Zwitterionic carriers, complexes, and pharmaceuticals and uses thereof

Abstract

The present application relates to a kind of zwitterionic carrier, complex and its medicine and application, the carrier described in the present application has better biocompatibility and biodegradability, and exhibits the function of drug delivery across multiple biological barriers.The carrier can load protein drugs through non-covalent interaction to effectively protect protein activity, and has small molecular weight, relatively simple and clear structure, can be controllably synthesized, scale production, and exhibits good industrialization prospect.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical delivery, and more particularly to a zwitterionic carrier, a complex thereof, and its drug and applications. Background Technology

[0002] The development of therapeutic biological macromolecules such as proteins, peptides, and antibodies has been rapid, and they have already been approved for clinical treatment of a variety of diseases. However, the routes of administration for these biological macromolecules are limited by biological barriers, with most relying on invasive delivery methods, such as injections. There is a lack of universally applicable carriers that facilitate the penetration of biological barriers by these macromolecules. Summary of the Invention

[0003] To address the aforementioned issues, this application provides a zwitterionic carrier for the delivery of biopharmaceutical drugs. The zwitterionic carrier described in this application exhibits excellent biocompatibility and biodegradability, enabling it to carry biopharmaceutical drugs across biological barriers and achieve non-invasive drug delivery. Furthermore, the assembly of the zwitterionic carrier with the protein drug is non-covalent, effectively protecting the protein's activity. Compared to traditional polymer carriers, the zwitterionic carrier described in this application has advantages such as small molecular weight, relatively simple and well-defined structure, and controllable synthesis and scale-up production, demonstrating promising industrialization prospects.

[0004] One aspect of the present invention provides a zwitterionic carrier, comprising a zwitterionic region and a region providing assembly driving force, wherein the zwitterionic region is a zwitterionic polypeptide chain formed by alternating cationic and anionic amino acids.

[0005] Furthermore, the regions providing the assembly driving force are carbon chains and cationic peptide segments.

[0006] Furthermore, the carbon chain is a hydrophobic carbon chain.

[0007] Furthermore, the hydrophobic carbon chain is a hydrophobic segment with a carbon atom backbone.

[0008] Furthermore, the carbon chain is a hydrocarbon chain.

[0009] Furthermore, the zwitterionic carrier has the structure abc or acb, where a is the zwitterionic region, c is the carbon chain, and b is the cationic peptide segment.

[0010] Furthermore, the cationic amino acid is selected from one or both of lysine and arginine; and / or the anionic amino acid is selected from one or both of glutamic acid and aspartic acid.

[0011] Furthermore, the zwitterionic region contains 8-30 amino acids.

[0012] Furthermore, the zwitterionic region contains 12-33 amino acids.

[0013] Furthermore, when the number of amino acids in the zwitterionic region is odd, the terminal amino acid is a cationic amino acid (arginine K or lysine R).

[0014] Furthermore, the number of amino acids alternating between cationic and anionic amino acids is at least one.

[0015] Furthermore, the zwitterionic polypeptide region contains 2 to 16 minimal repeating units, wherein the minimal repeating units are combinations of cationic and anionic amino acids in the form of A1B1 or A1A2B1B2, wherein A1 and A2 are the same cationic amino acid or different cationic amino acids, and B1 and B2 are the same anionic amino acid or different anionic amino acids.

[0016] Further, the minimum repeating unit comprises lysine and glutamic acid; or the minimum repeating unit comprises lysine and aspartic acid; or the minimum repeating unit comprises arginine and glutamic acid; or the minimum repeating unit comprises arginine and aspartic acid; or the minimum repeating unit comprises two lysines and two glutamic acid; or the minimum repeating unit comprises two lysines and two aspartic acid; or the minimum repeating unit comprises two arginines and two glutamic acid; or the minimum repeating unit comprises two arginines and two aspartic acid.

[0017] Furthermore, the carbon chain includes a hydrophobic alkyl chain, wherein the main chain of the hydrophobic alkyl chain has 8-18 carbon atoms.

[0018] Furthermore, the cationic peptide contains 1-8 cationic amino acids.

[0019] Furthermore, the cationic amino acid is selected from any one or more of lysine, arginine, and histidine.

[0020] Furthermore, the zwitterionic region has a straight-chain structure or a branched-chain structure.

[0021] Furthermore, the number of terminal branches of the branch structure is any one of 2, 4, 8, 16 and 32.

[0022] Furthermore, the zwitterionic carrier contains hydrophilic amino acids and hydrophobic amino acids, and the ratio of the hydrophilic amino acids to the hydrophobic amino acids is 1.0-4.0.

[0023] Furthermore, the ratio of the number of amino acids in the hydrophobic polypeptide chain to the number of amino acids in the zwitterionic polypeptide chain is 5:(4~20).

[0024] A second aspect of the present invention is to provide a complex comprising a biomacromolecule drug and a zwitterion carrier described above, wherein the biomacromolecule drug and the zwitterion carrier are non-covalently assembled.

[0025] Furthermore, the molecular weight of the biopharmaceutical is 3-300 kD.

[0026] Furthermore, the biomolecular drug is selected from any one of proteins, peptides, nucleic acids, and polysaccharides.

[0027] Furthermore, the biopharmaceutical is selected from any one of antibodies, antibody fragments, antigen-binding fragments, cytokines, biological enzymes, antigens, and growth factors.

[0028] Furthermore, the biopharmaceutical is selected from tumor cell necrosis factor α (TNF-α) inhibitors, interleukin 23 inhibitors, interleukin 12 / interleukin 23 (IL-12 / 23) inhibitors, and α4β7 integrin antagonists.

[0029] Furthermore, the biological macromolecular drug is selected from any one of adalimumab, infliximab, ustekinumab, vedelizumab, aflibercept, PD-L1 antibody, immunoglobulin, GLP-1 and its analogues, insulin, glucagon, interferon, trypsin, thrombin, lysozyme, protease inhibitors and catalase.

[0030] Furthermore, the mass ratio of the zwitterionic carrier to the biomolecular drug is (1~16):1.

[0031] Furthermore, the preparation method of the complex includes the following steps: mixing the zwitterionic carrier with a biological macromolecular drug to obtain the complex.

[0032] Furthermore, the mass ratio of the zwitterionic carrier to the biomolecular drug is (1~16):1.

[0033] A third aspect of the present invention is to provide the application of the zwitterionic carrier or complex described herein in the preparation of drugs for transbiological delivery.

[0034] Furthermore, the biological barriers include the intestinal barrier, the eye barrier, the mucosal barrier, the mucus barrier, the intestinal mucosa, the intestinal mucus, the lung mucosa, the lung mucus, the oral mucosa, and the nasal mucosa.

[0035] A fourth aspect of the present invention is to provide the application of the zwitterionic carrier or complex described herein in an oral formulation.

[0036] A fifth aspect of the present invention is to provide a medicament for treating digestive tract diseases, comprising the zwitterionic carrier or complex described in the present invention.

[0037] Furthermore, the digestive tract diseases include inflammatory bowel disease, colon cancer, rectal cancer, and colorectal cancer.

[0038] Furthermore, the therapeutic targets of the drug are selected from tumor cell necrosis factor α (TNF-α), interleukin 23 (IL-23), interleukin 12 / interleukin 23 p (IL-12 / 23), and α4β7 integrin.

[0039] A sixth aspect of the present invention is to provide a medicament for treating diabetes, comprising the zwitterion carrier or complex described herein.

[0040] A seventh aspect of the present invention is to provide a medicament for treating obesity, comprising the zwitterion carrier or complex described in the present invention.

[0041] The present invention also provides a method of administration of a formulation, comprising orally administering to a patient a formulation containing the zwitterionic carrier or complex described in the present invention.

[0042] Specifically, the diseases for which the formulation is applicable are selected from Crohn's disease, ulcerative colitis, colon cancer, rectal cancer, colorectal cancer, obesity, and diabetes.

[0043] A seventh aspect of the present invention is to provide the use of the zwitterionic carrier or complex described herein in an ocular disease treatment formulation.

[0044] An eighth aspect of the present invention is to provide the use of the zwitterionic carrier or complex described herein in oral or nasal inhalation or nasal delivery.

[0045] A ninth aspect of the present invention is to provide the application of the zwitterionic carrier or complex described herein in atomizing agents, sprays, and inhalers.

[0046] Compared with the prior art, the zwitterionic carrier provided by the present invention has at least the following beneficial effects:

[0047] 1) The zwitterionic carrier described in this invention has a well-defined structure, controllable synthesis, high preparation efficiency and yield, is easy to separate and purify, is suitable for mass production, and meets the needs of industrial production;

[0048] 2) The zwitterionic carrier described in this invention can stably and reversibly assemble with biological macromolecules without affecting the structure and activity of the biological macromolecules, thereby ensuring the therapeutic effect;

[0049] 3) The zwitterionic carrier described in this invention can effectively protect biological macromolecules from being decomposed in vivo and help active ingredients reach the lesion site;

[0050] 4) The zwitterionic carriers described in this invention can efficiently penetrate various biological barriers, providing new solutions for the treatment of gastrointestinal diseases, eye diseases, lung diseases, respiratory diseases, and even brain diseases. Detailed Implementation

[0051] To further illustrate the technical means and effects of the present invention in achieving the intended purpose, the following detailed description, in conjunction with preferred embodiments, provides a detailed explanation of the specific implementation methods, steps, structures, features, and effects of the zwitterionic carrier proposed in accordance with the present invention.

[0052] The foregoing and other technical contents, features, and effects of the present invention will be clearly presented in the following detailed description of preferred embodiments with reference to the accompanying drawings. Through the description of the specific embodiments, a more in-depth and specific understanding can be gained of the technical means and effects adopted by the present invention to achieve the intended purpose. However, the accompanying drawings are only provided for reference and illustration and are not intended to limit the present invention.

[0053] To illustrate the technical solutions of the invention in more detail, specific embodiments are listed below to demonstrate the technical effects; it should be emphasized that these embodiments are used to illustrate the invention and not to limit the scope of the invention.

[0054] Description of the detection instruments, detection methods, detection parameters, and reagent kits used in this example:

[0055] Particle size measurement instrument: Nano particle size analyzer and ZETA potentiometer, model Nano ZS90.

[0056] Average particle size: Samples were tested using a nanoparticle size analyzer and a ZETA potentiometer, model Nano ZS90, and the Z-average value was recorded based on the instrument readings.

[0057] PDI: PDI is a dimensionless value reflecting the width of particle size distribution, ranging from 0 to 1. The smaller the value, the more uniform the particle size, the more concentrated the particle size distribution, and the higher the particle uniformity. The complex formed by the zwitterionic carrier and the biopharmaceutical drug described in this invention is mainly used for oral delivery. The PDI value is for reference only, and a PDI less than 0.3 is preferred.

[0058] Diffusion coefficient: Diffusion coefficient = (sample fluorescence intensity value - blank well fluorescence intensity value) × dilution factor ÷ (original solution fluorescence intensity value - blank well fluorescence intensity value). The background value is approximately 0.05. A diffusion coefficient of not less than 0.05 is considered to have a permeation-enhancing effect. The larger the diffusion coefficient, the better the permeation-enhancing effect. (Reference: S. Hu, X. Pei, L. Duan, Z. Zhu, Y. Liu, J. Chen, T. Chen, P. Ji, Q. Wan, J. Wang, A mussel-inspired film for adhesion to wet buccal tissue and efficient buccal drug delivery, Nat. Commun. 12 (2021) 1689.)

[0059] Example 1:

[0060] The desired zwitterionic peptide fragment is synthesized by solid phase peptide synthesis (SPPS), and then alkyl groups with carboxyl or amino groups are branched onto the amino acid sequence through an amide reaction to obtain a zwitterionic carrier with the specified sequence.

[0061] The prepared zwitterionic supports are as follows:

[0062] NO.2. <![CDATA[K8-(CH2) 18 -(KE) 16 K]]> NO.3. <![CDATA[(KEKE)8-K4-K2-(CH2) 11 CH3]]> NO.4. <![CDATA[(KEKE)4-K2-(CH2) 11 CH3]]> NO.5. <![CDATA[CH3(CH2) 11 -K8-(KE) 16 K]]> NO.6. <![CDATA[R4-(CH2) 12 -(KE) 10 K]]> NO.7. <![CDATA[R6-(CH2) 16 -(KE) 10 K]]> NO.8. <![CDATA[K4-(CH2) 18 -(RD) 10 R]]> NO.9. <![CDATA[K4-(CH2) 18 -(RD)6R]]> NO.10. <![CDATA[K4-(CH2) 18 -(KE) 10 K]]>

[0063] In this context, "-" represents the connection formed by the reaction of amino and carboxyl groups.

[0064] Example 2:

[0065] The zwitterionic carrier from Example 1 was mixed with a solution of protein IgG (molecular weight), and the pH was adjusted to prepare a complex solution. The particle size of the complex was measured to determine its homogeneity. The state of the complex solution was observed to evaluate for any visible precipitation or turbidity. The detection and observation results are as follows:

[0066] Example 2.1 <![CDATA[K4-(CH2) 18 -(KE) 10 K]]> 235.7 Clear and without sediment Example 2.2 <![CDATA[K8-(CH2)6-(KE) 16 K]]> 180.5 Clear and without sediment Example 2.3 <![CDATA[(KEKE)8-K4-K2-(CH2) 11 CH3]]> 443.1 Clear and without sediment Example 2.4 <![CDATA[(KEKE)4-K2-(CH2) 11 CH3]]> 1013.5 Clear and without sediment Example 2.5 <![CDATA[CH3(CH2) 11 -K8-(KE) 16 K]]> 164.5 Clear and without sediment Example 2.6 <![CDATA[R4-(CH2) 12 -(KE) 10 K]]> 202.3 Clear and without sediment Example 2.7 <![CDATA[R6-(CH2) 16 -(KE) 10 K]]> 250.7 Clear and without sediment Example 2.8 <![CDATA[K4-(CH2) 18 -(RD) 10 R]]> 274.9 Clear and without sediment Example 2.9 <![CDATA[K4-(CH2) 18 -(RD)6R]]> 228.4 Clear and without sediment Example 2.10 <![CDATA[K4-(CH2) 18 -(KE) 10 K]]> 238.2 Clear and without sediment

[0067] The results showed that

[0068] Example 3: Calculation of the diffusion coefficient of the complex in the mucus layer using an in vitro intestinal mucus layer permeation simulation experiment.

[0069] The transwell chambers were placed in a culture plate. 1 mL of 20 mg / mL mucin solution was added to the upper chamber to simulate the intestinal mucus layer. The sample to be tested was then added to the upper layer. PBS buffer was added to the lower chamber, and the plate was incubated at 37°C for 4 h. After incubation, the lower PBS solution was collected, and its fluorescence intensity was measured. The diffusion coefficient of the complex in the simulated intestinal mucus was calculated using the formula: (sample fluorescence intensity - blank well fluorescence intensity) × dilution factor ÷ (original solution fluorescence intensity - blank well fluorescence intensity).

[0070] A higher diffusion coefficient indicates better penetration into simulated intestinal mucus, theoretically leading to better absorption of protein drugs during oral delivery. (Reference: S. Hu, X. Pei, L. Duan, Z. Zhu, Y. Liu, J. Chen, T. Chen, P. Ji, Q. Wan, J. Wang, A mussel-inspired film for adhesion to wet buccaltissue and efficient buccal drug delivery, Nat. Commun. 12 (2021) 1689.)

[0071] The diffusion coefficient of the complex prepared in Example 2 was measured in an in vitro intestinal mucus layer penetration simulation experiment as follows:

[0072] Example 3.1 <![CDATA[K4-(CH2) 18 -(KE) 10 K]]> 0.134 Example 3.2 <![CDATA[K8-(CH2)6-(KE) 16 K]]> 0.193 Example 3.3 <![CDATA[(KEKE)8-K4-K2-(CH2) 11 CH3]]> 0.107 Example 3.4 <![CDATA[(KEKE)4-K2-(CH2) 11 CH3]]> 0.095 Example 3.5 <![CDATA[CH3(CH2) 11 -K8-(KE) 16 K]]> 0.172 Example 3.6 <![CDATA[R4-(CH2) 12 -(KE) 10 K]]> 0.189 Example 3.7 <![CDATA[R6-(CH2) 16 -(KE) 10 K]]> 0.152 Example 3.8 <![CDATA[K4-(CH2) 18 -(RD) 10 R]]> 0.133 Example 3.9 <![CDATA[K4-(CH2) 18 -(RD)6R]]> 0.086 Example 3.10 <![CDATA[K4-(CH2) 10 -(KE) 10 K]]> 0.117

[0073] The results showed that the complex formed by the aforementioned zwitterionic carrier and IgG could penetrate the mucus layer. The presence of the mucus layer and the epithelial barrier layer are key factors hindering the transintestinal delivery of orally administered macromolecular proteins. The continuously secreted mucus layer effectively captures and rapidly clears pathogens and foreign particles, preventing them from penetrating the epithelium. Orally administered drugs are also easily intercepted in the mucus layer, thus reducing bioavailability. Therefore, zwitterionic carriers can carry IgG through the intestinal mucus layer, overcoming the key barrier to the absorption of oral macromolecular drugs and achieving the effect of oral delivery of macromolecular drugs.

[0074] Obviously, the above embodiments are merely examples for clear illustration and are not intended to limit the implementation.

[0075] While the foregoing disclosure has discussed some inventive embodiments that are currently considered useful through various examples, it should be understood that such details are for illustrative purposes only, and the appended claims are not limited to the disclosed embodiments. Rather, the claims are intended to cover all modifications and equivalent combinations that conform to the substance and scope of the embodiments of this application.

[0076] Similarly, it should be noted that, in order to simplify the description of this application and thus aid in the understanding of one or more embodiments of the invention, the foregoing description of the embodiments of this application sometimes combines multiple features into a single embodiment. However, this disclosure method does not imply that the subject matter of this application requires more features than those mentioned in the claims. In fact, the embodiments contain fewer features than all the features of the single embodiments disclosed above.

[0077] In some embodiments, numbers describing the quantity of components and attributes are used. It should be understood that such numbers used in the description of embodiments are sometimes modified by the terms "approximately," "approximately," or "generally." Unless otherwise stated, "approximately," "approximately," or "generally" indicates that the numbers are allowed to vary by ±. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may be changed according to the characteristics required by individual embodiments.

[0078] Finally, it should be understood that the embodiments described in this application are merely illustrative of the principles of the embodiments of this application. Other modifications may also fall within the scope of this application. Therefore, alternative configurations of the embodiments of this application are considered as examples and not limitations, and are regarded as consistent with the teachings of this application. Accordingly, the embodiments of this application are not limited to the embodiments explicitly described and illustrated in this application.

Claims

1. A zwitterionic carrier, characterized in that, It includes a zwitterionic region and a region that provides assembly driving force. The zwitterionic region is a zwitterionic polypeptide chain formed by alternating cationic and anionic amino acids, and the region that provides assembly driving force is a carbon chain and a cationic peptide segment.

2. The zwitterionic carrier according to claim 2, characterized in that, The zwitterionic carrier has the structure abc or acb, where a is the zwitterionic region, c is the carbon chain, and b is the cationic peptide segment.

3. The zwitterionic carrier according to any one of claims 1 or 2, characterized in that, The cationic amino acid is selected from one or both of lysine and arginine; and / or the anionic amino acid is selected from one or both of glutamic acid and aspartic acid, wherein the number of alternating cationic and anionic amino acids is at least 1; the zwitterionic region contains 8-30 amino acids.

4. The zwitterionic carrier as described in any one of claims 1 or 2, characterized in that, The carbon chain includes a hydrophobic alkyl chain, and the main chain of the hydrophobic alkyl chain has 8-18 carbon atoms.

5. The zwitterionic carrier as described in claim 2 or 3, characterized in that, The cationic peptide contains 1-8 cationic amino acids, wherein the cationic amino acids are selected from any one or more of lysine, arginine, and histidine.

6. The zwitterionic carrier as described in claim 1, characterized in that, The zwitterionic region has a straight-chain structure or a branched-chain structure.

7. A complex, characterized in that, Includes a biological macromolecular drug and the zwitterionic carrier according to any one of claims 1-14, wherein the biological macromolecular drug and the zwitterionic carrier are non-covalently assembled.

8. The complex according to claim 7, characterized in that, The molecular weight of the biological macromolecular drug is 3-300 kDa.

9. The complex according to claim 7, characterized in that, The biological macromolecular drug is selected from any one of proteins, peptides, nucleic acids, and polysaccharides.

10. The complex according to any one of claims 7-9, characterized in that, The mass ratio of the zwitterionic carrier to the biomolecular drug is (1~16):

1.

11. The use of the zwitterionic carrier as described in any one of claims 1-6 or the complex as described in any one of claims 7-10 in the preparation of drugs for transbiological delivery.

12. The application as described in claim 11, characterized in that, The biological barriers include the intestinal barrier, eye barrier, mucosal barrier, mucus barrier, intestinal mucosa, intestinal mucus, lung mucosa, lung mucus, oral mucosa, and nasal mucosa.

13. The use of the zwitterionic carrier as described in any one of claims 1-6 or the complex as described in any one of claims 7-9 in the preparation of oral formulations, ocular disease treatment formulations, oral or nasal inhaled or nasal drugs, nebulizers, sprays, and inhalers.