Long-acting microspheres containing Thymosin Alpha 1, a composition comprising the same, and a method for preparing the same

Abstract

The present invention relates to long-acting microspheres, a composition comprising the same, or a method for manufacturing the same, wherein the long-acting microspheres comprise thymosin alpha 1 or a pharmaceutically acceptable salt thereof; a biodegradable polymer; and a drug encapsulation extender selected from the group consisting of protamine sulfate and human serum albumin, wherein the weight ratio of the thymosin alpha 1 or the pharmaceutically acceptable salt thereof to the drug encapsulation extender is 5:1 to 40:1.

Description

Technology Field

[0001] The present invention relates to thymosin alpha 1-containing long-acting microspheres, a composition comprising the same, and a method for manufacturing the same. Background Technology

[0003] Thymosin alpha 1 (Tα1) is an endogenous immunomodulatory peptide composed of 28 amino acids that enhances cell-mediated immune responses by promoting the maturation and activation of T cells and increasing the function of antigen-presenting cells. Recently, water-soluble immunomodulatory peptides such as Tα1 have garnered attention as therapeutic agents for various infectious and immunodeficiency diseases. Although this peptide exhibits excellent biological activity, it has a very short in vivo half-life and is rapidly degraded or eliminated after administration, posing a problem that requires frequent, repeated administration to maintain a sustained therapeutic effect. For example, existing aqueous Tα1 formulations require subcutaneous injections at least twice a week, which can lead to reduced patient compliance and decreased therapeutic efficiency.

[0004] Peptide drugs are generally very water-soluble and suitable for injectable formulations, but they have low physicochemical stability, are prone to aggregation or denaturation during storage, and are difficult to maintain a sustained therapeutic effect due to rapid in vivo degradation. Therefore, there is a growing demand for formulations that can preserve the biological activity of peptides while providing stable, long-term release within the body.

[0005] Various attempts have been made to extend the in vivo half-life of peptide drugs containing Tα1 and to ensure sustained efficacy. However, when developing long-acting formulations of water-soluble peptides, problems such as low encapsulation efficiency, high initial burst release, peptide denaturation or loss during emulsification and solidification processes, microsphere aggregation, and reduced injectability frequently occur. Although sustained-release systems such as PLGA (polylactide-glycolide copolymer)-based microspheres, liposomes, polymer-peptide conjugates, and lipid / polymer nanoparticle-based formulations have been reported for peptide drugs, it is difficult to achieve stable release for more than 2 to 4 weeks due to the unique characteristics of Tα1, such as high hydrophilicity, low molecular weight, and structural instability.

[0006] Korean Patent Application No. 10-2024-0183066 and Korean Patent Application No. 10-2024-0072693 disclose sustained-release microsphere formulations containing peptides such as semaglutide and terzepatide, respectively. These documents describe a general approach to making enteric-acting peptide formulations using biodegradable polymers, but they do not address specific technical problems related to thymosin alpha 1, nor do they propose a solution to achieve high encapsulation efficiency and a stable one-month sustained-release profile specialized for Tα1.

[0007] Therefore, there is still a continuous demand for long-acting thymosin alpha 1 injectable formulations that overcome the limitations of existing technologies and improve patient compliance and overall therapeutic effects by providing high encapsulation efficiency, controlled initial release, and drug release levels lasting for at least one month.

[0009] [Prior Literature]

[0010] Republic of Korea Patent Application No. 10-2024-0183066

[0011] Republic of Korea Patent Application No. 10-2024-0072693 The problem to be solved

[0013] The present invention has the following objectives to solve the above technical problem:

[0014] First, the purpose is to provide long-acting microspheres that significantly improve the encapsulation efficiency of the hydrophilic drug thymosin alpha 1, and to this end, to minimize drug loss during the manufacturing and release processes by using a specific drug encapsulation extender in an optimal weight ratio.

[0015] Second, the purpose is to provide long-acting microspheres that effectively suppress the excessive release of the drug during the initial administration and subsequently exhibit a stable and continuous release pattern.

[0016] Third, the purpose is to provide a pharmaceutical composition that can maintain a therapeutically effective concentration for at least 4 weeks with only one administration per month, thereby increasing patient convenience and maximizing therapeutic effects.

[0017] Fourth, the purpose is to provide high-quality, long-acting microspheres having an average particle diameter suitable for injection administration and a uniform particle size distribution, and a method for manufacturing the same. means of solving the problem

[0019] One aspect of the present invention provides a long-acting microsphere comprising: thymosin alpha 1 or a pharmaceutically acceptable salt thereof; a biodegradable polymer; and a drug encapsulation extender selected from the group consisting of protamine sulfate and human serum albumin, wherein the weight ratio of the thymosin alpha 1 or the pharmaceutically acceptable salt thereof to the drug encapsulation extender is 5:1 to 40:1.

[0020] Another aspect of the present invention is a method for producing long-acting microspheres comprising thymosin alpha 1 or a pharmaceutically acceptable salt thereof, wherein

[0021] (a) a step of preparing a first aqueous phase comprising thymosin alpha 1 or a pharmaceutically acceptable salt thereof, and one or more drug encapsulating extenders selected from the group consisting of protamine sulfate and human serum albumin;

[0022] (b) a step of preparing an oil phase containing a biodegradable polymer dissolved in an organic solvent;

[0023] (c) a step of emulsifying the first aqueous phase into the oil phase to form a first emulsion;

[0024] (d) a step of forming a second emulsion through a membrane emulsification device using the first emulsion as the dispersed phase and the second aqueous phase as the continuous phase; and

[0025] (e) A method for manufacturing long-lasting microspheres is provided, comprising the step of removing the organic solvent from the second emulsion to solidify the microspheres. Effects of the invention

[0027] The long-lasting microspheres according to the present invention exhibit the following excellent effects.

[0028] First, the present invention can maximize the encapsulation efficiency into microspheres by including protamine sulfate and / or human serum albumin as a drug encapsulation extender in a specific weight ratio of 5:1 to 40:1 relative to thymosin alpha1, thereby forming an effective complex with thymosin alpha1, a hydrophilic drug.

[0029] Second, by stabilizing the position of the drug within the microsphere through the interaction between the drug and the drug encapsulation extender, the problematic initial over-release phenomenon can be effectively suppressed, such as controlling the cumulative release rate within 24 hours to less than 47%. This enhances the safety of drug administration and enables the realization of a predictable pharmacokinetic profile.

[0030] Third, thanks to high encapsulation efficiency and controlled release characteristics, a once-a-month administration cycle can be achieved with just a single injection, maintaining efficacy for at least four weeks. This has an excellent effect in improving treatment compliance by dramatically reducing the pain and discomfort patients experience from frequent injections.

[0031] Fourth, through a manufacturing method using a membrane emulsification device, microspheres with a uniform average particle diameter of 20 μm to 50 μm can be reproducibly manufactured, which minimizes pain during injection and enables stable administration.

[0032] In conclusion, the present invention has the effect of providing an innovative long-acting injectable platform technology that can significantly improve the quality of life of patients in the treatment of various diseases, such as anticancer adjuvant therapy, chronic viral hepatitis, and immunodeficiency. Brief explanation of the drawing

[0034] The present invention is described in detail with reference to preferred embodiments with reference to the attached drawings. FIGS. 1a to 1c are scanning electron microscope (SEM) images showing the surface morphology of long-acting microspheres containing thymosin alpha 1 prepared according to one embodiment and a comparative example of the present invention. Figure 2 is a graph showing the cumulative release rate over time of long-lasting microspheres according to one embodiment and a comparative example of the present invention. Figure 3 is a scanning electron microscope (SEM) image for evaluating the degradation characteristics of microspheres on the third day of release according to one embodiment and a comparative example of the present invention. Figure 4 is a scanning electron microscope (SEM) image for evaluating the degradation characteristics of microspheres on day 21 of release according to one embodiment and a comparative example of the present invention. Specific details for implementing the invention

[0035] The present invention aims to solve the problems of the aforementioned prior art and is centered on providing long-acting microspheres that use a specific drug loading enhancer in an optimal ratio to dramatically increase the encapsulation efficiency of thymosin alpha 1 and effectively suppress initial release.

[0037] Components of long-acting microspheres

[0038] The long-acting microspheres according to the present invention (hereinafter referred to as ‘microspheres of the present invention’) essentially comprise (1) thymosin alpha 1, which is an active ingredient, or a pharmaceutically acceptable salt thereof, (2) a biodegradable polymer that serves as a matrix for encapsulating the drug, and (3) a drug encapsulation extender that controls the drug encapsulation efficiency and release characteristics.

[0040] (1) Thymosin alpha 1 or a pharmaceutically acceptable salt thereof

[0041] The active ingredient of the present invention is thymosin alpha 1. In this specification, 'thymosin alpha 1' includes all forms derived from nature, synthetic, or produced by genetic engineering methods. Furthermore, the present invention includes not only thymosin alpha 1 itself but also pharmaceutically acceptable salt forms thereof. 'Pharmaceutically acceptable salt' means a non-toxic salt that maintains the biological activity of thymosin alpha 1 and may include, but is not limited to, acid addition salts with inorganic or organic acids such as acetates, hydrochlorides, sulfates, etc., or base addition salts such as sodium salts, potassium salts, calcium salts, etc.

[0043] (2) Biodegradable polymer

[0044] The microspheres of the present invention utilize a biodegradable polymer as a matrix that encapsulates a drug and releases the drug as it gradually decomposes within the body. A "biodegradable polymer" refers to a polymer that can be broken down into low-molecular-weight substances by hydrolysis or enzymatic degradation under physiological conditions within the body, and ultimately metabolized or excreted from the body.

[0045] The biodegradable polymers that can be used in the present invention may be selected from the group consisting of, but are not limited to, polylactic acid (PLA), polylactide, poly(lactic-co-glycolic acid) (PLGA), polylactide-co-glycolide, polyphosphazine, polyiminocarbonate, polyphosphoester, polyanhydride, polyorthoester, polycaprolactone (PCL), polyhydroxyvalate, polyhydroxybutyrate, polyamino acid, and copolymers or combinations thereof. Preferably, polylactide (PLA) and / or poly(lactic-co-glycolide) (PLGA), which have been approved by the U.S. FDA and proven safe for human use, may be used.

[0046] In particular, PLGA is a copolymer of lactide and glycolide, and has the advantage of being able to precisely control the degradation rate of the polymer, i.e., the drug release rate, by adjusting the ratio of the two monomers, the molecular weight, and the type of end group (e.g., carboxylic acid end group or ester end group). For example, the higher the proportion of hydrophilic glycolide, the faster the degradation and the more accelerated the drug release, while the higher the proportion of lactide, the slower the degradation and the more delayed the release. Additionally, the larger the molecular weight, the slower the degradation, and if it has a carboxylic acid end group, degradation occurs faster than if it has an ester end group due to an autocatalytic effect. In the present invention, by utilizing these characteristics, an optimal PLGA can be selected to suit a desired drug release period (e.g., one month), or two or more PLGAs with different characteristics can be combined and used. Poly(lactide-co-glycolide) may have a molar ratio of glycolide to lactide of about 60:40 to about 90:10, 60:40 to about 85:15, about 60:40 to about 80:20, about 60:40 to about 75:25, about 65:35 to about 0:10, about 70:30 to about 90:10, about 75:25 to about 90:10, about 65:35 to about 85:15, and about 70:30 to about 80:20, but is not limited to the above examples. Preferably, the molar ratio of glycolide to lactide in poly(lactide-co-glycolide) may be about 75:25.

[0047] In the microspheres of the present invention, the weight ratio of thymosin alpha 1 or its pharmaceutically acceptable salt to the biodegradable polymer is preferably 1:15 to 1:20. If the drug content is too low (i.e., the polymer ratio exceeds 1:20), the total amount of microspheres required for a single administration becomes excessively large, which may cause pain or discomfort at the injection site. Conversely, if the drug content is too high (i.e., the polymer ratio is less than 1:15), the physical structure of the microspheres becomes unstable, which may increase the initial release or make it difficult to achieve the desired release pattern. Therefore, the above weight ratio is an important factor in balancing drug efficacy and formulation stability.

[0049] (3) Drug encapsulation extender

[0050] The microspheres of the present invention comprise a drug encapsulation extender comprising one or more selected from the group consisting of protamine sulfate (PS) and human serum albumin (HSA) as one component. This extender effectively prevents hydrophilic thymosin alpha 1 from escaping from the inner aqueous phase (W1) to the outer aqueous phase (W2) during the W / O / W emulsification process, thereby maximizing encapsulation efficiency.

[0051] Protamine Sulfate (PS): Protamine is a peptide that carries a strong positive charge. It forms an ionic complex with thymosin alpha 1, which can carry a negative charge at physiological pH. Since this complex reduces water solubility and increases molecular weight, its diffusion through the hydrophobic oil phase to the outer aqueous phase during emulsification is significantly inhibited. Thanks to this strong ionic bond, PS exhibits a high enhancement of encapsulation efficiency even in very small amounts.

[0052] Human Serum Albumin (HSA): HSA is a protein that binds to and transports various substances within the blood, possessing multiple hydrophobic binding pockets within its molecule. Thymosin alpha 1 can reversibly bind to these HSA pockets through hydrophobic interactions. Additionally, HSA plays a role in physically inhibiting drug diffusion by increasing the viscosity of the internal phase. Through these mechanisms, HSA contributes to enhancing drug encapsulation efficiency and controlling initial release.

[0053] What is even more important in the present invention is the optimized weight ratio between thymosin alpha 1 and the drug encapsulation extender. Through in-depth research by the inventors, it was discovered that simply having a large amount of the extender is not necessarily beneficial.

[0054] The microspheres of the present invention are characterized by a weight ratio of thymosin alpha 1 or a pharmaceutically acceptable salt thereof to a drug encapsulation extender of 5:1 to 40:1. More preferably, the best effect is exhibited when the weight ratio is 6:1 to 8:1.

[0055] If the ratio of the filler is too low (e.g., exceeding 40:1), the effect of increasing encapsulation efficiency is negligible. Conversely, if the ratio of the filler is too high (e.g., less than 5:1), the osmotic pressure and viscosity of the internal aqueous phase increase excessively, causing porous defects in the internal structure during the microsphere solidification process. This porous structure weakens the mechanical strength of the microspheres and acts as a channel for the drug to easily escape, resulting in a counterproductive effect that actually causes a rapid increase in initial release.

[0056] Therefore, the specific weight ratio range defined in this invention is a key technical configuration for achieving low initial release by maintaining the stable non-porous structure of the microspheres while achieving high encapsulation efficiency. This is an important aspect demonstrating the inventive step of this invention, which cannot be easily predicted by simple experiments.

[0057] In a preferred embodiment, the drug encapsulation extender is protamine sulfate. Protamine sulfate exhibits a particularly excellent effect in improving encapsulation efficiency through strong ionic bonding with thymosin alpha 1.

[0059] Physical and Release Characteristics of Long-Lasting Microspheres

[0060] (1) Particle size

[0061] The microspheres of the present invention have a specific particle size distribution suitable for injection administration. The average particle diameter of the microspheres (D50, 50% cumulative particle diameter based on volume) is preferably 20 μm to 50 μm. In a specific example, it may be 20 μm to 35 μm, and in a more specific example, it may be 22 μm to 30 μm.

[0062] Within the above range, it is not phagocytosed by macrophages in the body, and the increased specific surface area can suppress the increase in initial release, prevent needle blockage during injection, and prevent pain induction.

[0064] (2) Drug release characteristics

[0065] The microspheres of the present invention exhibit very well-controlled initial release characteristics. Specifically, when a release test is performed in a phosphate-buffered saline (PBS) solution at pH 7.4 under 37°C conditions, the cumulative release rate is less than 47% within 24 hours. This means that the problem of high initial release, which was a problem of the prior art, has been successfully resolved. Low initial release is essential for ensuring safety by preventing a rapid rise in blood drug concentration at the beginning of administration and for sustaining drug efficacy for a long period.

[0066] Furthermore, the microspheres of the present invention are designed so that the therapeutic effect can be sustained for at least 4 weeks, i.e., more than 1 month, with a single administration. This is achieved by the drug being steadily released within a therapeutically effective concentration range over a certain period as the polymer matrix gradually degrades after a low initial release.

[0068] Pharmaceutical composition and uses

[0069] The present invention also provides a pharmaceutical composition comprising the long-acting microspheres described above and pharmaceutically acceptable excipients. The microspheres of the present invention are generally provided in the form of lyophilized powder and are prepared into an injectable suspension by mixing with an excipient, such as water for injection or a suitable suspension solvent, immediately before use. Excipients may additionally include isotonic agents (e.g., mannitol, sodium chloride), buffers, suspending agents (e.g., sodium carboxymethylcellulose), preservatives, etc.

[0070] The pharmaceutical composition of the present invention is useful for the prevention or treatment of various diseases in which the immunomodulatory function of thymosin alpha 1 is effective. Specifically, it can be used as an adjuvant therapy to enhance the immune response upon influenza vaccination in elderly patients with impaired immune function, as an adjuvant anticancer therapy, for the improvement or treatment of chronic viral hepatitis (e.g., chronic hepatitis B or C), immunodeficiency, various infectious diseases, autoimmune diseases, senile immunosuppression, or chronic fatigue.

[0071] In particular, since the efficacy of this composition lasts for at least 4 weeks with a single administration, it can be used as a long-acting injectable with an administration cycle of at least once a month. This contributes significantly to improving overall treatment efficacy by maximizing patient convenience and increasing treatment compliance.

[0073] Method for manufacturing long-acting microspheres

[0074] The present invention also provides a method for reproducibly manufacturing the long-acting microspheres. A preferred manufacturing method is based on a W / O / W dual emulsion method and is characterized in particular by the use of a membrane emulsification device. The manufacturing process comprises the following steps.

[0076] (a) Preparation stage for the first award (W1)

[0077] A first aqueous phase is prepared by dissolving thymosin alpha 1 or a pharmaceutically acceptable salt thereof and a drug encapsulation extender (protamine sulfate and / or human serum albumin) in water or a buffer solution. At this stage, the weight ratio of thymosin alpha 1 to the extender is an important variable determining the characteristics of the final microspheres, and as previously described, it is preferable to adjust it to 5:1 to 40:1. The first aqueous phase may additionally contain a hydrophilic surfactant. Examples of said surfactant include polyvinyl alcohol (PVA), Poloxamer 188 or 407, or polyvinylpyrrolidone (PVP).

[0079] (b) Paid (O) preparation stage

[0080] An oil phase is prepared by completely dissolving a biodegradable polymer (e.g., PLGA) in an organic solvent. As the organic solvent, solvents that dissolve the polymer well, do not mix with water, and have good volatility, such as dichloromethane (DCM), chloroform, and ethyl acetate, may be used. It is preferable to adjust the weight ratio of thymosin alpha 1 to the biodegradable polymer used in this step to 1:15 to 1:20. The biodegradable polymer used may be selected from the group consisting of polylactic acid, polylactide, polylactic-co-glycolic acid, poly(lactide-co-glycolide) (PLGA), polyphosphazine, polyiminocarbonate, polyphosphoester, polyanhydride, polyorthoester, polycaprolactone, polyhydroxyvalate, polyhydroxybutyrate, polyamino acid, and combinations thereof.

[0082] (c) Step of forming the first emulsion (W1 / O)

[0083] The first aqueous phase prepared in (a) above is added to the oil phase prepared in (b), and the mixture is stirred at high speed using a homogenizer or an ultrasonic grinder to form a first W / O type emulsion in which fine droplets of the first aqueous phase are dispersed in the oil phase. At this stage, since the size of the first aqueous phase droplets affects the drug distribution within the final microsphere, it is important to make them uniformly small.

[0085] (d) Step of forming the second emulsion (W1 / O / W2)

[0086] A second emulsion is formed by using the first emulsion formed in (c) above as the dispersed phase and the second aqueous phase (W2) in which the surfactant is dissolved as the continuous phase. One of the key features of the present invention is that this step is performed using a membrane emulsification device. A membrane emulsification device is a device that pushes the dispersed phase (first emulsion) into the continuous phase (second aqueous phase) through a membrane in which fine pores are uniformly distributed to form droplets of very uniform size. Using this method allows for the production of microspheres with a much narrower particle size distribution compared to general stirring methods, which is highly advantageous for ensuring the uniformity of quality of the final product. This contributes to controlling the average particle diameter of the final microspheres to 20 μm to 50 μm.

[0087] Hydrophilic surfactants used in the second aqueous phase may include polyvinyl alcohol (PVA), Poloxamer 188 or 407, or polyvinylpyrrolidone (PVP), among which polyvinyl alcohol is most preferable in terms of emulsion stabilization. The surfactant helps ensure that W / O droplets are stably dispersed within the second aqueous phase without aggregating with each other.

[0089] (e) Microsphere solidification and organic solvent removal step

[0090] The second emulsion (W1 / O / W2) formed in (d) above is stirred to remove the organic solvent and solidify the polymer droplets. The removal of the organic solvent is mainly carried out by vacuum evaporation or extraction into the continuous phase. In this process, the polymer dissolved in the oil phase precipitates, forming a solid microsphere containing a first aqueous droplet containing a drug and an extender inside.

[0091] Subsequently, the formed microspheres are collected through centrifugation or filtration and washed several times with sterile water for injection to remove surfactants or unencapsulated drugs remaining on the surface. Finally, by completely removing residual moisture through a freeze-drying process, long-acting microspheres in the form of a stable powder suitable for long-term storage can be obtained.

[0092] As previously explained, the microspheres obtained through this manufacturing method exhibit excellent release characteristics (less than 47% release within 24 hours) and long-term persistence (at least 4 weeks).

[0094] The present invention will be described in detail below by way of examples. However, these examples are merely illustrative, and the scope of the present invention is not limited by the examples.

[0096] Example 1. Preparation of microspheres containing thymosin alpha 1 and 1 mg of HSA

[0097] 0.1 mL of 1M acetic acid and 0.9 mL of purified water were mixed, and 0.4 mg of poloxamer 188, 35 mg of thymosin alpha 1, and 1 mg of HSA (human serum albumin) were dissolved in the mixture to prepare the first aqueous phase. 600 mg of the biodegradable polymer poly(L-lactic-co-glycolic acid) (PDLG 7502A, Corbion) was weighed and dissolved in 3.0 mL of dichloromethane to prepare the oil phase. Additionally, 4,000 mg of Poly(vinyl alcohol) (PVA) was dissolved in 400 mL of purified water to prepare the second aqueous phase. The first aqueous phase was added to the oil phase and treated with a homogenizer at 10,000 rpm for 1 minute to prepare the first emulsion. A secondary emulsion was prepared by simultaneously feeding the prepared primary emulsion as the dispersed phase and the second aqueous phase as the continuous phase through a membrane emulsification device. The process for preparing the secondary emulsion was carried out while maintaining a temperature of 25°C. Upon completion of the secondary emulsion preparation, the mixture was stirred at 40°C for 3 hours to solidify the microspheres and simultaneously remove the organic solvent. The microspheres, after the organic solvent was removed and solidified, were repeatedly washed several times with purified water using a harvester, and finally freeze-dried to obtain microspheres containing thymosin alpha 1 in powder form.

[0099] Example 2. Preparation of microspheres containing 5 mg of thymosin alpha 1 and HSA

[0100] Microspheres containing thymosin alpha 1 of Example 2 were obtained by carrying out the same procedure as in Example 1, except that 5 mg of HSA was used instead of 1 mg of HSA.

[0102] Example 3. Preparation of microspheres containing thymosin alpha 1 and PS 1 mg

[0103] Microspheres containing thymosin alpha 1 of Example 3 were obtained by carrying out the same procedure as in Example 1, except that 1 mg of PS (protamine sulfate) was used instead of 1 mg of HSA.

[0105] Example 4. Preparation of microspheres containing thymosin alpha 1 and PS 5 mg

[0106] Microspheres containing thymosin alpha 1 of Example 4 were obtained by carrying out the same procedure as in Example 1, except that 5 mg of PS (protamine sulfate) was used instead of 1 mg of HSA.

[0108] Comparative Example 1. Preparation of microspheres containing thymosin alpha 1

[0109] Microspheres containing thymosin alpha 1 of Comparative Example 1 were obtained by carrying out the same procedure as Example 1, except that the first aqueous phase was prepared without using 1 mg of HSA in Example 1.

[0111] Comparative Example 2. Preparation of microspheres containing thymosin alpha 1

[0112] Microspheres containing thymosin alpha 1 of Comparative Example 2 were obtained by carrying out the same procedure as in Example 1 above, except that 10 mg of HSA was used instead of 1 mg of HSA.

[0114] Comparative Example 3. Preparation of microspheres containing thymosin alpha 1

[0115] Microspheres containing thymosin alpha 1 of Comparative Example 3 were obtained by carrying out the same procedure as in Example 1 above, except that 10 mg of PS was used instead of 1 mg of HSA.

[0117] The compositions of Examples 1 to 4 and Comparative Examples 1 to 3 are as shown in Table 1 below:

[0118] division Ingredient name Example 1 Example 2 Example 3 Example 4 Comparative Example 1 Comparative Example 2 Comparative Example 3 First Prize Ta1 (mg) 35 HSA (mg) 1 5 - - - 10 - PS (mg) - - 1 5 - - 10 Poloxamer 188 (mg) 0.4 1M Acetic acid (mL) 0.1 Purified water (mL) 0.9 Paid PDLG 7502A (mg) 600 DCM (mL) 3 Second Prize PVA (mg) 4,000 Purified water (mL) 400

[0119] Ta1: Thymosin alpha 1, PS: Protamine sulfate, HSA: Human serum albumin, PVA: Poly (vinyl alcohol), DCM: Dichloromethane.

[0120] The information on the raw materials and reagents used in the preparation of the above examples and comparative examples is as shown in Table 2 below:

[0121] turn Product Name manufacturing company purpose note 1 Thymosin alpha 1 (Ta1) Chengdu Shengnuo Biopharm Co., Ltd. drugs Netherlands 2 poly(L-lactic-co-glycolic acid) (PDLG 7502A)(Trade name: PURASORB) Corbion Biodegradable polymer (for the purpose of manufacturing microspheres) Lactide : Glycolide molar ratio = 75:25 End group : Acid Intangible viscosity: 0.16-0.24 dL / g 3 Protamine sulfate (PS) Merck Drug encapsulation extender 4 Human serum albumin (HSA) Sigma Drug encapsulation extender 5 Sodium azide Sigma Microbial growth inhibitors during release testing 6 Poly(vinyl alcohol) (PVA) Sigma surfactants Molecular weight: 20,000-30,000, 88% hydrolyzed 7 Poloxamer 188 Sigma Non-ionic stabilizer 8 Acetic acid Daejeonghwageum pH adjuster 9 Dichloromethane Deoksan Pharmaceutical solvent

[0123] Test Example 1: Surface Shape Analysis

[0124] A certain amount of the microspheres prepared in the above examples and comparative examples were taken and fixed onto a carbon tape attached to a stage, and gold (Au) was coated using an ion sputter coater (STP-20, COXEM), and SEM images were analyzed using a scanning electron microscope (JSM-7600F, (JEOL), EM-40, (COXEM)). As a result, microspheres without or in an appropriate ratio of extender (Examples 1, 2, 3, 4) showed a stable structure with a smooth surface and good spherical shape (see Figs. 1b-1c). On the other hand, microspheres with an excessive ratio of extender (Comparative Examples 2, 3) exhibited a porous structure with numerous holes observed on the surface. This is analyzed to be because the excessive amount of extender relative to thymosin alpha 1 increased the osmotic pressure of the internal aqueous phase, causing structural defects as the internal water rapidly escaped during the process of removing the organic solvent and solidifying the microspheres. This porous structure suggests that it weakens the mechanical strength of microspheres and can serve as a pathway for rapid drug release.

[0126] Test Example 2: Particle Size and Particle Size Distribution Analysis

[0127] The particle size and uniformity of the particle size distribution of each microsphere prepared in the above examples and comparative examples were evaluated. Particle size is an important factor determining the ease of administration of the injectable (passability through the needle) and behavior within the body (e.g., avoidance of phagocytosis by macrophages). Using a particle size distribution analyzer (Mastersizer 3000+, Malvern), the volume-averaged particle diameter (D50) and the span value, which represents the width of the particle size distribution, of the microspheres in each example were measured. The span value is calculated as (D90-D10) / D50, and a smaller value indicates a more uniform particle size distribution.

[0128] As a result, as shown in Table 3 below, the microspheres prepared in all examples (Examples 1-4) had an average particle diameter (D50) between 22.9 μm and 28.6 μm. This confirms that they are of an appropriate size to pass through a standard subcutaneous or intramuscular injection needle without clogging.

[0129] division D(50) (um) Span value Example 1 22.9 1.3 Example 2 28.5 1.3 Example 3 28.6 1.1 Example 4 25.8 1.2 Comparative Example 1 15.2 1.1 Comparative Example 2 32.7 1.1 Comparative Example 3 24.8 1.0

[0131] In addition, the span values ​​were low, ranging from 1.1 to 1.3 in all examples, demonstrating that the manufacturing method of the present invention, particularly the process using a membrane emulsifier, can reproducibly produce microspheres with a very uniform and narrow particle size distribution. This is very important for enabling accurate dosage control during injection administration and ensuring quality consistency between batches.

[0133] Test Example 3: Analysis of drug encapsulation efficiency

[0134] The degree of improvement in drug encapsulation efficiency, one of the key effects of the present invention, was quantitatively evaluated. A certain amount of microspheres prepared in each example and comparative example was taken and completely dissolved in a solvent containing dimethylformamide (DMF) and trifluoroacetic acid (TFA). Subsequently, the amount of thymosin alpha 1 actually encapsulated within the microspheres was quantitatively analyzed using high-performance liquid chromatography (HPLC). Encapsulation efficiency (EE) was calculated according to the following formula.

[0135] Encapsulation Efficiency (EE%) = (Actual amount of drug encapsulated / Theoretical amount of drug added during manufacturing) × 100

[0136] The results are shown in Table 4:

[0137] Analysis of drug encapsulation efficiency division Encapsulation efficiency (EE%) Example 1 48.0 Example 2 67.6 Example 3 47.4 Example 4 95.1 Comparative Example 1 30.2 Comparative Example 2 37.4 Comparative Example 3 35.0

[0138] The encapsulation efficiency of Comparative Example 1, a control group that did not use a drug encapsulation extender, was only 30.2%. This means that most of the hydrophilic drug thymosin alpha 1 was lost to the external aqueous phase during the manufacturing process, clearly demonstrating the problems faced by conventional technology.

[0139] In Examples 1 to 4, in which an appropriate amount of drug encapsulation extender was added, the encapsulation efficiency increased significantly. In particular, in Example 2 (HSA) and Example 4 (PS), where the extender content was 5 mg, very high encapsulation efficiencies of 67.6% and 95.1% were achieved, respectively. This demonstrates that the drug encapsulation extender according to the present invention very effectively prevents the loss of thymosin alpha 1 during the emulsification process. In particular, when protamine sulfate (PS) was used, the encapsulation efficiency exceeded 95%, indicating that almost the entire theoretical dosage was successfully encapsulated within the microspheres.

[0140] In Comparative Example 2 (HSA) and Comparative Example 3 (PS), where the ratio of the filler was outside the specific range (3.5:1), the encapsulation efficiency actually decreased sharply to 37.4% and 35.0%, respectively. This is directly related to the porous structure observed in the SEM results of Test Example 1. Due to the excessive amount of filler, a porous structure was formed during the microsphere solidification process, and the drug inside was lost to the outside through the pores, resulting in a decrease in the final encapsulation efficiency.

[0141] These results experimentally demonstrate that, beyond simply adding a filler, finding an optimized weight ratio (5:1 to 40:1) between thymosin alpha 1 and the filler is critically important for simultaneously achieving high encapsulation efficiency and a stable microsphere structure.

[0143] Test Example 4: Evaluation of Emission Characteristics

[0144] The drug release patterns of the microspheres prepared in the above examples and comparative examples were evaluated to confirm the initial release inhibition effect and the potential for long-term sustained release. 50 mg of microspheres from each example and comparative example were taken, dispersed in phosphate-buffered saline (pH 7.4) containing 0.01% sodium azide, sealed, and stirred at 100 rpm in a 37°C constant temperature water bath.

[0145] At each time point on days 1, 2, 3, 7, 14, 21, and 28, a portion of the supernatant was taken, filtered through a 0.45 μm syringe filter, and quantitatively analyzed using HPLC (Alliance e2695, Waters). An equal volume of fresh eluent was replenished at each collection. The release characteristics of thymosin alpha-1 were calculated as a cumulative release rate based on the drug concentration measured at each time point.

[0147] The analysis conditions for the above measurements are as shown in Table 5 below:

[0148] item condition Column types C18(100mm x 4.6mm ID 5um) column temperature 25℃ Infusion amount 50 uL flow velocity 1.0 mL / min wavelength 210 nm Mobile phase A: 0.1% TFA in DW, B: ACN

[0150] The results of the analysis of the above emission characteristics are shown in Table 6 and Figure 2:

[0151] Point in time (day) Cumulative release rate of thymosin alpha 1 (%) Example 1 Example 2 Example 3 Example 4 Comparative Example 1 Comparative Example 2 Comparative Example 3 1 46.4 27.4 37.1 23.7 49.9 64.7 79.3 2 60.0 44.9 57.3 38.9 82.0 94.1 97.2 3 69.9 53.0 69.3 49.2 91.3 95.2 97.8 7 87.9 66.5 78.1 60.4 93.6 98.3 98.2 14 98.7 74.1 82.7 66.0 99.8 100.7 101.1 21 99.3 77.8 92.8 68.7 100.1 101.8 101.8 28 99.5 78.6 93.5 72.9 100.2 101.5 102.1

Claims

Claim 1 A long-acting microsphere comprising: thymosin alpha 1 or a pharmaceutically acceptable salt thereof; a biodegradable polymer; and a drug encapsulation extender selected from the group consisting of protamine sulfate and human serum albumin, wherein the weight ratio of the thymosin alpha 1 or the pharmaceutically acceptable salt thereof to the drug encapsulation extender is 5:1 to 40:

1. Claim 2 A long-acting microsphere according to claim 1, characterized in that the drug encapsulation extender is protamine sulfate. Claim 3 A long-acting microsphere according to claim 1, characterized in that the weight ratio of the thymosin alpha 1 or its pharmaceutically acceptable salt to the drug encapsulation extender is 6:1 to 8:

1. Claim 4 A long-lasting microsphere according to any one of claims 1 to 3, wherein the biodegradable polymer is selected from the group consisting of polylactic acid, polylactide, polylactic-co-glycolic acid, polylactide-co-glycolide (PLGA), polyphosphazine, polyiminocarbonate, polyphosphoester, polyanhydride, polyorthoester, polycaprolactone, polyhydroxyvalrate, polyhydroxybutyrate, polyamino acid, and combinations thereof. Claim 5 A long-acting microsphere according to any one of claims 1 to 3, characterized in that the weight ratio of the thymosin alpha 1 or its pharmaceutically acceptable salt to the biodegradable polymer is 1:15 to 1:

20. Claim 6 A long-lasting microsphere according to any one of claims 1 to 3, characterized in that the microsphere has an average particle diameter (D50) of 20 μm to 50 μm. Claim 7 A long-acting microsphere according to any one of claims 1 to 3, characterized in that the microsphere exhibits a cumulative release rate of less than 47% within 24 hours when measured at 37°C in a phosphate buffered saline (PBS) solution with a pH of 7.

4. Claim 8 A pharmaceutical composition comprising long-acting microspheres according to any one of claims 1 to 3 and pharmaceutically acceptable excipients, for use as an adjuvant therapy to enhance the immune response upon influenza vaccination in elderly patients with impaired immune function, as an adjuvant anticancer therapy, for the improvement or treatment of chronic viral hepatitis, immunodeficiency, infectious diseases, autoimmune diseases, immunodeficiency in the elderly, or chronic fatigue. Claim 9 A pharmaceutical composition according to claim 8, wherein the above pharmaceutical composition has an effect that lasts for at least 4 weeks with a single administration and has an administration cycle of at least once a month. Claim 10 A method for preparing long-acting microspheres comprising thymosin alpha 1 or a pharmaceutically acceptable salt thereof, comprising: (a) preparing a first aqueous phase comprising thymosin alpha 1 or a pharmaceutically acceptable salt thereof and a drug encapsulation extender selected from the group consisting of protamine sulfate and human serum albumin; (b) preparing an oil phase comprising a biodegradable polymer dissolved in an organic solvent; (c) emulsifying the first aqueous phase into the oil phase to form a first emulsion; (d) forming a second emulsion through a membrane emulsification device using the first emulsion as the dispersed phase and the second aqueous phase as the continuous phase; A method for manufacturing long-acting microspheres comprising the step of (e) removing the organic solvent from the second emulsion to solidify the microspheres, wherein the weight ratio of the thymosin alpha 1 or a pharmaceutically acceptable salt thereof to the drug encapsulation extender is 5:1 to 40:

1. Claim 11 delete Claim 12 A method of manufacturing according to claim 10, characterized in that the weight ratio of the thymosin alpha 1 or its pharmaceutically acceptable salt to the biodegradable polymer is 1:15 to 1:

20. Claim 13 A method of manufacturing, wherein the biodegradable polymer in claim 10 is selected from the group consisting of polylactic acid, polylactide, polylactic-co-glycolic acid, polylactide-co-glycolide (PLGA), polyphosphazine, polyiminocarbonate, polyphosphoester, polyanhydride, polyorthoester, polycaprolactone, polyhydroxyvalrate, polyhydroxybutyrate, polyamino acid, and combinations thereof. Claim 14 A method of manufacturing, wherein, in any one of claims 10 to 13, the microspheres have an average particle diameter (D50) of 20 μm to 50 μm. Claim 15 A method of manufacturing, wherein in any one of claims 10 to 13, the second aqueous phase comprises a hydrophilic surfactant. Claim 16 A method of preparation according to claim 15, wherein the hydrophilic surfactant is polyvinyl alcohol, poloxamer 188 or 407, or polyvinylpyrrolidone. Claim 17 A method of manufacturing, characterized in that, in any one of claims 10 to 13, the microspheres exhibit a cumulative release rate of less than 47% within 24 hours when measured under 37°C conditions in a phosphate buffered saline (PBS) solution with a pH of 7.

4. Claim 18 A method of manufacturing according to any one of claims 10 to 13, wherein the microspheres have a therapeutic effect that lasts for at least 4 weeks with a single administration and have an administration cycle of at least once a month.

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