or mixed connective tissue diseases are the most common.

Non-Sjbgren’s syndrome is divided into four main categories. These are primary lacrimal gland deficiencies, secondary lacrimal gland deficiencies, obstruction of lacrimal gland canals and reflex hyposecretion. Primary lacrimal gland deficiencies are known as age-related dry eye, congenital alacrima, family dysautonomia (Riley Day Syndrome); secondary lacrimal gland deficiencies are known as tear gland infiltration, denervation and ablation.

Evaporative dry eye is characterized by pathologically high levels of tear evaporation. It may be caused by internal conditions or exposure to environmental factors. An example of an internal reason is the decrease in the rate of blinking caused by driving, watching television, reading and computer work. In contrast, environmental factors are directly effective on the outer surface. These are central heating, dry climate, air pollution, wind, chemical burns and contact lens wear.

The most common cause of evaporative dry eye resulting from dysfunction of meibomian glands is squamous debris, terminal gland obstruction, and chronic inflammation of the eyelid margin behind the gray line, which may be accompanied by qualitative or quantitative changes in glandular secretion. Meibomian gland dysfunction can be defined by looking at the increased viscosity of the epidemic and the inability to express fat from the glands on the slit lamp according to the morphological characteristics of the eye canal obstruction.

Meibometry is used to measure the amount of fat in the cover margin reservoir, and meibography to measure the degree of cloth release for qualitative classification and evaluation. Its causes can be local, systemic, or syndromic.

Risk Factors of Dry Eye Disease

Risk factors that may cause dry eye disease are listed below:

• It is known that it is more common in elderly individuals with a decrease in the capacity of the lacrimal glands.

• It is observed at a higher rate in females compared to males with the decrease in estrogen and androgen levels after menopause.

• Humidity, temperature, airflow, and air quality play an important role in environmental factors. Meanwhile, people in dusty, windy and sun-exposed topical climate regions have a high tendency to dry eye disease.

• Computer use, watching television, driving at night cause many ocular disorders, and complaints such as fatigue, burning, redness, foreign body sensation, watering, blurred vision may occur in the eye.

• Dietary imbalances, omega-3 fatty acids and omega-6 fatty acid ratios found in foods primarily affect anti-inflammatory activities in the body. On the other hand, vitamin A deficiency can damage the lacrimal gland and cause dry eye disease by disrupting the development of goblet cells.

• Effects such as Sjogren’s syndrome (especially in women), menopause, aging (of both sexes) and anti androgenic drug use may lead to a decrease in the synthesis of these hormones and cause dry eye disease.

• It has been reported that systemic drugs, anticholinergics, diuretics, antihistamines, antiarrhythmics, P-blockers, postmenopausal hormone supplements and antiandrogenics used in the treatment of systemic disease occurring with advancing age in humans may cause dry eye disease by suppressing tear production or worsen the existing disease.

• The use of contact lenses disrupts the dynamics of tears and causes ocular discomfort and dryness. More than half of the individuals using lenses have been reported to be uncomfortable with dry eye disease symptoms.

• Autoimmune diseases, hepatitis-C, human immunodeficiency virus (HIV), radiation therapy and bone marrow transplantation are among the other important risk factors for dry eye disease.

Treatment of Dry Eye Disease

The goals in the treatment of ocular surface diseases are to relieve symptoms, to increase the quality of clear vision and life, to repair the damage to the ocular surface, to normalize the hemostatic level of the tear film and to correct the underlying cause.

Keratoconjunctivitis sicca treatment is diverse. The goal of treatment is to relieve dry eye symptoms, improve patient comfort, restore the ocular surface and tear film to normal, and prevent damage to the cornea when possible. Treatment can range from training, environmental or dietary changes, artificial tear substitutes, punctal plugs, and topical and/or systemic anti-inflammatory drugs to surgery.

Treatment options according to the degree of dry eye severity;

1 ^st Grade

• Training and advice, regulation of environmental factors

• Discontinuation of systemic drugs that may cause dry eye

• Artificial tears, drops and gels

• Treatment of eyelid disorders

2 ^nd Grade

If the 1 ^st degree treatments are not sufficient: • Anti-inflammatory drugs (corticosteroids, cyclosporine)

• Tetracyclines (for meibomianitis and rosacea)

• Punctal plugs

• Drugs that stimulate tear secretion should be added.

3 ^rd Grade

If the 2 ^nd degree treatments are not sufficient:

• Autologous serum

• Contact lens

• Permanent punctal occlusion should be added.

4 ^th Grade

If the 3 ^rd degree treatments are not sufficient:

• Systemic anti-inflammatory drugs

• Surgery (valve surgery; tarsorrhaphy; mucous membrane, salivary gland, amniotic membrane transplantation) should be added.

Artificial tears: Artificial tears are lubricant eye drops used to treat dryness and irritation associated with missing tear production in the keratoconjunctivitis sicca. Lubricant tears are available as OTC (over the counter) products and are usually the first line of treatment. Mild disease conditions require the application of lubricant drops four times a day, while in severe cases (10-12 times a day) more frequent application is necessary. These OTC products mainly vary in terms of their content, indications and availability of preservatives. Substances such as cellulose and polyvinyl derivatives, chondroitin sulfate and sodium hyaluronate determine their viscosity, retention times and adhesion to the ocular surface.

The increase in the viscosity of the tear drops prolongs the duration of action; however, it results in temporary blurred vision. Preservatives are added to multi-dose artificial tear containers to reduce the risk of bacterial contamination and extend shelf life. Many ophthalmic products contain preservatives and the risk of side effects increases as the frequency of daily administration and duration of use increase. The clinician should consider the sensitivity of the patient to preservatives, the frequency of use, the severity of the disease, the preservative-free contamination risk, and the cost when recommending artificial tear product.

Allergan’s “Refresh Tears” and Alcon’s “Tears Naturale” and “Bion Tears” are a few preservative-free artificial tears found on the market. Many ophthalmologists use treatments such as cyclosporine, corticosteroids, and tetracycline from other eyes, with artificial tears in moderate and severe dry eye shapes to reduce indications and symptoms. Lubricant tear ointments can be used throughout the day, but they are usually used before bedtime after application due to poor vision. In addition, an artificial tear such as Lacrisert containing hydroxy propyl cellulose can be used every morning.

Autologous serum eye drops: The autologous serum eye drops contain different tear components, such as hepatocyte growth factor, epidermal growth factor, vitamin A, and fibronectin, which are important for maintaining a healthy ocular surface. All these ingredients are not available in commercial products and the use of these eye drops for dry eye treatment is controversial.

Non-steroidal anti-inflammatory drugs and antibiotics: Non-steroidal anti-inflammatory drops containing drugs such as diclofenac sodium and ketorolac reduce inflammation associated with dry eye disease. Ophthalmic ointments containing antibiotics such as erythromycin and bacitracin are used to treat meibomian gland dysfunction.

Topical ophthalmic aqueous tetracycline solution has been developed for chronic dry eye disease. Tetracyclines are primarily used for anti-inflammatory effects rather than antibacterial effects in dry eye disease.

Punctal plug: A small medical device called a “punctal plug” is placed in the eye in order to prevent the obstruction of the canal and thus the nasolacrimal drainage of the eyes and thus to prevent the eyes from drying out. Clinical studies have shown that punctal plugs with occlusion plugs improve the symptoms and findings of dry eye disease. Punctal plugs are usually reserved for people with moderate to severe keratoconjunctivitis sicca, and artificial tears are required after insertion of an artificial plug.

Corticosteroids: Topical corticosteroids such as loteprednol etabonate, dexamethasone, prednisolone and fluoromethoIone have been shown to be effective in the inflammatory conditions associated with keratoconjunctivitis sicca and have been approved by the Food and Drug Administration (FDA) for the treatment of inflammatory conditions of the conjunctiva, cornea and anterior world. It is generally recommended for short-term use as it leads to adverse effects such as ocular infection, glaucoma and cataract for long-term use.

Cyclosporine: Cyclosporine A is effective in several ocular immune pathologies. Systemic administration of the drug is used in the treatment of local ophthalmic conditions involving cytokines such as corneal graft rejection, autoimmune uveitis and dry eye disease; however, it causes serious kidney and cardiovascular complications. Local application avoids various side effects related to systemic delivery, which gives this drug a broad safety profile. Topical ciclosporin A is the first FDA-approved drug indicated for the treatment of patients with a lack of aqueous production dry eye and is better for long-term treatment. 0,05% ophthalmic topical emulsion is marketed as “Restasis” and in India, as Cyclomune by Sun Pharma.

Cyclosporine A, one of the immunosuppression calcineurin inhibitors and used topically in the treatment of dry eye disease, is a very specific immunomodulator that prevents the activation of T lymphocytes and significantly reduces the levels of inflammatory cytokines in the conjunctival epithelium with an increase in goblet cells. It also inhibits mitochondrial- mediated pathways of aptoctosis.

The experimental study demonstrating the use of topical cyclosporine in the treatment of moderate, mild, and severe dry eye disease that does not respond to artificial tear therapy concluded that topical cyclosporine had beneficial effects in all categories of dry eye disease. The topical formulation of cyclosporine A is prepared using different vegetable oils, resulting in poor local tolerance and low bioavailability by patients due to the high hydrophobic properties. The use of colloidal carriers such as micelles, nanoparticles, and liposomes is a promising approach to achieving better tolerance and ocular bioavailability.

Vitamin A: Vitamin A is an important food naturally found in the tear film of healthy eyes. Vitamin A plays an important role in the production of the mucin layer, the most intrinsically lubricating layer of the tear film, which is very important for a healthy tear film. Vitamin A deficiency leads to mucin layer loss and goblet cell atrophy. Vitamin A drops protect the eyes from free radicals, toxins, allergens and inflammation. Topical retinoic acid treatment with vitamin A systemic administration was investigated in the treatment of xerophthalmia. One or more retinoids in effective amounts alone can be dispensed in a pharmaceutically acceptable ophthalmic vehicle and topically administered for effective treatment of dry eye disorders.

Omega-3 fatty adds: Today, oral support with essential fatty acids is recommended by ophthalmologists. Essential fatty acids are the precursors of eicosanoids, hormones that act locally play a role in inflammatory processes. Essential fatty acids may benefit patients with dry eye disease by reducing inflammation and changing the composition of meibomian lipids. Clinicians can recommend the intake of the omega-3 fatty acid diet to relieve dry eye disease.

Topical administration of a specific fatty acid has been shown to be beneficial in the treatment of the symptoms of dry eye disease for the first time at the Massachusetts Eye Research Institute. Topical alpha-linolenic acid therapy has been found to significantly reduce dry eye and inflammation changes at both eye and molecular levels. Therefore, topical administration of alpha-linolenic acid may be a new therapy to treat clinical signs and inflammatory changes in the dry eye.

Dry eye disease is a multifactorial disease of the tear and eye surface and can cause visual impairment, possible damage to the eye surface and symptoms of tear film imbalance. Artificial tear formulations applied topically to the eye are the most common treatment method for dry eye disease. However, an ideal artificial tear preparation that can fully treat the disease is not available in the present art. The preparations are inadequate in severe dry eye patients and at the same time, the preservatives they contain create sensitivity in the eye and do not remain in the eye for a long time after being dripped.

Patent document W02008016095 Al relates to a therapeutic agent comprising the active substance Nrf2, including epigallocatechin gallate, for keratoconjunctival disease. The therapeutic agent can be used in the treatment of dry eye disease. In addition, the therapeutic agent can be taken orally and can also be in the form of tablets. However, there are some disadvantages that negatively affect the functional benefits associated with EGCG: short halflife and high sensitivity to light and heat. Furthermore, EGCG is unstable in a neutral and alkaline environment and therefore undergoes gastrointestinal impairment in oral use. EGCG shows poor stability and very low oral bioavailability despite its high solubility in water.

It is well known that nanoparticles carry and provide drugs that are unstable in biological fluids and are not easily dispersed along the mucosal barrier. Oral nanoparticles are promising drug delivery systems due to improved bioavailability, targetability, bioadhesion and controlled drug release in the gastrointestinal tract. They can pass directly and/or adhere to the mucosa, which is a prerequisite step before the translocation of the particles. Therefore, bioadhesion plays a key role in the distribution of drugs throughout the epithelium and prevents hepatic first-pass metabolism and enzymatic degradation in the gastrointestinal tract.

Patent document US 7,919,072 Bl also relates to a positively charged oral drug transport system consisting of chitosan, poly-glutamic acid and at least one bioactive agent. The nanoparticular system of the invention is obtained by ionic gelation method. Epigallocatechin gallate is used as a bioactive agent. It is also mentioned that nanoparticles can be taken orally in the tablet. The use of the drug in the form of tablet nanoparticles containing the active substance epigallocatechin gallate in the treatment of dry eye is not mentioned in the said document.

Patent document US 2017/0087200 Al relates to the composition of green tea extract containing green tea extract encapsulated in chitosan nanoparticles used in the treatment of hepatic fibrosis. Chitosan nanoparticles are obtained by using penta sodium triphosphate as a crosslinking agent by ionic gelation method. In the said invention nanoparticles containing the active substance epigallocatechin gallate is used in the treatment of hepatic fibrosis.

Brief Description and Purposes of the Invention

With the invention, nanoparticle formulations were synthesized using the ionic gelation method with chitosan, a biodegradable, biocompatible, natural and renewable polymer, sodium tripolyphosphate, a non-toxic cross linker, and EGCG, a water-soluble active substance used in the treatment of dry eye disease.

Deficiencies such as short half-life and low bioavailability of epigallocatechin gallate are solved by ionic gelation method using encapsulation technology in the invention. Average particle size, zeta potential, chemical structure, surface morphologies, thermal properties were found with the characterization studies of the synthesized nanoparticles and a polymeric carrier system with appropriate properties was obtained.

It is known that pH and waiting time effects the particle size, the particle size decreases with the increase of pH in the acidic medium, and the size does not change in the acidic medium during the waiting period.

It was determined in the ionic gelling method that no nanoparticle formation occurred in the basic environment and gelling was performed in the acidic environment (pH 3).

Nanosized systems have a significant advantage in terms of cell uptake and their uptake by cells is 15-250 times higher than micron-sized systems. The particle sizes of the EGCG- loaded mannitol chitosan nanoparticles obtained are 196,5 ± 21,03 nm and provide a significant advantage.

The stability of colloidal distributions for nanoparticles is ensured when the zeta potential value (generally, ±30 mV) is high. When the zeta potential value decreases, the charged nanoparticle suspension cannot maintain its steady state, in this case, storing the formulations in a lyophilized form and diluting them before use provides significant advantages for stability. The zeta potentials of the EGCG-loaded mannitol chitosan nanoparticles obtained were found to be -24,57 ± 0,55 mV. The lyophilization of the obtained nanoparticles and the high zeta potential provide an advantage for stabilization and stability.

It can be concluded that global structures are formed, pores are encountered in between and encapsulation occurs when the surface morphology of the synthesized EGCG-loaded mannitol chitosan nanoparticles is examined with scanning electron microscopy. Meanwhile, it is seen that SEM images (Figure 2) and particle size measurements made with zeta sizer are in harmony.

Thermal gravimetric analysis is a characterization technique used to determine the thermal behavior of the substance or reaction products. This technique involves quantitatively measuring the weight changes that occur by applying a controlled increasing temperature program to a substance. It was concluded that chitosan and mannitol-free chitosan nanoparticles at the appropriate temperature experienced more mass loss than mannitol chitosan nanoparticles. Mannitol used as a cryoprotectant and sodium tripolyphosphate used as a cross linker in the lyophilization process provide a more stable structure in these differences (Figure 3).

Fourier Transform Infrared Spectroscopy is an important characterization technique for the illumination of the chemical structure. This technique measures the vibration frequencies of the bonds in the chemical structure and provides information about the functional groups. The chemical structure was illuminated by comparing the frequency changes of pure chitosan, mannitol-free chitosan nanoparticles, mannitol chitosan nanoparticles, EGCG-loaded mannitol chitosan nanoparticles and EGCG.

Mannitol used as a cryoprotectant before lyophilization was found to be a better lyophilized powder for tablet printing of mannitol lyophilized nanoparticles from mannitol-free lyophilized nanoparticles.

It has been concluded that tablets can be printed with EGCG-loaded mannitol chitosan nanoparticles used as active ingredient and tablet excipients suitable for epigallocatechin gallate-loaded chitosan nanoparticle tablet formulation and a product that can be used for dry eye disease has been designed.

Definition of the Illustrative Figures

Figure 1. Synthesis of EGCG-loaded chitosan nanoparticles according to the ionic gelling method. 1) Sodium tripolyphosphate solution 2) EGCG 3) Chitosan solution 4) Magnetic stirrer 5) EGCG-loaded chitosan nanoparticles are shown.

Figure 2. A) EGCG-loaded mannitol chitosan nanoparticles B) Mannitol chitosan nanoparticles C) Mannitol chitosan nanoparticles SEM images

Figure 3. A) Pure Chitosan B) Mannitol chitosan nanoparticles C) EGCG-loaded mannitol chitosan nanoparticles D) Mannitol-free chitosan nanoparticles thermal gravimetric analysis results. Detailed Description of the Invention

Epigallocatechin gallate is a phenolic compound in the structure of green tea. It is known to have an inhibitory effect on inflammation associated with autoimmune disorders. In the invention, the nanoparticular system was formed with epigallocatechin gallate, the main polyphenol of green tea consumed as a beverage, and tablet formulation was developed for oral use in the treatment of dry eyes. Epigallocatechin gallate-loaded mannitol chitosan nanoparticles were synthesized with the invention. The nanoparticular system has been developed to increase the stability and absorption of EGCG.

Table 1: Chemical formulae and molecular weights of the compounds used in the invention

The ionic gelling method is a method in which reversible physical cross-linking agents are used by electrostatic interaction instead of chemical cross-linking to prevent possible toxicity of reagents and other undesirable effects. Sodium tripolyphosphate is a polyethylene that can interact with cationic chitosan with electrostatic forces and is often used as a crosslinker. Chitosan is dissolved in an aqueous acidic solution to obtain the cation of chitosan in the ionic gelling method, as illustrated in Figure 1. It is added to the EGCG chitosan solution desired to be loaded. The resulting solution is then added dropwise by stirring continuously with polyanionic sodium tripolyphosphate solution. Depending on the interaction between mutually charged species, chitosan undergoes ionic signaling and collapses to form spherical particles. It is lyophilized by washing after centrifugation. The formed nanoparticles have a weak mechanical force and have a global structure.

Within the scope of the invention, epigallocatechin gallate-loaded chitosan nanoparticles were synthesized using chitosan and crosslinking sodium tripolyphosphate with the ionic gelling method, which is one of the nanoparticle synthesis methods. In vitro characterization studies of synthesized nanoparticles were carried out.

The method of synthesis of epigallocatechin gallate-loaded mannitol chitosan nanoparticles according to the ionic gelling method of the invention generally comprises the following steps as follows;

• Homogenization of 20-100 mg chitosan by mixing in a magnetic stirrer at room temperature 25°C overnight with the addition of 50 mL of 0,175% v/v acetic acid solution and adjusting to pH 3,

• Adding 5-50 mg of epigallocatechin gallate into the chitosan solution and mixing in a magnetic stirrer for 15-45 minutes,

• Preparing an aqueous solution of 0.1% w/v sodium tripolyphosphate and adjusting the pH to 3,

• Taking 1 mL of the sodium tripolyphosphate solution at a time with the help of an automatic pipette at 10-60 seconds intervals and adding it completely by mixing it into the chitosan-EGCG solution,

• Mixing in a magnetic stirrer at room temperature 25°C for 4 hours,

• Centrifuging at 4000-16000 rpm for 5-45 minutes,

• Washing with water and taking into a freeze dryer vial with the help of vortex after centrifugation,

• Addition of 60-300 mg mannitol as cryoprotectant, • Lyophilization for 15-60 hours.

The steps of the synthesis of EGCG-loaded mannitol chitosan nanoparticles in the 2,2: 1 chitosan/sodium tripolyphosphate ratio according to the ionic gelling method are as follows:

• 50 mg chitosan was homogenized by mixing at 400 rpm in a magnetic stirrer at room temperature 25°C overnight with the addition of 50 mL of 0,175% v/v acetic acid solution in a 100 mL beaker and adjusted to pH 3 with concentrated acetic acid.

• The weighed 25 mg of EGCG was added into the chitosan solution and stirred in a magnetic stirrer at 400 rpm for 30 minutes.

• An aqueous solution of 0,1% w/v sodium tripolyphosphate was prepared and adjusted to pH 3 with concentrated acetic acid.

• Sodium tripolyphosphate solution was taken 1 mL at a time with the help of an automatic pipette at 30 second intervals and added to the chitosan-EGCG solution on a 400 rpm magnetic stirrer.

• Stirred in a magnetic stirrer at 400 rpm at room temperature 25°C for 4 hours. Centrifuged at 9000 rpm for 20 minutes.

• Washed with distilled water and taken into a freeze dryer vial with the help of vortex after centrifugation.

• 145,46 mg of mannitol was added in a 2: 1 ratio as cryoprotectant.

• It was lyophilized in the specified lyophilization program for 30 hours.

Table 2: Lyophilization program

Table 3: In-vitro release kinetics of pure EGCG and EGCG-loaded mannitol chitosan nanoparticles EGCG was found to be the Korsmeyer-Peppas Model and EGCG-loaded mannitol chitosan nanoparticles were found to be the Higuchi Model according to the calculations made in in- vitro release kinetics studies of pure EGCG and EGCG-loaded mannitol chitosan nanoparticles.

Pre-formulations were formed by selecting appropriate excipients for tablet formulation. Dust controls of the pre-formulations (moisture content, cluster density, compressed density, Housner ratio, Carr index, angle of repose assays) and necessary tablet controls (appearance, tablet weight and weight uniformity assays, thickness, diameter and hardness assays, friability test, disintegration test) were performed. As a result, tablets weighing 200 mg were printed and necessary tablet controls were performed by deciding on the appropriate formulation and performing the necessary powder controls of epigallocatechin gallate-loaded mannitol chitosan nanoparticles and excipients.

The required powder mass is prepared as much as the number of tablets to be printed in the tablet printing process. The calculated amount of excipient and active substances are weighed using a precision scale. The powders are mixed in a cube mixer and homogenized. The appropriate staple-seal set is selected for the desired tablet feature. White, round, biconvex tablets containing EGCG-loaded mannitol chitosan nanoparticles are printed on the tablet press by making the necessary adjustments on the tablet device.

1 ^st Pre-Formulation

Rp

Lactose monohydrate (98% w/w)

Magnesium stearate (1% w/w)

Lyophilized product (1% w/w)

The required powder mass was prepared for 100 tablets. The calculated amount of excipient and active substance were weighed using a precision scale. The weighed lactose monohydrate and lyophilized product were homogenized in a 200 rpm cube mixer for 15 minutes. The lubricant was stirred at 200 rpm for 2 min until homogeneous by adding magnesium stearate. The appropriate staple-seal set was selected to have tablet weights of 200 mg. Tablets were printed on the tablet press by making the necessary adjustments on the tablet device.

2 ^nd Pre-Formulation

Rp

Lactose monohydrate (49% w/w)

Microcrystalline cellulose PHI 02 (49% w/w)

Magnesium stearate (1% w/w)

Lyophilized product (1% w/w)

The required powder mass was prepared for 100 tablets. The calculated amount of excipient and active substance were weighed using a precision scale. The weighed lactose monohydrate, microcrystalline cellulose PHI 02 and lyophilized product were homogenized in a 200 rpm cube mixer for 15 minutes. The lubricant was stirred at 200 rpm for 2 min until homogeneous by adding magnesium stearate. The appropriate staple-seal set was selected to have tablet weights of 200 mg. Tablets were printed on the tablet press by making the necessary adjustments on the tablet device.

The results of the two formulations were evaluated and the EGCG-loaded mannitol chitosan nanoparticle tablet formulation was started according to the 2 ^nd formulation.

Materials used for the 200 mg weight tablet formulation of EGCG-loaded mannitol chitosan nanoparticle and their proportions;

Rp

Lactose monohydrate (49% w/w)

Microcrystalline cellulose PHI 02 (49% w/w)

Magnesium stearate (1% w/w)

EGCG-loaded lyophilized product (1% w/w)

The required powder mass was prepared for 100 tablets. The calculated amount of excipient and active substance were weighed using a precision scale. The weighed lactose monohydrate, microcrystalline cellulose PHI 02 and EGCG-loaded lyophilized product were homogenized in a 200 rpm cube mixer for 15 minutes. The lubricant was stirred at 200 rpm for 2 min until homogeneous by adding magnesium stearate. The appropriate staple-seal set was selected to have tablet weights of 200 mg. Tablets were printed on the tablet press by making the necessary adjustments on the tablet device.

DUST CONTROLS

Appearance Assay

Pre-formulation tablets and EGCG-loaded mannitol chitosan nanoparticle tablets were visually checked. Appearance characteristics of 10 tablets;

• White colored

• Round,

• Biconvex and

• Uncoated.

Table 4: Moisture content of final powder mixtures at 105°C (%)

Table 5: Cluster density, compressed density, Hausner ratio and Carr indices of the excipients and formulations used in tablets Table 6: Powder flow characteristics of the excipients and their formulations used in tablets according to angle of repose assay

TABLET CONTROLS

Table 7: Weights and weight uniformity results of pre-formulation tablets and EGCG-loaded mannitol chitosan nanoparticle tablets

Table 8: Thickness, diameter and hardness results of pre-formulation tablets and EGCG- loaded mannitol chitosan nanoparticle tablets

Table 9: Friability results of pre-formulation tablets and EGCG-loaded mannitol chitosan nanoparticle tablets Table 10: Disintegration results of pre-formulation tablets and EGCG-loaded mannitol chitosan nanoparticle tablets