WO2023055321A1 - Formulations oculaires de micro-émulsion anti-inflammatoire dérivées de l'huile de nigella sativa - Google Patents

Formulations oculaires de micro-émulsion anti-inflammatoire dérivées de l'huile de nigella sativa Download PDF

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WO2023055321A1
WO2023055321A1 PCT/TR2022/050988 TR2022050988W WO2023055321A1 WO 2023055321 A1 WO2023055321 A1 WO 2023055321A1 TR 2022050988 W TR2022050988 W TR 2022050988W WO 2023055321 A1 WO2023055321 A1 WO 2023055321A1
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eye
drug
oil
ocular
microemulsion
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PCT/TR2022/050988
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English (en)
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Gülşah GEDIK
Görkem TIRANBEŞLI
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T.C. Trakya Üni̇versi̇tesi̇
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Priority claimed from TR2021/015389 external-priority patent/TR2021015389A2/tr
Application filed by T.C. Trakya Üni̇versi̇tesi̇ filed Critical T.C. Trakya Üni̇versi̇tesi̇
Publication of WO2023055321A1 publication Critical patent/WO2023055321A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/47—Euphorbiaceae (Spurge family), e.g. Ricinus (castorbean)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears

Definitions

  • the invention relates to liquid eye formulations containing nigella sativa oil and thymoquinone developed for use in the treatment of dry eye disease and the method of preparing the same.
  • the eye is a small, complex multidivisional organ. Its anatomy, physiology, and biochemistry are highly resistant to xenobiotics. The real challenge is to bypass these protective barriers of the eye and deliver the drug to the site in sufficient quantities to treat ophthalmic diseases.
  • the human eye consists of three layers that are suitable for transmitting and refracting light.
  • the outer layer is called the hard layer or eyelid. This layer bumps to form the transparent layer.
  • the hard layer which has a white and wire structure, is a membrane that protects the eye.
  • the vascular layer which is a multi-vessel connective tissue, makes the eyeball a completely dark room with a cell cover on both sides. In the anterior part of this, the eyelash body muscles and the eyelash region are located.
  • the protrusions in the form of small pyramids filled with blood to keep the hanging ligament, which is multi-core in the eyelash region, are called eyelash extensions.
  • the vein layer in the anterior section changes color to form a central hole (pupil), a diaphragm called iris.
  • the iris whose color varies from person to person, includes the muscle strands that are used to enlarge and shrink the pupil.
  • the retina layer forms the third and very thin layer of the eye.
  • the center of the hole, whitish small blister (optic nerve disc) located on the back of this is where the optic nerve enters and is called the blind spot. A little beyond the blind spot lies the yellow spot. This is the visual area where the external images are best shaped.
  • the optic nerve entering the posterior pole of the eye spreads in the form of many nerve wires towards the vascular layer and ends with neurons arranged in three layers.
  • the neurons in the first layer (multipolar neurons) run along the cylinder axis in the optic nerve; their anterior extension adjoins the cylinder axes of the vision neurons of the second layer.
  • This layer contains neurons in the form of cones and rods, one end of which enters the red part of the retina layer. The free ends of the cones and rods are directed to the vein layer. The rays coming into the vascular layer break and affect the nerve endings of the retina cells.
  • the main obstacles and determining factors in ocular drug release are the physicochemical properties of the drug, its excretion from the lacrimal fluid, corneal barriers, and extracorneal absorption.
  • Drug transport along the corneal epithelium is mainly via paracellular or transcellular. Hydrophilic drugs primarily penetrate the paracellular pathway, lipophilic drugs prefer passive or facilitated diffusion through transcellular pathway intercellular spaces. The lipophilic property, resolution, molecular size and shape, load, and degree of ionization also affect the path and rate of penetration in the cornea.
  • liquid dosage forms such as solution, suspension, and liposome are either drained from the conjunctival sac into the nasolacrimal canal or cleared from the precorneal area resulting in poor bioavailability of drugs.
  • Drugs are mainly eliminated from the precorneal lacrimal fluid by solution drainage, lacrimation, and inefficient absorption into the conjunctivae of the eye. These factors and the corneal barrier limit the penetration of the topically administered drug into the eye and less of the administered drug reaches the intraocular tissue. The following briefly informs the basic eye barriers.
  • the cornea is a clear and curved tissue that is specialized to focus the light in the foremost part of the eye and to protect the eye from external factors.
  • the anterior surface of the cornea is the basic refractive component of the eye and the other is the lens.
  • the cornea and lens allow light from the external environment to reach the retina effectively.
  • the cornea is an oval structure that forms 1/6 of the outer surface of the eye. When measured from the outside, its horizontal diameter is approximately 12.6 mm, its vertical diameter is approximately 11.7 mm, and its thickness is 0.5 mm in the middle and 1.2 mm in the edge.
  • the cornea must be transparent for clear diffraction of light. Therefore, it does not contain blood vessels in its structure.
  • the cornea forms a mechanical barrier that limits the path of drug molecules.
  • the stroma which consists of an extracellular matrix consisting of a stratified order of collagen fibrils, is characterized by the high water content that makes this layer impermeable to lipophilic molecules.
  • the molecules appear to have an amphiphilic nature, characterized by the presence of hydrophilic and lipophilic properties of the same structure.
  • the negatively charged cornea is more permeable to cations than anions at physiological pH.
  • the cornea consists of 5 layers: epithelium, Bowman's layer, stroma, Descemet’s membrane, and endothelium.
  • Corneal epithelium The cornea is of ectodermal origins such as hair, nails, and skin embryologically, so it is constantly renewed. This layer is the outermost layer of the cornea. It consists of five layers of cells. The cells at the bottom and the cells surrounding the cornea proliferate. These cells move upwards to replace the older cells so that the corneal epithelium is regenerated approximately every other week.
  • the corneal epithelium is natural lipophilic and acts as a selective membrane for small molecules, but also prevents diffusion of macromolecules by the paracellular pathway. Lipophilic drugs can pass through the corneal epithelium as transcellular and hydrophilic drugs as paracellular.
  • Outer limiting layer (Bowman's membrane): It occurs as a result of irregular compression of collagen fibers.
  • the epithelial cells are firmly attached to the Bowman's membrane, providing structural support to the cornea. It is a protective layer against trauma and bacterial invasion. It is not a true membrane and epithelial cells adhere firmly to the Bowman's membrane and provide support to the cornea.
  • Corneal stroma It is a transparent structure that forms the thickness of the cornea. It consists of up to 100 layers formed by regularly sorted collagen fibers and has high hydrophilic properties. The cells called keratocytes are rarely involved in their content. When this layer is damaged, healing often adversely affects vision, leading to loss of transparency and/or changes in corneal slope. It also provides transparency to the cornea.
  • Inner limiting layer (Descemet’s membrane): It is a basal membrane secreted by corneal endothelial cells. The corneal endothelium is damaged and edema develops in the cornea after its damage or disease.
  • Corneal endothelium It is the innermost layer of the cornea. It consists of cells arranged as a single layer in the form of a hexagon. The cells stick together in tight bonds. It acts as a semipermeable membrane. Thanks to the pump enzymes on the lateral sides of the cells, the water content of the corneal stroma is kept constant. These cells are not in contact with blood or lymph fluid but with intraocular fluid and are of different origin from vascular endothelium as the embryological origin.
  • the tear is an ultra-thin aqueous-mucin gel layer with a thickness of approximately 7-10 pm, covering the cornea and conjunctival epithelium, protecting the eye surface, containing antimicrobial proteins such as electrolytes, water, mucin, vitamin A, lysozyme-lactoferrin. Its tasks include making the cornea a smooth optical surface by eliminating small surface epithelial irregularity, wetting and protecting the cornea and conjunctiva sensitive surface epithelium, inhibiting the growth of microorganisms with mechanical washing and antimicrobial activity, and providing necessary nutrients to the cornea.
  • the physical pH of the tear is 7.35 on average, ranging from 5.20 to 8.35, with an osmolarity of 302 mosm/L and a volume of 7 pl. It is produced at 1.2 pL per minute on average.
  • the refractive index is 1.136. Changes in blood glucose are also reflected in tears. Tears are isotonic under normal conditions.
  • Mucin layer The tear forms the innermost layer of the film layer. It covers the surface of corneal and conjunctival epithelial cells on the surface. Its thickness is about 0.5 microns. The mucin layer is mostly secreted by the goblet cells in the conjunctiva, the conjunctiva, and corneal epithelial cells, and very little by Crypts of Henle and Glands of Manz. Mucin layer functions are;
  • Aqueous layer The tear is located in the middle of the film layer. It is the layer with the most thickness, about 8 microns thick.
  • the aqueous layer contains electrolytes, proteins, growth factors, vitamins, antibacterial molecules, cytokines, immunoglobulins, and hormones. This content ensures that the ocular surface is nourished and protected. Functions of the aqueous layer are;
  • Lipid layer It is located on the outermost part of the tear and is 1 micron thick. Its most important task is to prevent the evaporation of the aqueous layer. It also prevents the tear from spreading to the skin and is thought to have antibacterial properties. Functions of the lipid layer are;
  • Topical administration is mostly conducted in the form of ointments, gels, emulsions or suspensions to the eye compartments and is used in the treatment of anterior segment diseases. Topical administration has remained the most preferred method due to its ease of administration and low cost. In most topically administered drugs, the area of action is usually different layers of the cornea, conjunctiva, sclera and other tissues such as the iris and ciliary body of the anterior segment.
  • blood-aqueous barrier and blood-retinal barrier are the main obstacles to targeting anterior segment and posterior segment ocular drug, respectively. Although it is ideal to administer the drug to the retina through systemic administration, it is still a challenge due to the blood-retina barrier; the blood-retina barrier prevents the drug from passing through the blood through the retina.
  • 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 the 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 conducted in the treatment of xerophthalmia.
  • One or more effective retinoids alone can be dispensed in a pharmaceutically acceptable ophthalmic vehicle and topically administered for effective treatment of dry eye disease.
  • 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 recommend the intake of the omega-3 fatty acid diet to relieve dry eye disease.
  • omega-3 gel capsules marketed specifically for dry eyes include Thera Tear and Bio Tears. Rasid et al. demonstrated for the first time at the Massachusetts Eye Research and Surgery Institution that topical administration of a specific fatty acid is beneficial in the treatment of symptoms of dry eye disease.
  • Topical a-linolenic acid therapy has been found to significantly reduce dry eye and inflammation changes at both eye and molecular levels. Therefore, topical administration of a-linolenic acid may be a new therapeutic method to treat clinical signs and inflammatory changes in dry eye disease.
  • the periocular pathway includes subconjunctival, sub-tenons, retrobulbar, and peribulbar administration and is less invasive than the intravitreal pathway.
  • the drug is administered by passing the conjunctival epithelial barrier, which is a rate-limiting barrier for the permeability of water-soluble drugs.
  • Drug solutions are administered close to sclera and high retinal and vitreal concentrations are obtained.
  • intravitreal injection offers different advantages as molecules are administered directly to the vitreous. Unlike other routes, higher drug concentrations are achieved for the intravitreal injection vitreous and retina.
  • the comeal pathway plays an important role in diseases that affect the atrial segment, such as inflammation, infection, and glaucoma.
  • Corneal epithelium acts as a speed limiting barrier.
  • the blood-retina barrier prevents the drug substance from coming out of the blood and reaching tissues such as the retina.
  • This barrier consists of the retinal pigment epithelium and the retinal walls.
  • the transport of drug substance from the tear to other tissues is mainly influenced by two factors; the time of contact of the carrier system with the cornea and the drug substance permeability of the cornea.
  • the drug substance passes through the cornea by passive diffusion, facilitated diffusion, or active transport. It is not connected to passive diffusion carriers.
  • the cornea is a very tight barrier and its permeability rate is 10-7-10- 5 cm/sec.
  • the bioavailability of topically administered drug substances cannot exceed 5%, even for small molecules.
  • high lipophilic substances log P>3 are more difficult to pass than substances with a log P (lipid-water partition coefficient) of 2-3. Because their passage through the lipophilic epithelium slows when it comes to hydrophilic stroma.
  • Corneal permeability is a combination of passive diffusion and active transport. Active transport plays an important role, especially in hydrophilic compounds. Eye studies are difficult to carry out in humans. Therefore, in vivo studies are carried out in rabbits and pigs.
  • the tear fluid is regenerated every 6 minutes.
  • Various traumas such as inflammation, ulceration, burns disrupt the integrity of the cornea.
  • the eye becomes vulnerable to microorganisms and the passage of crops from the cornea with impaired integrity also changes.
  • Protein-bound drug substances cannot pass through the corneal epithelium due to the size of the protein-drug substance complex and thus their absorption does not occur.
  • Some drug substances can also undergo metabolism in the precorneal area.
  • tears contain lysosomal enzymes and can cause the drug substance to break down.
  • Solubility and partition coefficient One of the most important factors affecting eye absorption is known to be the solubility of the drug substance in oil or water.
  • the stroma For a substance with high solubility in oil, the stroma, for a substance with high solubility in water, the corneal epithelium acts as a speed limiting membrane. Therefore, lipophilic substances easily pass through the corneal epithelium, but resistance to hydrophilic substances is observed. Corneal epithelium is 50% and stroma and endothelium are 25% resistant to partially lipophilic substances.
  • Molecular weight and chemical form of the drug substance In general, it has been determined that compounds greater than 500 g/mol are less absorbed from the eye. It has been reported that those with greater molecular weight do not have significant effects on bioavailability. Changing the chemical shape, for example, to base, salt, or ester, affects solubility and hence bioavailability. The prodrug approach is based on this principle.
  • Topical drug targeting to the eye sets a suitable basis for the administration of ophthalmic drugs in the treatment of various eye diseases, and therefore topical administration is the most popular and accessible route of administration.
  • topical drug administration to the eye
  • there are many advantages of topical drug administration to the eye there are many anatomical, physiological and biological factors that limit the entry of drugs to the eye to protect the eye, which have a disadvantage of low bioavailability in drug targeting.
  • Eye capacity can only remain in a limited volume. Excess fluid, both normally produced and externally delivered, is rapidly discharged by the eye.
  • the preferred topical ocular drug administration systems for the patient-compliant may be in the form of eye drops without causing blurred vision and irritation.
  • Anterior-eye structures of the eye e.g. conjunctiva and eyelids
  • Topical administration can be used to administer pharmacological agents to the anterior ocular, corneal and anterior/posterior regions. Topical ocular administration to treat disorders of the anterior structures of the eye offers four main advantages over other routes of drug administration:
  • Topical drug administrations to the eye are considered a noninvasive route due to absorption from the conjunctiva because the drug entering the conjunctiva reaches the general bloodstream rather than the intraocular segments.
  • the nasolacrimal canal is known to contribute to systemic absorption of the drug. Since systemic absorption does not constitute an obstacle for targeting the posterior segment, the amount of intraocular drug obtained is generally below the effective concentration.
  • Ophthalmic solutions are sterile solutions administered by instillation in the eye. At the same time, other pharmaceutical factors such as antimicrobial agent need, osmolarity, buffering, viscosity and appropriate packaging are carefully examined in these dosage forms.
  • Polymers solutions increase the viscosity of the solution when polymer is added to the drug substance solution, so they cannot pass through the biological membranes and increase the bioavailability of the drug substance by prolonging the contact time with the cornea since they can remain in the eye for a long time.
  • different solution type systems can be prepared. These are mainly viscous solutions, bioadhesive hydrogels and in situ gelling systems.
  • Emulsions Ocular emulsions are usually prepared by dissolving or dispersing the drug substance in an oil phase, adding surface-active agent and mixing with water to form an oil emulsion in water.
  • the drug substance may be added to the phase in which it is soluble at the beginning of the emulsion production process or added to the formulation after the emulsion has been prepared by a suitable dispersion process.
  • Ophthalmic suspensions are used when the drug substance is not dissolved in the desired solvent or the solution is not stable.
  • a disadvantage of the suspensions may be that appropriate dosing cannot be performed because the concentration of the drug is dispersed in the solvent.
  • the insoluble drug substance in the suspensions should be in a micronized form in order not to cause corneal irritation or scratching.
  • the suspensions are commonly formulated by dispensing micronized drug powder (diameter less than 10 pm) in an appropriate aqueous solvent. Suspensions with a particle size above 10 pm in diameter may result in a foreign body sensation in the eye and cause reflex tear formation and damage to the eye tissue. Reduction in particle size generally increases patient comfort and the acceptability of suspension formulations.
  • Ophthalmic ointments prolong the contact time of the drug with the eye surface compared to many ophthalmic solutions, but ophthalmic ointments have disadvantages that cause blurred vision and ophthalmic opacification. Ophthalmic ointments can be partially sterilized, or alternatively they must be produced from sterile substances in the aseptic medium. Filtration through a suitable membrane or dry heat sterilization is often used.
  • Ophthalmic gels consist of mucoadhesive polymers, which provide local administration of the drug substance into the eye. These polymers can prolong the contact time of the drug with biological tissues and thus improve ocular bioavailability. The selection of the polymer plays a critical role in the release kinetics of the drug from the dosage form. Examples are carboxymethylcellulose, carbopol, polycarbophil, and sodium alginate. Inserts
  • Ophthalmic inserts are solid dosage forms of appropriate size and shape placed in the conjunctival fornix, lacrimal puncture, or cornea. This solid dosage form can be classified as corrosive (soluble) and non-abrasive (insoluble). Inserts ensure that the drug dose administered to the eye is given correctly and can significantly increase the bioavailability in the eye.
  • Conventional contact lenses provide a drug release for several hours.
  • Conventional hydrogel soft contact lenses are capable of absorbing the drug and minimize its absorption through the lacrimal fluid or conjunctiva.
  • Implants are commonly used to prolong the spread of drugs, especially in ocular fluids and tissues in the posterior. Implants are generally divided into two categories as biodegradable and non-biodegradable according to their degradation characteristics.
  • Colloidal system dosage forms include liposomes, nanoparticles, microemulsions, nanoemulsions, etc. Colloidal dosage forms have advantages such as providing sustainable and controlled release of the drug in the targeted area, reducing the frequency of administration of the drug, and overcoming blood-ocular barriers. Encapsulation of drugs with these colloidal carriers significantly increases permeability in the targeted area and prevents breakdown by ocular enzymes. Nanosuspensions are sub-micron colloidal systems consisting of a water-insoluble drug suspended in a suitable dispersion medium stabilized by surface-active agents. They are effective in increasing the solubility of the drug and thus its bioavailability. The load on the surface of the nanoparticles in the nanosuspensions facilitates adhesion to the cornea.
  • Nanoparticles are particles with a diameter of less than 1 pm, containing various biodegradable/non-degradable polymers, lipids, phospholipids or metals.
  • Liposomes are defined as small artificial vesicles that can be produced from natural non-toxic phospholipids and cholesterol. They are also promising systems in ocular drug targeting due to their size, amphiphilic properties and biocompatibility characteristics. Niosomes are more preferred in topical targeting than other vesicular systems because they are chemically more stable compared to liposomes. They can carry both lipophilic and hydrophilic drugs and also have low toxicity because they are not ionic. Dendrimers are macromolecular compounds consisting of a series of branches around the inner core. Nanometer size ranges are used in drug targeting due to its ease of preparation and functionality, and its ability to display multiple copies of surface groups for biological recognition operations.
  • microemulsion was first used by Hoar in the 1940s. Later, in a study conducted by J.H. Schulman in 1959, a single-phase system was obtained by titrating a milky emulsion with hexanol and the term ‘microemulsion’ was used for this system.
  • Microemulsions are optically isotropic and thermodynamically stable colloidal systems formed using oil, water and surfaceactive agent (SAA) and also auxiliary surface-active agent (ASAA) in some microemulsions.
  • SAA surfaceactive agent
  • ASAA auxiliary surface-active agent
  • Microemulsions are systems with small droplet size, thermodynamically stable, transparent and increase the bioavailability of the drug substance. It also overcomes toxicity problems by increasing the absorption and clinical effects of drug substances with low bioavailability problems.
  • Industrial production and sterilization are simple and inexpensive, and microemulsions, which provide convenience for soluble lipophilic drugs that depend on the lipophilic phase used, are the preferred drug delivery systems with their ease of increasing the solubility of the drug substance, their ease of preparation and administration and their long shelf life.
  • the microemulsions are of low viscosity, the droplet size is larger than the micellar structure, and the radius of the internal phase droplets varies between 10 and 100 nm.
  • microemulsions formed without the use of ASAA are called self-emulsifying microemulsions.
  • Self-emulsifying systems are biocompatible systems consisting of drug, oil and surface-active agent; mostly containing one or more auxiliary surface-active agents. Generally, the lipophilic properties of the drug substance in these systems are distributed between the aqueous and fatty excess and the distribution coefficient affects the release rate.
  • Self-emulsifying drug carrier systems are effective in improving the solubility and absorption of the drug in drugs with solubility and bioavailability problems. Hydrophobic drugs can be dissolved in these systems. Microemulsions provide all possible requirements due to their long durability (thermodynamic stability), low inter-surface tension and almost spontaneous formation, low viscosity, high dissolution capacity and small droplet size.
  • Organic phase The selection of oil for microemulsions is important as it both significantly affects the stability of the microemulsion and is necessary to dissolve the large amount of lipophilic drug that should be given. However, there is usually no simple oil selection that perfectly meets both conditions.
  • the organic phase not only dissolves the lipolytic drug, but also has several advantages in increasing the absorption of the lipophilic drug from the target site. Typically structures with excessively long hydrocarbon chains prevent the formation of microemulsions. Because this chain structure prevents it from penetrating the interface film. Shorter chain oils can penetrate deeper into the hydrophobic tails of the surface-active agents and thus form a more stable interface film required for microemulsion formation. However, the dissolution capacity with the organic phase increases with the length of the chain.
  • oils such as medium or long chain triglycerides (MCT, LCT) are also preferred instead of non-polar oils.
  • organic phases should not be multipolar because they prevent the formation of microemulsions. MCT, which is more water-soluble than LCT, is often preferred because they enable lipophilic drugs to dissolve at high concentrations.
  • the most commonly used external phases are soybean oil, castor oil, triglycerides where 95% of fatty acids are made from 8-10 carbon atoms, Migliol 812s (glycerol trials, caprylic and caprylic acids), fatty acids such as isopropyl myristate, oleic acid, and sucrose esters such as mono-, di- or tri-palmitates of sucrose. Since these excipients are well tolerated by the eye, their purity should be high in order to prevent contamination with potentially irritating substances.
  • Aqueous phase Water is the main component in the formation of microemulsion. In O/W type microemulsions, it functions as a continuous phase and unlike its immiscibility with oil, oil droplets are formed by hydrogen bonding with SAA and ASAA and oil type microemulsion is formed in water.
  • the aqueous phase buffers should be selected to minimize the difficulties during the preparation of the microemulsion systems and also to maximize the stability of the microemulsion.
  • microemulsions should contain several additives, such as antibacterial and isotonic agents. They may affect the available area of the microemulsions and should therefore be examined in the presence of other components of the microemulsions.
  • Salinity affects phase diagrams when ionic surface-active agents are added and reduces the phase inversion temperature (PIT) of non-ionic surface-active agents.
  • PIT phase inversion temperature
  • the preparation of microemulsions is very sensitive to temperature if the PIT is close to operating conditions. It is also important to adjust the initial pH at 7-8 to reduce the hydrolysis of phospholipids and triglycerides to fatty acids that may reduce the pH of the microemulsion.
  • preservatives cannot be included in microemulsions.
  • the surface should not interact with the drug substances and should not result in complexes, and should not be absorbed in nanofluids that will significantly reduce bacterial activity in bases.
  • Thiomersal and chlorobutanol can be used at concentrations of 0.01-0.2%; combinations increase their properties.
  • SAAs reduce the tension between the aqueous and oily phases, enabling the formation of emulsions.
  • the preferred adsorption of SAA allows changes in the physicochemical properties of the interface due to its amphiphilic nature. While the SAA concentration is 0.1% by weight in emulsions, the interface between the aqueous and oily phase is at least 10% in microemulsions due to the increase in the area. This high SAA concentration may cause ocular toxicity. Therefore, it may be better to reduce the amount of SAA and choose a preparation treatment that is not self-emulsifying. Generally, ionic SAAs are too toxic to be used for ocular administration. Therefore, non-ionic SAAs should be preferred.
  • SAAs are readily soluble in water due to the presence of ether functional groups.
  • the most commonly used SAAs in the preparation of microemulsifiers are poloxamers, polysorbates, polyethylene glycol and tyloxapol. PEG 200 is preferred due to its lowest viscosity among PEGs.
  • poloxamers polyoxyethylene glycol blocks are hydrophilic, while polyoxypropylene blocks are hydrophobic. The highest amount of oxy ethylene is found in pol oxamer 188 (a/a 82).
  • Amphoteric SAAs such as lecithin are of interest due to their low toxicity. Before use, the source of this compound must also indicate the degree of purification (change of fatty acid content), such as the physical properties, compositions and concentrations of phospholipids. Thus, for example, lecithin obtained from egg yolk contains 32% phosphatidylcholine, 22% phosphatidylethanolamine and 19% phosphatidylinositol. Miranol (MHT), another amphoteric SAA, is well tolerated in eye formulations. In addition, cationic SAAs such as stearylamine are useful due to the potential increase in the duration of stay through the binding of fat streaks to the epithelium.
  • ASAAs have three functions in the formation of microemulsions; (a) providing very low interface stresses required for the formation and thermodynamic stability of microemulsions, (b) being able to change the curvature of the interface depending on the relative importance of the apolar groups, and (c) affecting the fluidity of the interface film. If the surface film is too hard, it prevents microemulsion formation and causes a more viscous double brittle phase.
  • ASAA provides fluidity and is equivalent to a branched SAA. It also allows for a lower concentration of SAA in formulations. The presence of unsaturated boundaries on the hydrocarbon chain of the SAAs equally increases the viscosity of the film.
  • ASAAs used are generally composed of small molecules such as alcohols with low molecular weight, glycols with a length of carbon chains ranging between C2 and CIO. Some alcohols, such as pentanol and hexanol, are not used in pharmaceutical administrations due to their extremely irritating nature. Short-chain amines can also be used as ASAA.
  • Winsor I There are four types of emulsions, Winsor I, Winsor II, Winsor III and Winsor IV. There are two phases, emulsion and oil, in the phase called Winsor I. There is excess waste oil at the top of this phase, emulsion is formed at the bottom.
  • Winsor II also has 2 phases, water and emulsion, where the emulsion forms at the top and there is waste water at the bottom.
  • Winsor III there are 3 phases, these phases are W/O, O/W and the transition emulsion phase.
  • the emulsion is formed in the middle.
  • the concentrations in which the SAA/ASAA-Oil-Water system gives the most appropriate and durable emulsion are determined by triangular phase diagrams.
  • the appropriate microemulsion area of the triangular phase diagram is a method used to calculate the amount of components in the formulation. It also helps to examine and prevent stability problems. When there are more than three components in the system, the number of variables can be reduced to three by keeping one or more of the components at a fixed rate.
  • the diagram consists of the balance of W/O emulsion, lamellar or hexagonal liquid crystals, W/O microemulsion, O/W emulsion and O/W microemulsion.
  • the droplets of O/W and W/O type microemulsions were surrounded by a film consisting of SAA and ASAA molecules.
  • the hydrophobic part of the amphiphilic molecule is directed to the outer phase and the hydrophilic part is directed into the water droplet in the microemulsion, the outer phase of which is oil (W/O).
  • W/O oil
  • the outer phase of which is water (O/W) the hydrophobic part of the amphiphilic molecule is directed into the oil droplet and the hydrophilic parts are directed towards the outer phase.
  • the water, oil and SAA ratio is equal. This type of microemulsion is multi cornered, and the oil and water phases are separated by a layer with a high amphiphile. Transitional microemulsions are formed by the dispersion of SAA in water and oil as a single layer.
  • the first process in the formulation of microemulsion drug delivery systems is the plotting of triangular phase diagrams.
  • the aqueous phase, fatty phase, SAA and ASAA in the microemulsion formulation are mixed appropriately and the components in the formulation can be determined after determining the most suitable microemulsion area. Titration is the most common and easy method for drawing phase diagrams that are closely related to the solubility of the components.
  • One of the characteristics of microemulsions prepared with non-ionic surface-active agents is that their structures are sensitive to temperature changes. Each system is characterized by a narrow temperature variation, and since it is known that the emulsifying properties of the substances change with temperature, the PIT system has been developed as well as the HLB system.
  • the emulsifiers are rated according to the temperature at which they convert the O/W emulsion to the W/O emulsion. It is suggested in the literature that it is preferable to define an HLB temperature based on PIT. This temperature is a characteristic of the SAA-oil- water phase diagram. It is a PIT temperature for non-ionic SAAs, and a SAA below this temperature is preferably broken down as fat-inflated micelles in the water phase, and above this temperature, preferably as inverted micelles inflated in the water in the oil phase.
  • HLB Hydrophilic-Lipophilic Balance
  • HLB values of SAAs and ASAAs range from 0 to 20. As this value approaches 20, the emulsifier gains hydrophilic properties. Emulsifiers with HLB values less than 8 are lipophilic and those with large HLB values are hydrophilic.
  • the aqueous phase, fatty phase, SAA, and ASAAs are mixed appropriately; the resulting system may be O/W microemulsion, W/O microemulsion, O/W macroemulsion, W/O macroemulsion, lamellae, or hexagonal crystalline fluid.
  • One of the methods used in the preparation of microemulsion is high pressure homogenization.
  • transparent microemulsions are obtained by passing the prepared macroemulsions through a high pressure homogenizer.
  • PIT is used.
  • W/O emulsion is prepared at 59-80°C with suitable components to prepare O/W microemulsion, phase separation occurs when the formed emulsion is cooled to room temperature, and this temperature with phase separation is called phase separation temperature.
  • Microemulsion can be obtained by adding ASAA to the system after the phase separation phase.
  • Microemulsions of the systems prepared without ASAA can also be created by using a high pressure homogenizer.
  • the most appropriate amount of water for microemulsions can be determined by titration method or trial and error method. The trial and error method is not very preferred due to excessive material loss and empirical method (77).
  • the most important issue in the preparation of microemulsions is that the substances that make up the system, especially the SAAs they contain at a high rate (20% on average), do not exhibit toxic and irritant effects.
  • Microemulsions are transparent because of their small droplet size and pass the wavelength of visible light. While transparency is provided with a small amount of SAA in W/O systems, the amount of SAA required in O/W microemulsions is higher. Impairment of microemulsion stability is easily understood by observing changes in physical appearance. Accordingly, optical examinations of the glassy microemulsions could be performed with scanning electron microscopy (TEM), albeit limited to direct imaging.
  • TEM scanning electron microscopy
  • the density measured in the narrow-angle scattering method affects the structural correlation and particle size data of the droplet in proportion to the density of the substances that cause scattering, inter-droplet formations, and structural differences between the droplets.
  • the beam interacts with the colloidal particle with the Brown movement.
  • the intensity of the correlation function gives the diffusion coefficients of the beam-dispersing particles and hence the diameters of the microemulsion droplets according to the Stokes- Einstein equation.
  • Zeta potential analysis Zeta potential analyses are also performed in microemulsion systems. While small droplet O/W type emulsions show low resistance, conductivity is observed when droplet coagulation is observed in W/O type emulsions. The increase in resistance over time is a sign of aggregation, that is, instability. The system is diluted in bidistilled water with a conductivity of 50 pS.cm-1 and the analyzes are evaluated according to the Helmholtz-Smoluchowski equation. In ocular microemulsions, the loads of internal phase droplets affect their ocular absorption. Positive charged droplets applied to the cornea, which has a negative load, adhered to the tissues and increased the release of microemulsion with the increase in the length of stay in the eye.
  • Rheological properties The viscosity of the system is of great importance in terms of the ease of use and increase in bioavailability of microemulsions. Single-point viscosimeters and multi-point viscosimeters are used in the measurement of viscosity. Rotation-type rheometers using the cone-table method, one of the multipoint viscometers, are frequently used for rheological analysis. Macroemulsions generally show pseudo-plastic flow, while microemulsions show Newtonian flow. The microemulsion formulation must have certain rheological properties, especially in topical administration.
  • Microemulsions have been developed as new drug carrier systems to increase the ocular absorption of ophthalmic drugs. Most of the formulation strategies aim to maximize ocular drug permeability by extending drug residence time in the cornea and conjunctival area, as well as minimizing pre-comeal drug loss. Microemulsions are promising dosage forms for eye use as they have many advantages related to ophthalmic administration. Microemulsions are of particular interest in administering hydrophobic drugs to the cornea due to the possibility of the drug being loaded on the fat particle. O/W microemulsions have been evaluated as a suitable system for ocular administration for dissolution, increase of absorption and also prolongation of the release profile of low-soluble drugs.
  • microemulsions Since the droplet size of the microemulsions is very small (usually below 150 nm), these systems provide more penetration of the drug into the deeper layers of the eye by releasing the drug in a controlled manner.
  • microemulsions have excellent advantages, limitations in the selection of SAA/ASAA and potential toxicity associated with higher concentrations of SAA/ASAA often restrict the use of microemulsions.
  • Kumar et al. developed a voriconazole-loaded ocular microemulsion formulation in 2014 and evaluated the traditional formulation with the formulation they developed in in vitro release and ex vivo transition studies.
  • Tear hyperosmolarity may occur due to increased evaporation or decreased aqueous secretion.
  • the increase in protein and electrolyte concentration causes a decrease in the volume of tears, which initially leads to irritation on the ocular surface and later to inflammation.
  • Tear film instability diagnosed with decreased dryness time is also related to increased evaporation.
  • Dry eye is a common condition in the ophthalmology clinic. According to recent studies, the prevalence of dry eye is estimated to be between 5-30% in various age groups. While 7.1 million people over the age of 40 in the United States have been reported to experience ocular discomfort due to dry eye syndrome, other studies on prevalence have reported that 6% of the population over the age of 40 and 15% of the population over the age of 65 have dry eye. The frequency of dry eyes increases with age and is significantly more frequent, especially in women. In addition, the incidence of dry eye increases significantly in various systemic diseases.
  • Aqueous insufficiency not related to Sjogren syndrome may occur due to reasons such as aqueous insufficiency caused by aging, congenital absence of tear gland (Congenital Alakrima), deterioration in sympathetic and parasympathetic innervation of the tear gland, infiltration and ablation of the tear gland.
  • aqueous insufficiency caused by aging, congenital absence of tear gland (Congenital Alakrima), deterioration in sympathetic and parasympathetic innervation of the tear gland, infiltration and ablation of the tear gland.
  • tear release from the tear gland is normal, but the dry eye clinic develops as a result of evaporation from the ocular surface.
  • Tear hyperosmolarity activates a series of inflammatory events on the ocular surface and damages the surface epithelium by releasing inflammatory mediators into the tear.
  • Epithelial damage includes cell death by apoptosis, loss of goblet cells, and impaired mucin expression, leading to tear film instability. This imbalance exacerbates ocular surface hyperosmolarity and completes the vicious cycle.
  • Tear film instability can be initiated by a variety of etiologies, including xerophthalmia, ocular allergy, topical preservative use, and contact lens wear prior to tear hyperosmolarity.
  • Epithelial damage caused by the dry eye stimulates the corneal nerve endings, leading to compensatory reflex lacrimal tear secretion due to increased eye opening and closure.
  • a normal mucin deficiency on the ocular surface contributes to the symptoms by increasing the frictional resistance between the eyelids and the eyeball.
  • High reflex input in this period has been put forward as the basis of a neurogenic inflammation in the gland.
  • Primary causes of tear hyperosmolarity are lacrimal insufficiency and/or watery tear flow due to increased evaporation from the tear film. This is indicated by the arrow in the top middle of the figure.
  • Valve lipid quality may be altered by the action of esterase and lipase enzymes released in normal valve products with increasing numbers of blepharitis (inflammation of the eyelid, eyelid). Reduced watery tear flow results from impaired conduction of lacrimal fluid into the conjunctival sac. It is unclear whether this is a feature of normal aging. However, it can be induced by some systemic drugs such as antihistamines and antimuscarinic agents.
  • the first things to do in the treatment of dry eye are to reveal and treat the underlying causes, to relieve symptoms, to reduce tear osmolarity, to correct the stability of the tear layer and to reverse eye surface damage. Regardless of the cause of dry eye syndrome, the patient must be informed about the treatment. Treatment should be aimed at improving symptoms, ensuring the integrity of the ocular surface and preventing complications. If there is a systemic disease causing dry eye, it should be treated. It should always be kept in mind that dry eye treatment is a chronic, generally resistant and very difficult disease to treat. Since dry eye symptoms have a wide range and are common in many ophthalmological diseases, there may be misconceptions in dry eye treatment. Since there have been studies in recent years suggesting that this disease is an inflammatory syndrome, treatment protocols have started to shift to anti-inflammatory and immunomodulatory areas.
  • Ophthalmic anti-inflammatory agents are products formulated to reduce pain or treat inflammation by administration to the eyes. Antiinflammatory agents act against one or more mediators, which cause inflammation and reduce eye irritation and swelling. Anti-inflammatory eye drops are also used to relieve pain after surgery.
  • Restasis ophthalmic emulsion contains 0.05% cyclosporine as drug substance and castor oil, glycerin, polysorbate 80, carbomer, water and sodium hydroxide as excipients. Restasis eye drops contain Cyclosporine, an immunosuppressive drug. Cyclosporine can increase reduced tear production with inflammation in the eyes. Restasis eye drops are used to treat the chronic dry eye that may be caused by inflammation. Xiidra® Ophthalmic Solution
  • Xiidra ophthalmic solution contains 5% lifitegrast as drug substance, sodium chloride, sodium phosphate, sodium thiosulfate, sodium hydroxide, hydrochloric acid and water as excipients.
  • Xiidra works by blocking a specific protein on the surface of the cells in our body. This protein may cause your eyes to not produce enough tears or to produce tears that are not in the right consistency to keep your eyes healthy.
  • Xiidra eye drops are used to treat the symptoms of dry eye disease.
  • Ophthalmic lubricants are used to treat dry and irritated eyes.
  • Nigella sativa is found in the Ranunculaceae family. It is also called black seed, black cumin, and fertility grain.
  • N Nigella sativa
  • 12 Nigella species are grown, among which the seeds of Nigella sativa L., Nigella damascena and Nigella arvensis are used as food and medicine.
  • Nigella sativa L. is the most widely cultivated and traded species in Turkey.
  • Nigella sativa is a cultivated plant whose medicinal use dates back to ancient civilizations.
  • nigella sativa whose seeds are used as both food and folk medicine, has been increasing all over the world with recent pharmacological studies. Seeds, a rich source of the plant's drug substances, have long been used as a traditional remedy in the Middle and Far East. It has traditionally been used in the treatment of asthma, bronchitis, inflammation, eczema, fever, influenza, hypertension, cough, headache, dizziness, diabetes, kidney and liver dysfunctions, nervous disorders, rheumatism, cancer and gastrointestinal disorders. In studies investigating the pharmacological effects of the components of nigella sativa seeds, antiinflammatory, antimicrobial, antibacterial, anticarcinogenic, antitumoral, antioxidant and immune system strengthening effects have been reported.
  • Nigella sativa seeds generally contain fixed oils, essential oils, protein, amino acid, carbohydrates, fibers, alkaloids, tannins, saponins, minerals, ascorbic acid, thiamine, niacin, pyridoxine, and folic acid.
  • Saturated fatty acids in fixed oil include myristic acid, palmitic acid and stearic acid, and unsaturated fatty acids include oleic acid, linoleic acid, eicosadienoic acid, arachidonic acid, and linolenic acid.
  • the essential oil containing the most important pharmacologically active components also contains nigellone, carvacrol, p-cement, d-limonene, a and P-pinene.
  • Nigella sativa seeds contain 34-39% oil, more than 30% of which is fixed oil and 0.5-1.5% essential oil. TQ constitutes approximately 18-24% of this essential oil.
  • carvacrol, nigellimin-N-oxide, nigellicine and nigellidine are included.
  • Nigella sativa seed also contains 29-37% carbohydrate, 20-23% protein, and the trace amounts of vitamins, minerals, and eight of nine essential amino acids. It has been published that there are 4 main active pharmacologically drug substances in nigella sativa seed essential oil, TQ, DTQ, THQ or nigellon and THY.
  • TQ 2-isopropyl-5-methyl-l,4- benzoquinone structure and molecule formula C10H12O2.
  • Various pharmacologically active chemical components have been isolated from the essential oils of nigella sativa seed; in addition to TQ, DTQ, THQ and TMY, there are p-cement, d-limonene, a-pinene, P-pinene, trans-anethole, carvacrol and nigellone.
  • TQ, p-cement, longifoline, carvacrol, a-cubebene, a- pinene, limonene, P-pinene and sabinene are the main components of nigella sativa seed essential oil.
  • Linoleic acid, oleic acid, palmitic acid are the main fatty acids in fixed oil.
  • Nigella sativa seed is quite high in content of unsaturated fatty acids, and the vast majority of unsaturated fatty acids are linolenic acids, while oleic acids make up less of them. It contains saturated fatty acids palmitic, stearic, eicosadienoic and myristic acid.
  • Nigella sativa seed has a rich vitamin and mineral content.
  • vitamins Bl thiamine
  • B2 riboflavin
  • B3 niacin
  • B6 pridoxin
  • B9 folic acid
  • a and y-tocopherol retinol
  • minerals such as potassium, iron, copper, calcium, zinc, phosphorus, magnesium and selenium.
  • TQ which is found in 18-24% of the essential oil of nigella sativa seed and has a molecular weight of 164.2 g/mol, is a main active phenolic compound and is widely used in the treatment of many diseases with some pharmacological effects. In in vitro and in vivo studies on TQ, it was observed that it had multifaceted biological activity properties and its toxicological effects were comprehensively examined. Enol and keto form structures are found in tautomeric form and keto form is responsible for its pharmacological properties. TQ is a hydrophobic molecule so it enters the cell by passive diffusion. It has high sensitivity to light. Its stability decreases at high pH levels and minimal degradation occurs at acidic pH levels.
  • TQ anti-inflammatory antimicrobial
  • antibacterial antibacterial
  • anticancer nervous system
  • respiratory system respiratory system
  • antidiabetic and antioxidant.
  • TQ, THQ DTQ, TMY in nigella sativa oil and these main components with pharmacological properties such as p-cement, nigellidine and nigellicine are stated in the literature.
  • Inflammation is mainly regulated by two enzymes: cyclooxygenase (COX) and lipoxygenase (LOX).
  • COX cyclooxygenase
  • LOX lipoxygenase
  • PG prostaglandins
  • LT leukotrienes
  • TQ inhibits both the COX and LOX pathways. This reduces the formation of reactive oxygen species (ROS).
  • ROS reactive oxygen species
  • nigella sativa seed fixed oil and TQ inhibited the inflammation chain as a potential anti-inflammatory agent at some stages and inhibited eicosanoid production and membrane lipid peroxidation.
  • Nigella sativa seed fixed oil and TQ have been shown to inhibit the COX and 5-LOX pathways of the calcium ionophore- stimulated arachidonic acid metabolism in the white blood cells of stimulated mice. These results have shown that the anti-inflammatory effect may not only be due to the TQ in the oil, but also due to the other components, especially C20:2 fatty acid.
  • Khan proved the anti-inflammatory effect of the aqueous extract of plant seeds with its inhibitory feature on claw edema in animal models.
  • mice In mice, edema was created with carrageenan, and it was observed that edema was partially eliminated by oral nigella sativa extract and completely eliminated by intraperitoneal administration.
  • nigella sativa essential oil and its active component, TQ had a dose-dependent anti-inflammatory effect and prevented edema and granuloma formation in mice with edema in the claws and ears with carrageenan.
  • the anti-inflammatory activities of DTQ, THQ, TMY, and TQ compounds were investigated in vitro using COX-1 and COX-2. The results showed that all products had more significant inhibitory activity compared to indomethacin versus at least one COX form.
  • DTQ, THQ, TMY and TQ contribute collectively to the anti-inflammatory activity of nigella sativa, and shows that a new nonsteroidal anti-inflammatory drug can be developed by advancing the studies.
  • Some synthetic drugs used to treat inflammation cause changes in biological activities and have an adverse effect.
  • Studies to date have proven that TQ has a cytoprotective effect and this effect has been mainly associated with the anti-inflammatory effect of TQ.
  • Nigella sativa components have been shown to have anti-inflammatory effects in many inflammatory diseases, including experimental allergic encephalomyelitis (EAE), colitis, and arthritis. When the EAE animal received TQ, they showed that they had higher glutathione levels, perivascular inflammation with no disease symptoms compared to animals not treated with EAE.
  • TQ can correct oxidative stress-related damage to many organ systems such as kidney, liver, heart, lung, and stomach.
  • Many beneficial effects of TQ such as anticarcinogenic, anti-inflammatory, analgesic, hypoglycemic, and strengthening the immune system have been suggested and reported to be safe.
  • Toxic effects of TQ could only be demonstrated at very high doses.
  • TQ shows selective cytotoxic effect at high doses. It has been reported that TQ can effectively kill tumor cells without showing cytotoxicity in normal cells such as osteoblast, mouse kidney cells, human lung fibroblasts and vero cells.
  • TQ did not show toxic and lethal effects in oral administration in rats at doses of 10-100 mg/kg.
  • the maximum tolerable dose of TQ was 22.5 mg/kg in male rats, 15 mg/kg in female rats, and 250 mg/kg in male and female rats.
  • the oral lethal dose (LD50) of TQ in mice was reported as 2.4 g/kg and the IP LD50 value in rats as 10 mg/kg.
  • TQ did not change serum ALT and lactate dehydrogenase (LDH) biochemical parameters at 4, 8, 12.5, 25, and 50 mg/kg IP doses, and showed toxic effects in IP injection at doses above 50 mg/kg, and the IP LD50 value was 90.3 mg/kg.
  • LDH serum ALT and lactate dehydrogenase
  • Khader et al. found in their study that ROS formed during biotransformation of TQ in the body can increase oxidative stress. It can consume antioxidant enzymes in high doses of TQ and cause DNA damage in hepatocytes.
  • B.H. Ali et al. reported low toxicity in their study on nigella sativa seed and oil. They reported that the route of administration affected the toxicity results of TQ.
  • nigella sativa seeds are beneficial for health as well as nutrition.
  • Many pharmacological effects such as anti-inflammatory, antioxidant, antimicrobial, anticancer were determined by biological activity studies.
  • Thymoquinone (TQ) is one of these drug substances.
  • the main pharmacologically active key substances in the structure of nigella sativa essential oil include TQ, dithymoquinone (DTQ), thymohydroquinone (THQ), and thymol (TMY).
  • TQ isolated from nigella sativa seeds has an antiinflammatory effect by reducing the formation of reactive oxygen species (ROS) by inhibiting the cyclooxygenases (COX) and lipoxygenase (LOX) enzyme pathways that initiate the inflammation process.
  • ROS reactive oxygen species
  • the dosage form should extend the contact time with the conjunctiva, be simple to administer to the patient, not irritate the eye, and have appropriate properties for the eye.
  • Microemulsions have been developed as new drug carrier systems to increase the ocular absorption of ophthalmic drugs. Most of the formulation strategies aim to maximize ocular drug permeability by extending drug residence time in the cornea and conjunctival area, as well as minimizing pre-comeal drug loss. Microemulsions are promising dosage forms for eye formulations as they have many advantages related to ophthalmic administration. Microemulsions are of particular interest in administering hydrophobic drugs to the cornea due to the possibility of the drug being loaded into the oil droplet. O/W (oil-in-water) microemulsions have been evaluated as a suitable system for ocular administration for dissolution, increase of absorption and also prolongation of the release profile of low-soluble drugs.
  • Dry eye disease has been defined as a tear film disorder resulting from tear deficiency or excessive tear evaporation, leading to ocular surface damage and ocular discomfort symptoms in the interpalpebral area.
  • This definition has changed and the dry eye is defined as a complex inflammatory syndrome of the lacrimal functional unit.
  • topical drugs in dry eye treatment Therefore, innovative drug formulations of anti-inflammatory and herbal origin are needed for the treatment of dry eye disease.
  • ocular microemulsion formulations which are among the innovative drug forms, have been prepared by using the basic drug substances with anti-inflammatory properties in nigella sativa oil and by additionally loading Thymoquinone (TQ).
  • TQ Thymoquinone
  • the products known in the art are products containing either artificial tears or cyclosporine.
  • nigella sativa oil and thymoquinone were used in this invention.
  • the primary object of the invention is to benefit from the rich content of nigella sativa oil, which has anti-inflammatory properties.
  • the ocular eye drops that will provide the treatment of inflammation which is a symptom of dry eye disease
  • these main components such as Thymoquinone, Thymohydroquinone, Dithymoquinone, Thymol as well as -cement, nigellidine and nigellicine with pharmacological properties in nigella sativa essential oil as nigella sativa oil and by adding Thymoquinone substance with proved the anti
  • ocular microemulsion formulations were developed using nigella sativa oil for the first time. It was determined as a prerequisite for obtaining it as a cold press in the selection of nigella sativa oil. Because it has been proven by the literature that the total phenolic component content of nigella sativa oil obtained with the cold press method is high and low oxidative stability is observed. Using the anti-inflammatory properties of thymoquinone, an ocular formulation was performed for the first time.
  • ocular microemulsion formulations were prepared. These are the phase titration method and the high speed homogenization method.
  • the surfaceactive agents it contains irritate the ocular surface Therefore, it is necessary to develop the best formulation by using the minimum amount of surface-active agent. Therefore, characterization and stability studies were conducted on the formulations developed using two different methods. As a result, the minimum surface-active agent content was determined in the ocular microemulsion formulations prepared according to the high speed homogenization method and the ideal formulation was selected as the formation method.
  • In vitro release studies were performed using thymoquinone-containing ocular microemulsion formulations dialysis membrane and pharmacokinetic evaluations were made in line with the data obtained in this study.
  • Tween 20 and Cremophor RH40 have been used as surface-active agents in formulations. Cremophor RH40; Polyoxyl 40 hydrogenated castor oil and Tween 20; Polyoxyethylene 20 sorbitan monolaurate.
  • the method validation of the Thymoquinone substance was performed for the first time in the literature with the LC- MS/MS device in this invention. Solubility studies of thymoquinone were performed for the first time in the literature in nigella sativa oil, Tween 80, Cremophor RH40, Cremophor ELP, propylene glycol, PEG 400 and glycerin. The cytotoxicity study on the ARPE-19 cell line for the use of Thymoquinone in eye formulations was conducted for the first time in the literature.
  • FIG. 1 Process Flow Diagram of the Formulation Prepared by High Speed Homogenization Method
  • the invention relates to eye drops effective in the treatment of dry eye disease containing nigella sativa oil and Thymoquinone (TQ).
  • TQ Thymoquinone
  • the invention also contains a surface-active agent in eye drops.
  • the surface-active agent is Polyoxyethylene 20 sorbitan monolaurate (Tween 20) or Polyoxyl 40 hydrogenated castor oil (Cremophor RH40).
  • the first formulation comprises 0.01% by weight of Thymoquinone, 1.98% by weight of nigella sativa oil, 2.97% by weight of Tween 20 and 95.04% by weight of distilled water.
  • the other formulation contains 0.01% by weight of Thymoquinone, 2.00% by weight of nigella sativa oil, 6.00% by weight of Cremophor RH40 and 91.99% by weight of distilled water.
  • the surface-active agents selected in the invention are not irritant to the eye, and transparent emulsions with high patient compliance were obtained by keeping the amount of water high. In addition, the cost of production has been reduced by using a high rate of water.
  • the amount of oil is sufficient to eliminate the lipid deficiency of the eye.
  • the amount of thymoquinone has been chosen to be non-toxic to the eye but effective. It is suitable for administration to the eye in both formulations. The residence time in the eye is long enough in both formulations. With the HET-CAM test, it has been shown that they are safe products for eye administration. Kinetic studies have demonstrated that both formulations exhibit controlled and extended release.
  • nigella sativa oil obtained by cold press was used for the oil phase that forms the microemulsion.
  • the oil phase was obtained with the anti-inflammatory drug substances (e.g. TQ, DTQ, THQ, THY, etc.) in nigella sativa oil in the treatment of dry eye disease.
  • anti-inflammatory drug substances e.g. TQ, DTQ, THQ, THY, etc.
  • Substances that are used as a surface-active agent in microemulsion and eye formulations and that do not show irritation by the literature are preferred. These are Cremophor RH40; Polyoxyl 40 hydrogenated castor oil and Tween 20; Polyoxyethylene 20 sorbitan monolaurate.
  • formulations were developed with 2 different methods. Distilled water was used as water phase. Thymoquinone was used as the drug substance. Thymoquinone, which is also present in trace amounts in nigella sativa oil, was also dissolved in nigella sativa oil (because it has lipophilic properties) and included in the formulation. Thus, it is aimed to help in the treatment of dry eye disease by benefiting from the anti-inflammatory feature of the formulation.
  • the method of preparing the eye drops of the invention comprises the process steps of: a) Obtaining the water phase by mixing the surface-active agent and distilled water with the high speed homogenizer in the speed range of 4000-16000 rpm, b) Obtaining the oil phase by mixing nigella sativa oil and thymoquinone with the mixer in the speed range of 4000-16000 rpm, c) Obtaining ocular microemulsion by mixing the water and oil phase with the mixer in the speed range of 4000-16000 rpm with the high speed homogenizer.
  • Microemulsion pre-formulation studies were prepared to be 10 mL using a high speed homogenization method. Homogenizer conditions were mixed at 4000 rpm for 30 seconds, 8000 rpm for 30 seconds, and 16000 rpm for 3 minutes for a total of 4 minutes. All formulations were prepared at room temperature ( Figurel). Selection of microemulsion formulations: The prepared preformulations were first observed and then placed in the stability cabinet (40°C under accelerated stability condition; 75% Humidity). The samples kept for 3 months were observed again and the droplet size, PDI (polydispersity index) and zeta potential values were examined with a zetasizer device. Microemulsion formulations were selected in line with the results obtained.
  • dry eye was defined as a tear film disorder resulting from tear deficiency or excessive tear evaporation, leading to ocular surface damage and ocular discomfort symptoms in the interpalpebral area.
  • this definition has changed and the dry eye is defined as a complex inflammatory syndrome of the lacrimal functional unit.
  • TQ anti-inflammatory, antimicrobial, antibacterial, anticancer, nervous system, respiratory system, antidiabetic, antioxidant.
  • a high speed homogenizer was used to obtain more stable oil microemulsion in water using less surface-active agents and more oil phases to form ocular microemulsion formulations.
  • Thymoquinone and formulations in the ARPE-19 cell line played an important role in determining the dosage amount by determining the dose (ICso) at which 50% of the cells remained alive by applying a certain concentration of MTT test to the cells.
  • the cytotoxicity study of TQ and formulations was performed with the MTT method on the ARPE-19 cell line and ICso values were determined.
  • TQ was administered to ARPE-19 cells at different doses and at different times, and according to the data obtained, the dose IC50 value at which 50% of the cells remained alive was found to be 54.152 pM at 24 hours and 609.48 pM at 48 hours. Cell viability was observed to increase at 24th and 48th hours due to low doses.
  • F3-1 and F7-9 formulations without TQ were administered to ARPE-19 cells at different doses and at different times.
  • the IC50 value for the dose F3-1 formulation in which 50% of the cells remained alive was found to be 19.434 pL at the 24th hour and 3.742 pL at the 48th hour.
  • the IC50 value for the F7-9 formulation was found to be 13.957 pL at the 24th hour and 4.644 pL at the 48th hour. It was determined that the effect of F3-1 formulation on cell viability was greater at the 24th hour at 20 pL, 10 pL, 5 pL and 2.5 pL doses, and cell viability decreased at the same doses at the 48th hour.
  • the formulation with the highest IC50 value may be preferred as F3-1.
  • F3-1-TQ and F7-9-TQ formulations containing TQ were administered to ARPE-19 cells at different doses and at different times.
  • the ICso value for the dose F3-1-TQ formulation in which 50% of the cells remained alive was found to be 197.32 pM at the 24th hour and 92.386 pM at the 48th hour.
  • ICso value for F7-9-TQ formulation was found to be 231.90 pM at the 24th hour and 45.15 pM at the 48th hour.
  • the effect of the F3- 1-TQ formulation on cell viability was found to be greater at the 24th hour at doses of 664 pM, 332 pM, 166 pM, and 83 pM, and cell viability decreased at the same doses at the 48th hour. However, at the lowest dose, 41.5 pM cell viability was observed to increase more at 48 hours.
  • the effect on cell viability was increased at 24th hour compared to the decreasing dose. At 48th hour, cell viability increased compared to the decreasing dose of 670.4 pM, 335.4 pM and 167.7 pM, but cell viability decreased at 83.8 pM, and then cell viability increased again at 41.9 pM.
  • Cytotoxicity study was performed to determine the cell viability and IC50 value of TQ-loaded formulations and the formulation that yielded the best ICso value was found to be F7-9-TQ formulation.
  • the reason for this is that when we examine the results of the F7-9-TQ formulation in the release study, it provides more controlled release compared to the F3-1-TQ formulation, accordingly, it compares droplet size and PDI values and the results are more stable, its viscosity is high and the duration of stay in the environment may be longer.
  • the interpretation that it can perform linearly extended release by complying with 1st degree and Korsmeyer-Peppas models was evaluated. Therefore, when the in vitro results of F7-9-TQ are evaluated, it is proven that cell viability decreases over time by increasing cell viability by slow, effective and controlled release in cell lines at first.
  • Hen's Egg Test on Chorioallantoic Membrane is another alternative method for animal testing for corrosive and/or severe ocular irritants using the chorioallantoic membrane of the embryo chicken egg. This assay evaluates damage to this membrane to determine the potential irritation of the conjunctiva. The acute effects of the test substance on small vessels and proteins of the soft tissue of the membrane are assumed to be similar to those caused by substances in the eyes of rabbits.
  • TQ-loaded ocular microemulsion formulations were tested using this method and compared with the results obtained with 0.9% NaCl solution as control. Bleeding, vascular lysis and coagulation formation were observed in the administration of F3-1-TQ and F7-9-TQ formulations.
  • F3-1-TQ formulation was administered to the chorioallantoic membrane
  • vascular lysis formation was observed after 266.4 seconds and IS was found to be 0.346.
  • IS was evaluated, its formulation was found to be non-irritating.
  • the F7-9- TQ formulation was administered to the chorioallantoic membrane, bleeding, vascular lysis and coagulation were not observed in any way and did not cause any irritation.
  • TQ-loaded ocular microemulsion formulations were shown to be safe for ocular administration.

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Abstract

L'invention concerne des formulations oculaires contenant de l'huile de nigelle (nigella sativa) et de la thymoquinone développées pour une utilisation dans le traitement d'un syndrome de l'œil sec, ainsi qu'un procédé de préparation de celles-ci.
PCT/TR2022/050988 2021-10-01 2022-09-15 Formulations oculaires de micro-émulsion anti-inflammatoire dérivées de l'huile de nigella sativa WO2023055321A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
TR2021/015389 TR2021015389A2 (tr) 2021-10-01 Çörek otu yaği kaynakli anti̇i̇nflamatuar etki̇li̇ mi̇kroemülsi̇yon göz formülasyonlari
TR2021015389 2021-10-01

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WO2023055321A1 true WO2023055321A1 (fr) 2023-04-06

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
"Master Thesis", 30 November 2019, TRAKYA UNIVERSITY, Turkey, article TIRANBESLI G.: "Çörek otu yağı kaynaklı antiinflamatuar etkili mikroemülsiyon göz formülasyonlarının hazırlanması ve değerlendirilmesi [Preparation and Evaluation of Anti-inflammatory Microemulsion Eye Formulations From Black Seed Oil]", pages: 1 - 239, XP009546008 *
KOCATÜRK TOLGA, ERKAN EROL, METEOĞLU İBRAHIM, EKICI MEHMET, KARUL BÜYÜKÖZTÜRK ASLIHAN, YAVAŞOĞLU İRFAN, ÇAKMAK HARUN, DAYANIR VO: "Effects of Topical Thymoquinone in an Experimental Dry Eye Model", TURKISH JOURNAL OF OPHTHALMOLOGY, GALENOS YAYINCILIK, ISTAMBUL, TURKEY, vol. 48, no. 6, 1 December 2018 (2018-12-01), Istambul, Turkey, pages 281 - 287, XP093060088, ISSN: 1300-0659, DOI: 10.4274/tjo.50146 *

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