A new preformulation

Abstract

This invention relates to the field of drug delivery systems, and more specifically, to a pre-formulation for long-acting drug delivery, its preparation method and application. The pre-formulation of this invention significantly improves the initial burst release of drugs, enabling stable release from several days to several months, resulting in better efficacy and safety. During storage, the bioactive substance particles in the pre-formulation do not aggregate, exhibiting good dispersion stability.

Description

[0001] This application claims priority to an earlier application filed by the applicant with the China National Intellectual Property Administration on January 29, 2026, with patent application number 202610127311.7 entitled "A Novel Preform". The entire contents of the earlier application are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of drug delivery systems, and more specifically, to a pre-formulation for long-acting drug delivery, its preparation method, and its application. Background Technology

[0003] In the field of long-acting drug delivery systems, injectable in-situ molded implants (also known as in-situ gels) are a representative pre-formulation, attracting significant attention due to their ability to achieve sustained drug release for weeks to months. This technology typically involves dissolving or dispersing the drug in a biocompatible solvent pre-dissolved in a biodegradable polymer (such as polylactic-co-glycolic acid copolymer, PLGA), preparing a liquid precursor. Once injected into the body, the solvent undergoes exchange diffusion with body fluids (external diffusion of the solvent and internal penetration of the non-solvent), leading to a decrease in polymer solubility. This, in turn, triggers in-situ precipitation and phase transition, forming a solid or semi-solid drug reservoir. This reservoir acts as a physical barrier, effectively regulating and delaying drug release to achieve long-acting therapeutic effects.

[0004] However, this classic technical approach still faces significant challenges in achieving precise control over drug release. Its fundamental limitation lies in the inherent mismatch between drug diffusion kinetics and the in-situ formation kinetics of the diffusion barrier. Specifically, after injection, drugs (especially water-soluble small molecules or peptides) immediately exhibit high diffusion rates; in contrast, the construction of a dense controlled-release barrier is a relatively slow phase transition process. This "asynchrony" in the timescale of drug diffusion and barrier construction—that is, barrier formation significantly lags behind the initial diffusion control requirements—leads to a severe initial burst release phenomenon caused by a large amount of drug being released driven by a high concentration gradient before the effective barrier is fully established.

[0005] Eligar, a long-acting formulation of leuprorelin widely used in clinical practice ®For example, as a classic representative of in-situ molding implants, this formulation is an injectable pre-prepared drug consisting of leuprolide acetate, polylactic-co-glycolic acid copolymer (PLGA), and N-methylpyrrolidone, primarily used for palliative treatment of advanced prostate cancer. After injection, the solvent N-methylpyrrolidone rapidly diffuses into the surrounding tissue, inducing phase separation of the PLGA and its in-situ solidification into a gel. However, due to the lack of effective drug diffusion barriers before the complete formation of the gel network, this type of formulation commonly exhibits an initial burst release phenomenon. Pharmacokinetic data show that, in the 45 mg specification of Eligard... ® For example, publicly available data shows that the average peak value in the early stages after the first injection and subsequent injections can reach approximately 82 ng / mL. -1 and 102 ng·mL -1 The effective therapeutic concentrations were respectively reached as high as 0.1 ng / mL. -1 The figures of approximately 820 and 1020 times higher indicate that this type of in situ molded implantable formulation may result in higher systemic exposure in the early stages of administration. (References: J Control Release, 2019, 304: 125-134; Eligard® Leuprolide Acetate for Injectable Suspension, (See https: / / www.accessdata.fda.gov / drugsatfda_docs / label / 2007 / 021731s005,021488s010,021379s010,021343s015lbl.pdf). This "burst" initial release can not only trigger dose-related toxicity and affect medication safety, but also cause ineffective drug dissipation, substantially shortening the effective duration of the formulation. Furthermore, to compensate for the initial losses, formulation design often necessitates increasing the drug loading, further increasing potential risks and production costs.

[0006] While existing research attempts to mitigate the burst release of in-situ gels through strategies such as optimizing polymer molecular weight, developing novel solvent systems, or introducing release modifiers, these methods often face challenges such as complex processes and poor batch-to-batch reproducibility. More importantly, current technologies struggle to strike a balance between preserving good injectability and achieving burst release inhibition. Therefore, in the field of biopharmaceutical drug delivery, such as peptides and proteins, developing novel long-acting injectable formulations that combine ultra-low burst release characteristics, stable release behavior, and robust process properties has become an urgent priority to meet the growing clinical demand. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention aims to provide a novel pre-formulation that can significantly reduce the initial burst release of drugs, along with its preparation method and application.

[0008] More specifically, in a first aspect, the present invention provides a pharmaceutical composition comprising the following components: Component I: at least one pharmaceutically acceptable biodegradable polymer, said pharmaceutically acceptable biodegradable polymer comprising block A and block B, wherein block A is a pharmaceutically acceptable water-insoluble biodegradable polymer, block B is a pharmaceutically acceptable hydrophilic substance charged under physiological pH conditions, and block A and block B are linked by amide bonds. Component II: at least one biocompatible solvent; And at least one bioactive substance.

[0009] According to embodiments of the present invention, the composition is a pre-formulation; in some embodiments, the pre-formulation is an injectable in-situ gel; in some embodiments, the composition is used for continuous administration of bioactive substances; in some embodiments, the composition can reduce the initial burst release of bioactive substances.

[0010] According to an embodiment of the present invention, block A is carboxyl-terminated; block B contains at least one amino group; according to an embodiment of the present invention, the amide bond is -C(O)NH-, which is formed by the condensation of the terminal carboxyl group (-COOH) of block A with at least one amino group of block B; According to an embodiment of the present invention, at least one amino group in block B is selected from amino groups that are reactive / active.

[0011] According to an embodiment of the present invention, the amide bond is formed by the condensation of the terminal carboxyl group (-COOH) of block A with one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino groups of block B.

[0012] According to an embodiment of the present invention, block B contains at least one group that is ionizable at physiological pH.

[0013] According to an embodiment of the present invention, the ionizable group is selected from one or more of amino, guanidinyl, imidazole, carboxyl, mercapto, phenolic hydroxyl, etc.

[0014] According to an embodiment of the present invention, block A is a polymer formed from one or more selected from lactic acid, glycolic acid, ε-caprolactone, orthoformate, orthoacetate, and polyols. The polymer may be a homopolymer or a copolymer. According to an embodiment of the present invention, block A is selected from one or more selected from polylactic acid, polyglycolic acid, polylactic-glycolic acid copolymer, and polycaprolactone.

[0015] According to an embodiment of the present invention, the weight-average molecular weight (Mw) of block A is 3.5k~54kDa, more preferably 3.5k~7kDa, 7k~14kDa, 14~24kDa, 24k~38kDa, or 38k~54kDa. kDa, for example, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 20.5, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24.0, 24.5, 25.0, 25.5, 26.0, 26.5, 27 0, 27.5, 28.0, 28.5, 29.0, 29.5, 30.0, 30.5, 31.0, 31.5, 32.0, 32.5, 33.0, 33.5, 34.0, 34.5, 35.0, 35.5, 36.0, 36.5, 37.0, 37.5, 38.0, 38.5, 39.0, 39.5, 40.0, 40.5, 41.0, 41.5, 42.0, 42.5, 43.0, 43.5, 44.0, 44.5, 45.0, 45.5, 46.0, 46.5, 47.0, 47.5, 48.0, 48.5, 49.0, 49.5, 50.0 kDa or any interval defined by any of the above values. In some embodiments, the viscosity of A is in the range of 0.01-1.00 dl·g. -1 For example, values ​​of 0.01, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, and 1.00 dl·g -1 Or any range defined by the above values, preferably 0.08 - 0.16 dl·g -1 0.16-0.24 dl·g -1 0.32 - 0.44 dl·g -1 0.45-0.60 dl·g -1 , 0.14 - 0.22 dl·g -1 0.32 - 0.44 dl·g -1 0.45 - 0.55 dl·g -1 0.25 - 0.35 dl·g -1 .

[0016] According to embodiments of the present invention, the molar ratio of lactic acid to glycolic acid in the polylactic-co-glycolic acid copolymer (PLGA) is from about 50:50 to about 100:1, for example, 50:50, 65:35, 75:25, 85:15. In some embodiments, the polylactic-co-glycolic acid copolymer is carboxyl-terminated. PLGA includes, but is not limited to, models such as PLGA501H, PLGA502H, PLGA503H, PLGA504H, PLGA752H, PLGA653H, and PLGA851H.

[0017] In some embodiments, the polylactic acid (PLA) is carboxyl-terminated, including but not limited to PLA 203H, PLA202H, etc.

[0018] According to an embodiment of the present invention, block B is a charged amino acid, a charged oligopeptide, or a pharmaceutically acceptable salt thereof.

[0019] According to an embodiment of the present invention, the charged oligopeptide is composed of amino acids linked by peptide bonds.

[0020] According to embodiments of the present invention, the amino acids constituting the charged oligopeptide contain at least one charged amino acid. In some embodiments, the amino acids constituting the charged oligopeptide contain a neutral amino acid; in some embodiments, the amino acids constituting the charged oligopeptide do not contain a neutral amino acid.

[0021] According to an embodiment of the present invention, the amino acids in the charged amino acids or charged oligopeptides may be natural amino acids or pharmaceutically acceptable synthetic amino acids.

[0022] According to an embodiment of the present invention, the number of amino acids in the charged oligopeptide is ≥2; preferably, the number of amino acids in the charged oligopeptide is 2 to 10; for example, the number of amino acids in the charged oligopeptide is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0023] According to embodiments of the present invention, the amino group in block B is selected from reactive amino groups, wherein the amino group may be selected from the N-terminal α-amino group (Nα) of block B and / or the amino group of the side chain of an amino acid residue, and more particularly, from the ε-amino group (Nε) of the side chain of an amino acid (e.g., lysine). According to embodiments of the present invention, the amide bond is formed by the condensation of the terminal carboxyl group (-COOH) of block A with one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) amino groups of block B. In some embodiments, when more than one amino group is present in block B, block A may form an amide bond with any one or two or more reactive amino groups in block B.

[0024] According to an embodiment of the present invention, the charged amino acid is selected from histidine (H), lysine (K), arginine (R), glutamic acid (E) or aspartic acid (D).

[0025] According to an embodiment of the present invention, the neutral amino acid is selected from serine (S), threonine (T), asparagine (N), glutamine (Q), tyrosine (Y), cysteine ​​(C), glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), proline (P), phenylalanine (F), or tryptophan (W).

[0026] According to an embodiment of the present invention, the charged oligopeptide contains 2 to 10 amino acids (2, 3, 4, 5, 6, 7, 8, 9, or 10), and at least one of these amino acids is selected from histidine (H), lysine (K), arginine (R), glutamic acid (E), and aspartic acid (D), while the other amino acids are selected from one or more of the listed charged amino acids and neutral amino acids. For example, the charged oligopeptide is selected from KKK, DDD, KDK, KGD, DLD, DDDD, DDDDD, KKKK, and KKKKK.

[0027] According to an embodiment of the present invention, in component I, block A is PLGA and block B is a charged amino acid or a pharmaceutically acceptable salt thereof; or block A is PLA and block B is a charged amino acid or a pharmaceutically acceptable salt thereof; or block A is PLGA and block B is a charged oligopeptide or a pharmaceutically acceptable salt thereof; or block A is PLA and block B is a charged oligopeptide or a pharmaceutically acceptable salt thereof.

[0028] According to an embodiment of the present invention, component I is PLGA-histidine (PLGA-H), PLGA-lysine (PLGA-K), PLGA-arginine (PLGA-R), PLGA-glutamic acid (PLGA-E), PLGA-aspartic acid (PLGA-D), PLA-histidine (PLA-H), PLA-lysine (PLA-K), PLA-arginine (PLA-R), PLA-glutamic acid (PLA-E), PLA-aspartic acid (PLA-D), or PLGA-lysine. PLGA-KKK, PLGA-DDD, PLGA-KDK, PLGA-KGD, PLGA-DLD, PLGA-DDDD, PLGA-KGD, PLGA-DLD, PLGA-DDDD, PLGA-DDDD Aspartic acid-aspartic acid-aspartic acid (PLGA-DDDDD), PLGA-lysine-lysine-lysine-lysine (PLGA-KKKK), PLGA-lysine-lysine-lysine-lysine (PLGA-KKKKK), PLA-lysine-lysine-lysine (PLA-KKK), PLA-aspartic acid-aspartic acid-aspartic acid (PLA-DDD), PLA-lysine-aspartic acid-lysine (PLA-KDK), PLA-lysine-glycine- One or more of the following: aspartic acid (PLA-KGD), PLA-aspartic acid-leucine-aspartic acid (PLA-DLD), PLA-aspartic acid-aspartic acid-aspartic acid-aspartic acid (PLA-DDDD), PLA-aspartic acid-aspartic acid-aspartic acid-aspartic acid (PLA-DDDDD), PLA-lysine-lysine-lysine-lysine-lysine (PLA-KKKK), and PLA-lysine-lysine-lysine-lysine-lysine (PLA-KKKKK).

[0029] In this invention, amino acids are represented using internationally recognized single-letter codes. When the term "oligopeptide" or the fragment "oligopeptide" is represented by a string of letter codes, the string represents a structural unit formed by sequentially connecting the amino acids corresponding to each letter code through peptide bonds, from the N-terminus (α-amino terminus) to the C-terminus (α-carboxyl terminus). The peptide bond is an amide bond (-C(O)NH-) formed by the condensation of the α-carboxyl and α-amino groups of adjacent residues. For example, "DDDDD" represents a pentapeptide formed by five aspartic acid residues sequentially connected by peptide bonds.

[0030] The block A (represented by a polymer abbreviation) - block B (represented by an amino acid letter code string) form represents an example of component I, meaning that block A is selected from the polymer, block B is selected from the amino acid or oligopeptide, and the terminal carboxyl group (-COOH) of block A condenses with at least one amino group of block B to form an amide bond. As defined above, the amino group in block B is selected from reactive / active amino groups, further selected from the N-terminal α-amino group (Nα) of block B and / or the amino group of the amino acid residue side chain, and even further selected from the ε-amino group (Nε) of the amino acid (e.g., lysine) side chain. The amide bond is formed by the condensation of the terminal carboxyl group (-COOH) of block A with one or more (e.g., two, three, four, five, six, seven, eight, nine, ten) amino groups of block B.

[0031] Those skilled in the art will understand that, for example, the term “PLGA-KKK” as defined above refers to a compound formed by the covalent linkage of the terminal carboxyl group of PLGA and trilysine (KKK) via an amide bond (-C(O)NH-). Since KKK contains multiple reactive amino sites (including N-terminal α-amino Nα and three side chain ε-amino Nε1, Nε2, Nε3 located on different residues), the PLGA-KKK can be selected from compounds formed by the carboxyl group of PLGA and any one or more of the reactive amino groups of KKK through an amide bond, for example selected from (1) PLGA connected to the N-terminal α-amino (Nα), i.e. (PLGA-Nα)-KKK, (2) PLGA connected to the first lysine side chain ε-amino (Nε1), i.e. K(ε-PLGA)-KK, (3) PLGA connected to the second lysine side chain ε-amino (Nε2), i.e. KK(ε-PLGA)-K, (4) PLGA connected to the third lysine side chain ε-amino (Nε3), i.e. KKK(ε-PLGA). Similarly, PLA-KKK is a compound formed by the covalent connection of the terminal carboxyl group of PLA and trilysine (KKK) through an amide bond. It can also include compounds formed by any one or more of the reactive amino groups (Nα, Nε1, Nε2, Nε3 amino sites) of PLA and KKK through an amide bond.

[0032] PLGA-KDK is a compound formed by the covalent linkage of the terminal carboxyl group of PLGA and the oligopeptide KDK via an amide bond (-C(O)NH-). KDK contains multiple reactive amino sites, including an N-terminal α-amino group Nα, and side-chain ε-amino groups Nε1 and Nε3 of lysine residues at positions 1 and 3. Therefore, PLGA-KDK can be selected from compounds formed by the linkage of the carboxyl group of PLGA and any one or more of the reactive amino groups of KDK via an amide bond, for example selected from (1) PLGA linked to Nα: (PLGA-Nα)-KDK; (2) PLGA linked to Nε1 of K at position 1: K(ε-PLGA)-DK; (3) PLGA linked to Nε3 of K at position 3: KDK(ε-PLGA). Similarly, PLA-KDK is a compound formed by the covalent connection of the terminal carboxyl group of PLA and the oligopeptide KDK through an amide bond. It can also include compounds formed by any one or more of the reactive amino groups (Nα, Nε1, Nε3 amino sites) of PLA and KDK through an amide bond.

[0033] The PLGA-KGD is a compound formed by the covalent linkage of the terminal carboxyl group of PLGA and the oligopeptide KGD via an amide bond (-C(O)NH-). KGD contains multiple reactive amino sites, including an N-terminal α-amino group (Nα) and a side chain ε-amino group (Nε1) of the lysine at position 1. Therefore, the PLGA-KGD can be selected from compounds formed by the carboxyl group of PLGA and any one or more of the reactive amino groups of KGD via an amide bond, for example, selected from (1) PLGA linked to Nα: (PLGA-Nα)-KGD; (2) PLGA linked to Nε1 at position 1: K(ε-PLGA)-GD. Similarly, the PLA-KGD is a compound formed by the covalent linkage of the terminal carboxyl group of PLA and the oligopeptide KGD via an amide bond, which can also include compounds formed by the amide bond of any one or more of the reactive amino groups (Nα, Nε1 amino sites) of KGD.

[0034] The PLGA-KKKKK is a compound formed by the covalent linkage of PLGA and polylysine (KKKKK) through an amide bond (-C(O)NH-). Since KKKKK contains multiple reactive amino sites, including the N-terminal α-amino group Nα and the five lysine residue side chains ε-amino groups Nε1–Nε5, the PLGA-KKKKK can be selected from compounds formed by the carboxyl group of PLGA and any one or more of the reactive amino groups of KKKKK through an amide bond, for example selected from (1) PLGA linked to Nα: (PLGA-Nα)-KKKKK; (2) PLGA linked to Nε(i) (i=1–5) of any lysine residue: K-…-K(ε-PLGA)-K-…-K (where K with ε-PLGA is the i-th lysine in the sequence). Similarly, PLA-KKKKK is a compound formed by the covalent connection of the terminal carboxyl group of PLA and pentalysine KKKKK through an amide bond. It can also include compounds formed by any one or more of the reactive amino groups (Nα, Nε1, Nε2, Nε3, Nε4, Nε5 amino sites) of PLA and KKKKK through an amide bond.

[0035] According to an embodiment of the present invention, the pharmaceutically acceptable biodegradable polymer in component I has the following structural examples:

[0036]

[0037] According to an embodiment of the present invention, the method for preparing the pharmaceutically acceptable biodegradable polymer includes the following steps: Using carboxyl-protected block B as a raw material, it is mixed and reacted with block A to cause at least one amino group of block B to undergo amidation coupling with the terminal carboxyl group of block A to form an amide bond (-C(O)NH-), thereby obtaining the pharmaceutically acceptable biodegradable polymer.

[0038] When block B contains two or more amino groups (i.e., multiple amidation reaction sites exist), one or more of the amino groups in block B can be selectively amidated and coupled to the terminal carboxyl group of block A. Those skilled in the art, using conventional techniques, can selectively protect the amino groups in block B that are not desired to undergo amidation before reacting them with block A. That is, block B with protected carboxyl groups and optionally protected amino groups can be used as a raw material and mixed with block A to obtain the pharmaceutically acceptable biodegradable polymer.

[0039] In some embodiments, block B, with its carboxyl group protected and its amino group optionally protected, is used as a starting material and mixed with block A to react, causing at least one amino group of block B to undergo amidation coupling with the terminal carboxyl group of block A, forming an amide bond (-C(O)NH-), thereby obtaining the pharmaceutically acceptable biodegradable polymer. The "amino group optionally protected" includes the following: choosing not to protect the amino group in block B; choosing to protect any one amino group in block B; or choosing to protect two or more amino groups (2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0040] According to an embodiment of the present invention, the carboxyl protecting group is selected from benzyl (Bn); the amino protecting group is selected from tert-butoxycarbonyl (Boc) and / or benzyloxycarbonyl (Cbz).

[0041] According to an embodiment of the present invention, the amino group in block B is selected from amino groups that are reactive / active, and further, may be selected from the N-terminal α-amino group (Nα) of block B and / or the amino group of the amino acid residue side chain, and even further, may be selected from the ε-amino group (Nε) of the amino acid (e.g., lysine) side chain.

[0042] According to an embodiment of the present invention, the condensation conditions are as follows: the addition of a catalyst, a base, and an organic solvent; the catalyst is selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBt); the organic solvent is selected from one or more of dichloromethane and N,N-dimethylformamide.

[0043] According to an embodiment of the present invention, based on a total composition mass of 100 wt%, the sum of the contents of each component is 100 wt%, and component I accounts for 0.1 wt% to 30 wt% of the total formulation; more preferably 0.5 wt% to 25 wt%; even more preferably 1 wt% to 20 wt%; for example, component I accounts for 0.1 wt%, 0.2 wt%, 0.5 wt%, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, 21 wt%, 22 wt%, 23 wt%, 24 wt%, 25 wt%, 26 wt%, 27 wt%, 28 wt%, 29 wt%, or 30 wt% of the total formulation.

[0044] According to an embodiment of the present invention, the biocompatible solvent of component II is selected from one or a mixture of alcohols, esters, ketones, amides, sulfoxides.

[0045] According to an embodiment of the present invention, component II is selected from one or more of benzyl alcohol, octanol, ethyl acetate, methyl acetate, ethyl lactate, ethyl benzoate, benzyl benzoate, triacetyl glycerol, tripropionate, tributyrate, acetyl triethyl citrate, triethyl citrate, tributyl citrate, acetyl tributyl citrate, diethyl phthalate, diethyl tartrate, methyl isobutyl ketone, dimethyl sulfoxide, dimethyl acetamide, N-methylpyrrolidone, and N-ethyl-2-pyrrolidone.

[0046] According to an embodiment of the present invention, component II is selected from one or more of benzyl alcohol, benzyl benzoate, ethyl benzoate, triacetylglycerol, glyceryl tribanilate, and acetyl triethyl citrate.

[0047] According to an embodiment of the present invention, based on a total composition mass of 100 wt%, the sum of the contents of each component is 100 wt%, and component II accounts for 40 wt% to 99.8 wt% of the total formulation; preferably 60 wt% to 98 wt%; preferably 65 wt% to 95 wt%; preferably 70 wt% to 90 wt%; for example, 40 wt%, 45 wt%, 50 wt%, 55 wt%, 60 wt%, 65 wt%, 70 wt%, 75 wt%, 80 wt%, 85 wt%, 90 wt%.

[0048] According to an embodiment of the present invention, the bioactive substance is a polypeptide or protein drug with a molecular weight of 1 kDa to 500 kDa. Exemplarily, the biologically active substance has a molecular weight of 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, 200 kDa, 210 kDa, 220 kDa, 230 kDa, 240 kDa, 250 kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, 300 kDa, 310 kDa, 320 kDa, 330 kDa, 340 kDa, 350 kDa, 360 kDa, 370 kDa, 380 kDa, 390 kDa, 400 kDa, 410 kDa, 420 kDa, 430 kDa, 440 kDa, 450 kDa, 460 kDa, 470 kDa, 480 kDa, 490 kDa, 500 kDa, or any interval defined by any of the above values.

[0049] According to an embodiment of the present invention, the bioactive substance is selected from one or more polypeptides or proteins selected from the following therapeutic types: anti-inflammatory drugs, local anesthetics, analgesics, antihypertensive drugs, hypoglycemic drugs, anti-infective drugs (including antibacterial drugs, antiviral drugs, antifungal drugs, etc.), immunomodulatory or immunosuppressive drugs (including anti-rejection drugs), antitumor drugs, antiepileptic or anticonvulsant drugs, hormonal drugs, drugs for treating bone diseases, complement inhibitors, receptor agonists, and hematopoietic growth factors. In some embodiments, the bioactive substance is selected from macromolecular enzymes, such as catalase and glucose oxidase, or from model proteins, such as β-lactoglobulin.

[0050] According to embodiments of the present invention, the bioactive substances are selected from anaraxin, etanercept, tocilizumab, ziconopeptide, cobreptide, nesiritide, adrenaline, adrenocorticotropic hormone, leuprorelin, leuprorelin acetate, goserelin, triptorelin, nafarelin, temorelin, galopeptide (growth hormone neuropeptide), colistin, polymyxin, daptomycin, lactoferrin peptide, insulin, insulin aspart, insulin lispro, insulin glargine, insulin detemir, liraglutide, smegglutide, dulaglutide, telpoxetine, exenatide, benaglutide, abiglutide, canagliflozin, loxenatide, liximab, pralaminide, enfuviride, ibalizumab, interferon-α, interferon-β, interferon-γ, recombinant human granulocyte colony-stimulating factor, trastuzumab, pertuzumab, and liraglutide. Tadalafil, octotuzumab, cetuximab, panitumumab, bevacizumab, nivolumab, pembrolizumab, ipilimumab, trastuzumab emtansine, brentuximab velituximab, detrastuzumab, interleukin-2, octreotide, octreotide acetate, lanreotide, edodetrotide, cyclosporine, vancomycin, bacitracin, micafungin, micaserine, sematralon, human growth hormone and its derivatives Longpex, semapaxitam, recombinant follicle-stimulating hormone β, prolactin, luteinizing hormone, parathyroid hormone, teriparatide, abapatide, salmon calcitonin, vosolide, zeleptokinin, sovatide, recombinant human erythropoietin, pefescine, pemoxatide, dapoxetine α, methoxy polyethylene glycol erythropoietin β (CERA), and one or more of their pharmaceutically acceptable salts, analogues or derivatives.

[0051] According to embodiments of the present invention, the bioactive substance accounts for 0.1 wt% to 40 wt% of the total amount of the formulation; preferably 0.1 wt% to 30 wt%; more preferably 0.5 wt% to 25 wt%; even more preferably 1 wt% to 20 wt%; and even more preferably 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, 21 wt%, 22 wt%, 23 wt%, 24 wt%, 25 wt%, 26 wt%, 27 wt%, 28 wt%, 29 wt%, or 30 wt%.

[0052] According to an embodiment of the invention, the composition further comprises one or more pharmaceutically acceptable excipients.

[0053] According to an embodiment of the present invention, the excipients are selected from one or more of the following: antibacterial agents, buffers, pH adjusters, antioxidants, metal ion chelating agents, and release regulators.

[0054] According to an embodiment of the present invention, based on a total composition mass of 100 wt%, the sum of the contents of each component is 100 wt%, and the pharmaceutically acceptable excipients account for no more than 5 wt% of the total formulation; preferably, the pharmaceutically acceptable excipients account for no more than 2.5 wt% of the total formulation; preferably, the pharmaceutically acceptable excipients account for no more than 1 wt% of the total formulation; preferably, the pharmaceutically acceptable excipients account for no more than 0.5 wt% of the total formulation. For example, the pharmaceutically acceptable excipients account for 0.1 wt%, 0.5 wt%, 1.0 wt%, 1.5 wt%, 2.0 wt%, 2.5 wt%, 3.0 wt%, 3.5 wt%, 4.0 wt%, 4.5 wt%, and 5.0 wt% of the total formulation.

[0055] According to an embodiment of the present invention, the cumulative release of the composition within 24 hours can be effectively controlled at a low level, for example, less than 30%.

[0056] According to an embodiment of the present invention, the bioactive substance is in particulate form, preferably colloidal particles, and can be prepared by methods such as air jet milling, grinding, ball milling, antisolvent precipitation, ultrasonic disruption, and emulsification evaporation.

[0057] According to an embodiment of the present invention, the particle size of the bioactive substance particles is 1 nm-20 μm, preferably 1 nm-15 μm, for example 1 nm, 10 nm, 50 nm, 100 nm, 150 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm.

[0058] According to embodiments of the present invention, the particle size of the bioactive substance in the composition is 1 nm-1000 nm; preferably, the particle size of the bioactive substance is ≤900 nm; in some embodiments, the particle size of the bioactive substance is ≤900 nm, ≤850 nm, ≤800 nm, ≤750 nm, ≤700 nm, ≤650 nm, ≤600 nm, ≤550 nm, ≤500 nm, ≤450 nm, ≤400 nm, ≤350 nm, ≤300 nm, ≤250 nm, ≤200 nm, ≤150 nm, ≤100 nm, ≤50 nm or ≤10 nm.

[0059] On the other hand, the present invention also relates to a method for preparing the composition described in any of the above claims, which, according to an embodiment of the present invention, includes the following steps: (i) Dissolve the biodegradable polymer of component I in the biocompatible solvent to obtain a clear and homogeneous solution; (ii) The bioactive substance is added to the solution of step (i) and stirred or homogenized until the bioactive substance is completely dissolved or uniformly dispersed to obtain the composition; According to an embodiment of the present invention, in the preparation method, optionally, pharmaceutically acceptable excipients are further added to the solution of step (i).

[0060] In some embodiments, in step (ii), the bioactive substance is in particulate form, preferably colloidal particles; the bioactive substance particles can be prepared by methods such as air jet milling, grinding, ball milling, antisolvent precipitation, ultrasonic disruption, emulsification and evaporation.

[0061] The particle size of the bioactive substance is 1 nm-20 μm, preferably 1 nm-15 μm, for example, 1 nm, 10 nm, 50 nm, 100 nm, 150 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm.

[0062] On the other hand, the present invention also provides a pharmaceutically acceptable biodegradable polymer comprising block A and block B, wherein block A is a pharmaceutically acceptable water-insoluble biodegradable polymer, and block B is a pharmaceutically acceptable hydrophilic substance charged under physiological pH conditions, wherein block A and block B are covalently linked by amide bonds. Block A and block B are as defined in the first aspect above; the pharmaceutically acceptable biodegradable polymer is as defined in the first aspect.

[0063] According to embodiments of the present invention, the composition is administered topically or by injection.

[0064] According to embodiments of the present invention, the composition of the present invention can be administered subcutaneously, intramuscularly, intra-articularly, intra-periodontally, or directly to a wound.

[0065] According to embodiments of the present invention, the composition can be administered by spraying, dipping, rinsing, applying, or dripping; or the composition can be applied by a dotting applicator or a roller applicator.

[0066] According to embodiments of the present invention, the composition of the present invention is administered in a single dose or in multiple doses.

[0067] According to embodiments of the present invention, the composition of the present invention can provide sustained and effective treatment for several days, weeks, or months after application.

[0068] According to an embodiment of the present invention, the present invention provides a formulation, the formulation further comprising a packaging material filled with the composition, the packaging material being selected from one or more of the following: vials, pre-filled syringes, and cartridges.

[0069] On the other hand, the present invention provides the use of the pharmaceutically acceptable biodegradable polymer in the preparation of a medicament. According to embodiments of the invention, the medicament is a pre-formulation, and in some embodiments, is an injectable in-situ gel; in some embodiments, the pharmaceutical composition is used for sustained administration of a bioactive substance; in some embodiments, the pharmaceutical composition can reduce the initial burst release of the drug.

[0070] On the other hand, the present invention also relates to the use of the compositions described in any of the foregoing in the preparation of a medicament for the continuous administration of a bioactive substance.

[0071] Terms and Abbreviations Unless otherwise stated, the definitions of groups and terms recorded in this application specification and claims, including their definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, and definitions of specific compounds in the examples, can be arbitrarily combined and combined with each other. Such combinations and combinations of group definitions and compound structures shall fall within the scope of this application specification.

[0072] The numerical values ​​recorded in this application specification and claims, when the range of the numerical values ​​is defined or can only be "integers", should be understood to record the two endpoints of the range and every integer within the range. For example, "integers from 0 to 10" should be understood to record every integer of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0073] When a range of values ​​is defined as a "number" or may include "integers" or "non-integers," it should be understood as recording the two endpoints of the range, every integer within the range, and every decimal within the range. For example, "integers from 0 to 10" should be understood as recording not only every integer of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, but also at least the sum of each of these integers with 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9.

[0074] The term "charged amino acid" refers to an amino acid whose side chain (R group) can ionize under physiological pH conditions, thereby carrying a net positive or negative charge. Those skilled in the art will understand that the charged amino acid includes histidine (H), lysine (K), arginine (R), glutamic acid (E), or aspartic acid (D).

[0075] The term "neutral amino acid" refers to an amino acid whose side chain does not ionize and has a net charge of zero at physiological pH (but some side chains may be polar). Those skilled in the art will understand that neutral amino acids are selected from serine (S), threonine (T), asparagine (N), glutamine (Q), tyrosine (Y), cysteine ​​(C), glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), proline (P), phenylalanine (F), or tryptophan (W).

[0076] The term "pre-formulation" refers to a liquid or semi-solid composition of polymers, biocompatible organic solvents, and bioactive substances that remains flowable before injection and transforms into a solid or semi-solid implant after injection into the body. Therefore, "pre-formulation" and "precursor solution" have the same meaning and are used interchangeably.

[0077] The term "injectable in-situ gel" refers to a solid or semi-solid drug reservoir with sustained-release function that forms in situ under physiological conditions through mechanisms such as solvent diffusion and phase transition after the pre-formulation is injected into a specific site in the body. Therefore, "injectable in-situ gel" and "in-situ gel" have the same meaning and can be used interchangeably.

[0078] The term "biodegradable" refers to the property of organic matter that, under natural or artificially controlled conditions, can be decomposed and transformed into small molecules such as carbon dioxide or water through the life processes of organisms.

[0079] The term "bioactive substance" refers to a compound or biological macromolecule that has the function of preventing, diagnosing, alleviating, or treating diseases. In this invention, "bioactive substance," "active ingredient," and "drug" have the same meaning and can be used interchangeably.

[0080] The term "water-insoluble" refers to a substance whose equilibrium solubility in deionized water is less than 1 mg / mL at 25 °C. -1 .

[0081] In this invention, the term "particle size," when used to describe a bioactive substance, refers to the particle size of the bioactive substance dispersed in a preformulation as solid particles. This size refers to the intensity-based Z-mean particle size measured by dynamic light scattering (DLS) in the biocompatible organic solvent environment. Unless otherwise stated, all particle sizes described in this invention are measured under these conditions. The average particle size described in this invention is measured within 24 hours after formulation preparation.

[0082] The term "initial burst release" refers to the rapid, uncontrolled over-release of a drug that occurs in the early stages after administration (usually within 24 hours).

[0083] The term "physiological pH" refers to pH 7.4 ± 0.2. Furthermore, the physiological pH value corresponds to the physiological pH value of human tissue fluid, which is approximately 7.35 to 7.45.

[0084] The abbreviations used in this invention have the following definitions: PLA: Polylactic acid, including carboxyl-terminated polylactic acid PLAH and ester-terminated polylactic acid PLAS; PGA: Polyglycolic acid; PLGA: Polylactic acid-glycolic acid copolymer; D, DDD, DDDD, DDDDD: Aspartic acid and oligomers; K, KKK, KKKK, KKKKK: Lysine and oligomers; H: Histidine; R: Arginine; E: Glutamic acid; S: Serine; T: Threonine; N: Asparagine; Q: Glutamine; Y: Tyrosine; C: Cysteine; G: Glycine; A: Alanine; V: Valine; L: Leucine; I: Isoleucine; M: Methionine; P: Proline; F: Phenylalanine; W: Tryptophan; AUC: Area under the curve during drug administration; C max Peak blood drug concentration.

[0085] Beneficial effects (1) The inventors unexpectedly discovered during the formulation development process that by using the biodegradable polymer of the present invention and dispersing it together with bioactive substances in a biocompatible solvent, a high-performance pre-formulation system can be constructed, thereby effectively solving the technical problem of severe burst release of drugs in the early stage of traditional in-situ molded pre-formulations.

[0086] Experimental data show that, using the preformulation of this invention, the cumulative release of the drug within 24 hours can be effectively controlled at a low level (less than 30%), which is far superior to the unmodified polymer control group.

[0087] (2) The formulation system of the present invention can also greatly improve the colloidal stability of bioactive substance particles during pre-formulation storage, effectively prevent particle aggregation, sedimentation or growth, and ensure the physicochemical stability of the formulation and the uniformity of the dosage during the shelf life. This stability further consolidates the reliability of its reduced burst release effect.

[0088] Compared to the passive approach of traditional formulations that involve "injection first, followed by in-situ barrier formation," this invention achieves an active approach of "pre-constructing an interfacial barrier within the pre-formulation before injection." During the process of the barrier gradually improving, densifying, and ultimately continuously constructing from the initial barrier layer after injection, drug release can be maintained at a controlled low-flux state, effectively suppressing initial burst release. This provides a new technical pathway for achieving ultra-low burst release, or even near-zero burst release, in long-acting injectable formulations.

[0089] (3) The formulation system of the present invention does not require changes to the basic structure of the prescription system. By simply adjusting the proportion of biodegradable polymers added, the drug release behavior can be flexibly adjusted over a wide range. This characteristic is beneficial for designing personalized long-term dosing regimens with differentiated release cycles based on the therapeutic needs of different bioactive substances and the target blood drug concentration characteristics, demonstrating good prescription controllability and application flexibility.

[0090] (4) The formulation system of the present invention exhibits good process robustness in terms of drug particle size, and can maintain stable diffusion behavior under a wide particle size distribution, thereby reducing the requirements for strict control of particle size during formulation development and production. This characteristic is beneficial to improving the process window of formulation design and providing support for achieving stable batch-to-batch consistency under large-scale production conditions, demonstrating good potential for industrial application. Attached Figure Description

[0091] Figure 1 The in vitro drug diffusion curves for formulations 1-3 in Example 1 are shown. Figure 2 Transmission electron microscope images of the adsorption layer on the surface of drug particles in Formulations 4 and 5 of Example 1. Figure 3 Zeta potential of drug particles in Formulations 4 and 5 of Example 1 Figure 4 To study the particle size changes of insulin preformulations with different polymer contents (Formulations 3, 5-8) in Example 1 during long-term storage. Figure 5Schematic diagram of the operation of a dissipative quartz crystal microbalance and Example 1: Polymer adsorption on the surface of insulin particles Figure 6 Zeta potential for preparing the biodegradable polymer of Example 1 Figure 7 The in vitro drug diffusion curves for formulations 9-12 in Example 2 are shown. Figure 8 The in vitro drug diffusion curves for formulations 13-16 in Example 3 are shown. Figure 9 The in vitro drug diffusion curves for formulations 17, 3, and 18 in Example 4 are shown. Figure 10 The in vitro drug diffusion curves for formulations 19-22 in Example 5 are shown. Figure 11 The in vitro drug diffusion curves for formulations 23-25 ​​and formulation 3 in Example 6 are shown. Figure 12 The in vitro drug diffusion curves for formulations 26-27, 3, 5, 7-8, and 28-29 in Example 7 are shown. Figure 13 The in vitro drug release curves for formulations 27, 3, 5, and 7 in Example 7 are shown. Figure 14 The in vitro drug diffusion curves for formulations 6 and 30-33 in Example 8 are shown. Figure 15 The in vitro drug diffusion curves for formulations 34-38 in Example 9 are shown. Figure 16 The in vitro drug diffusion curves for formulations 39-45 in Example 10 are shown. Figure 17 The in vitro drug diffusion curves for formulations 46-49 in Example 11 are shown. Figure 18 The in vitro drug diffusion curves for formulations 50-53 in Example 12 are shown. Figure 19 The in vitro drug diffusion curves for formulations 54-59 in Example 13 are shown. Figure 20 The in vitro drug diffusion curves for formulations 60-65 in Example 14 are shown. Figure 21 The in vitro drug diffusion curves for formulations 66-71 in Example 15 are shown. Figure 22 The in vitro drug diffusion curves for formulations 72-79 in Example 16 are shown. Figure 23 The in vitro drug diffusion curves for formulations 80-83 in Example 17 are shown. Figure 24 The in vitro drug diffusion curves for formulations 84-87 of Example 18 are shown. Figure 25The in vitro drug diffusion curves for formulations 88-91 in Example 19 are shown. Figure 26 The in vitro drug diffusion curves for formulations 92-96 of Example 20 are shown. Figure 27 The in vitro drug diffusion curves for formulations 97-100 in Example 21 are shown. Figure 28 The in vitro drug diffusion curves for formulations 101-104 in Example 22 are shown. Figure 29 The in vitro drug diffusion curves for formulations 105-108 in Example 23 are shown. Figure 30 The curves showing the changes in plasma concentration over time in rats after subcutaneous injection of insulin solution and in the control group, as shown in Example 24. Figure 31 Example 24: Plasma concentration changes over time for rats after subcutaneous injection of prescriptions 3, 6, 5, 7, and 8. Figure 32 The graphs showing the changes in blood glucose levels over time in rats after subcutaneous injection of the control group and prescriptions 3, 6, 5, 7, and 8 in Example 24 are shown. Figure 33 The curves showing the changes in plasma concentration over time in rats after subcutaneous injection of exenatide solution and in the control group in Example 25 are shown. Figure 34 Example 25: Plasma concentration change over time for rats after subcutaneous injection of formulation 109-111. Figure 35 The graphs show the changes in blood glucose levels over time in rats after subcutaneous injection of the control group and prescription 109-111 in Example 25. Figure 36 The curves showing the changes in plasma concentration over time in rats after subcutaneous injection of telpoprit solution and in the control group in Example 26 are shown. Figure 37 The curves showing the change in plasma concentration over time for rats after subcutaneous injection of formulations 74, 75, 112, and 113 in Example 26 are shown. Figure 38 The curves showing the changes in plasma concentration over time in rats subcutaneously injected with goserelin solution and in the control group in Example 27 are shown. Figure 39 Example 27: Plasma concentration change over time after subcutaneous injection of formulation 102 in rats. Detailed Implementation

[0092] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0093] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0094] Unless otherwise stated, percentages (%) in a prescription are understood as weight percentages (wt%).

[0095] Experimental equipment and measurement methods: 1. Drug diffusion experiment Slowly inject 100 µL of the injectable in-situ gel preformed preparation prepared in this invention into the bottom of a quartz dish containing 2 mL of deionized water, so that it covers the bottom of the dish. Then place the dish in a shaker at 37 °C and shake at 150 rpm. Take 0.2 mL of deionized water at 1, 2, 4, 6, 12 and 24 h and use high performance liquid chromatography to detect the mass of drug molecules transferred from the triacetylglycerol phase to the aqueous phase.

[0096] 2. In vitro release experiment Add approximately 50 µL of the injectable in-situ gel preformulation prepared in this invention to 2 mL of deionized water, place it on a shaker at 37°C and shake (speed set to 150 rpm). Take out all the deionized water each day and add 2 mL of fresh deionized water. Detect the drug concentration and calculate the cumulative drug release percentage using high performance liquid chromatography.

[0097] 3. Particle size distribution determination Equipment: Nanoparticle size and Zeta potential analyzer (Zetasizer Ultra, Malvern Panacol, UK) Assay method: The pre-formulation was diluted to 0.2 mg / mL using triacetylglycerol as the dispersion solvent. -1 Take 1 mL of the sample and place it in the universal dip cell kit provided with the Malvern particle size analyzer to determine the particle size distribution. The specific measurement conditions are as follows: the sample temperature is set to 25 ℃, the material property is selected as protein, the dispersion medium is triacetylglycerol, the detection angle is set to 173°, and the other parameters are set to the instrument's recommended default settings.

[0098] 4. Measurement of Zeta potential on particle surface Equipment: Nanoparticle size and Zeta potential analyzer (Zetasizer Ultra, Malvern Panacol, UK) Assay method: The pre-formulation was diluted to 0.2 mg / mL using triacetylglycerol as the dispersion solvent. -1Take 1 mL of the sample and place it in the universal dip cell kit provided with the Malvern particle size analyzer. Measure the zeta potential of the particle surface. The specific measurement conditions are as follows: the sample temperature is set to 25 °C, the material property is selected as protein, the dispersion medium is triacetylglycerol, the applied voltage is 10 mV, and the other parameters are set to the instrument's recommended default settings.

[0099] 5. Methods for evaluating the electronegativity of polymers Equipment: Nanoparticle size and Zeta potential analyzer (Zetasizer Ultra, Malvern Panacol, UK) Weigh 10 mg of polymer powder and dissolve it in 200 μL of dimethyl carbonate. Then, add 300 μL of PBS aqueous solution and perform ultrasonic emulsification with a probe (30 s, 30% amplitude). Using PBS aqueous solution as the dispersion solvent, take 10 μL of the emulsion and add it to 990 μL of PBS solution.

[0100] Measurement method: Take 1 mL of sample and place it in the universal dip cell kit equipped with the Malvern particle size analyzer. Measure the relative Zeta potential of the polymer. The specific measurement conditions are as follows: the sample temperature is set to 25 ℃, the material property is selected as polymer, the dispersion medium is PBS solution, the applied voltage is 10 mV, and the other parameters are set to the instrument's recommended default settings.

[0101] 6. Method for determining the mass of polymer adsorption on the surface of drug particles Equipment: Dissipative Quartz crystal microbalance (QSense Pro, Biolin Scientific, Sweden) Assay method: Weigh appropriate amounts of polymer powder and add triacetin to prepare a solution with a concentration of 1.0 mg / mL. -1 Solution. Place the blank gold-coated chip in the flow cell of the metal module, and equilibrate with air to obtain the frequency of the blank chip. Then remove the chip, place it in the center of the spin coater disk, and fix it under vacuum. 2.0 mg / mL -1 A concentrated drug colloidal particle dispersion (using dimethyl carbonate, which meets the requirements of good dispersion, low viscosity, and easy volatility for spin coating) was dropped onto the center of the chip. The spin coater was set to 4000 rpm, with 5 μL added at a time. Spin coating was performed at 4000 rpm for 30 seconds, repeated four times. After all drops were added, the chip was rotated at 4000 rpm for 100 seconds to ensure the dimethyl carbonate evaporated, resulting in a chip with drug colloidal particles spin-coated on its surface. The chip was placed in a flow cell of a metal module, and the oscillation frequency of channel 3 was recorded. Subsequently, pure triacetyl ester was introduced to equilibrate the baseline (the slope of the oscillation frequency within 10 min < 3 Hz·min). -1(This can be considered as reaching equilibrium). After equilibrium is reached, 1.0 mg / mL of the solution is added separately. -1 The polymer solutions were dispensed at 0.35 mL / min. -1 A flow rate was passed through a chip spin-coated with colloidal particles. After 20 minutes, pure triacetyl ester solvent was introduced again to remove non-specifically adsorbed free polymer molecules on the chip surface. The decrease in frequency compared to the equilibrium baseline was recorded, and the adsorbed weight of polymer on the surface of the colloidal particles was calculated.

[0102] 7. Transmission electron microscopy characterization Equipment: Transmission electron microscope (HT7800, Hitachi High Technology Corporation) Assay method: The pre-formulation was diluted to 2 mg / mL using triacetylglycerol as the dispersion solvent. -1 Next, 5 μL of the sample was deposited onto a carbon-coated copper grid, and excess sample was blotted off after 5 min. Then, 10 μL of 2% phosphotungstic acid staining solution was added, and after staining for 5 min, excess staining solution was blotted off. Then, 10 μL of water was added to the copper grid to wash off excess phosphotungstic acid staining solution. Finally, the copper grid was dried and imaging was performed. The acceleration voltage was 120 kV.

[0103] Unless otherwise stated, in vitro experiments were performed in three independent batches, each in parallel; results are expressed as mean ± standard deviation.

[0104] Preparation Example 1. Preparation method of biodegradable polymer block A-block B 1. Preparation of PLGA-DDD:

[0105] Step 1: Preparation of (S)-4-(benzyloxy)-2-((tert-butoxycarbonyl)amino)-4-oxobutyryl-L-aspartic acid dibenzyl ester (S)-4-(benzyloxy)-2-((tert-butoxycarbonyl)amino)-4-oxobutyric acid (11.1 g, 1.0 eq), L-aspartic acid dibenzyl ester hydrochloride (12.0 g, 1.0 eq), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (9.86 g, 1.5 eq), 1-hydroxybenzotriazole (6.95 g, 1.5 eq), triethylamine (3.46 g, 1.0 eq), and dichloromethane (110 mL, 10 V) were added sequentially to a reaction flask, and the mixture was stirred overnight at room temperature. The reaction solution was washed sequentially with 0.5 M dilute hydrochloric acid (55 mL, 5 V), NaHCO3 solution (55 mL, 5 V), and water (55 mL, 5 V). The organic phase was concentrated and purified by column chromatography to obtain the product.

[0106] Step 2: Preparation of dibenzyl((S)-2-amino-4-(benzyloxy)-4-oxobutyryl)-L-aspartic acid (S)-4-(benzyloxy)-2-((tert-butoxycarbonyl)amino)-4-oxobutyryl-L-aspartic acid dibenzyl ester (12.2 g, 1.0 eq) was added to 120 mL of 2 M ethyl hydrogen chloride solution, stirred for 2–3 h, concentrated, and 70 mL of methyl tert-butyl ether was slowly added to the residue. The mixture was stirred for 30 min, filtered, and the filter cake was washed with methyl tert-butyl ether. The solid was dried under vacuum at room temperature to obtain the product.

[0107] Step 3: Preparation of dibenzyl((S)-4-(benzyloxy)-2-((S)-4-(benzyloxy)-2-((tert-butoxycarbonyl)amino)-4-oxobutamido)-4-oxobutyryl)-L-aspartic acid (S)-4-(benzyloxy)-2-((tert-butoxycarbonyl)amino)-4-oxobutyric acid (5.56 g, 1.0 eq), dibenzyl((S)-2-amino-4-(benzyloxy)-4-oxobutyryl)-L-aspartic acid (9.55 g, 1.0 eq), EDCI (3.95 g, 1.2 eq), HOBt (2.79 g, 1.2 eq), triethylamine (1.74 g, 1.0 eq), and dichloromethane (100 mL, 10 V) were added sequentially to a reaction flask, and the mixture was stirred overnight at room temperature. The reaction solution was washed sequentially with 0.5 M dilute hydrochloric acid (50 mL, 5 V), NaHCO3 solution (50 mL, 5 V), and water (50 mL, 5 V). The organic phase was concentrated and purified by column chromatography to obtain the product.

[0108] Step 4: Preparation of dibenzyl((S)-2-((S)-2-amino-4-(benzyloxy)-4-oxobutyramido)-4-(benzyloxy)-4-oxobutyryl)-L-aspartic acid Dibenzyl((S)-4-(benzyloxy)-2-((S)-4-(benzyloxy)-2-((tert-butoxycarbonyl)amino)-4-oxobutamido)-4-oxobutyryl)-L-aspartic acid (9.0 g, 1.0 eq) was added to 90 mL of 2M hydrogen chloride in ethyl acetate solution. The mixture was stirred at room temperature for 1.0 h. After concentrating the reaction solution, 70 mL of methyl tert-butyl ether was slowly added, and stirring was continued for 1–2.0 h. The mixture was filtered, and the filter cake was washed with methyl tert-butyl ether and dried under vacuum at room temperature to obtain the product.

[0109] Step 5: Preparation of PLGA-DDD ester Dibenzyl((S)-2-((S)-2-amino-4-(benzyloxy)-4-oxobutamido)-4-(benzyloxy)-4-oxobutyryl)-L-aspartic acid (2.03 g, 2.6 eq), PLGA (14.5 g, 1.0 eq), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (391 mg, 2.0 eq), 1-hydroxybenzotriazole (275.6 mg, 2.0 eq), triethylamine (257 mg, 2.5 eq), and dichloromethane (217 mL, 15 V) were added sequentially to a reaction flask and stirred overnight at room temperature. The reaction mixture was washed sequentially with 0.5 M dilute hydrochloric acid (110 mL), sodium bicarbonate aqueous solution (110 mL), and water (110 mL). After concentration of the organic phase, the product was purified by column chromatography to obtain the PLGA-DDD ester product.

[0110] Step 6: Preparation of PLGA-DDD 12.0 g of PLGA-DDD ester, 1.0 g of Pd / C, and 180 mL of dichloromethane (15 V) were added sequentially to a reaction flask, and hydrogenated at room temperature and atmospheric pressure for 12 h. The reaction solution was filtered through a diatomaceous earth filter. 1.0 g of mercaptosilica gel was added to the filtrate, and the mixture was stirred for 1.0 h and then filtered. The filtrate was washed twice with 50 mL of water, the organic phase was concentrated, and the product was analyzed by HPLC to obtain the PLGA 502H-DDD product.

[0111] 2. Detection method for PLGA-DDD: (1) Detection of aspartic acid Chromatographic conditions: Column: Agela Venusil XBP C18(L) 4.6×250 mm, 5 μm, or a column with equivalent column efficiency. Mobile phase A: 0.1% perchloric acid aqueous solution Mobile phase B: Acetonitrile-methanol (1:4) Flow rate: 1.0 mL·min -1 Column temperature: 30 ℃ Wavelength: 330 nm Injection volume: 5 μL gradient:

[0112] Solution preparation: FDAA (DMF) solution: Take an appropriate amount of 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA), dissolve it in DMF and dilute it quantitatively to prepare a solution containing approximately 5 mg per 1 mL.

[0113] Reference solution: Accurately weigh approximately 10 mg of aspartic acid reference standard, place it in a 20 mL volumetric flask, and add 6 mol·L⁻¹ -1 Dissolve in 0.5 mL of hydrochloric acid solution, then use 6 mol·L⁻¹ -1 Adjust the pH to neutral with sodium hydroxide solution, dilute to the mark with water, and mix well. Accurately measure 1.0 mL of the above solution into a 10 mL volumetric flask, dilute to the mark with water, and mix well to obtain the reference solution (aspartic acid concentration approximately 0.05 mg / mL). -1 ).

[0114] Test solution (hydrolyzed sample): Accurately weigh approximately 20 mg of this product and place it in a 10 mL volumetric flask. Add 6 mol·L⁻¹ -1 Dissolve the sample in 1 mL of hydrochloric acid solution, place it in an oven at 110 °C for 24 h, remove it, cool it to room temperature, and then use 6 mol·L⁻¹ hydrochloric acid solution. -1 Adjust the pH to neutral with sodium hydroxide solution, dilute to the mark with water, and mix well. (The concentration of the test sample is approximately 2 mg / mL) -1 ) Test solution (unhydrolyzed sample): Accurately weigh approximately 15 mg of the test solution and place it in a 2 mL volumetric flask. Add 0.5 mL of DMF and sonicate to dissolve. Dilute to volume with water, shake well, and centrifuge (12000 rpm, 10 min). Collect the supernatant. (The concentration of the test solution is approximately 5 mg / mL.) -1 ) Derivatization process: Derivatization blank: Accurately measure 1 mL of water and place it in a 5 mL volumetric flask. Add 1 mL of FDA solution and 1 mL of 4.2% sodium bicarbonate aqueous solution sequentially. Shake well and incubate in a 60 °C water bath for 1 h. After cooling, add 0.4 mol·L⁻¹ -1 Dilute 1 mL of hydrochloric acid solution to the mark with acetonitrile, shake well, filter, and collect the filtrate.

[0115] Take the reference solution, the test solution (unhydrolyzed sample), and the test solution (hydrolyzed sample) and perform the same procedure as the derivatization blank.

[0116] (2) Detection of PLGA-DDD Chromatographic conditions: Column: Agela Vensil ASB C 18 4.6 × 250 mm, 5 μm or equivalent column efficiency Mobile phase A: pH 2.5, 10 mM KH2PO4 buffer Mobile phase B: Acetonitrile Flow rate: 1.0 mL·min -1 Column temperature: 25 ℃ Wavelength: 210 nm Injection volume: 10 μL gradient:

[0117] Solution preparation: Reference solution: Accurately weigh an appropriate amount of aspartic acid tripeptide reference standard, dissolve it in methanol, and quantitatively dilute it to prepare a solution containing approximately 0.3 mg per mL. Accurately measure 1 mL of this solution into a 10 mL volumetric flask and dilute with methanol-mobile phase A. (1:9) Dilute to the mark and mix well. (0.03 mg·mL) -1 ) Test solution: Accurately weigh approximately 60 mg of the test sample and place it in a 2 mL volumetric flask. Add 0.5 mL of acetonitrile, sonicate for 15 minutes to dissolve, and dilute to the mark with methanol-mobile phase A (1:9). Shake well, centrifuge (12000 rpm, 10 minutes), and collect the supernatant. (30 mg·mL⁻¹) -1 ).

[0118] 3. Preparation of K(ε-PLGA)-KK in PLGA-KKK-like structures:

[0119] Step 1: Preparation of benzyl N6-((benzyloxy)carbonyl)-N2-(N6-((benzyloxy)carbonyl)-N2-(tert-butoxycarbonyl)-L-lysine salt N6-(benzyloxycarbonyl)-L-lysine benzyl ester hydrochloride (10.0 g, 1.0 eq), N6-(benzyloxycarbonyl)-N2-(tert-butyloxycarbonyl)-L-lysine (9.35 g, 1.0 eq), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (5.65 g, 1.2 eq), 1-hydroxybenzotriazole (4.0 g, 1.2 eq), triethylamine (2.49 g, 1.0 eq), and dichloromethane (100 mL, 10 V) were added together and stirred overnight at room temperature. The reaction mixture was washed successively with 0.5 M dilute hydrochloric acid (50 mL, 5 V), NaHCO3 solution (50 mL, 5 V), and water (50 mL, 5 V). The organic phase was concentrated to give an oily substance.

[0120] Step 2: Preparation of benzyl N6-((benzyloxy)carbonyl)-N2-(N6-((benzyloxy)carbonyl)-L-lysine salt)-L-lysine salt 10.0 g, 1.0 eq) of benzyl N6-((benzyloxy)carbonyl)-N2-(N6-((benzyloxy)carbonyl)-N2-(tert-butoxycarbonyl)-L-lysine salt was dissolved in 50 mL of ethyl acetate, and 4 M ethyl hydrochloride solution was added and stirred for 2-3 h. A solid precipitated out, which was filtered, washed with ethyl acetate from the filter cake, and dried under vacuum at room temperature to obtain a white solid.

[0121] Step 3: Preparation of N6-((benzyloxy)carbonyl)-N2-(N6-((benzyloxy)carbonyl)-N2-(N2-((benzyloxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysyl)-L-lysine benzyl ester Benzyl N6-((benzyloxy)carbonyl)-N2-(N6-((benzyloxy)carbonyl)-L-lysyl)-L-lysine salt (3.0 g, 1.0 eq), N2-benzyloxycarbonyl-N6-tert-butoxycarbonyl-L-lysine (1.7 g, 1.0 eq), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.03 g, 1.2 eq), 1-hydroxybenzotriazole (729 mg, 1.2 eq), triethylamine (453 mg, 1.0 eq), and dichloromethane (30 mL, 10 V) were added sequentially to the reaction flask, and the mixture was stirred at room temperature for 4–5 h. The reaction solution was washed sequentially with 0.5 M dilute hydrochloric acid (15 mL, 5 V), NaHCO3 solution (15 mL, 5 V), and water (15 mL, 5 V). After concentration of the organic phase, the product was purified by column chromatography.

[0122] Step 4: Preparation of benzyl N6-((benzyloxy)carbonyl)-N2-(N6-((benzyloxy)carbonyl)-N2-(((benzyloxy)carbonyl)-L-lysyl)-L-lysine salt Add 50 mL of 2N ethyl hydrochloride solution to N6-((benzyloxy)carbonyl)-N2-(N6-((benzyloxy)carbonyl)-N2-(N2-((benzyloxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysyl)-L-lysine benzyl ester (4.8 g, 1.0 eq), stir for 2-3 h to precipitate a solid, filter, wash the filter cake with ethyl acetate, and dry under vacuum at room temperature to obtain a white solid.

[0123] Step 5: Preparation of K(ε-PLGA)-KK ester The following were added sequentially to the reaction flask: benzyl N6-((benzyloxy)carbonyl)-N2-(N6-((benzyloxy)carbonyl)-N2-(((benzyloxy)carbonyl)-L-lysyl)-L-lysine salt (2.82 g, 2.5 eq), PLGA (17.2 g, 1.0 eq), EDCI (463.9 mg, 2.0 eq), HOBt (326.9 mg, 2.0 eq), triethylamine (306.3 mg, 2.5 eq), dichloromethane (250 mL, 15 V), and... N,N -Dimethylformamide (25 mL, 1.5 V), stirred overnight at room temperature. The reaction solution was washed successively with 0.5 M dilute hydrochloric acid (125 mL, 7.5 V), sodium bicarbonate aqueous solution (125 mL, 7.5 V), and water (125 mL, 7.5 V). The organic phase was concentrated and purified by column chromatography to obtain K(ε-PLGA)-KK ester.

[0124] Step 6: Preparation of K(ε-PLGA)-KK 19.0 g of K(ε-PLGA)-KK ester, 4.75 g of Pd / C, and 700 mL of dichloromethane were added sequentially to a reaction flask and hydrogenated at room temperature and atmospheric pressure for 36 h. The mixture was filtered through diatomaceous earth, and 3.0 g of Pd / C was added to the filtrate. Hydrogenation was continued at atmospheric pressure for another 12 h. This process was repeated once to confirm complete reaction. 10.0 g of diatomaceous earth was added to the reaction solution, and the mixture was stirred for 30 min before filtration. Approximately 10.0 g of mercaptosilica gel was added to the filtrate, and the mixture was stirred for 30 min before filtration through diatomaceous earth. The filtrate was filtered three times through a polytetrafluoroethylene membrane (0.45 μm pore size), concentrated, dried under vacuum, and analyzed by HPLC to obtain K(ε-PLGA)-KK.

[0125] 4. PLGA-KKK testing method: Chromatographic conditions: Column: Agela Venusil ASB C18, 4.6 × 250 mm 5 µm or equivalent column. Mobile phase A: 0.1% perchloric acid aqueous solution Mobile phase B: Methanol Flow rate: 1.0 mL·min -1 Column temperature: 30 ℃ Wavelength: 330 nm Injection volume: 5 μL gradient:

[0126] Solution preparation FDAA solution: Take an appropriate amount of 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA), dissolve it in DMF and dilute it quantitatively to prepare a solution containing approximately 5 mg per 1 mL.

[0127] Reference solution: Take an appropriate amount of lysine reference standard, accurately weigh it, dissolve it in water and dilute it quantitatively to prepare a solution containing about 0.05 mg per 1 mL.

[0128] Test solution (hydrolyzed sample): Accurately weigh approximately 20 mg of this product and place it in a 10 mL volumetric flask. Add 6 mol·L⁻¹ -1 Dissolve the sample in 1 mL of hydrochloric acid solution, place it in an oven at 110 °C for 24 h, remove it, cool it, and then use 6 mol·L⁻¹ hydrochloric acid solution. -1 Adjust the pH to neutral with sodium hydroxide solution, dilute to the mark with water, and mix well. (The concentration of the test sample is approximately 2 mg / mL) -1 ) Test solution (unhydrolyzed sample): Accurately weigh approximately 15 mg of the test solution and place it in a 2 mL volumetric flask. Add 0.5 mL of DMF and sonicate to dissolve. Dilute to the mark with water, shake well, and centrifuge (12000 rpm, 10 minutes). Collect the supernatant. (The concentration of the test solution is approximately 5 mg / mL) -1 ) Derivatization process: Derivatization blank: Accurately measure 1 mL of water and place it in a 5 mL volumetric flask. Add 1 mL of FDA solution and 1 mL of 4.2% sodium bicarbonate aqueous solution sequentially, shake well, and incubate in a 60 °C water bath for 1 h. After cooling, add 0.4 mol·L⁻¹ -1 Dilute 1 mL of hydrochloric acid solution to the mark with acetonitrile, shake well, filter, and collect the filtrate.

[0129] Take the reference solution, the test solution (hydrolyzed sample), and the test solution (unhydrolyzed sample) and perform the same procedure as the derivatization blank.

[0130] 5. The block A-block B biodegradable polymers involved in this invention (including PLGA-charged amino acids, PLA-charged amino acids, PLGA-charged oligopeptides, and PLA-charged oligopeptides) can all be prepared by referring to the above method; the difference is that the reaction steps, materials, etc. are replaced or adjusted accordingly, as shown in the following examples.

[0131]

[0132]

[0133] Note 1: Both PLGA and PLA mentioned above are carboxyl-terminated; Note 2: The block A-block B biodegradable polymers used in the formulations of the following examples have the structures obtained in the preparation examples above. For example, the PLGA 502H-KKK structure specifically used in the formulation is shown as K(ε-PLGA 502H)-KK.

[0134] Preparation of injectable in situ gel Drug colloidal particles are prepared by air jet milling, grinding, ball milling, antisolvent precipitation, ultrasonic crushing, emulsification and evaporation or other techniques. The prepared drug colloidal particles are then uniformly dissolved or dispersed in an organic solvent containing biodegradable polymers to obtain the desired injectable in-situ gel.

[0135] Example 1: Preparation of Insulin In Situ Gel Preform and Investigation of its Pharmaceutical Efficacy Dissolve insulin in 0.012 mol·L⁻¹ -1 Prepare a solution of 20 mg / mL in hydrochloric acid aqueous solution. -1 For the drug aqueous solution, under stirring at 1200 rpm, slowly add 4.5 mL of acetone and 15 mL of dimethyl carbonate to 0.5 mL of the drug aqueous solution, followed by vortexing for 15 s. Centrifuge the resulting suspension at 3000 rpm for 5 min, then at 4000 rpm for 1 min, and discard the supernatant. After redispersing the insulin particles with dimethyl carbonate, centrifuge at 4000 rpm for 5 min, discard the supernatant, and dry the resulting precipitate at 25 ℃ under a vacuum of 50 mbar for about 30 min to obtain insulin particles.

[0136] According to the formulation shown in Table 1-1, PLGA 502, PLGA 502H, and the biodegradable polymer PLGA 502H-DDD of the present invention were dissolved in triacetylglycerol, and then the above-mentioned insulin particles were added and dispersed evenly to obtain an insulin injection type in situ gel preform.

[0137] Table 1-1 Formulations and initial particle sizes of insulin in situ gel preforms containing different PLGA polymers

[0138] Drug diffusion tests were conducted on appropriate amounts of the pre-formulation, and the results are shown in the figure. Figure 1 The results showed that prescription 1 had a cumulative insulin diffusion rate of over 60% within the first 24 hours, exhibiting typical severe initial burst release; prescription 2 showed a slower diffusion rate, but the cumulative diffusion rate over 24 hours was still close to 45%; unexpectedly, prescription 3 effectively suppressed the cumulative insulin diffusion rate to below 20% within the same time period, and the diffusion rate was low during the initial 2-hour burst release phase.

[0139] Secondly, after testing the average particle size of insulin particles in preforms prepared from different polymers, it was found that the average particle size of insulin particles in formulation 1 was 2540 nm, the average particle size of insulin particles in formulation 2 decreased to 909 nm, and the average particle size of insulin particles in formulation 3 decreased to 278 nm. This indicates that the biodegradable polymer PLGA 502H-DDD of the present invention can more effectively inhibit particle aggregation and form a more stable dispersion system.

[0140] According to the formulations shown in Table 1-2, PLGA 502 and the biodegradable polymer PLGA 502H-DDD of the present invention were dissolved in triacetylglycerol, and then the insulin particles mentioned above were added and dispersed evenly to obtain an insulin injection type in situ gel preform.

[0141] Table 1-2 Formulations and initial particle sizes of insulin in situ gel preforms containing different PLGA polymers

[0142] Transmission electron microscopy was used to observe the polymer adsorption layer on the surface of drug particles; the results are shown in [Figure number missing]. Figure 2 The results showed that the biodegradable polymer PLGA 502H-DDD of the present invention could form a continuous and clearly discernible adsorption layer on the surface of insulin particles; while no obvious coating structure was observed on the surface of the control group particles using PLGA 502, and polymer adsorption was not significant.

[0143] Further measurements were performed on the zeta potential of the colloidal particles in the injectable in-situ gel systems of formulations 4 and 5. The results are shown in […]. Figure 3 The results showed that the addition of the biodegradable polymer PLGA 502H-DDD to the preform significantly increased the absolute value of the Zeta potential on the surface of the drug particles; in contrast, the effect of PLGA 502 in the preform on increasing the Zeta potential on the surface of the drug particles was not significant.

[0144] According to the formulation ratios shown in Table 1-3, different masses of PLGA 502H-DDD were dissolved in triacetin, and then pre-prepared insulin particles were added and dispersed evenly to obtain insulin injection-type in situ gel pre-formulations with different polymer contents (formulations 3, 6, 5, 7, 8).

[0145] Table 1-3 Formulations and initial particle sizes of insulin in situ gel preforms with different contents of PLGA 502H-DDD

[0146] Place the above prescriptions at a temperature of 25°C. C±2 C. The drug was stored at a relative humidity (RH) of 40%±10% for 1 month, 2 months, and 3 months to assess its long-term storage stability. Results showed that the average particle size of the drug particles in each pre-formulated drug remained stable throughout the observation period, with fluctuations of less than 5%. (See attached figures). Figure 4 The above results demonstrate that the biodegradable polymer of the present invention can impart excellent physical stability to the in-situ gel preformulation over a wide concentration range.

[0147] Contrary to the inventors' expectations, the biodegradable polymer system of the present invention not only improves the initial burst release of drugs, but also ensures that the bioactive material particles in the preform remain well dispersed and do not aggregate significantly during storage, thus exhibiting excellent physicochemical stability.

[0148] The inventors used a dissipative quartz crystal microbalance to quantitatively investigate the effect of polymer structure on its adsorption on the surface of insulin. The results are shown in [Figure number missing]. Figure 5 The results showed that, under the condition of triacetylglycerol as solvent, the biodegradable polymer PLGA 502H-DDD of the present invention exhibited significant adsorption on the surface of insulin particles, and its adsorption mass was significantly higher than that of polymer PLGA502. This indicates that the biodegradable polymer structure design of the present invention can significantly enhance the interfacial adsorption of the polymer on the surface of drug colloidal particles, thereby making it easier to form a continuous and stable adsorption layer.

[0149] Furthermore, the inventors characterized the charge properties of the biodegradable polymer of this invention: 10 mg of polymer powder was weighed into a 1.5 mL centrifuge tube and dissolved in 200 μL of triacetin; then 300 μL of PBS aqueous solution was added, and an emulsion was prepared by ultrasonic emulsification (30 s, 30% amplitude) using a probe. Using PBS solution as the dispersion solvent, 10 μL of the emulsion was diluted in 990 μL of PBS solution. 1 mL of the sample was placed in the universal Dip Cell Kit equipped with a Malvern particle size analyzer to measure the Zeta potential. The results are shown in [Figure number missing]. Figure 6 The results showed that the biodegradable polymers of the present invention exhibited obvious positive or negative charge characteristics.

[0150] Example 2: Preparation of insulin in situ gel preform containing PLGA-charged oligopeptides and its pharmaceutical efficacy investigation. According to the formulations shown in Table 2-1, biodegradable polymers K(ε-PLGA 502H)-KK, K(ε-PLGA 502H)-DK, K(ε-PLGA 502H)-GD and PLGA 502H-DLD with different structures were dissolved in triacetylglycerol; then the insulin particles prepared in Example 1 were added and fully dispersed until uniform, thus obtaining insulin in situ gel preformulations containing PLGA-charged oligopeptides with different structures.

[0151] Table 2-1 Formulations and initial particle sizes of insulin in situ gel preforms containing PLGA-charged oligopeptides with different structures

[0152] An appropriate amount of the pre-formulation was used for an in vitro drug diffusion test. The results are shown in the figure. Figure 7 The results showed that insulin in situ gel preforms containing different types and sequences of PLGA-charged oligopeptides (such as KKK, KDK, KGD, and DLD) all exhibited similar and significant release inhibition effects.

[0153] Example 3: Preparation of an in situ gel preform of insulin containing PLA-charged oligopeptide and its pharmaceutical efficacy investigation. According to the formulation shown in Table 3-1, PLA 203S, PLA 203H, PLA 203H-DDD and K(ε-PLA203H)-KK were dissolved in triacetin, and then the above insulin particles were added and dispersed thoroughly until uniform, thus obtaining insulin in situ gel preformulation containing different PLA-charged oligopeptides.

[0154] Table 3-1 Formulations and initial particle sizes of insulin in situ gel preforms containing different PLA-charged oligopeptides

[0155] Preformulations prepared using PLA with common ester or carboxyl-terminated groups (PLA 203S, PLA 203H, corresponding to formulations 13 and 14) exhibited average drug particle sizes as high as 2770 nm and 700 nm, indicating particle aggregation. In contrast, formulations 15 and 16, prepared using the polymers described in this invention (PLA 203H-DDD, K(ε-PLA 203H)-KK), had average particle sizes of only 305 nm and 313 nm, respectively. These results demonstrate that the structural design of the biodegradable polymers of this invention also provides excellent interfacial stabilization in PLA systems. By forming an adsorption layer on the drug particle surface, the particle size is maintained at the nanoscale, significantly improving the physical stability of the preformulations.

[0156] Further drug diffusion tests were conducted on a suitable amount of the pre-formulation, and the results are shown below. Figure 8The results showed that when the water-insoluble segments of the biodegradable polymer were replaced by PLA instead of PLGA, the preformulations prepared using polymers with charged oligoamino acids as terminal segments (K(ε-PLA 203H)-KK and PLA 203H-DDD, corresponding to formulations 15 and 16) significantly reduced the diffusion rate of insulin into the aqueous phase and exhibited stronger controlled-release capability compared with ordinary ester- or carboxyl-terminated PLA (PLA 203S, PLA 203H, corresponding to formulations 13 and 14).

[0157] Example 4: Preparation of an in situ gel preform of insulin containing PLGA-charged polymer and its pharmaceutical efficacy investigation. According to the formulation shown in Table 4-1, PLGA 502H-D, PLGA 502H-DDD and PLGA 502H-DDDDD were dissolved in triacetin, and then the insulin particles prepared in Example 1 were added and dispersed evenly to obtain insulin in situ gel preformulation containing PLGA-charged amino acids and PLGA-charged oligopeptides containing different numbers of amino acids.

[0158] Table 4-1 Insulin in situ gel preforms containing PLGA-charged polymers and their initial particle sizes

[0159] A suitable amount of the pre-formulation was used for a drug diffusion test, and the results are shown in the figure. Figure 9 The results showed that when the hydrophilic B-segments used to construct the biodegradable polymers were a single charged amino acid residue (D), a trimer oligopeptide residue (DDD), and a pentamer oligopeptide residue (DDDDD), insulin-injectable in-situ gels with good moldability could still be successfully prepared. Further comparative analysis of diffusion curves revealed that preforms prepared from polymers modified with different numbers of hydrophilic amino acid segments (including PLGA502H-D, PLGA 502H-DDDDD, and PLGA 502H-DDDDD) all significantly inhibited the diffusion rate of insulin into the aqueous phase, and the cumulative diffusion amount remained at a low level within 24 hours.

[0160] Example 5: Preparation and pharmacological effect investigation of PLGA-charged oligopeptides containing different lactic acid:glycolic acid molar ratios in in situ gel-prepared insulin formulations. According to the formulation shown in Table 5-1, PLGA 503H-DDD, PLGA 653H-DDD, PLGA 752H-DDD and PLGA 851H-DDD (with lactic acid to glycolic acid molar ratios of 50:50, 65:35, 75:25 and 85:15, respectively) were dissolved in triacetin, and then the insulin particles prepared in Example 1 were added and fully dispersed until uniform, thus obtaining a series of PLGA-charged oligopeptide insulin in situ gel preforms with different lactic acid-glycolic acid copolymerization ratios.

[0161] Table 5-1 Formulations and initial particle sizes of PLGA-charged oligopeptides in situ gels containing different lactic acid:glycolic acid molar ratios

[0162] A suitable amount of the pre-formulation was used for a drug diffusion test, and the results are shown in the figure. Figure 10 The results showed that well-formed insulin-injectable in-situ gels could be successfully prepared when the water-insoluble segment PLGA of the biodegradable polymer had different molar amounts of lactic acid and glycolic acid. Further analysis of its drug diffusion behavior revealed that the series of polymers (PLGA 503H-DDD, 653H-DDD, 752H-DDD, and 851H-DDD) modified with negatively charged oligomeric aspartic acid segments (DDD) all exhibited significant and similar insulin diffusion inhibition capabilities. The cumulative release of each formulation within 24 hours was effectively controlled at a low level (below 30%), and the initial burst release was significantly improved.

[0163] Example 6: Preparation of insulin in situ gel preformulation containing PLGA-charged oligopeptides of different molecular weights and its pharmaceutical efficacy investigation According to the formulation shown in Table 6-1, PLGA 501H-DDD, PLGA 502H-DDD, PLGA 503H-DDD and PLGA 504H-DDD were dissolved in triacetin, and then the insulin particles prepared in Example 1 were added and fully dispersed until uniform, thus obtaining a series of PLGA-charged oligopeptide insulin in situ gel preforms with different molecular weights.

[0164] Table 6-1 Formulations and initial particle sizes of insulin in situ gel preforms containing PLGA-charged oligopeptides of different molecular weights

[0165] A suitable amount of the pre-formulation was used for a drug diffusion test, and the results are shown in the figure. Figure 11The results showed that when the molecular weight of the water-insoluble segment PLGA of the biodegradable polymer varied within a certain range (for example, the weight-average molecular weight (Mw) of PLGA was in the range of about 3.5 k to 54 kDa, of which the weight-average molecular weight (Mw) of PLGA501H, PLGA 502H, PLGA 503H and PLGA 504H were about 3.5 k to 7 kDa, 7 k to 17 kDa, 24 k to 38 kDa and 38 k to 54 kDa, respectively), the biodegradable polymers prepared from them (including PLGA 501H-DDD, PLGA 502H-DDD, PLGA 503H-DDD and PLGA 504H-DDD) could effectively inhibit the outward diffusion of insulin into the aqueous phase, and the cumulative diffusion amount within 24 h was less than 30%.

[0166] Example 7: Preparation of insulin in situ gel preforms containing different concentrations of PLGA-charged oligopeptides and its pharmaceutical efficacy investigation. According to the formulation shown in Table 7-1, different masses of PLGA 502H-DDD were dissolved in triacetin, and then the above-mentioned insulin particles were added and dispersed thoroughly until uniform, thus obtaining a series of PLGA-charged oligopeptide insulin in situ gel preforms containing different concentrations, wherein PLGA 502H-DDD accounted for 0.25 wt%, 0.5 wt%, 1 wt%, 4 wt%, 10 wt%, 18 wt%, 25 wt%, and 30 wt% of the total composition, respectively.

[0167] Table 7-1 Formulations and initial particle sizes of insulin in situ gel preforms containing different concentrations of PLGA-charged oligopeptides

[0168] A suitable amount of the pre-formulation was used for a drug diffusion test, and the results are shown in the figure. Figure 12 The results showed that even when the proportion of PLGA 502H-DDD in the insulin injectable in situ gel was only 0.5 wt%, it could significantly inhibit the outward diffusion of insulin into the aqueous phase. Within the content range studied in this example, as the content of PLGA 502H-DDD further increased, its inhibitory effect on the initial diffusion rate did not show a significant enhancement, indicating that the system has highly sensitive interfacial adsorption and diffusion blocking capabilities even at low content conditions. Furthermore, the in vitro release behavior of the injectable in situ gel under different PLGA 502H-DDD content conditions was evaluated, and the results are as follows: Figure 13As shown, the injectable in-situ gel prepared using PLGA 502H-DDD exhibits a significant negative correlation between drug release rate and polymer content. Specifically, as the PLGA 502H-DDD content increases, the overall insulin release rate gradually decreases, and the release period correspondingly lengthens. This result demonstrates that by adjusting the amount of the biodegradable polymer in the pre-formulation, the drug release kinetics can be effectively controlled without altering the formulation composition or preparation process. In summary, the injectable in-situ gel system obtained by this invention does not require changes to the basic structure of the formulation system; by simply adjusting the proportion of the biodegradable polymer, flexible regulation of drug release behavior can be achieved over a wide range. This characteristic facilitates the design of personalized long-term dosing regimens with differentiated release periods based on the therapeutic needs and target blood drug concentration characteristics of different bioactive substances, demonstrating excellent formulation controllability and application flexibility.

[0169] Example 8: Preparation of insulin in situ gel preforms containing PLGA- and PLA-charged oligopeptides in different solvents and its pharmaceutical efficacy investigation. According to the formulations shown in Table 8-1, PLGA 502H-DDD or PLA 203H-DDD were dissolved in different organic solvents, and then the insulin particles were added and dispersed thoroughly until uniform, thus obtaining a series of PLGA- and PLA-charged oligopeptide insulin in situ gel preforms containing different solvents.

[0170] Table 8-1 Formulations and initial particle sizes of insulin in situ gel preforms containing PLGA- and PLA-charged oligopeptides in different solvents.

[0171] A suitable amount of the pre-formulation was used for a drug diffusion test, and the results were as follows: Figure 14 As shown in the figure. The results indicate that all the listed biocompatible solvents can be used to prepare insulin in situ gel preforms containing PLGA 502H-DDD or PLA 203H-DDD charged oligopeptide modified polymers. The obtained preforms can effectively inhibit the outward diffusion of the drug into the aqueous phase, and the drug diffusion amount is less than 5% under the experimental conditions investigated.

[0172] Example 9: Preparation of PLA-charged oligopeptides in situ gel preforms containing different mixed solvents and investigation of their pharmaceutical effects. According to the formulation shown in Table 9-1, PLA 203H-DDD was dissolved in a mixed solvent composed of triacetylglycerol and benzyl benzoate in different proportions, and then the insulin particles were added and dispersed thoroughly until uniform, thus obtaining a series of PLA-charged oligopeptide insulin in situ gel preforms with different mixed solvent ratios.

[0173] Table 9-1 Formulations and initial particle sizes of insulin in situ gel preforms containing PLA-charged oligopeptides in different mixed solvents

[0174] A suitable amount of the pre-formulation was used for a drug diffusion test, and the results were as follows: Figure 15 As shown in the figure. The results indicate that, within the range investigated, insulin-injectable in-situ gels prepared using mixed solvents of triacetylglycerol and benzyl benzoate in different proportions (the proportions of the two solvents in the insulin-injectable in-situ gel ranged from 10 wt% to 86 wt%) could effectively inhibit the outward diffusion of the drug into the aqueous phase. Under the experimental conditions investigated, the drug diffusion amount was less than 15%.

[0175] Example 10: Preparation of insulin in situ gel preforms containing different drug loading amounts of PLGA-charged oligopeptides and its pharmaceutical efficacy investigation. According to the formulation shown in Table 10-1, PLGA 502H-DDD was dissolved in triacetin, and then the above insulin particles were added and dispersed thoroughly until uniform, thus obtaining a series of PLGA-charged oligopeptide insulin in situ gel preforms with different drug loadings, wherein the insulin particles accounted for 0.5 wt%, 1 wt%, 4 wt%, 10 wt%, 18 wt%, 25 wt% and 30 wt% of the total composition, respectively.

[0176] Table 10-1 Formulations and initial particle sizes of insulin in situ gel preforms containing PLGA-charged oligopeptides with different drug loading capacities.

[0177] A suitable amount of the pre-formulation was used for a drug diffusion test, and the results are shown in the figure. Figure 16 Experimental results show that, within the investigated range, increasing the insulin drug content in the injectable in-situ gel did not significantly affect its outward diffusion behavior into the aqueous phase. Under different insulin loading conditions, the obtained in-situ gel systems all exhibited low initial drug diffusion levels, and the diffusion behavior of the drug into the aqueous phase did not significantly accelerate with increasing drug loading. These results indicate that the charged oligopeptide-modified polymer in-situ gel system constructed in this invention has a wide drug loading range and can maintain good drug diffusion restriction performance even under high drug loading conditions, providing an effective formulation basis for high-dose drug delivery.

[0178] Example 11 Preparation of insulin in situ gel preform containing PLGA-charged oligopeptides of different particle sizes and its pharmaceutical efficacy investigation Insulin particles of different sizes were prepared according to the method shown in Table 11-1. PLGA 502H-DDD was dissolved in triacetin according to the formulation shown in Table 11-2. Then, the insulin particles prepared according to Table 11-1 were added and fully dispersed until uniform, thus obtaining PLGA-charged oligopeptide insulin in situ gel preforms containing different particle sizes, wherein the average particle sizes of the insulin particles were 10 μm, 2 μm, 600 nm, and 150 nm, respectively.

[0179] Table 11-1 Preparation process of drug particles with different particle sizes

[0180] Table 11-2 Formulations of PLGA-charged oligopeptide in situ gel preforms containing insulin particles of different sizes

[0181] A suitable amount of the pre-formulation was used for a drug diffusion test, and the results are shown in the figure. Figure 17 Even when the size of insulin particles varies over a wide range (from nanometer to micrometer scale), the preformulations prepared using PLGA 502H-DDD polymer consistently and effectively inhibited drug diffusion into the aqueous phase. Within the investigated particle size range of approximately 150 nm to 10 μm, formulations under different particle size conditions exhibited similar low-level cumulative release behavior, with no significant differences observed between their release curves. These results demonstrate that the biodegradable polymer system constructed in this invention exhibits good adaptability to variations in drug particle size, and its burst release inhibition effect does not depend on precise control of the initial size of the drug particles. Therefore, the technical solution of this invention demonstrates good process robustness in terms of drug particle size, maintaining stable diffusion behavior under a wide particle size distribution, thereby reducing the requirements for strict particle size control during formulation development and production. This characteristic is beneficial for improving the process tolerance of formulation design and supports stable batch-to-batch consistency under large-scale production conditions, demonstrating good potential for industrial application.

[0182] Example 12 Investigation of different drug particle preparation methods Insulin particles were prepared according to the methods shown in Table 12-1, including grinding, ball milling, ultrasonic disruption, and emulsification evaporation. PLGA 502H-DDD was dissolved in triacetin according to the formulation shown in Table 12-2, and then the insulin particles prepared according to Table 12-1 were added and fully dispersed until uniform, thus obtaining the PLGA-charged oligopeptide insulin in situ gel preform.

[0183] Table 12-1 Preparation process of drug particles with different particle sizes

[0184] Table 12-2 Formulations and initial particle sizes of PLGA-charged oligopeptide in-situ gel preforms for insulin particles prepared by different methods

[0185] A suitable amount of the pre-formulation was used for a drug diffusion test, and the results are shown in the figure. Figure 18 The results showed that insulin particles with different particle size characteristics obtained using different particle preparation methods could effectively inhibit drug diffusion into the aqueous phase when used to prepare injectable in-situ gels. Regardless of whether the insulin particles originated from different particle preparation processes such as grinding, ball milling, ultrasonic disruption, or emulsification and evaporation, the resulting pre-formulations all exhibited stable initial burst release inhibition effects, and no significant changes in diffusion behavior due to differences in particle preparation methods were observed. These results indicate that the biodegradable polymer described in this invention is applicable to various drug particle preparation methods and has good adaptability to differences in drug particle size introduced by different preparation processes; its diffusion inhibition performance is not dependent on a specific particle preparation method. Therefore, the technical solution of this invention exhibits good process flexibility and production robustness in particle preparation, allowing for flexible selection of appropriate particle preparation processes based on factors such as the physicochemical properties of the active pharmaceutical ingredient, available equipment conditions, and production scale, without affecting the release behavior and quality consistency of the final formulation. This characteristic is beneficial for achieving stable batch-to-batch quality control under process scale-up, process transfer, and large-scale production conditions, demonstrating good industrial feasibility and application value.

[0186] Example 13: Preparation of in-situ gel preform containing catalase and its medicinal effects. Catalase with a molecular weight of approximately 250 kDa was dissolved in 0.012 mol·L⁻¹ -1 Hydrochloric acid aqueous solution, prepared to a concentration of 20 mg / mL -1 Catalase aqueous solution. Under stirring at 1200 rpm, 4.5 mL of acetonitrile and 15 mL of dimethyl carbonate were slowly added to 0.5 mL of catalase solution, followed by vortexing for 15 s. The resulting suspension was centrifuged at 3000 rpm for 5 min, then at 4000 rpm for 1 min. The supernatant was discarded. Dimethyl carbonate was added to redisperse the catalase particles, and the mixture was centrifuged at 4000 rpm for 5 min. The supernatant was discarded, and the resulting precipitate was dried at 25 ℃ under a vacuum of 50 mbar for approximately 30 min to obtain catalase particles.

[0187] According to the formulation shown in Table 13-1, PLGA 502H-DDD and K(ε-PLGA 502H)-KK were dissolved in triacetin, and then the above catalase particles were added and dispersed thoroughly until uniform to obtain catalase injection type in situ gel preform.

[0188] Table 13-1 Formulation and initial particle size of in situ gel preform containing catalase

[0189] A suitable amount of the pre-formulation was used for a drug diffusion test, and the results are shown in the figure. Figure 19 The results showed that, regardless of whether PLGA 502H-DDD or K(ε-PLGA 502H)-KK was used to prepare an injectable in-situ gel preform of catalase, the cumulative outward diffusion of catalase with a molecular weight of approximately 250 kDa was less than 5% in a 24-hour diffusion test. This example demonstrates that for macromolecular enzyme drugs (such as catalase), the biodegradable polymers of this invention can effectively inhibit their diffusion into the aqueous phase, and this diffusion-blocking effect is independent of the charge type carried by the B-segment; that is, regardless of whether the B-segment is positively or negatively charged, stable diffusion inhibition performance can be maintained. Furthermore, even under high drug loading conditions (30 wt%), the above-mentioned diffusion inhibition effect can still be maintained.

[0190] Example 14 Preparation of in-situ gel preform containing glucose oxidase and its medicinal effects investigation Glucose oxidase with a molecular weight of approximately 160 kDa was dissolved in 0.012 mol·L⁻¹ -1 Prepared in hydrochloric acid aqueous solution at a concentration of 20 mg / mL -1 Aqueous solution of glucose oxidase. Add 4.5 mL of acetonitrile and 15 mL of dimethyl carbonate slowly to 0.5 mL of glucose oxidase solution while stirring at 1200 rpm, then vortex for 15 s. Centrifuge the resulting suspension at 3000 rpm for 5 min, then at 4000 rpm for 1 min, and discard the supernatant. Redisperse the glucose oxidase particles with dimethyl carbonate, centrifuge at 4000 rpm for 5 min, discard the supernatant, and dry the resulting precipitate at 25 ℃ under 50 mbar vacuum for approximately 30 min to obtain glucose oxidase particles.

[0191] According to the formulation shown in Table 14-1, PLGA 502H-DDD and K(ε-PLGA 502H)-KK were dissolved in triacetin, and then the above glucose oxidase particles were added and fully dispersed until uniform to obtain glucose oxidase injection type in situ gel preform.

[0192] Table 14-1 Formulation and initial particle size of in situ gel preform containing glucose oxidase

[0193] A suitable amount of the pre-formulation was used for a drug diffusion test, and the results are shown in the figure. Figure 20 The results showed that, regardless of whether PLGA 502H-DDD or K(ε-PLGA 502H)-KK was used as the biodegradable polymer for preparing glucose oxidase injectable in-situ gel preforms, the cumulative outward diffusion was less than 5% in the 24-h diffusion test. This example demonstrates that, for another type of macromolecular enzyme—glucose oxidase—the biodegradable polymer of this invention can also effectively inhibit its outward diffusion into the aqueous phase, and this diffusion blocking effect is independent of the charge type carried by the B segment. Furthermore, even under high drug loading conditions (30 wt%), the resulting in-situ gel system still exhibited stable and controllable diffusion behavior.

[0194] Example 15 Preparation of β-lactoglobulin-containing in situ gel preform and its medicinal effects investigation β-lactoglobulin (molecular weight approximately 18.4 kDa) was dissolved in deionized water to prepare a solution of 50 mg / mL. -1 Drug aqueous solution. Under stirring at 1200 rpm, 1.8 mL of tetrahydrofuran and 10 mL of dimethyl carbonate were slowly added to 0.2 mL of β-lactoglobulin solution, followed by vortexing for 15 s. The resulting suspension was centrifuged at 4000 rpm for 5 min, and the supernatant was discarded. Dimethyl carbonate was added to redisperse the β-lactoglobulin particles, and the mixture was centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and the resulting precipitate was dried at 25 °C and 50 mbar vacuum for approximately 30 min to obtain β-lactoglobulin particles.

[0195] According to the formulation shown in Table 15-1, PLGA 502H-DDD and K(ε-PLGA 502H)-KK were dissolved in triacetin, and then the above-mentioned β-lactoglobulin particles were added and dispersed thoroughly until uniform to obtain the β-lactoglobulin injectable in situ gel preform.

[0196] Table 15-1 Formulation and initial particle size of β-lactoglobulin-containing in situ gel preforms

[0197] A suitable amount of the pre-formulation was used for a drug diffusion test, and the results are shown in the figure. Figure 21The results showed that, regardless of whether PLGA 502H-DDD or K(ε-PLGA 502H)-KK was used as the biodegradable polymer for preparing β-lactoglobulin injectable in-situ gel preforms, the cumulative outward diffusion was less than 5% in the 24-hour diffusion test. These results indicate that the biodegradable polymer system constructed in this invention can effectively inhibit the outward diffusion of β-lactoglobulin into the aqueous phase. Furthermore, within a wide drug loading range (1 wt%-30 wt%), the resulting in-situ gel systems exhibited stable and low release levels, and no significant differences in diffusion behavior due to changes in drug loading were observed.

[0198] Example 16 Preparation of in-situ gel preform containing telpolide and its medicinal effects investigation Dissolve telpolide (molecular weight approximately 4.8 kDa) in deionized water to prepare a solution of 100 mg / mL. -1 The drug aqueous solution was stirred at 1200 rpm, and 0.9 mL of acetone and 10 mL of dimethyl carbonate were slowly added to 0.1 mL of telpoeptide solution, followed by vortexing for 15 s. The resulting suspension was centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and the telpoeptide particles were redispersed with dimethyl carbonate. The suspension was then centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and the resulting precipitate was dried at 25 ℃ under a vacuum of 50 mbar for about 30 min to obtain telpoeptide particles.

[0199] According to the formulation shown in Table 16-1, PLGA 502H-DDD, PLA 203H-DDD, and K(ε-PLA 203H)-KK were dissolved in their respective organic solvents, and then the above-mentioned telpoeptide particles were added and fully dispersed until uniform, thus obtaining the telpoeptide injectable in-situ gel preform.

[0200] Table 16-1 Formulation and initial particle size of telpolide-containing in situ gel preforms

[0201] A suitable amount of the pre-formulation was used for a drug diffusion test, and the results are shown in the figure. Figure 22 The results showed that, regardless of whether PLGA or PLA was used as the main chain to construct the biodegradable polymers of the present invention (such as PLGA 502H-DDD, PLA 203H-DDD and K(ε-PLA203H)-KK), and regardless of whether benzyl benzoate or triacetyl ester was used as the solvent, when preparing the telpoide injectable in situ gel preform, the cumulative outward diffusion amount was less than 15% in the 24-hour diffusion test.

[0202] Example 17 Preparation of canagliflozin-containing in-situ gel preform and its pharmaceutical effects Canagliflozin (molecular weight approximately 4.4 kDa) was dissolved in deionized water to prepare a solution of 100 mg / mL. -1 Drug aqueous solution. Under stirring at 1200 rpm, 0.9 mL of acetone and 10 mL of dimethyl carbonate were slowly added to 0.1 mL of canagliflozin solution, followed by vortexing for 15 s. The resulting suspension was centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and the canagliflozin particles were redispersed with dimethyl carbonate. The mixture was then centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and the resulting precipitate was dried at 25 ℃ under 50 mbar vacuum for approximately 30 min to obtain canagliflozin particles.

[0203] According to the formulation shown in Table 17-1, PLGA 502H-DDD was dissolved in triacetin, and then the above-mentioned canagliflozin particles were added and dispersed thoroughly until uniform, thus obtaining the canagliflozin injectable in situ gel preform.

[0204] Table 17-1 Formulation and initial particle size of canagliflozin-containing in-situ gel preformulation

[0205] A suitable amount of the pre-formulation was used for a drug diffusion test, and the results are shown in the figure. Figure 23 The results showed that, within a wide range of drug loading from 1 wt% to 20 wt% and polymer content from 2 wt% to 20 wt%, the cumulative outward diffusion of canagliflozin injectable in situ gel preforms prepared using PLGA 502H-DDD was consistently below 15% in the 24-h diffusion test.

[0206] Example 18 Preparation of exenatide-containing in-situ gel preformulation and its pharmaceutical effects investigation Exenatide (molecular weight approximately 4.2 kDa) was dissolved in deionized water to prepare a solution of 50 mg / mL. -1 Drug aqueous solution. Under stirring at 1200 rpm, 1 mL of acetonitrile was added to every 0.05 mL of drug aqueous solution, followed by slow addition of 3.15 mL of dimethyl carbonate, and then vortexed for 15 s. The resulting suspension was centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and the exenatide particles were redispersed with dimethyl carbonate, centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and the resulting precipitate was dried at 25 ℃ and 50 mbar vacuum for about 30 min to obtain exenatide particles.

[0207] According to the formulation shown in Table 18-1, PLGA 502H-DDD and K(ε-PLGA 502H)-KK were dissolved in triacetin, and then the above exenatide particles were added and fully dispersed until uniform to obtain the exenatide injectable in situ gel preform.

[0208] Table 18-1 Formulation and initial particle size of exenatide-containing in-situ gel preformulation

[0209] A suitable amount of the above pre-formulation was used for a drug diffusion test, and the results are shown in the figure. Figure 24 The results showed that, regardless of whether PLGA502H-DDD or K(ε-PLGA 502H)-KK was used as the biodegradable polymer for preparing exenatide injectable in-situ gel preform, the cumulative outward diffusion amount remained consistently below 15% during the 24-hour diffusion test. These results indicate that the biodegradable polymer system constructed in this invention exhibits excellent diffusion inhibition effects on the peptide drug exenatide.

[0210] Example 19 Preparation of in-situ gel preform containing smegglutinin and its pharmaceutical effects Smegglutinin (molecular weight approximately 4.1 kDa) was dissolved in deionized water to prepare a solution of 100 mg / mL. -1 Drug aqueous solution. Under stirring at 1200 rpm, 0.9 mL of acetone and 10 mL of dimethyl carbonate were slowly added to each 0.1 mL solution of semaglutide, followed by vortexing for 15 s. The resulting suspension was centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and the semaglutide particles were redispersed with dimethyl carbonate. After centrifugation at 4000 rpm for 5 min, the supernatant was discarded, and the resulting precipitate was dried at 25 ℃ and 50 mbar vacuum for about 30 min to obtain semaglutide particles.

[0211] According to the formulation shown in Table 19-1, PLGA 502H-DDD or K(ε-PLGA 502H)-KK were dissolved in triacetin, and then the above-mentioned smegglutide particles were added and dispersed thoroughly until uniform to obtain the smegglutide in-situ gel preform.

[0212] Table 19-1 Formulation and initial particle size of in-situ gel preformulation containing smegglutinin

[0213] A suitable amount of the above pre-formulation was used for a drug diffusion test, and the results are shown in the figure. Figure 25The results showed that, regardless of whether PLGA502H-DDD or K(ε-PLGA 502H)-KK was used as the biodegradable polymer for preparing semaglutide injectable in-situ gel preform, the cumulative outward diffusion amount was less than 5% in the 24-hour diffusion test. These results indicate that the biodegradable polymer system constructed in this invention can also effectively inhibit the diffusion of the peptide drug semaglutide.

[0214] Example 20: Preparation of liraglutide in situ gel preform and its pharmaceutical effects. Liraglutide (molecular weight approximately 3.8 kDa) was dissolved in 0.012 mol·L⁻¹ -1 Prepare a solution of 20 mg / mL in hydrochloric acid aqueous solution. -1 Liraglutide aqueous solution. Under stirring at 1200 rpm, 4.5 mL of acetone and 15 mL of dimethyl carbonate were slowly added to 0.5 mL of liraglutide solution, followed by vortexing for 15 s. The resulting suspension was centrifuged at 3000 rpm for 5 min, then at 4000 rpm for 1 min, and the supernatant was discarded. The liraglutide particles were redispersed with dimethyl carbonate, centrifuged at 4000 rpm for 5 min, and the supernatant was discarded. The resulting precipitate was dried at 25 ℃ under 50 mbar vacuum for approximately 30 min to obtain liraglutide particles.

[0215] According to the formulation shown in Table 20-1, PLGA 502H-DDD or K(ε-PLGA 502H)-KK were dissolved in triacetin, and then the above-mentioned liraglutide particles were added and dispersed thoroughly until uniform, thus obtaining the liraglutide in-situ gel preform.

[0216] Table 20-1 Formulation and initial particle size of liraglutide-containing in situ gel preformulation

[0217] A suitable amount of the above pre-formulation was used for a drug diffusion test, and the results are shown in the figure. Figure 26 The results showed that regardless of whether PLGA502H-DDD or K(ε-PLGA 502H)-KK was used as the biodegradable polymer for preparing liraglutide injectable in-situ gel preformulation, the cumulative outward diffusion amount remained consistently below 15% during the 24-hour diffusion test. These results indicate that the biodegradable polymer system constructed in this invention exhibits excellent diffusion inhibition effects on the polypeptide drug liraglutide.

[0218] Example 21 Preparation of leuprolide acetate in situ gel preform and its medicinal effects investigation Leuprolide acetate (molecular weight approximately 1.3 kDa) was dissolved in deionized water to prepare a solution of 50 mg / mL. -1 Drug aqueous solution. Slowly add 1 mL of acetonitrile to each 0.05 mL of drug aqueous solution and vortex to mix. Then slowly add 3.15 mL of dimethyl carbonate, followed by vortexing for 15 s. Centrifuge the resulting suspension at 4000 rpm for 5 min, discard the supernatant, redisperse the leuprolide acetate granules with dimethyl carbonate, centrifuge at 4000 rpm for 5 min, discard the supernatant, and dry the resulting precipitate at 25 ℃ and 50 mbar vacuum for approximately 30 min to obtain leuprolide acetate granules.

[0219] According to the formulation shown in Table 21-1, PLA 203H-DDD and K(ε-PLA 203H)-KK were dissolved in benzyl benzoate, and then the above-mentioned leuprolide acetate particles were added and fully dispersed until uniform to obtain the leuprolide acetate in-situ gel preform.

[0220] Table 21-1 Formulation and initial particle size of leuprolide acetate in situ gel preforms

[0221] A suitable amount of the above pre-formulation was used for a drug diffusion test, and the results are shown in the figure. Figure 27 The results showed that by selecting different types of biodegradable polymers, including PLA 203H-DDD and K(ε-PLA 203H)-KK, and combining them with a benzyl benzoate solvent system, when used to prepare leuprolide acetate injectable in-situ gel preforms, the cumulative outward diffusion amount remained consistently below 5% during the 24-hour diffusion test. These results indicate that, under the conditions of using PLA as the polymer backbone and benzyl benzoate as the solvent, the biodegradable polymer system constructed in this invention can also effectively inhibit the diffusion of leuprolide small molecule peptide drugs.

[0222] Example 22 Preparation of in-situ gel preform containing goserelin and its medicinal effects Dissolve goserelin (molecular weight approximately 1.2 kDa) in deionized water to prepare a solution of 50 mg / mL. -1 Drug aqueous solution. Slowly add 1 mL of acetonitrile to each 0.05 mL of drug aqueous solution and vortex to mix. Then slowly add 3.15 mL of dimethyl carbonate, followed by vortexing for 15 s. Centrifuge the resulting suspension at 4000 rpm for 5 min, discard the supernatant, redisperse the goserelin granules with dimethyl carbonate, centrifuge at 4000 rpm for 5 min, discard the supernatant, and dry the resulting precipitate at 25 ℃ and 50 mbar vacuum for approximately 30 min to obtain goserelin granules.

[0223] According to the formulation shown in Table 22-1, K(ε-PLA 203H)-KK was dissolved in benzyl benzoate, and then the above-mentioned goserelin particles were added and dispersed thoroughly until uniform to obtain the goserelin in situ gel preform.

[0224] Table 22-1 Formulation and initial particle size of in-situ gel preformulation containing goserelin

[0225] A suitable amount of the above pre-formulation was used for a drug diffusion test, and the results are shown in the figure. Figure 28 The results showed that when K(ε-PLA203H)-KK was used as a biodegradable polymer to prepare a goserelin injectable in-situ gel preform, the cumulative outward diffusion amount remained consistently below 5% during the 24-hour diffusion test. These results indicate that the biodegradable polymer system constructed in this invention has a good diffusion inhibition effect on the peptide drug goserelin.

[0226] Example 23 Preparation of octreotide acetate in situ gel preform and its pharmaceutical effects. Octreotide acetate (molecular weight approximately 1.0 kDa) was dissolved in deionized water to prepare a 50 mg / mL solution. -1 To prepare the drug solution, add 300 μL of acetonitrile to 0.05 mL of the solution, then slowly add 3 mL of dimethyl carbonate, followed by vortexing for 15 s. Centrifuge the resulting suspension at 10,000 rpm for 5 min, discard the supernatant, redisperse the octreotide acetate particles with dimethyl carbonate, and centrifuge again at 10,000 rpm for 5 min. Dry the resulting precipitate at 25 ℃ and 50 mbar vacuum for approximately 30 min to obtain octreotide acetate particles.

[0227] According to the formulation shown in Table 23-1, PLGA 502H-DDD was dissolved in triacetin, and then the above-mentioned octreotide acetate particles were added and dispersed thoroughly until uniform to obtain the octreotide acetate in situ gel preform.

[0228] Table 23-1 Formulation and initial particle size of octreotide acetate in situ gel preformulation

[0229] A suitable amount of the above pre-formulation was used for a drug diffusion test, and the results are shown in the figure. Figure 29The results showed that, with the PLGA 502H-DDD content increasing from 4 wt% to 30 wt%, the cumulative outward diffusion of octreotide acetate injectable in situ gels prepared with different formulations was less than 15% within 24 hours. This indicates that even for the small molecular weight cyclic peptide drug octreotide, the biodegradable polymers of the present invention (e.g., PLGA 502H-DDD) can effectively inhibit its outward diffusion into the aqueous phase, and this diffusion inhibition effect remains stable over a wide range of polymer content and formulation composition, demonstrating good formulation adaptability and parameter robustness.

[0230] Example 24: In vivo pharmacokinetic and pharmacodynamic studies of an insulin-containing in situ gel preformulation. 1) Animal grouping SPF-grade male SD rats, weighing 300-350 g, were selected and acclimatized for one week under a 12-hour diurnal cycle and an environmental temperature of 25°C. They were then randomly divided into 7 groups. (1) Insulin solution group (2) Control group of insulin in situ gel pre-prepared formulation (containing 18 wt% PLGA 502) (3) Insulin in situ gel pre-prepared experimental group (Formulation 3, containing 1 wt% PLGA 502H-DDD) (4) Insulin in situ gel pre-prepared experimental group (Formulation 6, containing 2 wt% PLGA 502H-DDD) (5) Insulin in situ gel pre-prepared experimental group (Formulation 5, containing 4 wt% PLGA 502H-DDD) (6) Insulin in situ gel pre-prepared experimental group (Formulation 7, containing 10 wt% PLGA 502H-DDD) (7) Insulin in situ gel pre-prepared experimental group (Formulation 8, containing 18 wt% PLGA 502H-DDD) Table 24-1 Formulation and initial particle size of insulin-containing in situ gel preformulation

[0231] 2) Modeling and drug administration Rats were fasted for 12 hours before the experiment (with free access to water) and then given a single intraperitoneal injection of 50.0 mg / kg. -1 Streptozotocin was used to induce type 1 diabetes. Fasting blood glucose levels remained consistently above 16.7 mmol / L over three consecutive days of measurement. -1 The rats were identified as type 1 diabetes rats.

[0232] The administration regimen is as follows: Rats in the insulin solution group received a subcutaneous injection of 6.0 IU / kg.-1 The subcutaneous injection dose of insulin solution and insulin in situ gel pre-prepared control group (containing 18 wt% PLGA 502) was 50.0 IU·kg. -1 The subcutaneous injection dose of the insulin in situ gel pre-prepared experimental group (Formula 3) was 50.0 IU·kg. -1 The subcutaneous injection dose of insulin in situ gel pre-prepared groups (Prescription 6, Prescription 5, Prescription 7, and Prescription 8) was 100.0 IU·kg. -1 .

[0233] 3) Pharmacokinetic and pharmacodynamic evaluation A small sample of whole blood was collected via the jugular vein and placed in a blood collection tube. The blood collection tube was then pumped at 1600... g Centrifuge for 15 min, collect the supernatant plasma, and store at -80 ℃. The insulin concentration in the plasma at each time point is determined by high performance liquid chromatography-tandem mass spectrometry.

[0234] The in vivo pharmacokinetic and pharmacodynamic results of the insulin in situ gel preform prepared in this invention are shown in the appendix. Figure 30-32 As shown. Among them, Figure 30 The left image shows the plasma insulin concentration-time curve after subcutaneous injection of insulin solution in rats, while the right image shows the plasma insulin concentration-time curve after subcutaneous injection of insulin in situ gel pre-prepared control group (containing 18 wt% PLGA 502). Figure 31 Plasma insulin concentration-time curves after subcutaneous injection of insulin in situ gel pre-formulations containing different concentrations of PLGA 502H-DDD into rats (Formulation 3, Formulation 6, Formulation 5, Formulation 7, Formulation 8); Figure 32 The curves showing the changes in blood glucose levels over time after subcutaneous injection of insulin in situ gel pre-prepared control group (containing 18 wt% PLGA 502) and insulin in situ gel pre-prepared experimental groups with different concentrations of PLGA 502H-DDD (prescription 3, prescription 6, prescription 5, prescription 7, and prescription 8) in rats.

[0235] The results showed that after administration of insulin solution, the plasma insulin concentration rapidly reached its peak (2810.40 mIU·L⁻¹) within 1 hour. -1 However, it dropped to 9.4 mIU·L within 8 hours. -1 The effective hypoglycemic effect lasted only 3 hours; the plasma insulin concentration in the insulin in situ gel pre-prepared control group (containing 18 wt% PLGA 502) reached 1736.55 mIU·L within 1 hour after administration. -1 However, plasma insulin levels (>10.8 mIU·L) -1The effect lasted only 1 day. In contrast, the insulin in situ gel preformulation experimental group showed superior controlled-release properties. Within 2 hours after administration, the plasma insulin concentrations of formulations 3, 6, 5, 7, and 8 in the insulin in situ gel preformulation experimental group increased to 110.90 mIU·L⁻¹. -1 107.74 mIU·L -1 97.51 mIU·L -1 100.50 mIU·L -1 and 39.37 mIU·L -1 No significant burst release was observed in any of the experimental groups. Simultaneously, all experimental groups showed a significant prolongation of effective plasma insulin levels (>10.8 mIU·L⁻¹). -1 The duration of insulin's effect was maintained at 4, 12, 18, 24, and 30 days, respectively. With increasing PLGA 502H-DDD content in the preform, the stability of the plasma insulin concentration-time curve was further improved. Furthermore, the PLGA 502H-DDD system significantly prolonged the effective hypoglycemic effect of insulin. The effective hypoglycemic effects of formulations 3, 6, 5, 7, and 8 in the insulin in situ gel preform experimental groups were 5, 12, 18, 24, and 30 days, respectively; in contrast, the effective hypoglycemic effect of the insulin in situ gel preform control group (containing 18 wt% PLGA 502) was only about 1 day. These results indicate that by adjusting the PLGA 502H-DDD content in the in situ gel preform, the in vivo release behavior of insulin and the duration of its hypoglycemic effect can be effectively controlled.

[0236] Example 25: In vivo pharmacokinetic and pharmacodynamic studies of exenatide injectable in situ gel preformulation 1) Animal grouping SPF-grade male SD rats, weighing 300-350 g, were selected and acclimatized for one week under a 12-hour diurnal cycle and an environmental temperature of 25°C. They were then randomly divided into 5 groups: (1) Exenatide solution group (2) Exenatide in situ gel pre-prepared control group (containing 10 wt% PLGA 502) (3) Exenatide in situ gel preformulation experimental group (Formulation 109, containing 1 wt% K(ε-PLGA 502H)-KK) (4) Exenatide in situ gel preformulation experimental group (Formulation 110, containing 2 wt% K(ε-PLGA 502H)-KK) (5) Exenatide in situ gel preformulation experimental group (Formulation 111, containing 10 wt% K(ε-PLGA 502H)-KK) Table 25-1 Formulation and initial particle size of exenatide-containing in situ gel preformulation

[0237] 2) Modeling and drug administration Rats were fed a high-fat diet for 4-6 weeks, fasted for 12 hours, and then given a single intraperitoneal injection of 35.0 mg / kg. -1 Streptozotocin was used to establish a type 2 diabetes model. Fasting blood glucose levels were measured in rats two weeks after model establishment; fasting blood glucose levels remained consistently above 16.7 mmol / L. -1 The rats were identified as rats with type 2 diabetes.

[0238] The administration regimen is as follows: Rats in the solution group were administered 100 μg / kg subcutaneously. -1 The subcutaneous injection dose of exenatide solution and exenatide in situ gel pre-formulation control group (containing 10 wt% PLGA 502) was 4 mg / kg. -1 The subcutaneous injection dose of exenatide in situ gel pre-formulation experimental groups (Formulation 109, Formulation 110) was 5 mg / kg. -1 The subcutaneous injection dose of exenatide in situ gel pre-formulation experimental group (Formulation 111) was 10 mg / kg. -1 .

[0239] 3) Pharmacokinetic and pharmacodynamic evaluation A small sample of whole blood was collected via the jugular vein and placed in a blood collection tube. The blood collection tube was then pumped at 1600... g Centrifuge for 15 min, collect the supernatant plasma, and store at -80 ℃. Quantitatively determine the exenatide concentration in plasma at each time point using high performance liquid chromatography-tandem mass spectrometry.

[0240] The in vivo pharmacokinetic and pharmacodynamic results of the exenatide in situ gel preform prepared in this invention are attached. Figure 33-35 As shown. Among them, the appendix Figure 33 Plasma drug concentration-time curve after subcutaneous injection of exenatide solution in rats (left), and plasma drug concentration-time curve after subcutaneous injection of exenatide in situ gel pre-prepared control group (containing 10 wt% PLGA 502) in rats (right). Figure 34 The plasma drug concentration-time curves are shown for rats after subcutaneous injection of different concentrations of exenatide in situ gel preforms (Formula 109, Formula 110, Formula 111) at different concentrations of K(ε-PLGA 502H)-KK in Example 25. Figure 35Blood glucose level-time curves after subcutaneous injection of exenatide in situ gel pre-prepared control group (containing 10 wt% PLGA 502) and exenatide in situ gel pre-prepared exenatide with different concentrations of K(ε-PLGA 502H)-KK (prescription 109, prescription 110, prescription 111) in rats.

[0241] The results showed that the plasma drug concentration in the exenatide solution group rapidly reached its peak (151.75 ng / mL) within 2 hours after administration. -1 ), and decreased to 1.09 ng·mL within 8 h. -1 This indicates that it is rapidly eliminated from the body. In the exenatide in situ gel pre-formulation control group (containing 10 wt% PLGA 502), the plasma exenatide concentration rapidly increased to 1957.39 ng·mL 2 hours after administration. -1 The next day it dropped to 4.68 ng·mL -1 Over the following 8 days, the blood drug concentration ranged from 1.63 to 4.68 ng / mL. -1 The levels fluctuated within a certain range. However, in the experimental group (Formula 109), the blood drug concentration reached 135.11 ng / mL 2 hours after administration. -1 With increasing K(ε-PLGA 502H)-KK concentration, the plasma drug concentrations in prescription 110 and prescription 111 groups reached 43.36 ng·mL at 2 h after administration. -1 and 58.24 ng·mL -1 The drug concentrations were maintained until day 12 and day 27, respectively, showing more stable blood drug concentrations compared to the solution group.

[0242] Furthermore, the K(ε-PLGA 502H)-KK system significantly prolonged the duration of exenatide's effective hypoglycemic effect. In the exenatide in-situ gel pre-formulation control group (containing 10 wt% PLGA 502), blood glucose levels showed a phased decrease and increase after administration: the average blood glucose level on day 5 decreased to 8.92 ± 1.24 mmol·L⁻¹. -1 The hypoglycemic effect lasted until day 7, while the effect only lasted for 2 days. In contrast, the K(ε-PLGA 502H)-KK system showed a more significant and sustained hypoglycemic effect. In the exenatide in-situ gel pre-formulation experimental group (formulation 109), the mean blood glucose level on day 5 after administration decreased from 18.16 ± 1.19 mmol·L. -1 It decreased significantly to 7.44 ± 1.41 mmol·L⁻¹ -1 The effect was maintained until day 12. In the exenatide in-situ gel pre-formulation experimental group (Formulation 110), blood glucose levels decreased to 6.32 ± 1.0 mmol / L on day 3 after administration. -1This continued until day 18. Further increasing the concentration of K(ε-PLGA 502H)-KK in the pre-formulation to 10 wt% (i.e., formulation 111) resulted in a decrease in blood glucose levels to 6.56 ± 1.17 mmol·L on day 2 post-administration. -1 Furthermore, its effective blood sugar-lowering effect can be maintained until day 27, with blood glucose levels ranging from 4.76 to 9.38 mmol / L. -1 The blood sugar level fluctuated within a certain range, demonstrating good blood sugar lowering effect and long-lasting effect.

[0243] Example 26 In vivo pharmacokinetic study of telpolide injectable in situ gel preformulation 1) Animal grouping SPF-grade male SD rats, weighing 250-300 g, were selected and acclimatized for one week under a 12-hour diurnal cycle and an environmental temperature of 25°C. They were then randomly divided into 6 groups. (1) Telbopeptide solution group (2) Control group of telpoeptide in situ gel pre-prepared formulation (containing 10 wt% PLA 203) (3) Telborpeptide in situ gel preformulation experimental group (Formulation 112, containing 1 wt% K(ε-PLA 203H)-KK) (4) Telbopeptide in situ gel preformulation experimental group (Formulation 74, containing 2 wt% K(ε-PLA 203H)-KK) (5) Telbopeptide in situ gel preformulation experimental group (Formulation 75, containing 5 wt% K(ε-PLA 203H)-KK) (6) Telborpeptide in situ gel preformulation experimental group (Formulation 113, containing 10 wt% K(ε-PLA 203H)-KK) Table 26-1 Formulation and initial particle size of telpolide-containing in situ gel preformulation

[0244] 2) Administration The administration regimen is as follows: Rats in the solution group were administered 100 μg / kg subcutaneously. -1 The subcutaneous injection dose of telpolide solution and telpolide in situ gel pre-preparation control group (containing 10 wt% PLA 203) was 4 mg / kg. -1 The subcutaneous injection dose of telpolide in situ gel pre-formulation experimental groups (formulations 112, 74, 75, and 113) was 4 mg / kg. -1 .

[0245] 3) Pharmacokinetic evaluation A small sample of whole blood was collected via the jugular vein and placed in a blood collection tube. The blood collection tube was then pumped at 1600... gCentrifuge for 15 min, collect the supernatant plasma, and store at -80 ℃. Quantitatively determine the concentration of telpoide in plasma at each time point using high performance liquid chromatography-tandem mass spectrometry.

[0246] The in vivo pharmacokinetic results of the telpolide in situ gel preform prepared in this invention are attached. Figures 36-37 As shown. Among them, the appendix Figure 36 The left image shows the plasma drug concentration-time curve after subcutaneous injection of telpoeptide solution in rats, while the right image shows the plasma drug concentration-time curve after subcutaneous injection of telpoeptide in situ gel pre-prepared control group (containing 10 wt% PLA 203). Figure 37 The plasma drug concentration-time curves are shown for rats in Example 26 after subcutaneous injection of different concentrations of K(ε-PLA 203H)-KK in telpoeptide in situ gel preforms (formulations 112, 74, 75, 113).

[0247] The results showed that the plasma drug concentration in the telpotassium solution group rapidly increased to a peak (139.66 ng / mL) within 2 hours after administration. -1 ), and decreased to 4.29 ng·mL on day 3. -1 In the control group of the telpoprit in situ gel pre-formulation (containing 10 wt% PLA203), plasma drug concentration rapidly increased after administration, reaching 4475.83 ng·mL on day 1. -1 It then rapidly decreased to 31.86 ng / mL on day 5. -1 and maintained at 1.90–8.32 ng·mL on days 7–17. -1 Within the range. In contrast, the experimental group showed a more gradual and sustained in vivo exposure. The plasma drug concentration in the prescription 112 group was 273.98 ng / mL on day 1 after administration. -1 As the concentration of K(ε-PLA 203H)-KK increased, the plasma drug concentrations in prescription groups 74, 75, and 113 decreased to 88.38, 20.17, and 7.23 ng·mL, respectively, on day 1. -1 The concentrations were maintained for 24, 26, and 40 days, respectively. Overall, all experimental groups showed more stable and sustained blood drug concentration-time characteristics compared to the solution group and the in-situ gel pre-prepared control group.

[0248] Example 27 In vivo pharmacokinetic study of goserelin injectable in situ gel preformulation 1) Animal grouping SPF-grade male SD rats, weighing 250-300 g, were selected and acclimatized for one week under a 12-hour diurnal cycle and an environmental temperature of 25°C. They were then randomly divided into three groups: (1) Goserelin solution group (2) Control group of goserelin in situ gel pre-prepared formulation (containing 10 wt% PLA 203) (3) Goserelin in situ gel pre-prepared experimental group (Formulation 102, containing 10 wt% K(ε-PLA 203H)-KK) Table 27-1 Formulation and initial particle size of goserelin-containing in-situ gel preformulation

[0249] 2) Administration The administration regimen is as follows: Rats in the solution group were administered 100 μg / kg subcutaneously. -1 The subcutaneous injection dose of the control group (containing 10 wt% PLA 203) of goserelin solution and goserelin in situ gel pre-formulation was 4 mg / kg. -1 The subcutaneous injection dose of goserelin in situ gel pre-formulation experimental group (Formulation 102) was 4 mg / kg. -1 .

[0250] 3) Pharmacokinetic evaluation A small sample of whole blood was collected via the jugular vein and placed in a blood collection tube. The blood collection tube was then pumped at 1600... g Centrifuge for 15 min, collect the supernatant plasma, and store at -80 ℃. Quantitatively determine the concentration of goserelin in plasma at each time point using high performance liquid chromatography-tandem mass spectrometry.

[0251] The in vivo pharmacokinetic results of the goserelin in situ gel preformulation prepared in this invention are attached. Figures 38-39 As shown. Among them, the appendix Figure 38 The left image shows the plasma drug concentration-time curve after subcutaneous injection of goserelin solution into rats, and the right image shows the plasma drug concentration-time curve after subcutaneous injection of goserelin in situ gel pre-prepared control group (containing 10 wt% PLA 203). Figure 39 The plasma drug concentration-time curve is shown for rats after subcutaneous injection of the goserelin in situ gel preformulation (Formulation 102) of K(ε-PLA 203H)-KK in Example 27.

[0252] The results showed that the plasma drug concentration in the goserelin solution group rapidly reached its peak (76.29 ng / mL) within 15 minutes after administration. -1 ), and decreased to 2.70 ng·mL in 2 h. -1 This indicates that it is rapidly eliminated from the body. In the control group of the goserelin in situ gel pre-formulation (containing 10 wt% PLA 203), the plasma goserelin concentration rapidly increased to 922.62 ng / mL 2 hours after administration. -1 The concentration decreased to 1.26 ng·mL on day 1. -1The experimental group (prescription 102) achieved a blood drug concentration of 12.00 ng / mL 2 hours after administration. -1 The release time was extended to 29 days, showing a relatively stable blood drug concentration.

[0253] The exemplary embodiments of the present invention have been described above. However, the scope of protection of the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A pharmaceutical composition comprising the following components: Component I: At least one pharmaceutically acceptable biodegradable polymer, said pharmaceutically acceptable biodegradable polymer comprising block A and block B, wherein, Block A is a pharmaceutically acceptable water-insoluble biodegradable polymer, and block B is a pharmaceutically acceptable hydrophilic substance with a charge under physiological pH conditions. Block A and block B are connected by amide bonds. Component II: at least one biocompatible solvent; And, at least one bioactive substance; The composition is a preformulation.

2. The pharmaceutical composition according to claim 1, characterized in that, The composition remains fluid before injection and can be transformed into a solid or semi-solid implant after injection into the body. Preferably, the composition is an injectable in situ gel.

3. The composition according to claim 1, characterized in that, Block A is carboxyl-terminated; block B contains at least one amino group; the amide bond is -C(O)NH-, which is formed by the condensation of the terminal carboxyl group (-COOH) of block A with at least one amino group of block B; preferably, block A is a polymer formed from one or more selected from lactic acid, glycolic acid, ε-caprolactone, orthoformate, orthoacetate, and polyols. Preferably, block A is selected from one or more of polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, and polycaprolactone.

4. The composition according to any one of claims 1-3, characterized in that, Block B contains at least one group that is ionizable at physiological pH; Preferably, the ionizable group is selected from one or more of amino, guanidinyl, imidazole, carboxyl, mercapto, and phenolic hydroxyl groups; Preferably, block B is a charged amino acid, a charged oligopeptide, or a pharmaceutically acceptable salt thereof; Preferably, the amino acids constituting the charged oligopeptide contain at least one charged amino acid; Preferably, the amino acids in the charged amino acids or charged oligopeptides can be natural amino acids or pharmaceutically acceptable synthetic amino acids; Preferably, the number of amino acids in the charged oligopeptide is ≥2; preferably, the number of amino acids in the charged oligopeptide is 2~10. Preferably, the charged amino acid is selected from histidine (H), lysine (K), arginine (R), glutamic acid (E), or aspartic acid (D); Preferably, the neutral amino acid is selected from serine (S), threonine (T), asparagine (N), glutamine (Q), tyrosine (Y), cysteine ​​(C), glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), proline (P), phenylalanine (F), or tryptophan (W). Preferably, in component I, block A is PLGA and block B is a charged amino acid or a pharmaceutically acceptable salt thereof; or block A is PLA and block B is a charged amino acid or a pharmaceutically acceptable salt thereof; or block A is PLGA and block B is a charged oligopeptide or a pharmaceutically acceptable salt thereof; or block A is PLA and block B is a charged oligopeptide or a pharmaceutically acceptable salt thereof. Preferably, component I is PLGA-histidine (PLGA-H), PLGA-lysine (PLGA-K), PLGA-arginine (PLGA-R), PLGA-glutamic acid (PLGA-E), PLGA-aspartic acid (PLGA-D), PLA-histidine (PLA-H), PLA-lysine (PLA-K), PLA-arginine (PLA-R), PLA-glutamic acid (PLA-E), PLA-aspartic acid (PLA-D), or PLGA-lysine-lysine. PLGA-Lysine (PLGA-KKK), PLGA-Aspartic Acid-Aspartic Acid-Aspartic Acid (PLGA-DDD), PLGA-Lysine-Aspartic Acid-Lysine (PLGA-KDK), PLGA-Lysine-Glycine-Aspartic Acid (PLGA-KGD), PLGA-Aspartic Acid-Leucine-Aspartic Acid (PLGA-DLD), PLGA-Aspartic Acid-Aspartic Acid-Aspartic Acid-Aspartic Acid (PLGA-DDDD), PLGA-Aspartic Acid-Aspartic Acid-Aspartic Acid -Aspartic acid-aspartic acid (PLGA-DDDDD), PLGA-lysine-lysine-lysine-lysine (PLGA-KKKK), PLGA-lysine-lysine-lysine-lysine (PLGA-KKKKK), PLA-lysine-lysine-lysine (PLA-KKK), PLA-aspartic acid-aspartic acid-aspartic acid (PLA-DDD), PLA-lysine-aspartic acid-lysine (PLA-KDK), PLA-lysine-glycine-aspartic acid One or more of the following: PLA-KGD, PLA-aspartic acid-leucine-aspartic acid (PLA-DLD), PLA-aspartic acid-aspartic acid-aspartic acid-aspartic acid (PLA-DDDD), PLA-aspartic acid-aspartic acid-aspartic acid-aspartic acid (PLA-DDDDD), PLA-lysine-lysine-lysine-lysine-lysine (PLA-KKKK), and PLA-lysine-lysine-lysine-lysine-lysine (PLA-KKKKK); Preferably, component I accounts for 0.1 wt% to 30 wt% of the total formulation; more preferably, 0.5 wt% to 25 wt%; even more preferably, 1 wt% to 20 wt%.

5. The composition according to any one of claims 1-4, wherein the biocompatible solvent of component II is selected from alcohols, esters, ketones, amides, sulfoxides, or mixtures thereof; Preferably, component II is selected from one or more of benzyl alcohol, octanol, ethyl acetate, methyl acetate, ethyl lactate, ethyl benzoate, benzyl benzoate, triacetyl glycerol, tripropionate, tributyrate, acetyl triethyl citrate, triethyl citrate, tributyl citrate, acetyl tributyl citrate, diethyl phthalate, diethyl tartrate, methyl isobutyl ketone, dimethyl sulfoxide, dimethyl acetamide, N-methylpyrrolidone, and N-ethyl-2-pyrrolidone. More preferably, component II is selected from one or more of benzyl alcohol, benzyl benzoate, ethyl benzoate, triacetyl glycerol, glyceryl tribocylate, and acetyl triethyl citrate; Preferably, component II accounts for 40-99.8 wt% of the total formulation; more preferably 60 wt%-98 wt%; more preferably 65 wt%-95 wt%; more preferably 70 wt%-90 wt%.

6. The composition according to any one of claims 1-5, characterized in that, The bioactive substance is a polypeptide or protein drug with a molecular weight of 1 kDa to 500 kDa. Preferably, the bioactive substance is selected from one or more polypeptides or proteins selected from the following therapeutic types: anti-inflammatory drugs, local anesthetics, analgesics, antihypertensive drugs, hypoglycemic drugs, anti-infective drugs (including antibacterial drugs, antiviral drugs, antifungal drugs, etc.), immunomodulatory or immunosuppressive drugs (including anti-rejection drugs), antitumor drugs, antiepileptic or anticonvulsant drugs, hormonal drugs, drugs for the treatment of bone diseases, complement inhibitors, receptor agonists, and hematopoietic growth factors. Preferably, the bioactive substance is selected from macromolecular enzymes, such as catalase, glucose oxidase, or is a model protein, such as β-lactoglobulin; Preferably, the bioactive substance is selected from anaraxin, etanercept, tocilizumab, ziconopeptide, cobrote, nesiritide, adrenaline, adrenocorticotropic hormone, leuprorelin, leuprorelin acetate, goserelin, triptorelin, nafarelin, temorelin, galopeptide (growth hormone neuropeptide), colistin, polymyxin, daptomycin, lactoferrin peptide, insulin, insulin aspart, insulin lispro, insulin glargine, insulin detemir, liraglutide, smegglutide, dulaglutide, telpoxetine, exenatide, benaglutide, abiglutide, canagliflozin, loxenatide, liximab, pralaminide, enfuviride, ibalizumab, interferon-α, interferon-β, interferon-γ, recombinant human granulocyte colony-stimulating factor, trastuzumab, pertuzumab, and rituximab. Oxtuzumab, cetuximab, panitumumab, bevacizumab, nivolumab, pembrolizumab, ipilimumab, trastuzumab emtansine, brentuximab velituximab, detrastuzumab, interleukin-2, octreotide, octreotide acetate, lanreotide, edotreotide, cyclosporine, vancomycin, bacitracin, micafungin, micashermine, sematralon, human growth hormone and its derivatives, lonpectomycin, semapaxitam, recombinant follicle-stimulating hormone beta, prolactin, luteinizing hormone, parathyroid hormone, teriparatide, abapatide, salmon calcitonin, vosolidaide, zeleptolide, sovastatin, recombinant human erythropoietin, pefescine, pemoxatide, dapoxetine α, methoxy-polyethylene glycol erythropoietin beta (CERA), and one or more of their pharmaceutically acceptable salts, analogues or derivatives; Preferably, the bioactive substance accounts for 0.1 wt% to 40 wt% of the total amount of the formulation; more preferably, 0.1 wt% to 30 wt%; more preferably, 0.5 wt% to 25 wt%; and even more preferably, 1 wt% to 20 wt%.

7. The composition according to any one of claims 1-6, characterized in that, It further comprises one or more pharmaceutically acceptable excipients; Preferably, the excipients are selected from one or more of the following: antibacterial agents, buffers, pH adjusters, antioxidants, metal ion chelating agents, and release regulators; Preferably, pharmaceutically acceptable excipients account for no more than 5 wt% of the total formulation. Preferably, pharmaceutically acceptable excipients account for no more than 2.5 wt% of the total formulation. Preferably, pharmaceutically acceptable excipients account for no more than 1 wt% of the total formulation. Preferably, pharmaceutically acceptable excipients account for no more than 0.5 wt% of the total formulation.

8. The composition according to any one of claims 1-7, characterized in that, The composition is administered topically or by injection.

9. A method for preparing the composition according to any one of claims 1-7, characterized in that, Includes the following steps: (i) Dissolve the biodegradable polymer of component I in a biocompatible solvent to obtain a clear and homogeneous solution; (ii) Add the bioactive substance to the solution of step (i), and stir or homogenize until the bioactive substance is completely dissolved or uniformly dispersed to obtain the composition; In the preparation method, optionally, pharmaceutically acceptable excipients are further added to the solution of step (i).

10. Use of the composition according to any one of claims 1-7 in the preparation of a medicament for continuous administration of a bioactive substance.

Images