Sustained release formulation with increased initial release amount and manufacturing method therefor

Abstract

The sustained release formulation with an increased initial release amount according to the present invention exhibits a rapid release effect within 24 hours after administration and can exhibit a continuous sustained release effect thereafter, thereby achieving long-term drug efficacy persistence and an initial rapid release purpose at the same time. Found to have excellent bioavailability and be safe in the human body, the sustained-release formulation comprising microspheres containing leuprorelin according to the present invention thus can be effectively and safely used for the treatment of various luteinizing hormone-releasing hormone-related diseases such as endometriosis, uterine myoma, prostate cancer, pulmonary pre-menopausal cancer, and central precocious puberty.

Description

Extended-release formulation with increased initial release and method for manufacturing the same

[0001] The present invention relates to a sustained-release formulation of leuprorelin with increased initial release and rapid-release properties, and a method for producing the same.

[0002]

[0003] Luteinizing hormone-releasing hormone (LHRH), also known as gonadotropin-releasing hormone (GnRH), is a hypothalamic decapeptide (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) that regulates the reproductive system of vertebrates. LHRH agonists inhibit LH secretion by depleting LHRH receptors in the pituitary gland, thereby desensitizing pituitary receptors to LHRH stimulation. Additionally, LHRH agonists and antagonists have been shown to be effective in the treatment of endometriosis, fibroids, polycystic ovary syndrome, breast, ovarian and endometrial cancers in women, gonadotropic pituitary desensitization during medically assisted fertility protocols, benign prostate and polymorphism and prostate cancer in men, and precocious puberty in men or women.

[0004] Leuprorelin acetate, a representative LHRH agonist, has a short half-life when administered subcutaneously or intramuscularly. This leads to a rapid decline in blood concentrations after administration, which disappears within hours. This necessitates daily dosing to maintain efficacy, despite its injection form, resulting in patient discomfort.

[0005] In order to minimize the inconvenience caused by such frequent administration and to increase patients' compliance with medication, research has been conducted to apply drugs to injectable drug delivery systems to maintain drug release for a long period of time, and in particular, sustained-release formulations have been developed by manufacturing microspheres using biodegradable polymers.

[0006] However, although leuprorelin acetate requires sufficient drug release in the early stages of administration to exhibit pharmacological effects, the above-mentioned sustained-release formulations only achieve the long-term delayed release goal and have the disadvantage of not being able to release the drug at a high initial concentration.

[0007] In this way, the previously developed sustained-release leuprorelin formulations have limitations in that they cannot exhibit sufficient pharmacological effects, and there is a need for a new formulation that can simultaneously achieve the goals of long-term efficacy and rapid initial release.

[0008] The present inventors, while studying a new formulation that overcomes the initial release delay of sustained-release leuprorelin microspheres and simultaneously exhibits sustained release thereafter, have confirmed that effective initial release and sustained-release effects can be achieved simultaneously when a small amount of leuprorelin or a salt thereof is added to the exterior of sustained-release microspheres in which leuprorelin or a salt thereof is encapsulated, and thus completed the present invention.

[0009] Therefore, the purpose of the present invention is to provide a sustained-release formulation of leuprorelin with increased initial release and a method for producing the same.

[0010]

[0011] In order to achieve the above object, the present invention provides a method for producing a sustained-release formulation with increased initial release, comprising the steps of: 1) preparing a spray solution by dissolving leuprorelin or a salt thereof and a biodegradable polymer in an organic solvent; 2) spray-drying the spray solution to produce microspheres in which leuprorelin or a salt thereof is encapsulated; and 3) dispersing and washing the microspheres in a polyvinyl alcohol aqueous solution to produce microspheres from which residual solvent is removed; and further comprising a step of adding a lyophilized powder of leuprorelin or a salt thereof to the outside of the microspheres.

[0012] In addition, the present invention provides a sustained-release formulation with increased initial release produced by the above-described production method.

[0013] The present invention also provides sustained-release microspheres containing leuprorelin or a salt thereof and a biodegradable polymer, wherein leuprorelin or a salt thereof is encapsulated; and a sustained-release preparation having an increased initial release amount, wherein leuprorelin or a salt thereof is contained on the outside of the sustained-release microspheres.

[0014]

[0015] The sustained-release formulation with increased initial release according to the present invention exhibits a rapid release effect within 24 hours after administration and can exhibit a sustained sustained-release effect thereafter, thereby simultaneously achieving the purposes of long-term sustained efficacy and initial rapid release. The sustained-release formulation comprising microspheres containing leuprorelin according to the present invention has excellent bioavailability and has been confirmed to be safe for human use, and can be effectively and safely used to treat various luteinizing hormone-releasing hormone-related diseases such as endometriosis, uterine fibroids, prostate cancer, premenopausal breast cancer, and central precocious puberty.

[0016]

[0017] Figure 1 is a diagram showing the results of confirming the difference in release rate according to the difference in intrinsic viscosity of PLGA (Poly(lactide-co-glycolide)) used in the production of microspheres.

[0018] Figure 2 is a diagram showing the serum drug concentration over time for 0.5 or 1 day of administration and the serum drug concentration for 28 days (insert diagram) of formulations 3 and 4 containing a small amount of leuprorelin acetate (immediate-release type) on the outside of microparticles manufactured by the manufacturing method of the present invention, and formulations 7 and 8 not containing immediate-release leuprorelin as comparative manufacturing examples.

[0019]

[0020] The present invention relates to a sustained-release formulation with increased initial release and exhibiting both immediate-release and sustained-release characteristics, and a method for producing the same.

[0021] The sustained-release formulation of the present invention with increased initial release induces rapid release of leuprorelin, a drug for which initial release is important, within 24 hours, and can subsequently release the drug in a sustained manner, thereby having the advantage of simultaneously increasing patient compliance and drug efficacy.

[0022] The present invention provides a method for producing a sustained-release formulation with increased initial release, comprising the steps of: 1) preparing a spray solution by dissolving leuprorelin or a salt thereof and a biodegradable polymer in an organic solvent; 2) spray-drying the spray solution to produce microparticles in which leuprorelin or a salt thereof is encapsulated; and 3) dispersing and washing the microparticles in a polyvinyl alcohol aqueous solution to produce microparticles from which residual solvent is removed; and further comprising a step of adding a lyophilized powder of leuprorelin or a salt thereof to the outside of the microparticles.

[0023] The pharmacologically active ingredient of the present invention, leuprorelin or a salt thereof, may be characterized in that it is included in microparticles as a sustained-release formulation and further included on the outside of the microparticles to achieve initial rapid release.

[0024] In the present invention, the step 1) is a step of preparing a spray solution by dissolving leuprorelin or a salt thereof and a biodegradable polymer in an organic solvent.

[0025] Leuprorelin may be used interchangeably with leuprolide and may be represented by the following chemical formula 1.

[0026] [Chemical Formula 1]

[0027]

[0028]

[0029] In the present invention, the salt of leuprorelin may include any pharmaceutically acceptable salt without limitation, and may include, for example, an acid addition salt or a quaternary ammonium salt. The acid addition salt may include any acid addition salt formed from a free acid, and may be an addition salt derived from a free acid, including, for example, organic acids such as citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, glutamic acid, and aspartic acid, and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid. Most preferably, the salt of leuprorelin may be leuprolide acetate.

[0030] In the present invention, the biodegradable polymer is a polymer that is harmless to the human body by slowly decomposing when administered into the body, and may be at least one selected from the group consisting of polylactide, polyglycolide, polylactide-co-glycolide, polyorthoester, polyanhydride, polyhydroxybutyric acid, polycaprolactone, and polyalkyl carbonate. Preferably, polylactide-co-glycolide can be used, and when manufacturing sustained-release microspheres using the method of the present invention, PLGA having an intrinsic viscosity of 0.10 to 1.0 dl / g can be used, and preferably 0.14 to 0.22 dl / g can be used, and the molar ratio of lactide and glycol in PLGA can be 50 to 100: 50 to 0, 60 to 90: 40 to 10, or 70 to 80: 30 to 20, and preferably 75:25. For example, when manufacturing the western-type microspheres of the present invention, PLGA having a molar ratio of lactide and glycolide of 75:25 and an inherent viscosity of 0.14 to 0.22 dl / g can be used.

[0031] It was confirmed that the release (dissolution) rate can be maintained equally even when the intrinsic viscosity of these is changed from 0.14 to 0.22 dL / g, so polylactide-co-glycolide having an intrinsic viscosity of 0.14 to 0.22 dL / g can be used.

[0032] The biodegradable polymer may be included in an amount of 70 to 95 wt%, more preferably 80 to 95 wt%, and most preferably 85 to 95 wt%, based on the total weight of the microparticles.

[0033] The organic solvent used can be selected without limitation as a solvent in which the biodegradable polymer and leuprorelin or its salt can be dissolved, and can be selected according to the type of biodegradable polymer, and Class 3 and 4 solvents specified in the ICH guidelines can be selected in order to use a low-toxicity solvent.

[0034] In one embodiment of the present invention, glacial acetic acid, i.e., acetic acid, is selected and used as an organic solvent, which enables the provision of a formulation with higher safety compared to using class 2 methylene chloride as a solvent. Therefore, the organic solvent of the present invention may be acetic acid or glacial acetic acid, and preferably glacial acetic acid. The glacial acetic acid refers to acetic acid with a purity of 99.5% or higher.

[0035] In step 1) of the present invention, a spray solution is prepared by dissolving leuprorelin or a salt thereof and a biodegradable polymer in an organic solvent.

[0036] Microspheres containing leuprorelin or a salt thereof can be prepared by methods known in the art for preparing sustained-release injectables. Various methods are known, such as coacervation, melt extrusion, spray drying, solvent extraction, and solvent evaporation [double emulsion evaporation (W / O / W; water / oil / water) and single emulsion evaporation (o / w; oil / water)]. However, among these, preparation of microspheres by single and double emulsion evaporation methods, like the phase separation method, has the following disadvantages: difficulty in removing the organic solvent used to dissolve the biodegradable polymer; difficulty in the process due to changes in the solvent removal rate during mass production; allergic reactions due to gelatin used to increase the viscosity of the primary emulsion; possibility of drug denaturation and loss of activity due to strong shear force applied to form small microspheres during preparation of the primary emulsion; and limitations in drug encapsulation rate.

[0037] Therefore, in the present invention, among the methods for producing sustained-release injections known in the art, drug-encapsulated microspheres can be manufactured preferably through a spray-drying method, and step 2) of the present invention is a step of manufacturing microspheres encapsulated with leuprorelin or a salt thereof by spray-drying the spray solution.

[0038] The spray drying of the present invention is performed by supplying the spray liquid prepared in step 1) to a nozzle and evaporating the solvent with high-temperature air while spraying. The supply speed, temperature, time, and supply amount can be appropriately controlled depending on the type of biodegradable polymer used, the content in the microspheres, the content of leuprorelin or its salt, and the content of the solvent. More preferably, the spray drying can be performed using a spray dryer equipped with an ultrasonic nozzle, and the spray liquid can be sprayed into the drying chamber through the ultrasonic nozzle from the top of the sprayer while conveying the spray liquid at a flow rate of 1 to 10 ml per minute. The temperature during spray drying can be 100 to 150°C, 120 to 145°C, and preferably 130 to 140°C, and microspheres encapsulating leuprorelin or its salt can be manufactured through the spray drying step as described above.

[0039] The spray drying step of the present invention can be performed by adjusting the nozzle frequency according to the desired microparticle size, and can be preferably performed using an ultrasonic nozzle having a nozzle frequency of 40 to 80 kHz or 50 to 70 kHz. Using an ultrasonic nozzle having the above nozzle frequency can produce a sustained-release formulation with an increased initial release amount and an average particle size of 15 to 25 ㎛.

[0040] The above average particle size is smaller than that of the western-type microspheres, which are typically suitable for a 23-25G needle, and since subcutaneous injection is possible using a 26G needle, there is an advantage in that it can improve patient compliance with medication.

[0041] Step 3) of the present invention is a step of dispersing and washing the microspheres manufactured above in a polyvinyl alcohol aqueous solution to produce microspheres from which residual solvent has been removed. Through the dispersion process of step 3), any residual solvent remaining in the microspheres manufactured immediately after spray drying can be removed, and at the same time, the microspheres can be more effectively dispersed in an injection solution.

[0042] At this time, the polyvinyl alcohol aqueous solution may be used at a concentration of 0.05 to 2.0 (w / v)%, preferably 0.05 to 1.0 (w / v)%, or 0.1 to 0.5 (w / v)%, and more preferably 0.05 to 0.2 (w / v)%.

[0043] The molecular weight of the polyvinyl alcohol used may be, but is not limited to, 3,000 to 300,000 Da, more preferably 5,000 to 100,000 Da.

[0044] The dispersion step of the present invention can be performed by a method of stirring the polyvinyl alcohol aqueous solution and the microparticles using a stirrer, and the stirring can be performed at room temperature at 1000 to 3000 rpm for 1 minute to 5 hours, preferably 5 minutes to 3 hours, and more preferably 10 minutes to 2 hours.

[0045] Through the dispersion and washing steps described above, residual solvent can be removed and microspheres with improved injectability can be manufactured, and preferably, a final formulation having a residual amount of 0.5% (w / w) or less or 5,000 ppm, which is the management level recommended by the ICH Q3C (R8) guideline, can be produced. More preferably, the sustained-release preparation of the present invention manufactured by performing the dispersion and washing steps can be characterized by the removal of 99% or more, and more preferably 99.5% or more of the residual solvent, but is not limited thereto.

[0046]

[0047] In addition, the present invention provides a manufacturing method characterized by further comprising a step of adding a lyophilized powder of leuprorelin or a salt thereof to the outside of the microspheres.

[0048] The above step is a step for effectively increasing the initial release amount of leuprorelin or a salt thereof, and leuprorelin or a salt thereof is added to the outside of the microparticles in which leuprorelin or a salt thereof is encapsulated, manufactured through steps 1) to 3, so that leuprorelin or a salt thereof contained on the outside can be initially released.

[0049] More specifically, the addition may include a step of 4-1) suspending the microspheres manufactured in step 3) in water for injection and a water-soluble polyhydric alcohol, followed by freeze-drying and crushing to manufacture microspheres; and a step of 4-2) filling the freeze-dried powder of leuprorelin or a salt thereof into the microspheres of step 4-1).

[0050] Alternatively, the addition may include a step of suspending the microspheres manufactured in step 3) in water for injection containing leuprorelin or a salt thereof and a water-soluble polyhydric alcohol, and then freeze-drying and crushing the suspension.

[0051] That is, the process of adding leuprorelin or a salt thereof to the outside of microspheres in which leuprorelin or a salt thereof is encapsulated is a process of adding a lyophilized fragment or powder of leuprorelin or a salt thereof, separately lyophilized and then pulverized to fill the microspheres, or a process of resuspending the microspheres and leuprorelin or a salt thereof in water for injection, and then lyophilizing and pulverizing them together and adding them can be used without limitation.

[0052] At this time, a water-soluble polyhydric alcohol can be used for the freeze-drying process, and the water-soluble polyhydric alcohol can be mannitol, sorbitol or dulcitol.

[0053] Leuprorelin or its salt for immediate release added to the outside of the microspheres may be added in an amount of 2 to 8% (w / w), preferably 4 to 6% (w / w), based on the total weight of the active ingredient, i.e., leuprorelin or its salt (extended release) contained in the sustained-release microspheres. If the amount exceeds the above range, not only may excessive immediate release be induced, but also the duration may be reduced, making it difficult to achieve the efficacy for the desired period of time. If the amount is added in an amount below the above range, it is difficult to achieve a rapid increase in blood drug concentration for the initial 24 hours. In a preferred embodiment, 92 to 98% (w / w) of leuprorelin or its salt among the total active ingredient may be provided in a form encapsulated in the microspheres, and 2 to 8% (w / w) may be provided in a separate lyophilized powder form or an additional encapsulated form on the outside of the microspheres.

[0054] The above manufacturing method can produce a sustained-release formulation with improved bioavailability and increased initial release, which can be characterized by a high initial dissolution rate within 24 hours and a drug duration of about 30 to 65 days. In a preferred embodiment, the initial release within 24 hours in vitro or in vivo can be 10% or less, 8% or less, or 6% or less.

[0055] In the present invention, 'increased initial burst' means a C of about 30 to 50% or more compared to sustained-release leuprorelin microspheres in which leuprorelin or a salt thereof is not added to the outside of the microspheres. max It refers to a sustained-release formulation that shows an increase in value and a high initial release amount within 24 hours.

[0056] In addition to the processes of steps 1) to 4), additional processes necessary for manufacturing and recovering microparticles containing leuprorelin or a salt thereof of the present invention, such as redispersion, mixing, recovery, stirring, heating, and washing steps, may be performed as processes known to those skilled in the art, within the scope that does not impair the purpose and effect of the present invention.

[0057] The present invention provides sustained-release microspheres containing leuprorelin or a salt thereof and a biodegradable polymer, wherein leuprorelin or a salt thereof is encapsulated; and a sustained-release preparation having an increased initial release amount, wherein leuprorelin or a salt thereof is contained on the outside of the sustained-release microspheres.

[0058] The extended-release formulation with increased initial release may be manufactured by the method for manufacturing an extended-release formulation with increased initial release described above.

[0059] Leuprorelin or a salt thereof contained in the outer surface of the above-mentioned western-type microparticles may be contained in an amount of 2 to 8% (w / w), preferably 4 to 6% (w / w), of the total weight of the active ingredient.

[0060] In the above sustained-release formulation, leuprorelin or its salt can exist in two forms, including a form encapsulated in sustained-release microparticles and a form existing outside the microparticles, thereby achieving both initial rapid release and sustained release at the same time.

[0061] The sustained-release formulation of the present invention is preferably an injection, and is particularly preferably provided in the form of a prefilled syringe. Providing it in the form of a prefilled syringe increases convenience during the suspension preparation process and minimizes external contact, thereby reducing the risk of contamination.

[0062] If the western-type formulation of the present invention is an injection, it can be administered through various routes such as subcutaneous, intradermal, intravenous, intramuscular, or intraperitoneal.

[0063] The sustained-release formulation comprising microspheres containing leuprorelin or a salt thereof according to the present invention has excellent bioavailability, has been confirmed to be safe for humans, and exhibits an effective in vivo profile while containing 2 to 8% (w / w) of the total weight of the active ingredient outside the microspheres, so that it can be used alone or in combination with other known treatment methods for the treatment of various luteinizing hormone-releasing hormone-related diseases such as endometriosis, uterine fibroids, prostate cancer, premenopausal breast cancer, and central precocious puberty.

[0064]

[0065] The numerical values ​​described herein should be interpreted as including the equivalent range unless otherwise specified.

[0066]

[0067] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention and are not intended to limit the scope of the present invention.

[0068]

[0069] Example 1 - Method for preparing a sustained-release formulation of leuprorelin acetate

[0070] 1.1 Confirmation of release effect according to difference in PLGA's intrinsic viscosity

[0071] In the present invention, PLGA (Evonik, Resomer®752H, intrinsic viscosity 0.14 - 0.22 dL / g, hereinafter referred to as RG752H) was used as a biodegradable polymer. Since the intrinsic viscosity of RG752H is controlled in the range of 0.14 to 0.22 dL / g, an experiment was conducted to determine whether the drug release effect varies depending on the intrinsic viscosity of the biodegradable polymer used. Microspheres were prepared using leuprorelin acetate ((PPL, Sweden) (Formulation 1-1) or (Anigen Co., Ltd., South Korea) (Formulations 1-2 to 1-5)) and D,L-lactic acid, and varying the intrinsic viscosity of PLGA to 0.16 dl / g (Formulation 1-5), 0.18 dl / g (Formulation 1-4), 0.19 dl / g (Formulation 1-3), and 0.20 dl / g (Formulations 1-2, 1-1). The above components were dissolved in glacial acetic acid to prepare a spray solution, and the prepared spray solution was sprayed into a drying chamber through an ultrasonic nozzle and spray-dried at 135±5°C to prepare PLGA microspheres encapsulating the main component. To remove residual solvent in the microspheres after spray drying, the manufactured PLGA microspheres were dispersed in a 0.1 (w / v)% polyvinyl alcohol (PVA) solution, washed with water for injection, and filtered to remove residual PVA and glacial acetic acid.

[0072] The dissolution profiles of the manufactured formulations were evaluated based on their intrinsic viscosity. Accelerated release tests were conducted under the following test conditions, and samples collected from the release tests were subjected to HPLC analysis, with the results shown in Figure 1.

[0073] - Emission tester: Rotary type emitter (device number: LDT-PT-008)

[0074] - Emission temperature: 50℃

[0075] - Rotation speed: 20rpm

[0076] - Sampling: 1 hour, 4 hours, 6 hours, 24 hours

[0077] - Release solution: 10 mM lactic acid buffer, pH 3.2 (0.4% PVA + 0.1% Tween 80)

[0078] If the difference in the average release (dissolution) rates between the reference formulation and the comparative formulation is within ±15% at all release (dissolution) rate comparison points, and the similarity factor (f2) is 50 or more at all release (dissolution) rate measurement points, the formulations are judged to be equivalent. The similarity factor (f2) was calculated using the following equation.

[0079]

[0080] [Formula 1]

[0081]

[0082] - n: Number of points at which release (dissolution) (%) is measured

[0083] - Rt: Release (dissolution) rate of the reference formulation (%)

[0084] - Tt: Release (dissolution) rate of comparative formulation (%)

[0085]

[0086] As shown in Fig. 1, when comparing other formulations using microspheres (formulations 1-5) manufactured using RG752H, which has the lowest intrinsic viscosity of 0.16 dL / g, as the reference formulation, it was confirmed that the difference was within 15% at all release comparison points.

[0087] Additionally, the similarity factor (f2) values ​​ranged from 51 to 82, confirming that the release patterns of all experimental groups were equivalent.

[0088] Additionally, the average particle size (span value), content, residual solvent (acetic acid), and moisture content were confirmed for formulations 1-1 to 1-5.

[0089] Formulations 1-1 to 1-5 manufactured with different intrinsic viscosities of RG752H and the experimental values ​​measured from the formulations are shown in Table 1.

[0090]

[0091] Formulation 1-1 Sustained release RG752H Intrinsic viscosity Dispersion process solution PVA concentration Residual solvent (acetic acid) Average magnetic field (Span value) Drug and polymer content in microspheres Formulation 1-1 Sustained release 0.2 dL / g PVA 0.1% 0.32% 16.3 um (1.41) Drug: 3.75 mg (10%) Polymer: 33.75 mg (90%) Formulation 1-2 Sustained release 0.2 dL / g PVA 0.1% 0.32% 15.6 um (1.45) Drug: 3.75 mg (10%) Polymer: 33.75 mg (90%) Formulation 1-3 Sustained release 0.19 dL / g PVA 0.1% 0.26% 16.9 um (1.46) Drug: 3.75 mg (10%) Polymer: 33.75mg(90%) Formulation 1-4 Sustained release 0.18dL / gPVA 0.1%0.27%15.8um(1.45)Drug: 3.75mg(10%)Polymer: 33.75mg(90%) Formulation 1-5 Sustained release 0.16dL / gPVA 0.1%0.25%16.2um(1.44)Drug: 3.75mg(10%)Polymer: 33.75mg(90%)

[0092]

[0093] As can be seen in Table 1, there were no significant differences in residual solvent, average particle size, drug and polymer content in microspheres, and residual solvent in all experimental groups.

[0094] The above results indicate that when biodegradable polymers are used with the same composition, formulations having equivalent release patterns and quality can be manufactured even when their intrinsic viscosity varies by up to 0.04 dL / g within the manufacturer's specification range. Therefore, in the manufacturing examples of the present invention below, RG752H having an intrinsic viscosity of 0.20 dL / g or 0.21 dL / g was used for manufacturing microspheres.

[0095]

[0096] 1.2 Manufacturing Examples 1 to 6 Manufacturing Method

[0097] A spray solution was prepared by dissolving leuprorelin acetate (Anigen Co., Ltd., Republic of Korea), D,L-lactic acid, and PLGA (RG752H, intrinsic viscosity 0.20 dL / g or 0.21 dL / g) in glacial acetic acid. At this time, the drug was loaded into the microspheres at approximately 10% (w / w).

[0098] More specifically, a spray solution was prepared by dissolving leuprorelin acetate and PLGA in glacial acetic acid at a weight ratio of about 9 to 10 times that of the mixture, and leuprorelin acetate and PLGA were dissolved in glacial acetic acid at a weight ratio of 1:9 to 9.5. The prepared spray solution was sprayed into a drying chamber through a 60 kHz ultrasonic nozzle and then spray-dried at 135±5°C to prepare PLGA microspheres encapsulating the main ingredient.

[0099] To remove residual solvent within the microspheres after spray drying, the manufactured PLGA microspheres were dispersed in a 0.1–1.0 (w / v)% polyvinyl alcohol (PVA) solution dissolved in water for injection at 1,500 rpm for 20 minutes, and then filtered. Afterwards, the microspheres were washed again with water for injection and filtered to remove PVA and any glacial acetic acid remaining within the microspheres. The mannitol solution and the washed microspheres were suspended in water for injection, freeze-dried, and the dried microspheres were crushed through a 250 μm mesh sieve.

[0100] During the above process of suspending the washed microspheres, leuprorelin acetate was additionally dissolved in a mannitol solution at 5% (w / w) or less of the total active ingredient content (target dose), followed by lyophilization and crushing to produce a final formulation in which leuprorelin acetate was filled on the outside of the microspheres in addition to the active ingredient contained in the sustained-release microspheres.

[0101] Alternatively, a final formulation was prepared in which leuprorelin acetate was added immediately after removing PVA and residual glacial acetic acid from the microspheres and freeze-dried and crushed together, or by separately adding lyophilized leuprorelin acetate to the microspheres after freeze-drying and crushing the same, thereby filling the exterior of the microspheres with 5% (w / w) or less of leuprorelin acetate relative to the total active ingredient content.

[0102] The microspheres were manufactured with an average particle size of 20±3㎛ by setting the size of D10 to 7.0㎛ - 10.0㎛, D50 to 18.0㎛ - 23.5㎛, and D90 to 33.5㎛ - 45.5㎛.

[0103]

[0104] 1.3 Comparative Manufacturing Examples 1 and 2 Manufacturing Method

[0105] A spray solution was prepared by dissolving leuprorelin acetate (PPL, Sweden), D,L-lactic acid, and PLGA (RG752H, intrinsic viscosity 0.21 dL / g) in glacial acetic acid. At this time, the drug was loaded at 10% (w / w) in the microspheres. The prepared spray solution was sprayed into a drying chamber through an ultrasonic nozzle and then spray-dried at 135±5℃ to prepare PLGA microspheres loaded with the main ingredient. After spray-drying, the prepared PLGA microspheres were dispersed in distilled water (Formulation 7, Comparative Manufacturing Example 1) or 0.01 (w / v)% PVA solution (Formulation 8, Comparative Manufacturing Example 2) to remove the residual solvent in the microspheres, washed and filtered with water for injection to remove the residual PVA and glacial acetic acid, and lyophilized to prepare the final formulation of microspheres containing leuprorelin acetate.

[0106]

[0107] Manufacturing Examples 1 to 6 (Formulations 1 to 6) and Comparative Manufacturing Examples 1 to 2 (Formulations 7 to 8) manufactured by the above method are shown in Table 2.

[0108] Classification Slow-release main ingredient RG752H Intrinsic viscosity dispersion process solution PVA concentration Formulation 1 (Manufacturing example 1) Slow-release internal 3.638 mg external 0.180 mg 0.20 dL / g PVA 0.1% Formulation 2 (Manufacturing example 2) Slow-release internal 3.675 mg external 0.075 mg 0.21 dL / g PVA 0.1% Formulation 3 (Manufacturing example 3) Slow-release internal 3.675 mg external 0.075 mg 0.21 dL / g PVA 1.0% Formulation 4 (Manufacturing example 4) Slow-release internal 3.675 mg external 0.075 mg 0.21 dL / g PVA 0.1% Formulation 5 (Manufacturing example 5) Slow-release internal 3.675 mg external 0.176mg0.21dL / gPVA 0.1% Formulation 6 (Manufacturing Example 6) Sustained-release internal 3.675mg external 0.278mg0.21dL / gPVA 0.1% Formulation 7 (Comparative Manufacturing Example 1) Sustained-release internal 3.75mg0.21dL / gDW Formulation 8 (Comparative Manufacturing Example 2) Sustained-release internal 3.75mg0.21dL / gPVA 0.01%

[0109] Example 2. Particle size measurement of PLGA microspheres loaded with leuprorelin acetate

[0110] The most important factor determining the particle size is the nozzle frequency. Generally, the relationship between the droplet diameter and frequency of a spray follows the Lang formula (Equation 2).

[0111] [Formula 2]

[0112]

[0113] (T = Surface tension, ρ= Solution density, fa = Nozzle frequency)

[0114]

[0115] Through the above formula, the theoretical size of the microspheres according to the nozzle frequency was expected to be 35.7㎛ at 25KHz, 19.9㎛ at 60KHz, and 12.5㎛ at 120KHz. In order to manufacture microspheres with a particle size of 20㎛ based on the average particle size, an experiment was conducted by selecting a frequency of 60kHz.

[0116] The results of measuring the particle size of Formulation 1 (Manufacturing Example 1) among the microspheres produced by Peptron Co., Ltd. using an ultrasonic nozzle with a frequency of 60 kHz are shown in Table 3.

[0117] no.Diameter (㎛)Span valueD 10 D 50 D 90 Mean formulation 18.3220.2639.2222.101.538.3120.1738.8521.961.518.2920.5140.1022.501.55

[0118]

[0119] As shown in Table 3, the PLGA microspheres loaded with leuprorelin acetate, produced by setting the nozzle frequency to 60 kHz, were confirmed to have an average particle size of the early 20 ㎛ range. This is a smaller particle size than the sustained-release microspheres, which are typically suitable for 23-25G needles, and has the advantage of allowing subcutaneous injection using a 26G needle.

[0120]

[0121] Example 3. Removal of polyvinyl alcohol (PVA) used in the dispersion process and measurement of residual amount

[0122] The manufacturing method and sustained-release microsphere formulation of the present invention have the advantage of higher safety compared to the manufacturing process using class 2 methylene chloride by using class 3 glacial acetic acid, a low-toxicity solvent. Furthermore, the present invention includes a process of dispersing PLGA microspheres loaded with a main ingredient in a 0.1 to 1.0% (1,000 ppm to 10,000 ppm) PVA aqueous solution, stirring and filtering to obtain microspheres, and then washing and filtering the same with water for injection (WFI) in order to minimize the amount of solvent residue remaining in the finished pharmaceutical product. This process may be repeated several times as necessary.

[0123] In order to confirm whether the organic solvent and PVA were manufactured at a sufficiently low residual level through the above manufacturing process, a residue measurement experiment was performed. The test solution for measuring the PVA residual amount was prepared by dissolving the sample in 0.5 N sodium hydroxide and then neutralizing it with 1 N hydrochloric acid. The HPLC analysis conditions were as follows: a solution of sodium nitrate dissolved in acetonitrile as the mobile phase, a TSKgel G2500SWXL (7.8 * 300 mm, 5 μm) column, an RID detector, a column temperature of 37 °C, and a flow rate of 1 mL / min.

[0124] The results of measuring the residual PVA in formulations 1 to 4 manufactured by the method of the present invention by varying the PVA concentration of the dispersion process solution from 0.1 to 1.0% are shown in Table 4.

[0125] The amount of PVA remaining in the final formulation is on average less than 10 ppm, which indicates that more than 99% of the PVA used in the manufacturing process has been removed.

[0126]

[0127] Additionally, as confirmed in Table 4, it was confirmed that the organic solvent (residual solvent) remaining in the final formulation was removed by more than 99.5%. In particular, in the case of Formulation 2, the residual solvent was measured to be undetectable.

[0128] This means that formulations 1 to 4 of the present invention show a residual amount of 0.5% (w / w) or less than 5,000 ppm, which is the management level recommended by the ICH Q3C (R8) guideline.

[0129] Dispersion process solution PVA concentrationPVA residual amount measurement resultResidual solvent (acetic acid)Formulation 1PVA 0.1%9.38±0.24 ppm0.41%Formulation 2PVA 0.1%9.19±0.55 ppmN / AFormulation 3PVA 1.0%9.20±0.45 ppm0.31%Formulation 4PVA 0.1%9.38±0.34 ppm0.42%

[0130] In summary of the above results, compared to an example in which a maximum daily dose of 0.119 mg was used in a local sustained-release implant, the PVA residual amount of the sustained-release formulation of the present invention shows a significantly low value of about 10 ppm, and the residual solvent exists at less than 0.5%, so it can be seen that a safe formulation can be manufactured without toxicity issues due to residual PVA or residual solvent in the finished drug product.

[0131]

[0132] Example 4. Confirmation of the effect of Sokseobang

[0133] 4-1. Initial release evaluation (in vivo)

[0134] The sustained-release formulations 3 and 4 manufactured by the method of the present invention and the sustained-release formulations 7 and 8 that do not additionally contain leuprorelin acetate outside the microspheres were administered to rats and the initial release ability was confirmed. Twenty 10-week-old Sprauge-Dawley specific pathogen-free (SPF) rats were randomly divided into each experimental group (n=5) and administered subcutaneously as a single dose of 3 mg / kg. Thereafter, serum was obtained from each rat through jugular vein blood collection at 0.5, 1, 3, 6, and 24 hours and 2, 4, 7, 14, 21, and 28 days, stored at -80℃, and then the AUC of each experimental group was measured through LC-MS / MS analysis. C max , T max The results of confirming the AUC values ​​are shown in Table 5, and the results of measuring the serum concentration over time are shown in Figure 2.

[0135] Formulation 3 Formulation 4 Formulation 7 Formulation 8C max(ng / mL)80.1 ± 20.176.5 ± 14.249.54 ± 11.6347.02 ± 10.42T max (hr)0.6 ± 0.20.6 ± 0.21.00 ± 0.001.00 ± 0.00AUC 7-t (ng*day / ml)16.2 ± 6.418.0 ± 3.825.03 ± 3.8120.21 ± 1.99AUC 0-t (ng*day / ml)44.9 ± 11.551.8 ± 5.550.20 ± 5.1345.95 ± 3.56AUC INF (ng*day / ml)49.4 ± 13.255.1 ± 5.355.35 ± 5.7456.72 ± 9.33

[0136] - AUC 7-t : AUC-AUC from day 7 to the last sampling time 0-t : AUC from day 0 to the last sampling point

[0137] - AUC INF : AUC from day 0 to the point where the concentration becomes 0 (the point estimated by drawing a trend line)

[0138]

[0139] As can be seen in Table 5 and Figure 2, Formulations 3 and 4, which are sustained-release formulations containing 2% of the active ingredient outside the microspheres relative to the active ingredient content inside the microspheres, have higher C than sustained-release formulations 7 and 8. max The initial release within 24 hours was improved by about 35 to 40% or more. This result shows that the sustained-release formulation of the present invention can effectively improve the low initial release amount of existing sustained-release formulations.

[0140]

[0141] 4-2. In vivo pharmacokinetics and stability evaluation

[0142] Healthy postmenopausal women were administered 3.75 mg of leuprorelin acetate by a single subcutaneous injection of Formulation 1 and Formulation 2 of the present invention into the abdomen, and the pharmacokinetic characteristics and safety were confirmed. The pharmacokinetic evaluation group of Formulation 1 consisted of 36 subjects, and the pharmacokinetic evaluation group of Formulation 2 consisted of 38 subjects. The pharmacokinetic blood sampling time points were 0 (before administration), 0.5, 1, 1.5, 2, 4, 8, 12, 24, 72, 168, 240, 336, 504, 672, 840, and 1008 hr, for a total of 17 times.

[0143] In particular, as confirmed in Example 4-1, C max Since C is a parameter related to the initial release amount, Formulation 1 and Formulation 2 containing 4.8% or 2% of leuprorelin acetate, respectively, relative to the total leuprorelin acetate content on the outside of the microspheres were used to confirm the increase in initial release and bioavailability according to the content (%) of leuprorelin acetate added externally. max were compared and analyzed. C confirmed in Formulation 1 and Formulation 2 max And the AUC values ​​are shown in Table 6 below.

[0144]

[0145] Formulation 1 (n=36) Formulation 2 (n=38) C max (ng / mL)8096.6945712.325AUC 7-t (ng*day / ml)77663.32077218.699AUC t (ng*day / ml)162948.819142688.146

[0146] As can be seen in Table 6, Formulation 1, which contains 4.8% of the immediate-release active ingredient relative to the total active ingredient content outside the microparticles, has a C that is about 30% higher compared to Formulation 2, which contains 2% of the immediate-release active ingredient. max The value was indicated.

[0147] These results demonstrate that formulations containing less than 8% (w / w) of leuprorelin acetate, preferably 4 to 6% (w / w) of the total active ingredient content outside the microspheres, exhibit effective initial release and C in animals and humans. max It was confirmed that an increase in value can be induced. That is, by using the formulation of the present invention in which only less than 8% of the total active ingredient of leuprorelin acetate is included in the exterior of the sustained-release microspheres and the remaining leuprorelin acetate is encapsulated in the sustained-release microspheres, it is possible to manufacture a sustained-release microsphere preparation that overcomes the initial delayed release and exhibits excellent bioavailability and an effective therapeutic effect.

[0148]

[0149] Additionally, an in vivo safety evaluation of the formulation of the present invention was simultaneously performed, and as a result, no clinically significant adverse reactions of special interest or local adverse reactions were reported during the clinical trial, and no adverse reactions with clinically significant differences in frequency or pattern of occurrence between administration groups were identified in this clinical trial. Except for some cases collected as mild adverse reactions, no clinically significant changes were found in vital signs, clinical laboratory tests, physical examinations, 12-lead electrocardiograms, and BMD tests performed after administration of the investigational drug during the clinical trial period.

[0150]

[0151] In conclusion, it was determined that there were no clinically significant differences between the administration groups in safety evaluations including adverse reactions, local adverse reactions, concomitant medications, vital signs, physical examinations, clinical laboratory tests, 12-lead electrocardiograms, and BMD tests.

Claims

1. 1) A step of preparing a spray solution by dissolving leuprorelin or a salt thereof and a biodegradable polymer in an organic solvent; 2) a step of spray-drying the spray solution to produce microparticles encapsulating leuprorelin or a salt thereof; and 3) a step of dispersing and washing the microspheres in a polyvinyl alcohol aqueous solution to produce microspheres from which residual solvent has been removed; including; A method for producing a sustained-release formulation with increased initial release, characterized in that it further comprises a step of adding a lyophilized powder of leuprorelin or a salt thereof to the outside of the microspheres.

2. In paragraph 1, the addition 4-1) A step of suspending the microspheres manufactured in step 3) in water for injection and water-soluble polyhydric alcohol, and then freeze-drying and crushing them to manufacture microspheres; and 4-2) A method for producing a sustained-release formulation with increased initial release, comprising: a step of filling lyophilized powder of leuprorelin or a salt thereof into the microparticles of step 4-1); 3. In paragraph 1, the addition A method for producing a sustained-release formulation with increased initial release, comprising: a step of suspending the microspheres manufactured in step 3) above in water for injection containing leuprorelin or a salt thereof and a water-soluble polyhydric alcohol dissolved therein, and then freeze-drying and crushing the suspension; 4. A method for producing a sustained-release formulation with increased initial release, wherein, in paragraph 1, leuprorelin or a salt thereof added externally is added in an amount of 2 to 8% (w / w) based on the total weight of the active ingredient.

5. A method for producing a sustained-release formulation with increased initial release in the first paragraph, wherein the organic solvent is glacial acetic acid or acetic acid.

6. A method for producing a sustained-release formulation with increased initial release amount in the first paragraph, wherein the sustained-release formulation has 99% or more of the residual solvent removed.

7. A method for producing a sustained-release formulation with increased initial release in the second paragraph, wherein the water-soluble polyhydric alcohol is mannitol, sorbitol or dulcitol.

8. A method for producing a sustained-release formulation with increased initial release in the first paragraph, wherein the biodegradable polymer is at least one selected from the group consisting of polylactide, polyglycolide, polylactide-co-glycolide, polyorthoester, polyanhydride, polyhydroxybutyric acid, polycaprolactone, and polyalkyl carbonate.

9. A method for producing a sustained-release formulation with increased initial release in the first paragraph, wherein the sustained-release formulation with increased initial release has an average particle size of 15 to 25 μm.

10. A sustained-release preparation having an increased initial release amount manufactured by the manufacturing method of any one of claims 1 to 9.

11. Sustained-release microspheres containing leuprorelin or a salt thereof and a biodegradable polymer; and An extended-release formulation having an increased initial release amount, comprising leuprorelin or a salt thereof outside the extended-release microparticles.

12. A sustained-release formulation with increased initial release in claim 11, wherein leuprorelin or a salt thereof contained in the exterior of the microparticles is contained in an amount of 2 to 8% (w / w) of the total weight of the active ingredient.

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