Sustained release formulation with increased initial release and method of making same

Abstract

The sustained-release formulation of this invention, with its increased initial release, exhibits a rapid release effect within 24 hours after administration, followed by a sustained-release effect, thus simultaneously achieving both prolonged efficacy and rapid initial release. The sustained-release formulation of this invention, containing microspheres of leuprorelin, possesses excellent bioavailability and has been proven safe in humans; therefore, it 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.

Description

Technical Field

[0001] This invention relates to a leuprolide sustained-release formulation with both immediate and sustained-release properties and an increased initial release amount, as well as its preparation method. Background Technology

[0002] 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 induce pituitary receptor desensitization to LHRH stimulation by depleting LHRH receptors in the pituitary gland, thereby inhibiting LH secretion. Furthermore, LHRH agonists and antagonists have shown efficacy in treating endometriosis, fibroids, polycystic ovary syndrome, breast cancer, ovarian cancer, and endometrial cancer in women; gonadotropin-induced pituitary desensitization during assisted reproductive technologies; benign prostatic hyperplasia and prostate cancer in men; and precocious puberty in both men and women.

[0003] Leuprorelin acetate, a representative LHRH agonist, has a short half-life when administered subcutaneously or intramuscularly, resulting in a rapid decrease in blood concentration that disappears within hours. This means that despite being administered as an injectable drug, daily dosing is still necessary to maintain effectiveness, causing inconvenience and discomfort for patients.

[0004] As a way to minimize the inconvenience caused by the frequent dosing as described above and increase patient medication adherence, research is being conducted on applying drugs to injectable drug delivery systems to enable sustained drug release over a long period of time. In particular, sustained-release formulations in the form of microspheres prepared using biodegradable polymers have been developed.

[0005] However, although leuprorelin acetate needs to release sufficient drug at the beginning of administration in order to exhibit pharmacological effects, the sustained-release formulations described above only achieve the goal of delayed release over a long period of time, and have the disadvantage of not being able to release the drug at a high concentration at the beginning.

[0006] As mentioned above, the previously developed sustained-release formulations of leuprorelin have limitations in exhibiting sufficient pharmacological effects. Therefore, there is a need for a novel formulation that can simultaneously achieve both long-lasting efficacy and rapid initial release. Summary of the Invention

[0007] Technical issues In the process of researching novel formulations that overcome the initial release delay of sustained-release leuprolide microspheres and subsequently exhibit sustained-release properties, the inventors confirmed that adding a small amount of leuprolide or its salt to the outside of sustained-release microspheres encapsulated with leuprolide or its salt can simultaneously achieve effective initial release and sustained-release effects, thus completing the present invention.

[0008] Therefore, the purpose of this invention is to provide a leuprorelin sustained-release formulation with increased initial release and a method for preparing the same.

[0009] Technical solution To achieve the aforementioned objective, the present invention provides a method for preparing a sustained-release formulation with increased initial release, the method comprising the following steps: Step 1), preparing a spray by dissolving leuprorelin or its salt and a biodegradable polymer in an organic solvent; Step 2), preparing microspheres encapsulated with leuprorelin or its salt by spray drying the spray; and Step 3), preparing microspheres with residual solvent removed by dispersing the microspheres in an aqueous polyvinyl alcohol solution and washing them, further comprising the step of adding lyophilized powder of leuprorelin or its salt to the exterior of the microspheres.

[0010] Furthermore, the present invention provides a sustained-release formulation with increased initial release amount prepared by the aforementioned preparation method.

[0011] Furthermore, the present invention provides a sustained-release formulation with increased initial release, comprising: sustained-release microspheres encapsulated with leuprorelin or a salt thereof, comprising leuprorelin or a salt thereof and a biodegradable polymer; and leuprorelin or a salt thereof externally contained in the sustained-release microspheres.

[0012] The effects of the invention The sustained-release formulation of this invention, with its increased initial release, exhibits a rapid release effect within 24 hours after administration, followed by a sustained-release effect, thus simultaneously achieving both prolonged efficacy and rapid initial release. The sustained-release formulation of this invention, containing microspheres of leuprorelin, possesses excellent bioavailability and has been proven safe in humans; therefore, it 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. Attached Figure Description

[0013] Figure 1A graph showing the results of confirming the differences in release rates based on the differences in the intrinsic viscosity of the poly(lactide-co-glycolide) (PLGA) used in the microsphere preparation process.

[0014] Figure 2 The diagram illustrates the serum drug concentrations (inset) over 0.5 days or 1 day and 28 days after administration of microspheres containing a small amount of leuprolide acetate (immediate-release type) prepared by the preparation method of the present invention, for dosage forms 3 and 4, and dosage forms 7 and 8, which are comparative preparation examples and do not contain immediate-release leuprolide. Detailed Implementation

[0015] This invention relates to a sustained-release formulation exhibiting both immediate-release and sustained-release characteristics with an increased initial release amount, and a method for preparing the same.

[0016] The sustained-release formulation of the present invention, with its increased initial release, can induce rapid release of leuprorelin, a drug crucial for initial release, within 24 hours, followed by sustained release of the drug. Therefore, it has the advantage of simultaneously improving patient medication adherence and drug efficacy.

[0017] This invention provides a method for preparing a sustained-release formulation with increased initial release, characterized by comprising the following steps: Step 1), preparing a spray by dissolving leuprorelin or its salt and a biodegradable polymer in an organic solvent; Step 2), preparing microspheres encapsulated with leuprorelin or its salt by spray drying the spray; and Step 3), preparing microspheres with residual solvent removed by dispersing the microspheres in an aqueous polyvinyl alcohol solution and washing them, further comprising the step of adding lyophilized powder of leuprorelin or its salt to the outside of the microspheres.

[0018] The pharmacologically active ingredient of the present invention, leuprorelin or its salt, is characterized in that it is contained both as a sustained-release formulation within microspheres and on the outside of the microspheres to achieve initial rapid release.

[0019] In this invention, step 1) is the step of preparing a spray liquid by dissolving leuprorelin or its salt and a biodegradable polymer in an organic solvent.

[0020] Leuprorelin can be used interchangeably with leuprolide and can be represented by the following chemical formula 1.

[0021] Chemical Formula 1 In this invention, the salt of leuprolide can be unlimited to include all pharmaceutically acceptable salts, such as acid addition salts or quaternary ammonium salts. Acid addition salts can include all acid addition salts formed from free acids, such as free acid-derived addition salts of 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, methanesulfonic 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 leuprolide can be leuprolide acetate.

[0022] In this invention, the biodegradable polymer is a polymer that slowly decomposes and is harmless to the human body when administered in vivo. It can be one or more selected from the group consisting of polylactic acid, polyglycolic acid, polylactic-co-glycolic acid copolymer, polyorthoester, polyanhydride, polyhydroxybutyric acid, polycaprolactone, and polyalkyl carbonate. Preferably, polylactic-co-glycolic acid copolymer can be used. When preparing sustained-release microspheres using the method of this invention, PLGA with an intrinsic viscosity of 0.10 dl / g to 1.0 dl / g can be used, preferably 0.14 dl / g to 0.22 dl / g. The molar ratio of lactide to glycolide in the PLGA can be 50 to 100:50 to 0, 60 to 90:40 to 10, or 70 to 80:30 to 20, preferably 75:25. For example, in preparing the sustained-release microspheres of the present invention, PLGA with a molar ratio of lactide to glycolide of 75:25 and an intrinsic viscosity of 0.14 dl / g to 0.22 dl / g can be used.

[0023] Since it was confirmed that even when their intrinsic viscosity is changed to 0.14 dL / g to 0.22 dL / g, the same level of release (dissolution) rate can be maintained, polylactic acid-glycolic acid copolymers with intrinsic viscosity of 0.14 dL / g to 0.22 dL / g can be used.

[0024] The content of the biodegradable polymer relative to the total weight of the microspheres can be from 70% to 95% by weight, more preferably from 80% to 95% by weight, and most preferably from 85% to 95% by weight.

[0025] The organic solvents used can be selected without restriction from those capable of dissolving biodegradable polymers and leuprolide or its salts, and can be selected according to the type of biodegradable polymer. Class 3 and 4 solvents specified in the ICH guidelines can be selected to use low-toxicity solvents.

[0026] In one embodiment of the present invention, glacial acetic acid, i.e., acetic acid, is selected and used as the organic solvent, which provides a safer formulation compared to using class 2 dichloromethane as the solvent. Therefore, the organic solvent of the present invention can be acetic acid or glacial acetic acid, preferably glacial acetic acid. The glacial acetic acid refers to acetic acid with a purity of 99.5% or higher.

[0027] In step 1) of the present invention, the spray liquid is prepared by dissolving leuprorelin or its salt and biodegradable polymer in an organic solvent.

[0028] Microspheres containing leuprorelin or its salts can be prepared using methods known in the art for preparing sustained-release injections. These methods include various well-known techniques such as coagulation, 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, the preparation of microspheres using single and double emulsion evaporation methods, like phase separation methods, suffers from the following problems: difficulty in removing the organic solvent used to dissolve the biodegradable polymer; process difficulties due to variations in solvent removal rates during large-scale production; allergic reactions caused by gelatin used to increase the viscosity of the first emulsion; the possibility of drug denaturation and loss of activity due to the strong shear force applied during the preparation of the first emulsion to form the microsphere phase; and limitations in drug encapsulation efficiency.

[0029] Therefore, in this invention, preferably, microspheres encapsulated with drugs can be prepared by spray drying, a method known in the art for preparing sustained-release injections. Step 2) of this invention is the step of preparing microspheres encapsulated with leuprorelin or its salt by spray drying the spray liquid.

[0030] 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 during spraying. The appropriate supply rate, temperature, time, and supply volume can be adjusted according to the type of biodegradable polymer used, the content within the microspheres, the content of leuprorelin or its salt, and the solvent content. More preferably, the spray drying can be performed using a spray dryer equipped with an ultrasonic nozzle, the spray liquid can be delivered at a flow rate of 1 ml to 10 ml per minute, and the spray liquid can be sprayed into the drying chamber from the top of the sprayer through the ultrasonic nozzle. The temperature during spray drying can be 100°C to 150°C, 120°C to 145°C, preferably 130°C to 140°C, and microspheres encapsulated with leuprorelin or its salt can be prepared by the spray drying steps described above.

[0031] The spray drying step of the present invention can be performed by adjusting the nozzle frequency according to the target microsphere particle size. Preferably, an ultrasonic nozzle with a nozzle frequency of 40 kHz to 80 kHz or 50 kHz to 70 kHz can be used. If an ultrasonic nozzle with the nozzle frequency described above is used, a sustained-release formulation with an increased initial release amount and an average particle size of 15 μm to 25 μm can be prepared.

[0032] As mentioned above, the average particle size is smaller than that of sustained-release microspheres suitable for conventional 23-25G needles, and it can be administered subcutaneously using a 26G injection needle, thus having the advantage of improving patient medication adherence.

[0033] Step 3) of the present invention is a step of preparing microspheres with residual solvent removed by dispersing the prepared microspheres in an aqueous polyvinyl alcohol solution and washing them. Through the dispersion process in step 3), residual solvent in the microspheres prepared immediately after spray drying can be removed, while allowing them to be more effectively dispersed in the injection solution.

[0034] In this case, the concentration of the polyvinyl alcohol aqueous solution can be from 0.05 (w / v)% to 2.0 (w / v)%, preferably from 0.05 (w / v)% to 1.0 (w / v)% or from 0.1 (w / v)% to 0.5 (w / v)%, and more preferably from 0.05 (w / v)% to 0.2 (w / v)%.

[0035] The molecular weight of the polyvinyl alcohol used can be from 3,000 Da to 300,000 Da, more preferably from 5,000 Da to 100,000 Da, but is not limited thereto.

[0036] The dispersion step of the present invention can be carried out by stirring the polyvinyl alcohol aqueous solution and microspheres using a mixer. In this case, the stirring can be carried out at room temperature at 1000 rpm to 3000 rpm for 1 minute to 5 hours, preferably for 5 minutes to 3 hours, and more preferably for 10 minutes to 2 hours.

[0037] Through the dispersion and washing steps described above, residual solvents can be removed and microspheres with improved injection performance can be prepared. Preferably, a final dosage form that meets the management standards recommended by ICH Q3C(R8) guidelines, i.e., a residual amount of 0.5% (w / w) or less than 5000 ppm, can be produced. More preferably, the sustained-release formulation of the present invention prepared through the dispersion and washing steps is characterized by the removal of more than 99% of the residual solvent, more preferably, more than 99.5% of the residual solvent, but is not limited thereto.

[0038] Furthermore, the present invention provides a preparation method, characterized in that it further includes the step of adding lyophilized powder of leuprorelin or its salt to the outside of the microspheres.

[0039] The step described is for effectively increasing the initial release of leuprorelin or its salt by adding leuprorelin or its salt to the exterior of the microspheres encapsulated with leuprorelin or its salt prepared by steps 1) to 3), so that the leuprorelin or its salt contained on the exterior can be released initially.

[0040] More specifically, the addition may include the following steps: Step 4-1), suspending the microspheres prepared in Step 3) in water for injection and water-soluble polyol, then lyophilizing and pulverizing to prepare microspheres; and Step 4-2), filling the microspheres in Step 4-1) with lyophilized powder of leuprorelin or its salt.

[0041] Alternatively, the addition may include suspending the microspheres prepared in step 3) in water for injection containing leuprorelin or its salt and a water-soluble polyol, and then lyophilizing and pulverizing the suspension.

[0042] That is, the process of adding leuprorelin or its salt to the outside of microspheres encapsulated with leuprorelin or its salt, and adding lyophilized leuprorelin or its salt pulverized or powdered material, can be used without restriction. This can be done by lyophilizing leuprorelin or its salt separately, pulverizing it, and filling it into microspheres, or by resuspending the microspheres and leuprorelin or its salt in water for injection, and then lyophilizing, pulverizing, and adding them together.

[0043] In this case, water-soluble polyols can be used for the freeze-drying process. These water-soluble polyols can be mannitol, sorbitol, or galactitol.

[0044] Relative to the total weight of the active ingredient, i.e., leuprorelin or its salt (sustained-release) contained in the sustained-release microspheres, the amount of leuprorelin or its salt added to the outside of the microspheres for immediate release can be 2% (w / w) to 8% (w / w), preferably 4% (w / w) to 6% (w / w). If the amount exceeds this range, it will not only induce excessive immediate release but also reduce persistence, making it difficult to achieve efficacy within the target time. If the amount added is less than this range, it will be difficult to achieve a rapid increase in blood drug concentration within the initial 24 hours. As a preferred example, 92% (w / w) to 98% (w / w) of the total active ingredient, leuprorelin or its salt, can be provided in the form of encapsulation within the microspheres, and 2% (w / w) to 8% (w / w) of leuprorelin or its salt can be provided outside the microspheres in the form of a separate powder or additional encapsulation.

[0045] The preparation method described above can be used to prepare sustained-release formulations with improved bioavailability and increased initial release, characterized by a high initial dissolution rate within 24 hours and a drug duration of approximately 30 to 65 days. As a preferred example, in vitro ( in vitro ) or in the body ( in vivo The initial release rate within 24 hours can be less than 10%, less than 8%, or less than 6%.

[0046] In this invention, "increased initial burst" refers to the increase in C compared to sustained-release leuprorelin microspheres without the addition of leuprorelin or its salts to the exterior of the microspheres. max The value increases by approximately 30% to over 50%, and it exhibits a high initial release rate in sustained-release formulations within 24 hours.

[0047] Without hindering the purpose and effect of the present invention, in addition to the processes described in steps 1) to 4), the additional processes required for the preparation and recovery of microspheres encapsulated with leuprorelin or its salts, such as redispersion, mixing, recovery, stirring, heating, and washing steps, can be carried out using processes known to those skilled in the art.

[0048] The present invention provides a sustained-release formulation with increased initial release, comprising: sustained-release microspheres encapsulated with leuprorelin or a salt thereof, comprising leuprorelin or a salt thereof and a biodegradable polymer; and leuprorelin or a salt thereof externally contained in the sustained-release microspheres.

[0049] The sustained-release formulation with increased initial release can be prepared using the method described above for preparing sustained-release formulations with increased initial release.

[0050] The content of leuprorelin or its salt contained in the outer surface of the sustained-release microspheres may be 2% (w / w) to 8% (w / w) of the total weight of the active ingredient, preferably 4% (w / w) to 6% (w / w).

[0051] In the sustained-release formulation, leuprorelin or its salt may exist in two forms, including encapsulated in sustained-release microspheres and existing on the outside of the microspheres, thereby achieving both initial immediate release and sustained release simultaneously.

[0052] The sustained-release formulation of the present invention is preferably an injectable formulation, and more particularly, preferably, can be provided in the form of a pre-filled syringe. Providing it in the form of a pre-filled syringe increases the convenience of the suspension preparation process and reduces the risk of contamination by minimizing external contact.

[0053] When the sustained-release formulation of the present invention is an injection, it can be administered via various routes such as subcutaneous, intradermal, intravenous, intramuscular, or intraperitoneal.

[0054] The sustained-release formulation of the present invention comprising microspheres containing leuprorelin or its salts has excellent bioavailability and has been proven safe in humans. It contains 2% (w / w) to 8% (w / w) of the microspheres relative to the total weight of the active ingredient and exhibits effective in vivo characteristics. Therefore, it can be used alone or in combination with other known treatments in the treatment of various luteinizing hormone-releasing hormone-related diseases such as endometriosis, uterine fibroids, prostate cancer, premenopausal breast cancer, and central precocious puberty.

[0055] Unless otherwise stated, the values ​​described in this specification shall be interpreted as including equivalent ranges.

[0056] The following preferred embodiments are presented to aid in understanding the present invention. However, the embodiments described below are provided merely to facilitate a clearer understanding of the invention, and the scope of the invention is not limited to these embodiments.

[0057] Embodiments of the present invention Example 1. Preparation method of leuprolide acetate rapid-release formulation 1-1. Confirm the release effect based on the inherent viscosity difference of PLGA In this invention, PLGA (Evonik, Resomer® 752H, with an intrinsic viscosity of 0.14-0.22 dL / g, hereinafter referred to as RG752H) was used as the biodegradable polymer. Since the intrinsic viscosity of RG752H was controlled within the range of 0.14 dL / g to 0.22 dL / g, experiments were conducted to confirm whether the drug release effect changed with the intrinsic viscosity of the biodegradable polymer used. Microspheres were prepared using leuprorelin acetate (PPL, Sweden) (dosage form 1-1) or (ANYGEN CO., LTD., Korea) (dosage forms 1-2 to 1-5) and D,L-lactic acid, with the intrinsic viscosity of PLGA changed to 0.16 dL / g (dosage form 1-5), 0.18 dL / g (dosage form 1-4), 0.19 dL / g (dosage form 1-3), and 0.20 dL / g (dosage forms 1-2, 1-1). A spray solution was prepared by dissolving the component in glacial acetic acid. The prepared spray solution was then sprayed into a drying chamber through an ultrasonic nozzle, and spray-dried at 135±5℃ to prepare PLGA microspheres encapsulated with the main component. To remove residual solvent from the microspheres after spray drying, the prepared 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.

[0058] The dissolution modes with different intrinsic viscosities used in the prepared dosage forms were evaluated. Accelerated release tests were conducted under the following experimental conditions, and the samples collected during the release tests were analyzed by HPLC. The results are presented below. Figure 1 middle.

[0059] -Release Tester: Rotary type release device (Equipment No.: LDT-PT-008) - Release temperature: 50℃ - Speed: 20 rpm - Sampling: 1 hour, 4 hours, 6 hours, 24 hours - Release solution: 10mM lactate buffer, pH 3.2 (0.4% PVA + 0.1% Tween 80) At all release (dissolution) rate comparison time points, the average release (dissolution) rate difference between the reference and comparison dosage forms is within ±15%. Using all release (dissolution) rate measurement time points as comparison points, if the similarity factor (f2) is greater than 50, it is considered equivalent. The similarity factor (f2) is calculated using the formula shown below.

[0060] Formula 1 -n: Number of time points used to determine release (dissolution) (%) -Rt: Release (dissolution) rate (%) of the reference formulation -Tt: Release (dissolution) rate (%) of the comparison dosage form like Figure 1 As shown, when microspheres (dosage forms 1-5) prepared using RG752H with an intrinsic viscosity of 0.16 dL / g were used as reference dosage forms, it was confirmed that the differences were within 15% at all release comparison time points when compared with other dosage forms.

[0061] Furthermore, the similarity factor (f2) values ​​ranged from 51 to 82, thus confirming that the release patterns of all experimental groups were equivalent.

[0062] Furthermore, for dosage forms 1-1 to 1-5, the average particle size (span value), content, residual solvent (acetic acid), and moisture content were confirmed.

[0063] Table 1 shows the dosage forms 1-1 to 1-5 prepared by changing the intrinsic viscosity of RG752H and the experimental values ​​measured in the corresponding dosage forms.

[0064] Table 1 As shown in Table 1, it was confirmed that there were no significant differences in residual solvent, average particle size, drug and polymer content in microspheres, and residual solvent among all experimental groups.

[0065] The results indicate that formulations with equivalent release patterns and quality can be prepared by using biodegradable polymers of the same composition, even if their intrinsic viscosity is varied within the range of 0.04 dL / g as specified by the manufacturer. Therefore, in the following preparation examples of the present invention, microspheres were prepared using RG752H with an intrinsic viscosity of 0.20 dL / g or 0.21 dL / g.

[0066] 1-2. Preparation methods of Preparation Examples 1 to 6 A spray solution was prepared by dissolving leuprorelin acetate (ANYGEN CO., LTD., South Korea), D,L-lactic acid, and PLGA (RG752H, intrinsic viscosity 0.20 dL / g or 0.21 dL / g) in glacial acetic acid. Under these conditions, the drug loading within the microspheres was approximately 10% (w / w).

[0067] More specifically, a spray solution was prepared by dissolving leuprolide acetate and PLGA in glacial acetic acid at a weight ratio of approximately 9 to 10 times relative to these mixtures, wherein leuprolide acetate and PLGA were dissolved in glacial acetic acid at a weight ratio of 1:9 to 9.5. The prepared spray solution was then sprayed into a drying chamber through a 60 kHz ultrasonic nozzle and spray-dried at a temperature of 135 ± 5 °C to prepare PLGA microspheres encapsulated with the main component.

[0068] To remove residual solvent from the microspheres after spray drying, the prepared PLGA microspheres were dispersed in a 0.1–1.0 (w / v)% polyvinyl alcohol (PVA) solution dissolved in water for injection at 1500 rpm for 20 minutes and then filtered. The solution was then washed again with water for injection and filtered to remove PVA and residual glacial acetic acid from the microspheres. The mannitol solution and the washed microspheres were then suspended in water for injection, lyophilized, and the dried microspheres were pulverized through a 250 μm mesh.

[0069] In the process of the microspheres after suspension and washing, less than 5% (w / w) of leuprolide acetate is further dissolved in mannitol solution relative to the total active ingredient content (target dose), then lyophilized and pulverized to prepare a final dosage form in which leuprolide acetate is filled into the outside of the microspheres in addition to the active ingredient contained in the sustained-release microspheres.

[0070] Alternatively, a final dosage form can be prepared by removing PVA and residual glacial acetic acid from the microspheres, immediately adding leuprolide acetate and lyophilizing and pulverizing them together, or by adding lyophilized leuprolide acetate alone to the lyophilized microspheres and pulverizing them, thereby filling the outside of the microspheres with less than 5% (w / w) of leuprolide acetate relative to the total content of active ingredients.

[0071] The particle size of the microspheres was set to D10 of 7.0 μm-10.0 μm, D50 of 18.0 μm-23.5 μm, and D90 of 33.5 μm-45.5 μm, resulting in an average particle size of 20 ± 3 μm.

[0072] 1-3. Comparative preparation methods of preparation examples 1 to 2 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. This resulted in a drug loading of 10% (w / w) within the microspheres. The prepared spray solution was then sprayed into a drying chamber through an ultrasonic nozzle and spray-dried at 135 ± 5 °C to prepare PLGA microspheres encapsulated with the main component. To remove residual solvent from the microspheres after spray drying, the prepared PLGA microspheres were dispersed in distilled water (dosage form 7, comparative preparation example 1) or a 0.01% (w / v) PVA solution (dosage form 8, comparative preparation example 2), washed with water for injection, and filtered to remove residual PVA and glacial acetic acid before lyophilization to prepare microspheres containing leuprorelin acetate as the final dosage form.

[0073] Preparation Examples 1 to 6 (dosage forms 1 to 6) and Comparative Preparation Examples 1 to 2 (dosage forms 7 to 8) prepared by the method are shown in Table 2.

[0074] Table 2 Example 2. Determination of particle size of PLGA microspheres encapsulated with leuprolide acetate The most important factor in determining the microsphere size is the nozzle frequency. Typically, the relationship between the droplet diameter of the spray liquid and the frequency follows the Lang equation (Equation 2).

[0075] Formula 2 (T = surface tension, ρ = solution density, fa = nozzle frequency) Based on the above formula, the theoretical size of the microspheres according to the nozzle frequency is predicted to be 35.7 μm at 25 kHz, 19.9 μm at 60 kHz, and 12.5 μm at 120 kHz. In order to prepare microspheres with an average particle size of about 20 μm, a frequency of 60 kHz was selected for the experiment.

[0076] The particle size determination results of microspheres of dosage form 1 (preparation example 1) manufactured by Peptron, Inc. using an ultrasonic nozzle with a frequency of 60 kHz are shown in Table 3.

[0077] Table 3 As shown in Table 3, PLGA microspheres encapsulated with leuprolide acetate produced by setting the nozzle frequency to 60 kHz were confirmed to have an average particle size of 20 μm. This is smaller than the particle size of sustained-release microspheres suitable for conventional 23-25 ​​G needles, and has the advantage of being able to be administered subcutaneously using 26 G injection needles.

[0078] Example 3. Removal of polyvinyl alcohol (PVA) used in the dispersion process and determination of residual amount. The preparation method and sustained-release microsphere dosage form of this invention use glacial acetic acid, a Class 3 solvent with low toxicity, thus offering the advantage of higher safety compared to preparation processes using Class 2 dichloromethane. Furthermore, to minimize residual solvent in the finished drug product, this invention includes the following process: dispersing PLGA microspheres encapsulated with the main component in a 0.1% to 1.0% (1000 ppm to 10000 ppm) PVA aqueous solution, stirring and filtering to obtain microspheres, then washing them with water for injection (WFI) and filtering. This process can be repeated multiple times as needed.

[0079] Residual content determination experiments were performed to confirm whether the organic solvent and PVA were prepared at a sufficiently low residual level using the preparation process described above. The test solution for determining PVA residual content was prepared by adding 0.5N sodium hydroxide to a specified amount of sample to dissolve the sample, followed by neutralization with 1N hydrochloric acid. HPLC analysis conditions were as follows: a solution of sodium nitrate dissolved in acetonitrile was used as the mobile phase; a TSKgel G2500SWXL column (7.8*300mm, 5μm) was used; a RID detector was employed; the column temperature was set to 37℃; and the flow rate was set to 1mL / min.

[0080] The PVA concentration of the dispersion process solution was changed to 0.1% to 1.0%, and the results of PVA residue determination of dosage forms 1 to 4 prepared by the method of the present invention are shown in Table 4.

[0081] The residual PVA in the final dosage form was less than 10 ppm on average, which indicates that more than 99% of the PVA used in the preparation process has been removed.

[0082] Furthermore, as shown in Table 4, it was confirmed that more than 99.5% of the residual organic solvents in the final dosage form had been removed. In particular, in the case of dosage form 2, the residual solvent levels were undetectable.

[0083] This means that in dosage forms 1 to 4 of the present invention, the residual levels meet the management standards recommended by the ICH Q3C(R8) guidelines, namely 0.5% (w / w) or less than 5000 ppm.

[0084] Table 4 In summary, compared with the example of using a local sustained-release implant with a maximum daily dose of 0.119 mg, the rapid-release formulation of the present invention exhibits a significantly lower PVA residue level of approximately 10 ppm and a residual solvent content of less than 0.5%. Therefore, it can be seen that a safe dosage form can be prepared without the toxicity problems caused by residual PVA or residual solvents in the finished drug product.

[0085] Example 4. Confirmation of rapid-release and sustained-release effects 4-1. Initial release evaluation (in vivo) in vivo )) The immediate-release formulations 3 and 4, and the sustained-release formulations 7 and 8 (without further inclusion of leuprorelin acetate on the exterior of the microspheres), prepared by the method of this invention, were administered to rats to confirm initial release capacity. Twenty 10-week-old Sprague-Dawley specific pathogen-free (SPF) rats were randomly assigned to each experimental group (n=5) and administered a single subcutaneous dose of 3 mg / kg. Serum was then collected from each rat via jugular vein at 0.5 h, 1 h, 3 h, 6 h, 24 h, 2 days, 4 days, 7 days, 14 days, 21 days, and 28 days. The serum was stored at -80°C, and the AUC of each experimental group was determined by LC-MS / MS. C max T max The AUC values ​​are shown in Table 5, and the results of the serum concentration changes over time are shown in Table 6. Figure 2 middle.

[0086] Table 5 -AUC 7-t AUC from day 7 to the last sampling time point -AUC 0-t AUC from day 0 to the last sampling time point -AUC INF AUC from day 0 until the concentration reaches 0 (estimated time for plotting the trend). As shown in Table 5 and Figure 2 As shown, dosage forms 3 and 4 are rapid-release dosage forms containing 2% active ingredient on the outside of the microspheres, relative to the content of active ingredient in the microspheres. Compared with sustained-release dosage forms 7 and 8, their C maxThe initial release rate is increased by approximately 35% to 40% or more within 24 hours. This demonstrates that the immediate-release formulation of the present invention can effectively improve the low initial release rate of existing sustained-release formulations.

[0087] 4-2. In vivo pharmacokinetics and safety evaluation The pharmacokinetic properties and safety of leuprorelin acetate were confirmed by a single subcutaneous injection of dosage forms 1 and 2 of the present invention into the abdomen of healthy postmenopausal women. The pharmacokinetic evaluation group for dosage form 1 consisted of 36 participants, and the pharmacokinetic evaluation group for dosage form 2 consisted of 38 participants. Blood samples were collected at 0 (before administration), 0.5, 1, 1.5, 2, 4, 8, 12, 24, 72, 168, 240, 336, 504, 672, 840, and 1008 hours, for a total of 17 confirmations.

[0088] In particular, as described in Example 4-1, due to C max As parameters related to initial release, formulations 1 and 2, containing 4.8% or 2% leuprolide acetate on the exterior of the microspheres respectively, were used relative to the total leuprolide acetate content to C. max Comparative analysis was conducted to confirm the initial release increase and bioavailability based on the percentage of leuprolide acetate added externally. C was confirmed in dosage forms 1 and 2. max The AUC values ​​are shown in Table 6 below.

[0089] Table 6 As shown in Table 6, relative to the total content of active ingredients, formulation 1, which contains 4.8% immediate-release active ingredients on the outside of the microspheres, exhibits a C1% or higher increase of approximately 30% compared to formulation 2, which contains 2% immediate-release active ingredients on the outside of the microspheres. max value.

[0090] These results confirm that formulations containing less than 8% (w / w), preferably 4% (w / w) to 6% (w / w) leuprolide acetate on the exterior of the microspheres relative to the total active ingredient content can induce effective initial release and C in animals and humans. max The value increases. That is, if the dosage form of the present invention is used, in which less than 8% of the total active ingredient leuprolide acetate is contained on the outside of the sustained-release microspheres and the remaining leuprolide acetate is encapsulated in the sustained-release microspheres, it means that a fast-release microsphere formulation that exhibits effective therapeutic effects by overcoming the initial delayed release and having excellent bioavailability can be prepared.

[0091] Furthermore, the in vivo safety evaluation of the dosage form of the present invention was also conducted simultaneously. The results showed that no clinically significant adverse reactions of particular concern or local adverse reactions were reported during the clinical trial. No clinically significant differences in the frequency or pattern of adverse reactions were found between the treatment groups in this clinical trial. Apart from the 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 examinations performed after administration of the clinical trial drug during the clinical trial.

[0092] In conclusion, in the safety evaluations including adverse reactions, local adverse reactions, combined drugs, vital signs, physical examination, clinical laboratory tests, 12-lead electrocardiogram, and BMD, there were no clinically significant differences between the treatment groups.

Claims

1. A method for preparing a sustained-release formulation with increased initial release, characterized in that, Includes the following steps: Step 1) involves preparing the spray solution by dissolving leuprolide or its salt and a biodegradable polymer in an organic solvent; Step 2), microspheres encapsulated with leuprolide or its salts are prepared by spray drying the spray liquid; as well as Step 3) involves preparing microspheres with residual solvent removed by dispersing the microspheres in an aqueous polyvinyl alcohol solution and then washing them. It also includes the step of adding a lyophilized powder of leuprolide or its salt to the exterior of the microspheres.

2. The method for preparing a sustained-release formulation with increased initial release amount according to claim 1, characterized in that, The addition includes the following steps: Step 4-1): The microspheres prepared in step 3) are suspended in water for injection and water-soluble polyol, then lyophilized and pulverized to prepare microspheres; and Step 4-2), fill the microspheres of step 4-1) with lyophilized powder of leuprolide or its salt.

3. The method for preparing a sustained-release formulation with increased initial release amount according to claim 1, characterized in that, The addition includes suspending the microspheres prepared in step 3) in water for injection containing leuprolide or its salt and water-soluble polyol, and then lyophilizing and pulverizing the suspension.

4. The method for preparing a sustained-release formulation with increased initial release amount according to claim 1, characterized in that, The amount of the leuprolide or its salt added externally is 2% (w / w) to 8% (w / w) relative to the total weight of the active ingredient.

5. The method for preparing a sustained-release formulation with increased initial release amount according to claim 1, characterized in that, The organic solvent is glacial acetic acid or acetic acid.

6. The method for preparing a sustained-release formulation with increased initial release amount according to claim 1, characterized in that, The sustained-release formulation is one in which more than 99% of the residual solvent has been removed.

7. The method for preparing a sustained-release formulation with increased initial release amount according to claim 2, characterized in that, The water-soluble polyol is mannitol, sorbitol, or galactitol.

8. The method for preparing a sustained-release formulation with increased initial release amount according to claim 1, characterized in that, The biodegradable polymer is selected from one or more of the group consisting of polylactic acid, polyglycolic acid, polylactic-hydroxyacetic acid copolymer, polyorthoester, polyanhydride, polyhydroxybutyric acid, polycaprolactone, and polyalkyl carbonate.

9. The method for preparing a sustained-release formulation with increased initial release amount according to claim 1, characterized in that, The average particle size of the sustained-release formulation with increased initial release is 15 μm to 25 μm.

10. A sustained-release formulation with increased initial release, characterized in that, Prepared by the preparation method according to any one of claims 1 to 9.

11. A sustained-release formulation with increased initial release, characterized in that, Include: Sustained-release microspheres encapsulated with leuprolide or its salts, comprising leuprolide or its salts and a biodegradable polymer; and The sustained-release microspheres contain leuprolide or its salt on their exterior.

12. The sustained-release formulation with increased initial release according to claim 11, characterized in that, The content of leuprolide or its salt contained on the exterior of the microspheres is 2% (w / w) to 8% (w / w) relative to the total weight of the active ingredients.

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