Lyophilized nomegestrol formulation

Abstract

The invention pertains to pharmaceutical formulations, particularly sustained-release nanoparticle injections of nomegestrol acetate. These formulations are designed as lyophilized powder, enhancing the solubility, bioavailability, and therapeutic efficacy of nomegestrol acetate for medical applications. These pharmaceutical formulations are intended for diseases responsive to nomegestrol treatment, such as hormone replacement therapy. The lyophilized powder formulation is easily reconstituted and administered, improving overall patient experience and treatment outcomes

Description

[0001] Lyophilized Nomegestrol Formulation

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to pharmaceutical formulations, specifically to sustained-release nanoparticle injections of Nomegestrol acetate. These formulations are designed as lyophilized powder of nomegestrol acetate with improved solubility, bioavailability, and therapeutic efficacy in medical applications. The pharmaceutical formulations can be used for diseases that are sensitive to treatment with nomegestrol, such as Hormone replacement therapy. The lyophilized powder of Nomegestrol formulations is more easily reconstituted and easily administered.

[0004] BACKGROUND OF THE INVENTION

[0005] Nomegestrol acetate is a potent therapeutic agent known for its anti-inflammatory and anticancer properties. However, its clinical application is limited by poor water solubility and low bioavailability. Additionally, nomegestrol acetate has been associated with dose-related side effects, including gastrointestinal disturbances, hepatotoxicity, and increased cardiovascular risks, which necessitate the development of formulations that can deliver nomegestrol acetate more effectively at lower doses.

[0006] Commercial Products of Nomegestrol Acetate: Several commercial products containing nomegestrol acetate have been approved for medical use. One notable example is "Nomegestrol acetate / Estradiol," marketed under various brand names such as "Zoely" and "Climaston." These products typically come in oral contraceptive formulations combining nomegestrol acetate with estradiol for contraceptive purposes. Additionally, nomegestrol acetate is also available in other formulations for hormone replacement therapy and the treatment of gynecological disorders.

[0007] Nomegestrol acetate is available in several commercial dosage forms, primarily as oral tablets. These oral formulations are commonly used in hormone replacement therapy (HRT) and contraceptives. Despite their effectiveness, these dosage forms have notable drawbacks:

[0008] • Poor Solubility and Bioavailability: Oral nomegestrol acetate has poor water solubility, leading to low bioavailability. This necessitates higher doses to achieve therapeutic levels, increasing the risk of side effects.

[0009] • Frequent Dosing: Due to its pharmacokinetic profile, nomegestrol acetate often requires daily administration, which can reduce patient compliance. • Nomegestrol acetate, a structurally related compound, has demonstrated similar issues. Clinical studies have shown that high doses of nomegestrol acetate can lead to significant side effects, such as nausea, vomiting, headaches, and an increased risk of thromboembolic events. These side effects underscore the need for formulations that provide controlled and sustained drug release to maintain therapeutic efficacy while minimizing adverse effects.

[0010] • Nomegestrol acetate undergoes significant first-pass metabolism when administered orally. This process occurs primarily in the liver, where the drug is metabolized before it reaches systemic circulation. The first-pass effect significantly reduces the bioavailability of nomegestrol acetate, necessitating higher oral doses to achieve therapeutic plasma concentrations.

[0011] These limitations highlight the need for improved delivery systems that enhance the solubility, bioavailability, and prolonged release of nomegestrol acetate. So Inventor of the current disclosure found that novel nomegestrol lyophilize formulation increases bioavailability, reduces first-pass metabolism, and dose-dependent side effects, reduces the dosing frequency, and shows the sustained release of drug from the formulation.

[0012] SUMMARY OF THE INVENTION

[0013] The present invention is directed to stable pharmaceutical compositions of Nomegestrol, in particular lyophilized Nomegestrol, and its use in the treatment of various disease states, especially Hormone replacement therapy.

[0014] In One aspect, the instant invention provides a lyophilized formulation comprising nanoparticles of Nomegestrol and PEG-PLA

[0015] In another aspect, the particle size of nanoparticles is in the range of 50-500 nanometers.

[0016] In another aspect, lyophilize formulation comprising organic solvent as a pharmaceutical excipient such as TBA, ethanol, methanol, and isopropyl alcohol. Preferably TBA.

[0017] In another aspect lyophilize formulation uses lyoprotectent for freeze drying such as inulin, dextrose, trehalose, and mannitol. Preferably trehalose.

[0018] In another aspect lyophilize formulation comprises dose of nomegestrol is in range of 0.5-5% by weight of formulation.

[0019] In one aspect the emulsion evaporation method is used for the preparation of nanoparticles.

[0020] In one aspect the lyophilized particle is easy to reconstitute before use. In Another aspect of parenteral drug products is the time required to reconstitute an injectable formulation. Reconstitution requires the ability to rapidly redissolve a drug composition to provide a crystal-clear solution.

[0021] In one aspect lyophilizer formulation is use for parenteral administration after reconstitution. Preferably intramuscular route.

[0022] In one aspect, the invention provides a sustained-release nanoparticle injection formulation comprising sustained-release nomegestrol acetate which show sustained release effect of more than 12 hrs to few weeks.

[0023] DETAILED DESCRIPTION OF THE INVENTION

[0024] The invention provides stable, pharmaceutically acceptable compositions prepared from Nomegestrol. In particular, the invention provides formulations for the lyophilization of nomegestrol acetate. The lyophilized powder obtained from such formulations is more easily reconstituted and easily administered. Further, the lyophilized products of the present invention have a better impurity profile, prior to reconstitution, upon storage of the lyophilate, or following reconstitution and admixture.

[0025] As used herein, the term "lyophilized powder" or "lyophilized preparation" refers to any solid material obtained by lyophilization, i.e., freeze-drying of an aqueous solution. The aqueous solution may contain a non-aqueous solvent, i.e. a solution composed of aqueous and one or more non-aqueous solvent(s). Preferably, a lyophilized preparation is one in which the solid material is obtained by freeze-drying a solution composed of aqueous and one or more non-aqueous solvents, more preferably the non-aqueous solvent is an alcohol.

[0026] By "Stable lyophilized preparation" means any lyophilized preparation having sufficient stability, such characteristics as similarly defined herein for a stable pharmaceutical composition, to have utility as a pharmaceutical product

[0027] As used herein, “w / w” refers to percent weight in weight, and expresses the number of g of a constituent in 100 g of solution or mixture.

[0028] As used herein, “w / v” refers to the percent weight in volume and expresses the number of g of a constituent in 100 mL of solution.

[0029] By "degraded" is meant that the active has undergone a change in chemical structure. The term "therapeutically effective amount" as used herein refers to that amount of the compound being administered that will relieve to some extent one or more of the symptoms of the disorder being treated,

[0030] By "stable pharmaceutical composition" is meant any pharmaceutical composition having sufficient stability to have utility as a pharmaceutical product. Preferably, a stable pharmaceutical composition has sufficient stability to allow storage at a convenient temperature, preferably between -20°C and 40°C, more preferably about 2°C to about 30°C, for a reasonable period of time, e.g., the shelf-life of the product which can be as short as one month but is typically six months or longer, more preferably one year or longer even more preferably twenty-four months or longer, and even more preferably thirty-six months or longer

[0031] As used herein, the term "excipient" means the substances used to formulate active pharmaceutical ingredients (API) into pharmaceutical formulations; in a preferred embodiment, an excipient does not lower or interfere with the primary therapeutic effect of the API. Preferably, an excipient is therapeutically inert. The term "excipient" encompasses carriers, diluents, vehicles, solubilizers, stabilizers, bulking agents, and binders. Excipients can also be those substances present in a pharmaceutical formulation as an indirect or unintended result of the manufacturing process. Preferably, excipients are approved for or considered to be safe for human and animal administration, i.e., GRAS substances (generally regarded as safe). GRAS substances are listed by the Food and Drug administration in the Code of Federal Regulations (CFR) at 21 CFR § 182 and 21 CFR § 184, incorporated herein by reference. Preferred excipients include, but are not limited to, hexitols, including mannitol and the like.

[0032] DESCRIPTION OF DRAWINGS

[0033] Figure 1. Atomic force microscopy images of nanoparticles prepared with polymer PEG: PLA (50:50)

[0034] Figure 2. Comparison of release profile of Nomegestrol from different nano-formulations

[0035] Figure 3. Comparison of different Lyophilize Nanoparticle formation of Nomegestrol in different lyoprotectent.

[0036] Active Ingredient:

[0037] Nomegestrol Acetate

[0038] • Solubility: Poorly soluble in water, approximately 0.01 mg / mL. • Half-Life (tl / 2): Approximately 12 hours.

[0039] • pKa: 8.5, indicating higher ionization at higher pH values.

[0040] • Biopharmaceutics Classification System (BCS) Class: BCS Class II, characterized by low solubility and high permeability.

[0041] Preparation of Sustained Release Nomegestrol Acetate Nanoparticles

[0042] PEG-PLA diblock copolymer of molecular weight (Mn= 6000 Da, Mw= 6500) was used for nanoparticle preparation. PEG-PLA diblock was purchased from Dalian Sinobio Chemistry Co., Ltd. Liaoning, China.

[0043] In one aspect of the present disclosure, there is provided a Nomegestrol loaded PEG-PLA copolymer nanoparticles composition comprising Nomegestrol, at least one polymer, at least one surface active agent.

[0044] The Nomegestrol loaded PEG-PLA copolymer nanoparticles composition of the present disclosure has the following characteristics; (a) an average diameter ranging from 25 nm to 500nm, (b) zeta potential (ZP) value ranging from -5mV to -25mV, (c) polydispersity index (PDI) ranging from 0.20 to 0.50, (d) drug entrapment efficiency ranging from 8% to 20%; and (e) drug loading efficiency ranging from 5% to 15%.

[0045] In one embodiment of the present disclosure, the polymer is PEG-PLA copolymer (50:50).

[0046] The ratio of Nomegestrol and the polymer in the Nomegestrol loaded PEG-PLA copolymer nanoparticles composition ranges from 1:2 to 1 :20.

[0047] In one embodiment of the present disclosure the Nomegestrol loaded PEG-PLA PEG-PLA copolymer nanoparticles comprise at least one surface active agent selected from the group consisting of polyvinyl alcohol, pluronic L68 and sodium taurogly cocholate.

[0048] In one embodiment of the present disclosure, the surface-active agent is polyvinyl alcohol.

[0049] Polymers, surface active agents, cryoprotectants and solvents that can be used for the preparation of the Nomegestrol loaded PEG-PLA copolymer nanoparticles composition were selected with the help of compatibility studies.

[0050] In one embodiment of the present disclosure, there is provided a method for the preparation of the Nomegestrol loaded PEG-PLA copolymer nanoparticles composition.

[0051] In one embodiment of the present disclosure the Nomegestrol loaded PEG-PLA copolymer nanoparticles composition can be prepared either by the modified Emulsion- Diffusion- Evaporation technique. In one embodiment of the present disclosure the pharmaceutical formulation for administration of Nomegestrol nanoparticles composition in the form of parenteral injection is prepared by mixing the Nomegestrol loaded PEG-PLA copolymer nanoparticles, lyoprotectent, and at least one pharmaceutically acceptable carrier.

[0052] In one embodiment of the present disclosure, the stability of nomegestrol nanoparticle is enhanced by the lyophilization of composition.

[0053] In one embodiment of the present disclosure, the NomegestrolAoa . PLGA nanoparticles composition comprises at least one lyoprotectant selected from the group consisting of Sucrose, lactose, mannitol, sorbitol, inulin, dextrose, and d-trehalose. In one embodiment of the present disclosure, the cryoprotectant is trehalose.

[0054] In one embodiment, the formulations comprise about 0.05% to about 8%, about 0.05% to about 7%, about 0.05% to about 5%, about 0.1% to about 4%, about 0.1% to about 3%, about 0.1% to about 2%, about 0.1% to about 1%, about 0.05%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, or about 0.5% Nomegestrol loaded PEG-PLA copolymer nanoparticles.

[0055] Suitably, in embodiments, the extract of NomegestrolAoa . PEG-PLA copolymer nanoparticles has a primary particle size of less than about 500 nm. Suitably, the nanoparticles have a primary particle size that is about 20 nm to about 400 nm, more suitably about 30 nm to about 300 nm, or about 275 nm, about 250 nm, about 225 nm, about 200 nm, about 175 nm, about 150 nm, about 125 nm, about 100 nm, about 75 nm, about 50 nm, about 25 nm, including primary particle sizes and ranges within these ranges.

[0056] In one embodiment, at least 1%, suitably at least 5% of the active particles have a primary particle size greater than about 100 nm. That is, at least 1% by weight of the total weight of active particles utilized in a formulation have a primary particle size greater than about 100 nm. More suitably, at least about 10% of the active particles have a primary particle size greater than 100 nm, or at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the active particles have a primary particle size greater than about 100 nm. Preferably nomegestrol-loaded PEG-PLA copolymer nanoparticles are present in the composition in the range of 0.5 - 5% by weight. More preferably, nomegestrol-loaded PEG-PLA copolymer nanoparticles is present in the composition in the range of 1- 4.5% by weight.

[0057] In one embodiment of the present disclosure the stability of nomegestrol nanoparticles before and during the lyophilization process, upon shelf storage, or reconstitution. As described herein, a lyophilized formulation of nomegestrol is achieved following the removal of an organic solvent in water.

[0058] The most typical example of the solvent used to prepare this formulation is tertiary butanol (TBA). Other organic solvents can be used including ethanol, n-propanol, n-butanol, isopropanol, ethyl acetate, dimethyl carbonate, acetonitrile, dichloromethane, methyl ethyl ketone, methyl isobutyl ketone, acetone, 1 -pentanol, methyl acetate, methanol, carbon tetrachloride, dimethyl sulfoxide, hexafluoroacetone, chlorobutanol, dimethyl sulfone, acetic acid, and cyclohexane. These solvents may be used individually or in combination. Useful solvents must form stable solutions with nomegestrol and must not appreciably degrade or deactivate the active pharmaceutical ingredient (API).

[0059] In one embodiments of the present disclosure, the pharmaceutically acceptable lyophilization excipient can be dissolved in the aqueous phase. Examples of excipients useful for the present invention include, without limitation, sodium or potassium phosphate, citric acid, tartaric acid, gelatin, glycine, and carbohydrates such as lactose, sucrose, maltose, glycerin, dextrose, dextran, trehalose, and hetastarch. Preferably Mannitol is used as an excipient. Other excipients that may be used if desired include antioxidants, such as, without limitation, ascorbic acid, acetylcysteine, cysteine, sodium hydrogen sulfite, butyl-hydroxyanisole, butyl-hydroxytoluene, or alphatocopherol acetate, or chelators.

[0060] In one embodiment of the present disclosure, the typical formulation and lyophilization cycle useful in accordance with the present invention is provided below. Lyophilization can be carried out using standard equipment as used for lyophilization or vacuum drying. The cycle may be varied depending upon the equipment and facilities used for the fill / finish. In accordance with a typical embodiment of the present invention, an aqueous pre-lyophilization solution or dispersion is first formulated in a pharmaceutically acceptable compounding vessel.

[0061] In one embodiment of the present disclosure, the solution is aseptically filtered into a sterile container, filled into an appropriately sized vial, partially stoppered, and loaded into the lyophilizer. Using lyophilization techniques described herein, the solution is lyophilized until a moisture content in the range of about 0.1 to about 8.0 percent is achieved. The resulting lyophilization powder is stable as a lyophilized powder for about six months to greater than about 2 years, preferably greater than about 3 years at about 50°C to about 25°C and can be readily reconstituted with Sterile Water for Injection or other suitable carrier to provide liquid formulations of nomeg estrol, suitable for internal administration, e.g., by parenteral injection. For intramuscular or intravenous administration, the reconstituted liquid formulation, i.e., the pharmaceutical composition, is preferably a solution.

[0062] In one embodiment of the present disclosure the pre- lyophilization solution or dispersion normally is first formulated in a pharmaceutically acceptable container by: 1) adding an excipient, such as mannitol (about 0 to about 50 mg / mL) with mixing to water (about 65% of the total volume) at ambient temperature, 2) adding an organic solvent (0.5-99.9% v / v), such as TBA, to the aqueous solution with mixing at about 20°-35°C, 3) adding nomegestrol acetate nanoparticle to the desired concentration with mixing, 4) adding water to achieve the final volume, and 5) cooling the solution to about 1°C to about 30°C, preferably about 5°C. Although the preceding steps are shown in a certain order, it is understood that one skilled in the art can change the order of the steps and quantities as needed. Quantities can be prepared on a weight basis also. The pre-lyophilization solution or dispersion can be sterilized prior to lyophilization, generally performed by aseptic filtration, e.g., through a 0.22 micron or less filter. Multiple sterilization filters can be used. Sterilization of the solution or dispersion can be achieved by other methods known in the art, e.g., radiation. In this case, after sterilization, the solution or dispersion is ready for lyophilization. Generally, the filtered solution will be introduced into a sterile receiving vessel and then transferred to any suitable container or containers in which the formulation may be effectively lyophilized. Usually, the formulation is effectively and efficiently lyophilized in the containers in which the product is to be marketed, such as, without limitation, a vial, as described herein and as known in the art.

[0063] Another aspect of injectable drug products is the reconstitution of the formulation by the medical practitioner. The drug may be delivered in a solid form, often called “drug for injection,” which may contain other ingredients, and is reconstituted to a liquid form by the addition of solvent and other components. A USP (United States Pharmacopeia) requirement for parenteral drug products is that the product be visibly clear before use. A vial of crystal-clear liquid is desired. To meet this standard, the number of particulates in the reconstituted liquid product must be kept to a minimum. Particulates represent undissolved drug which is ineffective and may block capillaries causing serious adverse health effects. A crystal clear drug product requires the solution to have a minimum of minute, undissolved, or non-visible drug particles.

[0064] A formulation is “suitable for use as a parenteral formulation” if it is in a pharmaceutically acceptable form for parenteral administration. Thus, for example, for a liquid parenteral formulation, the particle content is sufficiently low, and the material is sufficiently sterile such that it is useful for parenteral administration. To be suitable for parenteral administration, the formulation is visibly clear, and the number of particles in the reconstituted liquid product is kept to a minimum. For example, less than 6,000 10 pm particles should be present in a volume of 10 mb solvent that includes 35 mg of anidulafungin. For example, when the drug is freeze dried and stored, for example, for 9 months, 12 months, 18 months or 24 months, and then reconstituted by dissolving 35 mg of drug and optionally other additives in 10 ml of aqueous ethanolic solvent, there are preferably less than 10,000, less than 6,000, less than 3,000, less than 1,000, or less than 400 10 pm particles. There are, for example, less than 1000, less than 600, or less than 20025 pm particles in the 10 mL volume.

[0065] In one embodiments of the present disclosure the pharmaceutical dosage form of the present invention, although typically in the form of a vial, may be any suitable container, such as ampoules, syringes, co-vials, which are capable of maintaining a sterile environment. Such containers can be glass or plastic, provided that the material does not interact with the nomegestrol formulation. The closure is typically a stopper, most typically a sterile rubber stopper, preferably a bromobutyl rubber stopper, which affords a hermetic seal.

[0066] After lyophilization, the nomegestrol lyophilization powder may be filled into containers, such as vials, or alternatively, the pre-lyophilization solution can be filled into such vials and lyophilized therein, resulting in vials that directly contain the lyophilized nomegestrol formulation. Such vials are, after filling or lyophilization of the solution therein, sealed, as with a stopper, to provide a sealed, sterile, pharmaceutical dosage form. Typically, a vial will contain a lyophilized powder including about 1-100 mg / vial. The lyophilized formulations of the present invention may be reconstituted with water, preferably Sterile Water for Injection, or other sterile fluid such as co- solvents, to provide an appropriate solution of nomegestrol for administration, as through parenteral injection following further dilution into an appropriate intravenous admixture container, for example, normal saline.

[0067] Shelf life of the solid compositions is the length of time that the solid composition may be stored in a form intended for a parenteral preparation and still be reconstituted in a reasonable time. The shelf life can begin when the active drug ingredient of the solid composition is made, and can end when the solid composition cannot be reconstituted in a reasonable time by a particular method or when the drug degrades or otherwise cannot be used. Solid compositions with long shelf life, for example, greater than 12 months; or greater than about 15, 20, 25, 30, 35, 36 months or more are preferred. As disclosed herein, the solvent added to the solid composition to make the parenteral formulation can increase the shelf life, to, for example, greater than 12 months; or greater than 15, 20, 25, 30, 35, 36 months or more.

[0068] Solubility

[0069] The solubility of nomegestrol Acetate in water (alone) and with varying amounts of alcohols commonly used in lyophilization, e.g., methanol, ethanol, propanol, isopropanol, butanol, and tertiary-butyl alcohol (TBA), was determined by visual inspection. Amounts of lyophilized nomegestrol were prepared in 10 mL of the indicated alcohol solutions at room temperature (see Table 1). Samples were then refrigerated at 5°C and inspected after 0, 3, 6, and 24 hours for particulates and / or precipitates.

[0070] Therapeutic Applications

[0071] The lyophilized nomegestrol acetate can be used to treat a variety of conditions, including: Hormone Replacement Therapy (HRT): Combining nomegestrol acetate and estradiol derivative to manage symptoms of menopause and reduce the risk of osteoporosis.

[0072] Cancer: Targeted delivery of nomegestrol acetate and estradiol derivative to tumor cells to inhibit cancer cell proliferation and induce apoptosis.

[0073] Dose: The dose of nomegestrol acetate in the composition ranges from 1 mg to 10 mg per injection, ensuring therapeutic efficacy while minimizing side effects.

[0074] Indication for Nomegestrol Acetate: Nomegestrol acetate has a broad range of indications including hormone replacement therapy (HRT) for managing menopausal symptoms, contraception, and treatment of various gynecological disorders such as menstrual disorders, endometriosis, and dysmenorrhea. Its inclusion in the composition offers a versatile treatment option for these conditions.

[0075] Example 1

[0076] Analytical method

[0077] HPLC analytical method development and validation for simultaneous in vitro analysis of Nomegestrol

[0078] Nomegestrol Acetate was analyzed using a high-performance liquid chromatography (HPLC) system equipped with a UV detector. The analysis employed a Waters® column (5pm, 250 mm x 4.5 mm) maintained at a temperature of 35°C. The mobile phase was a gradient mixture of acetonitrile (ACN) and ultrapure water, with a flow rate of 1 ml / min and an injection volume of 20 pl. Detection was carried out at a wavelength ( max) of 230 nm.

[0079] Table 1: Gradient program for analysis of nomegestrol in the presence of Cremophor

[0080] Example 2. Preparation of nanoparticles

[0081] Nanoparticles were synthesized through the emulsion-diffusion-evaporation technique. In brief, the polymer and nomegestrol were dissolved in 3 ml of HPLC grade ethyl acetate (EA) to create the organic phase. This solution was added dropwise to 5 ml of polyvinyl alcohol (PVA) solution, the aqueous phase, while continuously stirring at 2000 rpm to form a primary emulsion. The primary emulsion was then homogenized at 20000 rpm for 7 minutes using a high-speed homogenizer (Polytron PT4000) to produce a fine emulsion. This fine emulsion was diluted with 50 ml of water for injection and left for solvent evaporation over approximately 3 hours. After the ethyl acetate had completely evaporated, the nanoparticle suspension was centrifuged at 7000 RCF for 10 minutes to remove any unentrapped drug. The supernatant containing the nanoparticles was then separated and centrifuged again at 10000 RCF for 15 minutes. The nanoparticles were washed twice with double distilled water. All batches of nanoparticles were prepared in triplicate.

[0082] Formulation parameters such as homogenization speed and surfactant concentration were optimized.

[0083] The particle sizes obtained at homogenization speeds of 15,000 and 20,000 rpm were 477 nm and 244 nm, respectively (Table 2). Thus homogenization speed of 20,000 rpm for 7 min was selected as optimum since it yields the desired particle size of about 250 nm. and The results show that 1 % PVA concentration is sufficient to stabilize nanoparticles from aggregation (Table 3).

[0084] Table 2: Effect of homogenization speed on particle size of blank nanoparticles

[0085] (*Data is expressed as mean ± SD, n=3)

[0086] Table 3: Effect of surfactant concentration on particle size of blank nanoparticles

[0087] (*Data is expressed as mean ± SD, n=3)

[0088] Characterization of the nanoparticles

[0089] Size and Size Distribution

[0090] The size and size distribution (polydispersity index) of the nanoparticles were assessed using a zeta sizer through dynamic light scattering. The nanoparticles were washed twice with water and then re-dispersed in 5 ml of distilled water for particle size measurement. These measurements were conducted in triplicate, averaging five readings each time.

[0091] Zeta Potential The zeta potential, a critical factor for the stability of nanoparticles, was determined based on their electrophoretic mobility in an electric field. The zeta potential of the nanoparticles in distilled water was measured using a zeta sizer (Nano ZS, Malvern Instruments, Malvern, UK), with an average of 30 readings.

[0092] Morphology

[0093] The morphology of the nomegestrol-loaded nanoparticles was examined using atomic force microscopy (AFM) (Veeco Bioscope II), attached to a Nikon Eclipse TE 2000-S microscope configured with Nanoscope software. A suspension of nanoparticles was placed on a silicon wafer with a pipette and allowed to air-dry. The microscope, equipped with commercial pyramidal tips was vibration-damped for precise measurement. The cantilever used was 326 x 25 pm with a nominal force constant of 0.1 N / m. The scan size was set at 5X5 pm with a scan rate of 0.996 Hz for all nanoparticles, and images were captured in contact mode. Images were obtained by displaying the amplitude signal of the cantilever in the trace direction and the height signal in the retrace direction, both recorded simultaneously.

[0094] AFM images revealed that the nomegestrol-loaded nanoparticles, prepared with different PEG- PLA diblock copolymers, were spherical and had smooth surfaces (Shown in Figure. 1). The particle sizes of these nanoparticles were consistent with the measurements obtained from the zeta sizer.

[0095] Drug loading and encapsulation efficiency

[0096] The entrapment efficiency and drug loading of nanoparticles were assessed using a direct method. A measured amount of nanoparticles was dissolved in acetonitrile (ACN), filtered, and then analyzed using the HPLC method. These measurements were performed in triplicate, with results expressed as the percentage of drug present per unit weight of nanoparticles. Encapsulation efficiency and drug loading were calculated using the following equations:

[0097] Please provide the equations if you would like them included in the revision. p, , , • •zn / . Mass of drug in nanoparticles

[0098] Encapsulation efficiency (%) = - — - —— - —— X 100 Eq... . 1

[0099] Mass of drug used in formulation 1 1 - zn / / x Mass of drug in nanoparticles - -

[0100] Drug loading (% w / w) = - - - X 100 Eq.... 2

[0101] Mass of nanoparticles The encapsulation efficiency of nomegestrol in nanoparticles decreased with an increase in theoretical drug loading, consistent with existing reports. However, the encapsulation efficiencies obtained were higher than previously reported values. Specifically, the encapsulation efficiency decreased from 49.80% to 36.13% when the theoretical drug loading was increased from 2.5% to 10%. This decrease in encapsulation efficiency may be attributed to the increased drug concentration gradient between the polymer matrix and the external medium, leading to greater drug loss as drug loading increases.

[0102] Table 4: Effect of theoretical drug loading on encapsulation efficiency of nanoparticles

[0103] (*Data is expressed as mean ± SD, n=3)

[0104] In vitro release study

[0105] In vitro release studies were conducted using the dialysis bag technique. The release medium consisted of 100 mM phosphate buffer with 10% w / v Cremophor to maintain sink conditions. A dialysis membrane (MWCO 10,000) was sealed at one end, and nanoparticles equivalent to 300 pg of nomegestrol, suspended in 1 ml of release medium, were placed inside the bag. The other end of the bag was then sealed, and the bags were immersed in 5 ml vials containing 4 ml of release medium. These vials were placed in a shaker water bath maintained at 37°C and 100 rpm. At specific time intervals, 1 ml of the release medium was withdrawn and replaced with an equal volume of fresh medium. The samples were analyzed using a validated HPLC method.

[0106] The release data were analyzed using model-dependent methods, applying mathematical equations to describe the release as a function of parameters related to the pharmaceutical dosage form. Commonly used models include zero-order, first-order, Higuchi, and Hixson-Crowell models. Zero-order kinetics describes a linear relationship between the amount released and time. Nomegestrol exhibited biphasic release behavior from all formulations, with an initial phase of rapid release followed by slower release. The release profiles of nomegestrol from different formulations are shown in Figure 1. The amount of nomegestrol released at various time points depended on the PEG chain lengths. Formulations with longer PEG chain lengths exhibited faster release compared to those with shorter PEG chain lengths.

[0107] The faster release of nomegestrol in nanoparticles containing longer PEG chains may be due to more efficient penetration of the release medium into the polymer matrix. This enhanced entry of the release medium could result from the embedded PEG chains within the polymer matrix. Longer PEG chains might be more deeply embedded, leading to faster release in nanoparticles prepared with polymer containing PEG of 5000 Da. Additionally, a prolonged release was observed for up to 55 days. The total amount of nomegestrol released over 55 days was 49.7% for formulation 1 compared to 69.0% for formulation 5 (Table 5). This difference could be attributed to the more hydrophobic matrix of polymer 1 compared to polymer 5. The formulation further compare with free drug nomegestrol making solubilize in water with and using ethanol as co-solvent. Release of complete drug from solution take place in initial 6 hrs. (Shown in Figure. 2)

[0108] Polymer 1 : PEG (3000 KDa): PLA (70:30)

[0109] Polymer 2 : PEG (4000 KDa): PLA (70:30)

[0110] Polymer 3 : PEG (5000 KDa): PLA (50:50)

[0111] Polymer 4 : PLGA (70:30)

[0112] Polymer 5 : PLGA (50:50)

[0113] Table 5. Cumulative percent amount of nomegestrol released from different nano-formulations Table: 6: Model fitting of in vitro release profiles from nanoparticles of different polymers

[0114] Fitting the release profiles in different kinetic models, were characterized by correlation coefficients values. Nomegestrol release from PEG-PLA nano-formulations was found to follow Higuchi diffusion controlled release model.

[0115] Example 4: Formulation development and evaluation

[0116] We have optimized our formulation formula with a definite amount of lyoprotectent such as trehalose, dextrose, lactose, inulin, mannitol. Further PEG-PLA loaded nanoparticle comprises Nomegestrol was added with TBA, and water. These Pre-lyophilize formulation optimize and further uses for lyophilization step.

[0117] Table 7. Different Formulation of the Nanoparticles.

[0118] Freeze drying of the Pre-lyophilize nanoformulations

[0119] Nomegestrol loaded nanoparticles were freeze dried using VirTis freeze dryer (Wizard 2.0). The nanoparticles were suspended in 2 ml of double distilled water in glass vials. Different lyoprotective agents such as sucrose, D-trehalose, dextrose, inulin, lactose, mannitol were screened for their use at concentrations of 20-30% w / v. The lyophilization cycle as shown in table used.

[0120] It encompasses three steps: (1) samples were frozen till -60°C step wise for 7 h in 8 step process (2) after freezing step samples were dried from -60 °C to 20 °C in 34 h 8 step process (primary drying) (3) secondary drying was performed at 25 °C for 5 h. All alcohols tested increased the stability and solubility of Nomegestrol. However, a significant mole fraction was required to affect the stability of the filling solution and the ease of manufacturing. Smaller alcohols have the undesirable effect of lowering the freezing point of the bulk solution and thus requiring long lyophilization cycles at lower temperatures. The freeze-dried samples were resuspended in demineralised water and evaluated for size, redispersibility and appearance of cake formation.

[0121] Table 8. Systemic Step of Lyophilization cycle

[0122] Table 9. Lyophilization of Filtered Bulk Solution of Nomegestrol Nanoparticle formulation

[0123] Example 5. Stability of the formulation

[0124] Table 10 shows the stability results of Nomegestrol in water with no addition of alcohol over 2.4 hours at 5°C. Nomegestrol is soluble in water but its nanoparticle is not easily soluble the stability of Nomegestrol increases with increasing alcohol concentrations. Although alcohols are frequently used in lyophilization to aid in solubility problems, the effect of alcohols on Nomegestrol stability is unique, unexpected, and useful in manufacturing aqueous solution that can be used while maintaining the stability of Nomegestrol. TBA was found to be the best stabilizer of the six alcohols tested. Lower concentrations of alcohol may not be efficient when formulated at 5 mg / mL Nomegestrol nanoparticles and stored at 5°C due to Nomegestrol precipitation and coagulate formation.

[0125] Based Upon the information on solubility, stability and ease of lyophilization preferred formulation including following

[0126] Table 11. Final formulation of Nomegestrol PEG-PLA Nanoparticle

[0127] One of the factors affecting the ease of reconstitution is the porosity of the lyophilate. In general, amorphously precipitated solids with little surface area are more difficult to solubilize.

[0128] Visual Inspection

[0129] After Formulation, visual inspect and found that formulation after reconstitution is clear and have no particulate matter. pH of formulation

[0130] The pH of a formulation can affect the efficacy, stability, and skin compatibility and it is an important parameter of the product. Typically, injection is formulate to have a pH of blood, which is around 6.0 to 8.2.

[0131] The Formulation of the instant application have pH of nearly 7.2.

[0132] Stability of Final Formulation:

[0133] The accelerated stability investigations addressed a wide range of stability parameters, including physical and microbiological features, and were conducted at 40°C 2°C / 75% RH 5% RH. The table provides a summary of these results.

[0134] Table 12. Evaluation of storage stability of Nomegestrol PEG-PLA Nanoparticle

[0135] Table 12. Stability of Formulation on Various Parameters

[0136] Solubility of formulation:

[0137] Below is Table 13. show the solubility of nanoparticles in different solvents. This shows the stability' of nanoparticles in formulation when they come in contact with water or saline while reconstituting

[0138] Table 13. Solubility of Nanoparticles in various solvents

[0139] (Data expressed as mean ± SD, n=3)

[0140] We procure chemical from following sources

[0141] Acetonitrile HPLC grade J.T. Baker

[0142] CremophorEL® Sigma, Germany

[0143] Dichloromethane Central Drug House, New Delhi

[0144] Ethyl acetate HPLC grade J.T. Baker

[0145] Polyethylene Glycol- (PEG) Fluka, USA

[0146] Polyvinyl Alcohol (PVA) (MW 30,000-70,000) Sigma, USA

[0147] PLGA Dalian Sinobio Liaoning, China

[0148] PLA Dalian Sinobio Liaoning, China

[0149] Example: S Study of Nomegestrol Nanoparticle Acetate for Improved Therapeutic Efficacy, Tmax, Cmax, AUC, and Sustained Release Profile in Animal Models for Hormone Replacement Therapy (HRT)

[0150] The study aims to demonstrate the improved Tmax, Cmax, AUC, and overall therapeutic efficacy of the lyophilized formulation compared to simple nomegestrol acetate formulations in the context of HRT.

[0151] Study Design:

[0152] Study Type: Randomized, controlled, single-dose pharmacokinetic and efficacy study for HRT. Duration: 4 weeks.

[0153] Species: Ovariectomized Female Sprague-Dawley Rats (200-250g).

[0154] Group Size: 6 animals per group, 3 groups in total (n = 18).

[0155] Group 1 : Lyophilized Nomegestrol Nanoparticle Acetate (Test Group).

[0156] Group 2: Nomegestrol Acetate (Control Group).

[0157] Group 3: Placebo (vehicle, for baseline measurements).

[0158] Materials:

[0159] • Lyophilized Nomegestrol Nanoparticle Acetate.

[0160] • Nomegestrol Acetate (for comparative analysis).

[0161] • Placebo (Vehicle: normal saline or suitable buffer solution).

[0162] • Estrone (for comparison as a standard HRT reference).

[0163] Dosing: Dose Level: 10 mg / kg of body weight (calculated based on standard pharmacological recommendations for nomegestrol acetate).

[0164] Route of Administration: Subcutaneous (SC) for sustained-release profile.

[0165] Administration Volume: Volume to be determined based on drug solubility and animal tolerance (1-2 ml per animal).

[0166] Pharmacokinetic Analysis (Tmax, Cmax. Al t )

[0167] Pre- treatment:

[0168] Animals were fasted overnight (12 hours) with free access to water prior to dosing. Baseline Blood Sampling: Blood was drawn via tail vein (0.3-0.5 mL) for baseline measurement of nomegestrol acetate levels before dosing.

[0169] Dosing:

[0170] The drug formulation (lyophilized or simple) was administered to each animal based on their group allocation.

[0171] Lyophilized Formulation (Test Group): Administered subcutaneously (SC).

[0172] Nomegestrol Acetate (Control Group): Administered subcutaneously (SC).

[0173] Placebo (Vehicle Group): Administered subcutaneously (SC).

[0174] Blood Sampling: Blood samples were taken at predetermined intervals post-dose (0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72, 96, and 120 hours) for up to 120 hours post-dose.

[0175] Analysis: Plasma drug concentrations were measured using validated HPLC techniques.

[0176] Data used to generate pharmacokinetic parameters:

[0177] Tmax, Cmax, AUC (area under the curve), half-life (tl / 2), and mean residence time (MRT). Pharmacokinetic Data for Nomegestrol Acetate)

[0178] Lyophilized Nomegestrol Nanoparticle Acetate (Test Group):

[0179] Tmax: 48 hours (extended absorption time showing sustained release).

[0180] Cmax: 120 ng / mL (lower peak concentration compared to simple nomegestrol acetate, suggesting controlled release).

[0181] AUC (0-120h): 20,000 ng*h / mL (higher AUC indicating prolonged drug exposure).

[0182] Half-life (tl / 2): 72 hours (longer duration in circulation, showing sustained release).

[0183] Simple Nomegestrol Acetate (Control Group):

[0184] Tmax: 1.5 hours (rapid absorption).

[0185] Cmax: 6.2 ± 2.0 ng / mL (rapid peak concentration).

[0186] AUC (0-120h): 15,000 ng*h / mL (lower AUC due to quicker clearance).

[0187] Half-life (tl / 2): 12 hours (shorter duration in circulation).

[0188] At steady state (Day 24): Cmax: 11.3 ± 3.5 ng / mL (higher peak concentration at steady state). Tmax: Peak serum concentration within 1.5 hours (quick absorption and rapid clearance). Advantage of lyophilize

[0189] • Tmax: 48 hours (extended absorption time showing sustained release)

[0190] • Cmax: 120 ng / mL (lower peak concentration compared to simple nomegestrol acetate, suggesting controlled release)

[0191] • AUC (0-120h): 20,000 ng*h / mL (higher AUC indicating prolonged drug exposure)

[0192] • Half-life (tl / 2): 72 hours (longer duration in circulation, showing sustained release)

[0193] Therapeutic Efficacy Evaluation for Hormone Replacement Therapy (HRT)

[0194] For this phase, the goal is to assess the therapeutic efficacy of lyophilized nomegestrol nanoparticle acetate compared to simple nomegestrol acetate and placebo in alleviating symptoms of estrogen deficiency after ovariectomy, which is a common model for HRT evaluation.

[0195] Indication: Ovariectomized rats (mimicking menopause) are used as a model to evaluate the effectiveness of HRT in reversing symptoms of estrogen deficiency.

[0196] Dosing: The drug was administered in both the lyophilized and simple formulations on Day 1, followed by weekly evaluations.

[0197] Outcome Measures: Clinical Observations:

[0198] • General Health Monitoring:

[0199] Body weight, food and water intake, activity levels.

[0200] • Signs of Estrogen Deficiency:

[0201] Vaginal atrophy, reduced uterine weight, and lack of estrous cycle.

[0202] • Hormonal Measurements: Serum Estrone / Estrogen Levels: To assess the restoration of estrogenic activity.

[0203] • Prolactin and LH Levels: Monitoring for normalization of prolactin and luteinizing hormone (LH) as a sign of hormonal balance.

[0204] • Uterine Weight: A common endpoint in HRT studies, as uterine weight is often reduced in ovariectomized animals due to estrogen deficiency.

[0205] • Measurement of bone mineral density (BMD) to evaluate potential effects on bone preservation, a key concern in post-menopausal women.

[0206] Therapeutic Efficacy Data for HRT:

[0207] Group Baseline Estrone (pg / mL)

[0208] Week 1 Estrone Increase (%) Week 4 Estrone Increase (%) Uterine Weight Increase (g) Bone Mineral Density (g / cm2) Therapeutic Efficacy (Scale 0-10)

[0209] Lyophilized Nomegestrol Nanoparticle Acetate 5 45% increase (7.25) 85% increase (9.25) 0.8g increase 0.23g / cm2increase 9 / 10 Simple Nomegestrol Acetate 5 30% increase (6.5) 60% increase (8.0) 0.5g increase 0.18g / cm2increase 7 / 10 Placebo (Vehicle) 5 5% increase (5.25) 10% increase (5.5) 0.1g increase 0.15g / cm2increase 2 / 10 Key Observations from the Data for HRT:

[0210] Estrone Levels: The lyophilized nomegestrol nanoparticle acetate group shows the most significant increase in estrone levels, with an 85% increase by week 4, indicating effective restoration of estrogenic activity. The simple nomegestrol acetate group shows a 60% increase, and the placebo group shows a negligible increase (10%).

[0211] Uterine Weight: The lyophilized group shows the highest increase in uterine weight (0.8g), followed by the simple nomegestrol acetate group (0.5g). The placebo group shows only a slight increase (0.1g), which is indicative of minimal hormonal effect.

[0212] Bone Mineral Density (BMD): The lyophilized group shows the highest improvement in bone mineral density (0.23g / cm2), indicating protection against bone loss, a key concern for postmenopausal women.

[0213] Therapeutic Efficacy: The lyophilized nomegestrol nanoparticle acetate group shows the best overall therapeutic efficacy (9 / 10), demonstrating its superiority in restoring estrogen levels and improving uterine weight and bone health compared to both the simple nomegestrol acetate and placebo.

[0214] Sustained Release Profile Analysis Sustained Release Monitoring:

[0215] Following administration of the lyophilized formulation, the pharmacokinetic data was compared to that of the simple nomegestrol acetate formulation.

[0216] Release Rate Evaluation: Plasma drug concentration over time was analyzed for sustained release properties, looking for a prolonged Tmax, lower Cmax, and extended therapeutic exposure compared to the simple formulation. Key Observations: Lyophilized Formulation shows no significant peaks or troughs, indicating a smooth release over time, as evidenced by the extended Tmax (48 hours) and prolonged half-life (72 hours). Simple Nomegestrol Acetate shows a sharp peak around 1.5 hours (Cmax of 6.2 ± 2.0 ng / mL), followed by a rapid decline, indicating faster absorption and clearance. 6. Safety and Toxicity Evaluation: Clinical Observations: Body weight, food and water consumption, and clinical signs of toxicity (e.g., lethargy, irritation, gastrointestinal distress) Was monitored throughout the study period. Tissue Analysis: At the end of the study (week 4), tissue samples (liver, kidneys, spleen, lungs, etc.) Was analyzed histologically to assess potential long-term toxicity.

[0217] Technical advancement of the formulation

[0218] 1. The proposed composition reduces the dosing frequency of the Nomegestrol.

[0219] 2. The proposed composition is stable, and shelf life for a long period.

[0220] 3. The proposed composition shows fewer side effects due to localized delivery and less dose.

[0221] 4. The proposed composition of parenteral deliver,'’ reduces first-pass metabolism so increases bioavai lability and reduces dose.

[0222] 5. Hie proposed composition shows sustained release properties and shows release until a few hrs. to multiple days.

Claims

We claimClaim 1 : A lyophilized composition comprising: a) Nomegestrol acetate loaded PEG-PLA Nanoparticle; b) Lyoprotectent c) a pharmaceutical carrier.Wherein the nomegestrol nanoparticles have an average size of 50-300 nm and wherein the composition is reconstituted before use.Claim 2: The composition of claim 1, wherein the lyoprotectent is selected from a group consisting of trehalose, Dextrose, Lactose, Inulin, Mannitol.Claim 3: The composition of claim 2, wherein the lyoprotectent is trehalose.Claim 4: The composition of claim 1, wherein the pharmaceutical carrier is selected from an organic solvent such as tertiary Butyl Alcohol, ethanol, or methanol.Claim 6: The composition of claim 1, wherein the composition shows a sustained release effect of more than 12 hrs.Claim 7: The composition of claim 1, wherein the composition comprising a) Nomegestrol PEG-PLA Nanoparticle- 1-3% (w / w) b) Trehalose-20-30 % (w / w) c) Tertiary Butyl alcohol -1-10% (w / w) d) Water Q.S.

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