Selenium-containing polyamino acid, pharmaceutical composition and application

Abstract

Disclosed is a selenium-containing polyamino acid having a structure represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein A, B, D, E, m, n and x are as defined in the specification. Also disclosed is a pharmaceutical composition comprising a selenium-containing polyamino acid or a pharmaceutically acceptable salt thereof. Also disclosed is the use of the selenium-containing polyamino acid or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the selenium-containing polyamino acid or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating diseases selected from osteoarthritis, rheumatoid arthritis and periodontitis. Formula (I): E [-(B) m-(A) n-D] x.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of polymers, in particular to a selenium-containing polyamino acid, a pharmaceutical composition comprising the selenium-containing polyamino acid, and uses thereof. BACKGROUND

[0002] Osteoarthritis is one of the most common degenerative diseases, which is more common in obese and elderly populations. Studies have shown that as of 2020, about 654 million people aged 40 and above worldwide suffered from symptomatic knee osteoarthritis (Lu, H. et al. EClinical Medicine. 2020, 100587, 29); in China, the current total number of osteoarthritis patients is more than 100 million (Long, H.; Zeng, X.; Liu, Q.; Wang, H.; Vos, T.; Hou, Y.; Lin, C.; Qiu, Y.; Wang, K.; Xing, D.; Zhang, Y.; Zhou, M.; Lin, J. Burden of Osteoarthritis in China, 1990-2017: Findings from the Global Burden of Disease Study 2017. The Lancet Rheumatology 2020, 2(3), e164-e172). With the trend of population aging and the increase of sports population, the incidence of osteoarthritis will further expand. This has become one of the major threats to human health worldwide.

[0003] Traditionally, the drug treatment for osteoarthritis is usually through analgesic treatment by anti-inflammatory or hormone drugs, which usually requires long-term medication and has obvious side effects, and at the same time lacks means for early and middle stage intervention of osteoarthritis. Recently, a new method-viscoelasticity supplement therapy has gradually been used for the treatment of osteoarthritis. This method injects exogenous viscoelastic polymer into the joint cavity to maintain the lubricating ability of synovial fluid, thereby slowing down the disease progression to a certain extent. This method is safe and has certain therapeutic effect.

[0004] A series of natural polysaccharides such as hyaluronic acid or chitosan have been approved for marketing for viscoelasticity supplement therapy of joint cavity. However, there are some problems in natural polysaccharide-based viscoelasticity supplement of joint cavity, for example, there is a large expression of hyaluronidase in joint cavity, which will accelerate the degradation of hyaluronic acid injected into joint cavity, so that the effect is maintained for a short time, and frequent injection is needed. For example, patients often need to inject hyaluronic acid into the joint cavity once a week for several weeks. Such frequent injection will increase the risk of joint swelling, infection and local adverse reactions. In addition, natural polysaccharides are generally produced by fermentation or extracted from bird tissues, which is high in cost and has potential biological safety hazards [Zhang, Z.; Wei, X.; Gao, J.; Zhao, Y.; Zhao, Y.; Guo, L.; Chen, C.; Duan, Z.; Li, P.; Wei, L. Intra-Articular Injection of Cross-Linked Hyaluronic Acid-Dexamethasone Hydrogel Attenuates Osteoarthritis: An Experimental Study in a Rat Model of Osteoarthritis. Int. J. Mol. Sci. 2016, 17 (4), 411.].

[0005] Therefore, it is urgent to develop a new drug for viscoelasticity supplement therapy for joint cavity injection. SUMMARY

[0006] To solve the above and other problems, at least one embodiment of the present disclosure provides a selenium-containing polyamino acid or a pharmaceutically acceptable salt thereof, the selenium-containing polyamino acid having a structure shown in formula (I):

[0007] E[-(B) m -(A) n -D] x

[0008] Formula (I)

[0009] wherein A is independently for each occurrence a selenium-containing amino acid unit having a structure shown in formula (I-a):

[0010]

[0011] wherein R1, R2, R3, R4and R5are each independently for each occurrence selected from H and C 1～6 alkyl, the C 1～6 alkyl is optionally substituted with 1 to 3 groups independently selected from halogen, C 1～6 alkyl, C 1～6 alkoxy, C1～6 substituted by haloalkyl, phenyl, cyano, nitro, amino, hydroxy, and carboxy, R6is, at each occurrence, independently selected from the group consisting of C 1～6 alkyl-Se-, and C 1～6 alkyl-Se + (O - )-, the C 1～6 alkyl is optionally substituted with 1 to 3 substituents independently selected from the group consisting of halo, C 1～6 alkyl, C 1～6 alkoxy, C 1～6 haloalkyl, phenyl, cyano, nitro, amino, hydroxy, and carboxy;

[0012] B is, at each occurrence, independently an oxyethylene unit, an oxypropylene unit, or an amino acid unit selected from the group consisting of:

[0013]

[0014] D is selected from the group consisting of H and C 1～6 acyl;

[0015] E is a core moiety having x number of attachment sites, wherein the backbone of the core moiety comprises 1 to 40 carbon atoms and 1 to 12 heteroatoms independently selected from the group consisting of N, O, and S;

[0016] m is, at each occurrence, independently an integer greater than or equal to 0 and less than or equal to 4,000, and n is, at each occurrence, independently an integer greater than or equal to 5 and less than or equal to 4,000; and

[0017] x is an integer selected from the group consisting of 1 to 20.

[0018] In some forms, the selenium-containing polyamino acid, or pharmaceutically acceptable salt thereof, is of Formula (I), wherein R6is CH3-Se- or CH3-Se + (O - )-.

[0019] In some forms, the selenium-containing polyamino acid, or pharmaceutically acceptable salt thereof, is of Formula (I), wherein x is 1, and E is selected from the group consisting of C 1～6 alkylamino, and C 6～10 arylamino, wherein the C 1～6 alkyl or C 6～10 aryl is optionally substituted with 1 to 3 substituents independently selected from the group consisting of halo, C 1～6 alkyl, C 1～6 alkoxy, C 1～6 haloalkyl, phenyl, cyano, nitro, amino, hydroxy, and carboxy.

[0020] In some forms, in the selenium-containing polyamino acid or its pharmaceutically acceptable salt, x is an integer from 2 to 20, and E is a polyvalent core moiety formed by removing x hydrogen atoms from one or more amino groups selected from the group consisting of:

[0021]

[0022] In some forms, m is 0 in the selenium-containing polyamino acid or its pharmaceutically acceptable salt; and R1, R2, R3, R4 and R5 are all hydrogen.

[0023] In some forms, in the selenium-containing polyamino acid or its pharmaceutically acceptable salt, m is an integer greater than or equal to 3 and less than or equal to 500 each time it appears; and B is an oxyethylene unit.

[0024] In some forms, the selenium-containing polyamino acid or its pharmaceutically acceptable salt, x is 1 and E is benzylamino.

[0025] In some forms, in the selenium-containing polyamino acid or its pharmaceutically acceptable salt, m is an integer greater than or equal to 5 and less than or equal to 2000 each time it appears; and B is a non-selenium-containing amino acid unit derived from sarcosine, lysine, or trifluoroacetyllysine.

[0026] In some forms, x is 1 in the selenium-containing polyamino acid or its pharmaceutically acceptable salt; and E is benzylamino.

[0027] In some forms, the selenium-containing polyamino acid or its pharmaceutically acceptable salt has a content of 0.01 ≤ m / (m+n) ≤ 0.99.

[0028] In some forms, the selenium-containing polyamino acid or its pharmaceutically acceptable salt has a content of 0.05 ≤ m / (m+n) ≤ 0.95.

[0029] In some forms, in the selenium-containing polyamino acid or its pharmaceutically acceptable salt, A and B are arranged in any order in the molecular chain of the selenium-containing polyamino acid.

[0030] In some forms, the selenium-containing polyamino acid or its pharmaceutically acceptable salt contains 0.1% to 30% selenium.

[0031] At least one embodiment of this disclosure also discloses a pharmaceutical composition comprising any of the above-described selenium-containing polyamino acids or their pharmaceutically acceptable salts, and a pharmaceutically acceptable carrier.

[0032] In some forms, the pharmaceutical composition is an injectable aqueous solution.

[0033] In some forms, the injectable aqueous solution contains PBS as a solvent.

[0034] In some forms, the injectable aqueous solution also contains GSH as a reducing agent.

[0035] At least one embodiment of this disclosure also discloses the use of any of the above-mentioned selenium-containing polyamino acids or their pharmaceutically acceptable salts or pharmaceutical compositions in the preparation of medicaments for treating diseases selected from osteoarthritis, rheumatoid arthritis, and periodontitis. Attached Figure Description

[0036] To more clearly illustrate the technical solutions of the embodiments of this disclosure, the accompanying drawings of the embodiments will be briefly described below. Obviously, the drawings described below only relate to some embodiments of this disclosure and are not intended to limit this disclosure.

[0037] Figure 1 The 1H NMR spectrum (400MHz, solvent: CF3COOD / CDCl3 = 1:2 (v / v) of the polyselenomethionine homopolymer pSeMet60 according to an embodiment of this application is shown.

[0038] Figure 2 The proton NMR spectrum (400 MHz, solvent: CF3COOD / CDCl3 = 1:2 (v / v)) of polyethylene glycol-terminated polyselenomethionine polymer PEG-b-pSeMet60 according to an embodiment of this application is shown.

[0039] Figure 3 The 1H NMR spectrum (400MHz, solvent: CF3COOD / CDCl3 = 1:2(v / v)) of pSar50-b-pSeMet10, a block copolymer of polysarcosine and polyselenomethionine according to an embodiment of this application, is shown.

[0040] Figure 4 The 1H NMR spectrum (400MHz, solvent: CF3COOD / CDCl3 = 1:2(v / v)) of the block copolymer pTFALys50-b-pSeMet10 of polytrifluoroacetyllysine and polyselenomethionine according to an embodiment of this application is shown.

[0041] Figure 5 The 1H NMR spectrum (400MHz, solvent: CF3COOD / CDCl3 = 1:2 (v / v) of the random copolymer pTFALys50-r-pSeMet60 of polytrifluoroacetyllysine and polyselenomethionine according to an embodiment of this application is shown.

[0042] Figure 6 The 1H NMR spectrum (400MHz, solvent: D2O) of pLys50-b-pSeMet10, a block copolymer of polylysine and polyselenomethionine according to an embodiment of this application, is shown.

[0043] Figure 7 The 1H NMR spectrum (400MHz, solvent: D2O) of pLys50-r-pSeMet10, a random copolymer of polylysine and polyselenomethionine according to an embodiment of this application, is shown.

[0044] Figure 8A ~C shows the 1H NMR spectrum (400MHz, solvent: D2O) of the ultra-high molecular weight random copolymers of polysarcosine and selenomethionine oxide according to embodiments of this application, namely: (A) single-arm trackless polyamino acid UH-pSar-r-pSeMetO-2000, (B) double-arm polyamino acid UH-PSar-r-PSeMetO-4000, and (C) triple-arm trackless polyamino acid UH-PSar-r-PSeMetO-6000, wherein the molar content of the selenomethionine oxide polymer unit is 5%;

[0045] Figure 9 Size exclusion chromatography (SEC) of the hexafluoroisopropanol (HFIP) phase of polyselenomethionine homopolymer according to an embodiment of this application is shown, wherein: red indicates pSeMet60, blue indicates pSeMet120, and the HFIP phase contains 50 mM CF3CO2Na.

[0046] Figure 10 The DMF phase SEC of a random copolymer of polytrifluoroacetyl lysine and polyselenomethionine according to an embodiment of this application is shown, wherein: red represents pTFA50-r-pSeMet10, blue represents pTFA50-r-pSeMet10, and the DMF phase contains 0.1M LiBr;

[0047] Figure 11A~E shows the 1H NMR spectrum and DMF phase SEC of the ultra-high molecular weight polysarcosine-polyselenomethionine random copolymer according to embodiments of this application, which are: (A) DMF phase SEC of three-arm random polyamino acid UH-pSar-r-pSeMet-6000 (red), two-arm random polyamino acid UH-pSar-r-pSeMet-4000 (blue) and one-arm random polyamino acid UH-pSar-r-pSeMet-2000 (purple), wherein the DMF phase contains 0.1M LiBr, (B) 1H NMR spectrum of four-arm random polyamino acid UH-PSar-r-PSeMet-800; (C) 1H NMR spectrum of five-arm random polyamino acid UH-PSar-r-PSeMet-1000; (D) 1H NMR spectrum of eight-arm random polyamino acid UH-PSar-r-PSeMet-8000; and (E) DMF phase SEC of four-arm random polyamino acid UH-PSar-r-PSeMet-800, five-arm random polyamino acid UH-PSar-r-PSeMet-1000, and eight-arm random polyamino acid UH-PSar-r-PSeMet-8000, wherein the DMF phase contains 0.1M LiBr;

[0048] Figure 12 The catalytic activity test results of the ultra-high molecular weight polysarcosine-polyselenomethionine oxide copolymer according to the embodiments of this application are shown, wherein: cyan represents UH-PSar-PSeMetO-1.0%, yellow represents UH-PSar-PSeMetO-2.0%, brick red represents blank, and dark blue represents control ebselen (a commercially available GPx simulant), wherein the test concentrations of UH-PSar-PSeMet-1.0% and UH-PSar-PSeMet-2.0% are 5.0 mg / mL, and the test concentration of ebselen is 0.1 mg / mL;

[0049] Figure 13 The cytotoxicity of rat chondrocytes after incubation for 24 h with selenium-containing polyamino acids of different concentrations, compositions, and structures according to embodiments of this application is shown. The selenium-containing polyamino acids used are: pLys50-b-pSeMet10 (pLys-pSeMet-1), pLys50-b-pSeMet20 (pLys-pSeMet-2), pLys50-r-pSeMet10 (pLys-pSeMet-3), and pLys50-r-pSeMet20.

[0050] Figure 14The cytotoxicity of UH-PSar-PSeMetO-1.0%+GSH to (a) rat chondrocytes and (b) mouse chondrocytes ATDC5, according to embodiments of this application, is shown.

[0051] Figure 15 The following is an illustration of the metabolism of UH-PSar-PSeMetO-2.0% with different degrees of polymerization in the rat knee joint according to embodiments of this application: (a) shows a schematic flowchart of the metabolism detection method, and (b) shows fluorescence imaging of the rat knee joint at different time points, wherein the copolymers used have degrees of polymerization of 200, 2000, and 4000, and the materials are modified with the fluorescent dye Cy5.5.

[0052] Figure 16 The Mankin scores of a rat model of osteoarthritis after 4 weeks of treatment with different selenium-containing polyamino acids according to embodiments of this application are shown, where: n = 3, ***p < 0.001, scale bar is 100 μm;

[0053] Figure 17 The selenium content in various organs of rats at 4, 8, and 12 weeks after intra-articular injection of selenium-containing polyamino acids according to embodiments of this application is shown. Detailed Implementation

[0054] For the purposes of the detailed description below, it should be understood that this disclosure may take various alternative variations and sequences of steps unless expressly stated otherwise. Furthermore, except where indicated by + in any operational example or otherwise, all figures representing the amount of an ingredient as used, for example, in the specification and claims, should be understood to be modified in all cases by the term “about.” Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximations varying according to the desired performance to be obtained from this disclosure. At least not in an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be interpreted at least according to the number of significant figures reported and by applying ordinary rounding techniques.

[0055] Although the numerical ranges and parameters described in this disclosure are approximate, the values ​​listed in the specific examples are reported as precisely as possible. However, any numerical value inherently contains some error that is necessarily caused by the standard deviation found in their respective test measurements.

[0056] Furthermore, it should be understood that any numerical range described herein is intended to include all subranges falling within it. For example, the range “1 to 10” is intended to include all subranges between the minimum value 1 and the maximum value 10, i.e., a minimum value equal to or greater than 1 and a maximum value equal to or less than 10.

[0057] In this disclosure, unless otherwise expressly stated, the use of the singular includes the plural and the plural includes the singular. Furthermore, in this disclosure, unless otherwise expressly stated, “or” is used to mean “and / or,” even if “and / or” may be explicitly used in certain circumstances. Additionally, in this disclosure, unless otherwise expressly stated, “a” or “an” is used to mean “at least one.” For example, “a” polymer, “a” composition, etc., refer to any one or more of these articles.

[0058] Osteoarthritis is one of the most common degenerative diseases, prevalent among obese and middle-aged and elderly individuals, with the vast majority being symptomatic osteoarthritis of the knee. Not all osteoarthritis patients exhibit symptoms. However, those already diagnosed with symptomatic osteoarthritis need to actively seek treatment to prevent the damage within the joint from gradually worsening, eventually leading to joint deformity and loss of joint function.

[0059] Currently, clinical treatments for osteoarthritis include topical and oral analgesics and anti-inflammatory drugs. These methods can provide short-term relief of osteoarthritis symptoms, but they cannot reverse the progression of the disease, have low utilization rates, and pose potential risks of systemic side effects. Alternatively, intra-articular drug injection can be used to treat osteoarthritis. Intra-articular injection is an effective treatment strategy for osteoarthritis, but it is hampered by problems such as rapid drug diffusion and degradation, thus requiring frequent injections. For example, hyaluronic acid (HA) is one of the most commonly used joint lubricants in clinical practice and is a major component of synovial fluid. However, the high expression of hyaluronidase at the site of osteoarthritis accelerates its degradation, and patients typically need weekly injections for several weeks. In addition, anti-inflammatory drugs such as corticosteroids are commonly used clinically, but they also suffer from short residence time in the body after single injection and require repeated injections. Frequent intra-articular injections increase the risk of joint swelling and infection; the occurrence of local adverse reactions is also related to the physician's injection technique, the patient's post-injection activity, and the purity of the drug.

[0060] To address the above and other issues, this disclosure provides a selenium-containing polyamino acid and its application in the treatment of osteoarthritis. In some cases, the selenium-containing polyamino acid of this disclosure can be used to replace hyaluronic acid-based intra-articular injections commonly used in current literature and clinical practice. It exhibits a longer intra-articular degradation time (up to 12 weeks) in patients with osteoarthritis, significantly exceeding the degradation time of hyaluronic acid. During treatment based on this selenium-containing polyamino acid, patients can significantly reduce the frequency of intra-articular injections, requiring only one injection every 1-3 months, far lower than the frequency of hyaluronic acid injections, which are typically administered approximately once a week for five consecutive weeks. Furthermore, this invention utilizes the selenium-containing polyamino acid to mimic glutathione reductase, efficiently scavenging reactive oxygen species (ROS) and significantly reducing inflammation at the site of osteoarthritis. Under the same injection frequency and dosage, its therapeutic effect is superior to hyaluronic acid-based injections.

[0061] In some embodiments, this disclosure provides selenium-containing polyamino acids of formula (I) or pharmaceutically acceptable salts thereof:

[0062] E[-(B) m -(A) n -D] x

[0063] Formula (I)

[0064] in,

[0065] Each time A appears, it is an independent selenium-containing amino acid unit with the structure shown in formula (Ia):

[0066]

[0067] in,

[0068] R1, R2, R3, R4, and R5 are each independently selected from H and C each time they appear. 1～6 Alkyl, the C 1～6 Alkyl groups are optionally selected from 1 to 3 independently chosen from halogens, C 1～6 Alkyl, C 1～6 Alkoxy, C 1～6 Further substitution by substituents of haloalkyl, phenyl, cyano, nitro, amino, hydroxyl, and carboxyl groups, and

[0069] R6 is selected independently from C each time it appears. 1～6 Alkyl-Se- and C 1～6 Alkyl-Se + (O - )-, the C 1～6 Alkyl groups are optionally selected from 1 to 3 independently chosen from halogens, C 1～6 Alkyl, C 1～6 Alkoxy, C 1～6 The substituents of haloalkyl, phenyl, cyano, nitro, amino, hydroxyl and carboxyl groups are further substituted;

[0070] Each time B appears, it is independently an oxyethylene unit, a propylene oxide unit, or an amino acid unit selected from the following group:

[0071]

[0072] D is selected from H and C. 1～6 Acyl end group;

[0073] E is a core portion having x connection sites, wherein the main chain of the core portion comprises 1 to 40 carbon atoms and 1 to 12 heteroatoms independently selected from N, O and S;

[0074] Each time m appears, it is an independent integer greater than or equal to 0 and less than or equal to 4,000; each time n appears, it is an independent integer greater than or equal to 5 and less than or equal to 4,000; and

[0075] x is an integer selected from 1 to 20.

[0076] In some embodiments, the selenium-containing polyamino acids described in this application may be in reduced or oxidized form. For example, in formula (I) above, R6 may be C 1～6 Alkyl-Se- and C 1～6 Alkyl-Se + (O - )-, such as CH3-Se- or CH3-Se + (O - )-wait.

[0077] In other embodiments, the selenium-containing polyamino acid described in this application may be a single-arm polymer or a multi-arm polymer (e.g., a polymer having 2 to 20 arms, such as polymers having 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, or 2 to 6 arms).

[0078] On the one hand, as used herein, the term "single-arm polymer," also generally referred to as a linear polymer, refers to a polymer whose molecular chain is mainly composed of a series of repeating units linearly linked by covalent bonds. Single-arm polymers generally lack significant branching or cyclic structures, exhibiting a relatively linear morphology. Examples of single-arm polymers include, but are not limited to, polyethylene and polypropylene, whose molecular chains are composed of a large number of ethylene or propylene monomer units arranged linearly. In other words, a single-arm polymer can be understood as a linear polymer chain with a single, unbranched structure. In some examples, the selenium-containing polyamino acid represented by formula (I) above can be a single-arm polymer, i.e., the core portion E may have only a single linker site, and the polymer molecular chain may contain only a single -(B). m -(A) n -D. For example, in equation (I) above, x can be 1, and E can be selected from, for example, C. 1～6 Alkylamino and C 6～10 The core portion of the arylamino group, wherein the C 1～6 Alkyl or C 6～10 Aryl groups are optionally selected from 1 to 3 independently selected from halogens, C 1～6 Alkyl, C 1～6 Alkoxy, C 1～6The substituents of halogenated alkyl, phenyl, cyano, nitro, amino, hydroxyl, and carboxyl groups are further substituted. For example, E may include, but is not limited to, methylamino, ethylamino, n-propylamino, isopropylamino, n-butylamino, tert-butylamino, phenylamino, tolueneamino, ethylphenylamino, xyleneamino, trimethylamino, benzylamino, phenylethylamino, etc. For example, E may be benzylamino, etc. Such single-arm selenium-containing polyamino acid polymers can be formed by reacting one or more polymeric monomers, for example, using a substance having only a single functional group capable of initiating polymerization, as an initiator. For example, the selenium-containing polyamino acid polymers described in this application can be formed by polymerizing selenium-containing amino acid monomers (such as selenomethionine, etc.) and optionally one or more other comonomers (such as ethylene oxide, propylene oxide, various non-selenium-containing amino acids, etc.) using a monoamine (such as methylamine, ethylamine, n-propylamine, tert-butylamine, benzylamine, etc.) as an initiator.

[0079] On the other hand, as used herein, the term "multi-armed polymer" refers to a polymer whose molecular chain contains multiple polymer branches (i.e., arms). Examples of multi-armed polymers may include, but are not limited to, star polymers, dendritic polymers, hyperbranched polymers, etc. Alternatively, multi-armed polymers can be classified into polymers having two, three, four, five, six, eight, ten, twelve, fourteen, sixteen, eighteen, twenty, or more arms. In some examples, the core portion E of the selenium-containing polyamino acid shown in formula (I) above may have multiple (e.g., 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, or more) linkage sites, and its molecular chain may contain -(B) corresponding one-to-one with these linkage sites. m -(A) n -D polymer branches (i.e., arms). Such multi-arm selenium-containing polyamino acid polymers can be formed, for example, by reacting a substance having multiple functional groups (such as amino groups) capable of initiating polymerization with one or more polymeric monomers as an initiator. For example, the selenium-containing polyamino acid polymers described in this application can be formed by using polyamines selected from the group consisting of:

[0080]

[0081] As an initiator, a selenium-containing amino acid monomer (such as selenomethionine monomer, etc.) is polymerized with one or more other comonomers (such as ethylene oxide monomer, propylene oxide monomer, various non-selenium-containing amino acid monomers, etc.). In the resulting selenium-containing polyamino acid structure, the core moiety E can be a multivalent core moiety having multiple linkage sites formed by removing hydrogen atoms from one or more amino groups selected from the polyamines of the above group. For example, E can be a divalent group CH2(-NH-)2, a trivalent group CH(-CH2CH2NH-)3 or N(-CH2CH2NH-)3, or a multivalent group with a similar structure.

[0082] In other embodiments, the selenium-containing polyamino acid described in this application may be a homopolymer or copolymer.

[0083] On the one hand, as used herein, the term "homogeneous polymer" refers to a polymer formed by the polymerization of only a single monomer, such as polyethylene, polypropylene, polylactic acid, polyglutamic acid, etc. In some examples, the selenium-containing polyamino acid shown in formula (I) above can be a polyselenomethionine homopolymer, i.e., m can be 0. For example, in formula (I) above, m can be 0; R1, R2, R3, R4, and R5 can be independently selected from hydrogen and C. 1～6 Alkyl groups, etc. For example, in formula (I) above, m can be 0; R1, R2, R3, R4 and R5 can all be hydrogen.

[0084] On the other hand, as used herein, the term "copolymer" refers to a polymer formed by the polymerization of two or more monomers. Copolymers can be classified according to the types of monomers involved in the polymerization, such as binary copolymers, ternary copolymers, and more. As used herein, the term "binary copolymer" refers to a copolymer whose polymer molecular chain is composed of two comonomers; the term "ternary copolymer" refers to a copolymer whose polymer molecular chain is composed of three comonomers; and so on. In some embodiments, the selenium-containing polyamino acid described in this application may be a binary copolymer formed by copolymerizing selenomethionine monomer with another different comonomer, such as a binary copolymer formed by copolymerizing selenomethionine monomer with ethylene oxide or propylene oxide monomers, or a binary copolymer formed by copolymerizing selenomethionine monomer with various non-selenomethionine amino acid monomers, etc. Alternatively, the selenium-containing polyamino acid described in this application may be a ternary or more copolymer formed by copolymerizing selenomethionine monomer with two or more different comonomers, such as a ternary copolymer formed by copolymerizing selenomethionine monomer with ethylene oxide or propylene oxide monomer and another different non-selenomethionine amino acid monomer, a ternary copolymer formed by copolymerizing selenomethionine monomer with two other different non-selenomethionine amino acid monomers, or a multi-component copolymer formed by copolymerizing selenomethionine monomer with ethylene oxide or propylene oxide monomer and two or more other different non-selenomethionine amino acid monomers, etc.

[0085] Alternatively, copolymers can also be classified according to the arrangement of monomers in the copolymer's molecular weight as random copolymers (r), block copolymers (b), alternating copolymers (alt), graft copolymers (g), etc. As used herein, the term "random copolymer" refers to a copolymer in which different types of monomer units appear randomly in the copolymer molecular chain, with a small number of consecutive units of a single type of monomer unit and no regular arrangement order; the term "block copolymer" refers to a copolymer in which two or more types of monomer units each exist as long segments composed of only a single type of monomer unit (e.g., segments of hundreds or even thousands of monomer units), forming a block structure overall; the term "alternating copolymer" refers to a copolymer in which different structural units in the copolymer molecular weight are strictly alternating, and the molar fraction of each structural unit in the copolymer is basically the same; the term "graft copolymer" refers to a polymer in which the main chain of the copolymer molecular chain is composed of a single type of monomer unit, while the branches are composed of other types of monomer units. The copolymers described in this application may optionally be random copolymers, block copolymers, or any combination thereof.

[0086] In some aspects, for example, the selenium-containing polyamino acid described in this application may be a trackless copolymer or block copolymer of selenomethionine monomer and ethylene oxide monomer. For example, the selenium-containing polyamino acid described in this application may be PEG-terminated polyselenomethionine. In some examples, in formula (I) above, B may be an ethylene oxide (EO) unit; E may be benzylamino; m may be an integer greater than or equal to about 3 and less than or equal to about 500 independently. For example, in formula (I) above, m may be greater than or equal to about 3, greater than or equal to about 5, greater than or equal to about 10, greater than or equal to about 20, greater than or equal to about 30, greater than or equal to about 50, greater than or equal to about 75, greater than or equal to about 100, greater than or equal to about 125, greater than or equal to about 150, and less than or equal to about 450, less than or equal to about 400, less than or equal to about 350, less than or equal to about 300, less than or equal to about 250, less than or equal to about 200.

[0087] In other aspects, the selenium-containing polyamino acid described in this application may be a trackless copolymer or block copolymer of selenomethionine monomer and another non-selenium-containing amino acid comonomer. For example, the other non-selenium-containing amino acid comonomer may be selected from the following group:

[0088]

[0089] In some examples, the additional non-selenium amino acid comonomers used in this application may be selected from Sar, Lys, TFALys, or any combination thereof.

[0090] In other aspects, the selenium-containing polyamino acids described in this application may also be trackless copolymers or block copolymers of selenomethionine monomers with ethylene oxide monomers and one or more non-selenium-containing amino acid monomers (such as one or more non-selenium-containing amino acid monomers selected above).

[0091] In some examples, the selenium-containing polyamino acids described in this application may have a suitable degree of polymerization. For example, in formula (I) above, m may be an integer greater than or equal to about 0 and less than or equal to about 2000, such as greater than or equal to about 10, greater than or equal to about 20, greater than or equal to about 50, greater than or equal to about 100, greater than or equal to about 200, greater than or equal to about 500, greater than or equal to about 750, greater than or equal to about 1000, greater than or equal to about 1250, greater than or equal to about 1500, greater than or equal to about 1600, greater than or equal to about 1700, greater than or equal to about 1800, greater than or equal to about 1900, and less than or equal to about 2000; n may be an integer greater than or equal to about 2000. Integers greater than or equal to 5 and less than or equal to approximately 2000, for example, greater than or equal to approximately 5, greater than or equal to approximately 10, greater than or equal to approximately 20, greater than or equal to approximately 50, greater than or equal to approximately 100, greater than or equal to approximately 200, greater than or equal to approximately 500, greater than or equal to approximately 750, greater than or equal to approximately 1000, greater than or equal to approximately 1250, greater than or equal to approximately 1500, greater than or equal to approximately 1600, greater than or equal to approximately 1700, greater than or equal to approximately 1800, greater than or equal to approximately 1900, and less than or equal to approximately 2000; provided that the sum of m+n is not greater than 2000. In some respects, m and n can have any suitable ratio. For example, m / (m+n) can be between 0.01 and 0.99, such as 0.05–0.95, 0.05–0.90, 0.05–0.80, 0.05–0.60, 0.05–0.50, 0.05–0.30, 0.05–0.20, etc.

[0092] In some examples, when the selenium-containing polyamino acid described in this application is a copolymer of selenomethionine monomer and another non-selenium-containing amino acid monomer, the selenium-containing polyamino acid may optionally be a block copolymer or a random copolymer. For example, in formula (I) above, x may be an integer from 1 to 20, for example, an integer from 1 to 3; m may be an integer greater than 0 and less than or equal to 4,000; n may be an integer greater than 5 and less than or equal to 4,000. For example, in the molecular chain of the selenium-containing polyamino acid of formula (I) above, A and B may be arbitrarily selected within the range defined above and arranged in any order.

[0093] In some embodiments, the selenium-containing polyamino acids described in this application may have a suitable selenium content. For example, the selenium content in the selenium-containing polyamino acids may be about 0.1% to 30%.

[0094] In some embodiments, the selenium-containing polyamino acids described in this application may also exist as pharmaceutically acceptable salts. Examples of suitable pharmaceutically acceptable salts include, but are not limited to: inorganic salts, such as hydrochlorides, hydrobroms, sulfates, phosphates, etc.; organic salts, such as formates, acetates, trifluoroacetates, maleates, tartrates, etc.; sulfonates, such as methanesulfonates, benzenesulfonates, p-toluenesulfonates, etc.; amino acid salts, such as arginine salts, aspartate salts, glutamate salts, etc.; and metal salts, such as sodium salts, potassium salts, cesium salts, etc.; alkaline earth metal salts, such as calcium salts, magnesium salts, etc.; organic amine salts, such as triethylamine salts, pyridine salts, picolinate salts, ethanolamine salts, triethanolamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, etc.

[0095] At least one embodiment of this application also provides a pharmaceutical composition. The pharmaceutical composition may comprise any of the selenium-containing polyamino acids or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers may include, but are not limited to, solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic reagents, absorption-delaying reagents, salts, preservatives, pharmaceuticals, pharmaceutical stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and combinations thereof, as known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, pp. 1289-1329). In some examples, the pharmaceutically acceptable carrier may include solvents, diluents, gelling agents, dispersion media, etc.

[0096] In some embodiments, the pharmaceutical composition described in this application can be prepared as an injectable dosage form. For example, the pharmaceutical composition described in this application can be an injectable aqueous solution or an injectable hydrogel, such as an injectable aqueous solution. In some forms, the injectable aqueous solution may contain PBS or a similar carrier as a solvent. In other forms, the injectable aqueous solution may further contain GSH as a reducing agent.

[0097] At least one embodiment of this application also provides the use of any of the above-described selenium-containing polyamino acids or their pharmaceutically acceptable salts or pharmaceutical compositions in the preparation of treatments for various diseases. These diseases may include, but are not limited to, osteoarthritis, rheumatoid arthritis, and periodontitis. In some aspects, the selenium-containing polyamino acids or their pharmaceutically acceptable salts or pharmaceutical compositions containing them described in this application can be used as viscoelastic substances to provide lubrication to the joint cavity, or additionally to scavenge reactive oxygen species. Additionally, the selenium-containing polyamino acids or their pharmaceutically acceptable salts or pharmaceutical compositions prepared therefrom described in this application can be long-acting when used for intra-articular injection, for example, maintaining efficacy for a period exceeding 4 weeks, 5 weeks, 6 weeks, 8 weeks, or even 10 weeks or longer. Therefore, the selenium-containing polyamino acids or their pharmaceutically acceptable salts or pharmaceutical compositions prepared therefrom described in this application can at least partially or even completely replace currently used hyaluronic acid-based intra-articular injections in the literature and clinical practice, exhibiting a longer intra-articular degradation time in osteoarthritis, reaching 12 weeks or longer, far exceeding the degradation time of hyaluronic acid-based hydrogels. Therefore, when using medications made from selenium-containing polyamino acids to treat diseases, patients can significantly reduce the frequency of injections, requiring only one injection every 1 to 3 months, far lower than the frequency of hyaluronic acid injections (approximately once a week for five consecutive weeks). Furthermore, these selenium-containing polyamino acids can mimic glutathione reductase, effectively scavenging reactive oxygen species and significantly reducing inflammation at the site of osteoarthritis. Under the same injection frequency and dosage, its therapeutic effect is better than that of hyaluronic acid-based injections.

[0098] Examples

[0099] The embodiments of this application will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be construed as limiting the scope of this application. Unless otherwise specified, specific conditions in the examples are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used without specified manufacturers are all commercially available conventional products. Unless otherwise indicated, all quantities listed are described in parts by weight based on total weight. This application should not be construed as being limited to the specific embodiments described.

[0100] Preparation Example 1: Preparation of poly(L-selenomethionine) homopolymer

[0101] A 0.1 M ultra-dry dichloromethane solution was prepared from selenomethionine carboxyl ring anhydride (SeMet NCA) (50 mg, 0.23 mmol). After thorough stirring, a 0.5 M dichloromethane solution of benzylamine was added. The reaction was allowed to proceed at room temperature for 24 h, and the reaction mixture was then added to anhydrous diethyl ether. The precipitate was dried under vacuum for 24 h to obtain a white solid, which was the product poly( L -Selenomethionine).

[0102] Different poly(L-selenomethionine) homopolymers were prepared using the feed ratios shown in Table 1 and the method described above.

[0103] Table 1. Composition and parameters of selenium-containing polyamino acids

[0104]

[0105]

[0106] Note:

[0107] (1) The polymerization reactions were all carried out at room temperature (~25℃) and under a nitrogen atmosphere, and the infrared absorption spectrum IR was at ~1780cm. -1 The characteristic absorption at the site indicates a conversion rate higher than 95% for the treated reaction;

[0108] (2) The theoretical molecular weights cMn and D, calculated based on the feed ratio, were obtained by size exclusion chromatography / multi-angle light scattering / differential refractive index (SEC / MALS / RI) of the HFIP (5mM CF3CO2Na) phase. dMn and D were obtained by DMF (0.1M LiBr).

[0109] The phase was obtained by SEC / MALS / RI coupled measurement;

[0110] (3) DCM: dichloromethane; DMF: dimethylformamide.

[0111] Figure 1 The 1H NMR spectrum (400MHz, solvent: CF3COOD / CDCl3 = 1:2 (v / v) of the polyselenomethionine homopolymer pSeMet60 according to an embodiment of this application is shown. Figure 9 Size exclusion chromatography (SEC) of the HFIP phase of the polyselenomethionine homopolymer according to an embodiment of this application is shown, wherein: red indicates pSeMet60, blue indicates pSeMet120, and the HFIP phase contains 50 mM CF3CO2Na.

[0112] Preparation Example 2: Preparation of poly(selenomethionine) block copolymer

[0113] 2.1. Preparation of poly(ethylene glycol-L-selenomethionine) block copolymer PEG-b-pSeMet

[0114] SeMet NCA (50 mg, 0.23 mmol) was prepared into a 0.1 M ultra-dry dichloromethane solution. After thorough stirring, a 0.5 M benzylamine (BnNH2) dichloromethane solution was added. The reaction was allowed to proceed for 24 h, resulting in a colorless, transparent liquid. The precipitate was formed in anhydrous diethyl ether and dried under vacuum for 24 h to obtain a white solid.

[0115] Using the feed ratios shown in Table 1, different poly(ethylene glycol-L-selenomethionine) block copolymers were prepared by the method described above.

[0116] Figure 2 The 1H NMR spectrum (400MHz, solvent: CF3COOD / CDCl3 = 1:2 (v / v)) of polyethylene glycol-terminated polyselenomethionine polymer PEG-b-pSeMet60 according to an embodiment of this application is shown.

[0117] 2.2. Preparation of poly(sarcosine-L-selenomethionine) block copolymer pSar-b-pSeMet

[0118] Sarcosine carboxycyclic anhydride (Sar NCA) was dissolved in ultra-dry dichloromethane, and 17 μL of a 0.5 M benzylamine solution in dichloromethane (0.0086 mmol, 1.0 equivalent) was added. After 12 h, infrared absorption spectroscopy showed that Sar NCA was completely consumed. SeMet NCA (19 mg, 0.087 mmol, 10.0 equivalent) was weighed, dissolved in ultra-dry dichloromethane to prepare a 0.1 M solution, and added to the above reaction system. After 24 h, the system was a colorless and transparent liquid. The precipitate was formed in anhydrous diethyl ether and dried under vacuum for 24 h to obtain a white solid.

[0119] Using the feed ratios shown in Table 1, different poly(sarcosine-L-selenomethionine) homopolymers were prepared by the method described above.

[0120] Figure 3 The 1H NMR spectrum (400MHz, solvent: CF3COOD / CDCl3 = 1:2 (v / v)) of the block copolymer pSar50-b-pSeMet10 of polysarcosine and polyselenomethionine according to an embodiment of this application is shown.

[0121] 2.3. Preparation and deprotection of poly(L-(N-epsilon-trifluoroacetyl)lysine-L- selenomethionine) block copolymer pTFALys-b-pSeMet. Figure 4

[0122] Weigh 50 mg of TFALysNCA (0.19 mmol, 50.0 equivalents) and dissolve it in DMF. Add 17 μL of 0.5 M BnNH2 in DMF solution. After 24 h, infrared absorption spectroscopy showed that TFALysNCA was completely consumed. Weigh 8.3 mg of SeMetNCA (0.037 mmol, 10.0 equivalents) and dissolve it in dichloromethane. Mix thoroughly. After 24 h, the system was a colorless and transparent liquid. Precipitate formed in anhydrous diethyl ether and dried under vacuum for 24 h to obtain a white solid.

[0123] Using the feed ratios shown in Table 1, different poly(L-(N-ε-trifluoroacetyl)lysine-L-selenomethionine) block copolymers were prepared by the method described above.

[0124] Weigh 50 mg (approximately 0.19 mmol TFALys, 1.0 equivalent) of pTFALys50-b / r-pSeMet10 into a mixture of 20 mL methanol and 1 mL deionized water. Add anhydrous K₂CO₃ (250 mg, 1.8 mmol, 10 equivalent). Heat to 55 °C and stir vigorously for 4 h while sealed. Remove the solvent by rotary evaporation, and dissolve the crude product in 5% HCl solution. Dialyze the solution in 2 M NaCl solution for 24 h, then in deionized water for 48 h, and freeze-dry the sample solution. The product is a white, lumpy substance.

[0125] Figure 6 The 1H NMR spectrum (400MHz, solvent: CF3COOD / CDCl3 = 1:2(v / v)) of the block copolymer pTFALys50-b-pSeMet10 of polytrifluoroacetyllysine and polyselenomethionine according to an embodiment of this application is shown. Figure 10 The 1H NMR spectrum (400MHz, solvent: D2O) of pLys50-b-pSeMet10, a block copolymer of polylysine and polyselenomethionine according to an embodiment of this application, is shown. Preparation Example 3: Preparation of poly(selenomethionine) random copolymer The diagram shows the DMF phase SEC of a trackless copolymer of polytrifluoroacetyl lysine and polyselenomethionine according to an embodiment of this application, wherein: red represents pTFA50-r-pSeMet10, blue represents pTFA50-r-pSeMet10, and the DMF phase contains 0.1M LiBr.

[0126] Preparation of poly(L-(N-epsilon-trifluoroacetyl)lysine-L-selenomethionine) copolymer pTFALys-r-pSeMet and deprotection

[0127] Figure 5 Figure 7

[0128] Weigh 50 mg of TFALys NCA (0.19 mmol, 50.0 equivalent) and dissolve it in DMF. Weigh 8.3 mg of SeMet NCA (0.037 mmol, 10.0 equivalent) and dissolve it in dichloromethane, mixing thoroughly. Add 17 μL of 0.5 M BnNH2 in DMF solution. After 24 h, infrared absorption spectroscopy showed that TFALys NCA and SeMet NCA were completely consumed. After 24 h, the system was a colorless and transparent liquid. The precipitate was formed in anhydrous diethyl ether and dried under vacuum for 24 h to obtain a white solid.

[0129] The above product was deprotected using a method similar to that in Preparation Example 2, Section 2.3, to obtain a poly(L-lysine-L-selenomethionine) copolymer.

[0130] Preparation Example 4: Preparation of ultra-high molecular weight (UH) polymersThe 1H NMR spectrum (400MHz, solvent: CF3COOD / CDCl3 = 1:2 (v / v) of the random copolymer pTFALys50-r-pSeMet60 of polytrifluoroacetyllysine and polyselenomethionine according to an embodiment of this application is shown. 4.1. Preparation of UH-PSar-r-PSeMet-2000 The 1H NMR spectrum (400MHz, solvent: D2O) of pLys50-r-pSeMet10, a random copolymer of polylysine and polyselenomethionine according to an embodiment of this application, is shown.

[0131] 4.2. Preparation of PSeMet oxide PSeMetO

[0132] Figure 8A

[0133] Sar NCA (200 mg, 1.72 mmol, 1900.0 equivalents) and SeMet NCA (20 mg, 0.092 mmol, 100.0 equivalents) were weighed and dissolved in 5 mL of ultra-dry dichloromethane. 1.8 μL of a 0.5 M BnNH2 solution in dichloromethane and 9.0 μL of a 0.5 M benzoic acid solution in dichloromethane were added. After 48 h, infrared absorption spectroscopy showed complete consumption of NCA. The system was a colorless, transparent liquid. The precipitate was formed in anhydrous diethyl ether and dried under vacuum for 24 h to obtain a white solid, UH-PSar-PSeMet-2000. Other polymers with different SeMet ratios could be prepared under similar conditions by changing the feed ratio. The overall degree of polymerization of all UH-PSar-PSeMet-2000 was controlled to 2000 by adjusting the feed ratio.

[0134] 4.3. Preparation of two-armed random polyamino acid UH-PSar-r-PSeMet-4000

[0135] Weigh 100 mg of UH-PSar-PSeMet-2000 (0.063 mmol SeMet, 1.0 equivalent) and dissolve it in 500 μL of dH₂O. Add 8.0 μL of 30% H₂O₂ (~1.2 equivalent). Stir at room temperature for 6 h. The 1H NMR spectrum showed that SeMet was completely converted to SeMetO. After dialyzing and lyophilization, a white solid, UH-PSar-PSeMetO-2000, was obtained.

[0136] Figure 8BThe 1H NMR spectrum (400 MHz, solvent: D2O) of the ultra-high molecular weight random copolymer UH-pSar-r-pSeMetO-2000-5% of polysarcosine and selenomethionine oxide according to an embodiment of this application is shown. Figure 11 shows the DMF phase SEC of the ultra-high molecular weight random copolymer of polysarcosine and selenomethionine according to an embodiment of this application, wherein: red represents UH-pSar-r-pSeMet-6000, blue represents UH-pSar-r-pSeMet-4000, and purple represents UH-pSar-r-pSeMet-2000, and the DMF phase contains 0.1 M LiBr.

[0137] 4.3 Preparation of three-armed random polyamino acid UH-PSar-r-PSeMet-6000

[0138] Sar NCA (200 mg, 1.72 mmol, 1900.0 equivalents) and SeMet NCA (20 mg, 0.092 mmol, 100.0 equivalents) were weighed and dissolved in 5 mL of ultra-dry dichloromethane. 1.8 μL of a 0.25 M ethylenediamine solution in dichloromethane and 18.0 μL of a 0.5 M benzoic acid solution in dichloromethane were added. After 48 h, infrared absorption spectroscopy showed complete consumption of NCA. The system was a colorless, transparent liquid. The precipitate was formed in anhydrous diethyl ether and dried under vacuum for 24 h to obtain a white solid, UH-PSar-PSeMet-4000. Other polymers with different SeMet ratios could be prepared under similar conditions by changing the feed ratio. The overall degree of polymerization of all UH-PSar-PSeMet-4000 was controlled to 4000 by adjusting the feed ratio. Figure 8C The 1H NMR spectrum of the product is shown (400MHz, solvent: D2O). Figure 11A

[0139] Sar NCA (200 mg, 1.72 mmol, 1900.0 equivalents) and SeMet NCA (20 mg, 0.092 mmol, 100.0 equivalents) were weighed and dissolved in 5 mL of ultra-dry dichloromethane. 1.2 μL of a 0.25 M tris(2-aminoethyl)amine solution in dichloromethane and 27.0 μL of a 0.5 M benzoic acid solution in dichloromethane were added. After 48 h, infrared absorption spectroscopy showed complete consumption of NCA. The system was a colorless, transparent liquid. The precipitate was formed in anhydrous diethyl ether and dried under vacuum for 24 h to obtain a white solid, UH-PSar-PSeMet-6000. Other polymers with different SeMet ratios could be prepared under similar conditions by changing the feed ratio. All UH-PSar-PSeMet-6000 polymers were prepared with a total degree of polymerization of 6000 controlled by adjusting the feed ratio. Preparation of four-armed random polyamino acid UH-PSar-r-PSeMet-800 The 1H NMR spectrum of the product is shown (400MHz, solvent: D2O).

[0140] Figure 11B The DMF phase SEC of the ultra-high molecular weight polysarcosine-polyselenomethionine random copolymer prepared in Sections 4.1, 4.2 and 4.3 of Preparation Example 4 above is shown, wherein red represents the three-arm random polyamino acid UH-pSar-r-pSeMet-6000, blue represents the two-arm random polyamino acid UH-pSar-r-pSeMet-4000, and purple represents the single-arm random polyamino acid UH-pSar-r-pSeMet-2000 (purple) DMF phase SEC, wherein the DMF phase contains 0.1M LiBr.

[0141] 4.4 Preparation of five-armed random polyamino acid UH-PSar-r-PSeMet-1000

[0142] Sar NCA (200 mg, 1.72 mmol, 800.0 equivalents) and SeMet NCA (7.8 mg, 0.035 mmol, 16.0 equivalents) were weighed and dissolved in 5 mL of ultra-dry dichloromethane. 4.3 μL of a 0.5 M polypropylene imine triamine dendritic polymer (first generation) in dichloromethane and 124.0 μL of a 0.5 M benzoic acid solution in dichloromethane were added. After 48 h, infrared absorption spectroscopy showed complete consumption of NCA. The system was a colorless, transparent liquid. The precipitate was formed in anhydrous diethyl ether and dried under vacuum for 24 h to obtain a white solid, UH-PSar-PSeMet-800. Other polymers with different SeMet ratios could be prepared under similar conditions by changing the feed ratio. Figure 11C The 1H NMR spectrum of the product is shown (400MHz, solvent: D2O).

[0143] 4.5 Preparation of eight-armed random polyamino acid UH-PSar-r-PSeMet-8000

[0144] Sar NCA (200 mg, 1.72 mmol, 1000.0 equivalent) and SeMet NCA (7.8 mg, 0.035 mmol, 20.0 equivalent) were weighed and dissolved in 5 mL of ultra-dry dichloromethane. 3.4 μL of a 0.5 M tetraethylenepentamine solution in dichloromethane and 88.4 μL of a 0.5 M benzoic acid solution in dichloromethane were added. After 48 h, infrared absorption spectroscopy showed complete consumption of NCA. The system was a colorless, transparent liquid. The precipitate was formed in anhydrous diethyl ether and dried under vacuum for 24 h to obtain a white solid, UH-PSar-PSeMet-1000. Other polymers with different SeMet ratios could be prepared under similar conditions by changing the feed ratio. Figure 11D The 1H NMR spectrum of this product is shown (400MHz, solvent: D2O).

[0145] 4.6 Figure 11E

[0146] Sar NCA (200 mg, 1.72 mmol, 8000.0 equivalents) and SeMet NCA (7.8 mg, 0.035 mmol, 160.0 equivalents) were weighed and dissolved in 5 mL of ultra-dry dichloromethane. Then, 4.3 μL of a 0.05 M polypropyleneimine triamine dendritic polymer (second generation) in dichloromethane and 326.0 μL of a 0.5 M benzoic acid solution in dichloromethane were added. After 48 h, infrared absorption spectroscopy showed complete consumption of NCA. The system was a colorless, transparent liquid. The precipitate was formed in anhydrous diethyl ether and dried under vacuum for 24 h to obtain a white solid, UH-PSar-PSeMet-8000. Other polymers with different SeMet ratios could be prepared under similar conditions by changing the feed ratio. Test Examples: The 1H NMR spectrum of the product is shown (400MHz, solvent: D2O).

[0147] Cytotoxicity: The DMF phase SEC of the ultra-high molecular weight polysarcosine-polyselenomethionine random copolymer prepared in Sections 4.4, 4.5 and 4.6 of Preparation Example 4 above is shown, wherein: green represents four-arm random polyamino acid UH-PSar-r-PSeMet-800, blue represents five-arm random polyamino acid UH-PSar-r-PSeMet-1000, and red represents eight-arm random polyamino acid UH-PSar-r-PSeMet-8000, wherein the DMF phase contains 0.1M LiBr.

[0148] Figure 13

[0149] Figure 14

[0150] Extracted rat or mouse chondrocytes (ATDC5) were seeded into 96-well plates (2 × 10⁶ cells per well). 3 Cells (100 μL of culture medium). After incubation for 24 h, replace with 150 μL of fresh culture medium, and add the corresponding drug to the corresponding wells, and continue incubation for 24 h. Before detection, remove the original culture medium (reducing substances will affect the measurement results), and wash the plate three times with PBS. Add 100 μL of fresh culture medium and 10 μL of CCK-8 (Cell Counting Kit-8), incubate at 37 °C for 1 h, and measure the optical density (OD) values ​​at 450 nm (detection wavelength) and 630 nm (reference wavelength).

[0151] GPx catalytic activity of selenium-containing polyamino acids:The cytotoxicity of rat chondrocytes after incubation for 24 h with different concentrations of selenium-containing polyamino acids according to embodiments of this application is shown. The selenium-containing polyamino acids used are: pLys50-b-pSeMet10 (pLys-pSeMet-1), pLys50-b-pSeMet20 (pLys-pSeMet-2), pLys50-r-pSeMet10 (pLys-pSeMet-3), and pLys50-r-pSeMet20. Figure 12 The cytotoxicity of UH-PSar-PSeMetO-1.0%+GSH according to embodiments of this application against: (a) rat chondrocytes and (b) mouse chondrocytes ATDC5 is shown.

[0152] The results showed that UH-PSar-PSeMetO-1.0%+GSH did not exhibit significant cytotoxicity within the concentration range of 100 ng / mL to 2.00 mg / mL, and even at 1.00 μg / mL, it showed pro-cell proliferation properties. Furthermore, UH-PSar-PSeMetO-1.0%+GSH also did not show cytotoxicity against mouse chondrocyte ATDC5 cells within the same concentration range.

[0153] Therapeutic effect of selenium-containing polyamino acids in a rat osteoarthritis model (OA):

[0154] The catalytic activity of GPx containing selenium polyamino acids was determined by the NADPH colorimetric method.

[0155] The experimental groups consisted of UH-PSar-PSeMetO-1.0% (5.0 mg / mL) and UH-PSar-PSeMetO-2.0% (5.0 mg / mL), the control group consisted of the selenium-containing small molecule drug ebuselin (0.1 mg / mL), and the blank group consisted of PBS. In UH-PSar-PSeMet-x, 'x' represents the SeMet content in the polyamino acid. The working solution was prepared by thoroughly mixing 20 μL of 100 mM NADPH solution, 20 μL of 75 mM GSH solution, 10 μL of glutathione reductase GR solution, and 750 μL of PBS solution. The oxidation solution was prepared by diluting 21.5 μL of 70% peroxide tert-butanol (TBHP) solution with 4.0 mL of deionized water. 40 μL of sample solution, 80 μL of working solution, and 20 μL of oxidation solution were added sequentially, and the decrease in absorbance at 340 nm was measured at room temperature. The GPx catalytic activity of ibuselin was set to 100, and the GPx catalytic activity of selenium-containing polyamino acids was calculated as follows:

[0156] GPx catalytic activity = 100 × ((Rx-R0) × CSe / MSe) / ((Reb-R0) × Ceb / Meb) where Rx, CSe, and Mx are the slope, selenium content, and selenium molecular weight of the experimental group (containing selenium polyamino acids), respectively, and R0, Reb, Ceb, and Meb are the slope of the blank group, the slope of the control group (ibuselenoline), the concentration of ibuselenoline, and the molecular weight of ibuselenoline, respectively.

[0157] Figure 15 The catalytic activity test results of the ultra-high molecular weight polysarcosine-polyselenomethionine oxide copolymer according to the embodiments of this application are shown, wherein: cyan represents UH-PSar-PSeMetO-1.0%, yellow represents UH-PSar-PSeMetO-2.0%, brick red represents blank, and dark blue represents control ibuselin, wherein the test concentration of UH-PSar-PSeMet-1.0% and UH-PSar-PSeMet-2.0% is 5.0 mg / mL, and the test concentration of ibuselin is 0.1 mg / mL.

[0158] In the results, UH-PSar-PSeMetO-2.0% and UH-PSar-PSeMetO-1.0% were selected as templates to verify the GPx catalytic activity of selenium-containing polyamino acids, with a concentration of 5.0 mg / mL. The positive control was ibuselin at a concentration of 0.1 mg / mL. The slope of the UH-PSar-PSeMetO-1.0% group was 0.02220 ± 0.00007, the slope of the ibuselin group was 0.02517 ± 0.00015, and the slope of the Blank group was 0.01395 ± 0.00006. After conversion, at the same selenium content, the catalytic activity of UH-PSar-PSeMetO-1.0% was approximately 38% of that of ibuselin. With increasing selenium content, the GPx catalytic activity of selenium-containing polypeptides at the same mass concentration increases. The above results indicate that UH-PSar-PSeMetO exhibits significant GPx catalytic activity and can regulate ROS levels.

[0159] Biosafety

[0160] Adult SD rats (4 months old, weighing 300-500g, male) were selected as animal models of osteoarthritis (OA).

[0161] (1) The purchased 80 adult SD rats were randomly divided into 8 groups:

[0162] ①Sham Group;

[0163] ②UH-PSar-PSeMetO-5.0% group: 25μL of UH-PSar-PSeMetO-5.0% PBS solution (the solid content of UH-PSar-PSeMetO-5.0% is 2.0wt%) was injected into the knee joint;

[0164] ③UH-PSar-PSeMetO-5.0%+GSH group: 25μL of 80mM GSH solution of UH-PSar-PSeMetO-5.0% (the solid content of UH-PSar-PSeMetO-5.0% is 2.0wt%) was injected into the knee joint;

[0165] ④ UH-PSar-PSeMetO-2.0% + GSH group: 25 μL of 80 mM GSH solution of UH-PSar-PSeMetO-2.0% (the solid content of UH-PSar-PSeMetO-2.0% is 2.0 wt%) was injected into the knee joint;

[0166] ⑤ UH-PSar-PSeMetO-1.0% group: 25 μL of 80 mM GSH solution of UH-PSar-PSeMetO-1.0% (the solid content of UH-PSar-PSeMetO-1.0% is 2.0 wt%) was injected into the knee joint;

[0167] ⑥UH-PSar group: 25μL of UH-PSar in PBS solution was injected into the knee joint (UH-PSar Mn = 140kDa, degree of polymerization is comparable to UH-PSar-PSeMetO-1.0% / 2.0% / 5.0%, and its solid content in solution is 2.0wt%).

[0168] ⑦ UH-PSar+GSH group: 25 μL of 80 mM GSH solution of UH-PSar (the solid content of UH-PSar is 2.0 wt%) was injected into the knee joint;

[0169] ⑧PBS group: 25 μL of PBS was injected into the knee joint.

[0170] (2) The SD rats in the Sham group underwent sham knee surgery (i.e., skin incision and suturing) as a normal control group; the other 4 groups first underwent knee OA modeling surgery (i.e., anterior cruciate ligament transection and medial meniscectomy), and the groups were intervened in different ways 2 weeks after the operation. Four weeks after the intervention, the knee joints of the SD rats were harvested for subsequent testing.

[0171] (3) Four weeks after treatment, the knee joints of SD rats in each group were harvested. Soft tissue other than osteochondral tissue was removed as much as possible. After washing with physiological saline, gross observation, photography and ICRS cartilage damage grading system scoring were completed within 6 hours.

[0172] Four weeks after treatment, tissue samples from the knee joints of SD rats in each group were collected, fixed in formaldehyde, decalcified, dehydrated, and embedded in paraffin. Serial sections were prepared at a thickness of 5 μm, and the paraffin sections were retrieved from glass slides and baked overnight for later use. Histological evaluation was performed using hematoxylin and eosin (H&E), toluidine blue (TB), safranin O-Fixed Green (SO&FG) staining, type II collagen (ColII) immunohistochemical staining, and the Mankin score for cartilage injury histological assessment. Figure 17 The Mankin scores of a rat model of osteoarthritis are shown after 4 weeks of treatment with different selenium-containing polyamino acids according to embodiments of this application, where: n = 3, ***p < 0.001, and scale bar is 100 μm.

[0173] In terms of gross results, at 4 weeks post-surgery, compared to the Sham group, the PBS group showed very obvious osteophytes and bone defects in the medial tibial plateau region and tibial plateau. For example, the gray arrows in the PBS group indicate large and deep defects in the tibial plateau. Gross anatomical results showed that the cartilage structure of the rats was destroyed at 4 weeks post-surgery, and the surgery successfully induced early OA formation. The cartilage damage in the PSar and PSar+GSH groups was similar to that in the PBS group; in fact, their tibial osteophytes and bone defects were even more significant than those in the PBS group, indicating that they did not have an OA treatment effect. The cartilage damage in the UH-PSarPSeMetO-5.0% group was slightly better than that in the PBS, PSar, and PSar+GSH groups, but its tibial and femoral osteophytes and bone defects were still very obvious, with an uneven surface, wrinkled texture, and a relatively coarse, pale cartilage structure. The UH-PSar-PSeMetO-5.0%+GSH group had 80mM GSH added to the injection solution as a reducing agent in the early stages of treatment. Under the same treatment time and dosage, the cartilage damage in the UH-PSar-PSeMetO-5.0%+GSH group was less than that in the UH-PSar-PSeMetO-5.0% group, the cartilage surface was smoother, and the morphology was similar to that of the Sham group. The treatment effects of the UH-PSar-PSeMetO-2.0%+GSH group and the UH-PSarPSeMetO-1.0%+GSH group were similar to those of the UH-PSar-PSeMetO-5.0%+GSH group.

[0174] In the tissue section results, at 4 weeks post-surgery, the cartilage layer boundaries (such as the tidal line) in the PBS group were discontinuous and incomplete, with obvious defects. The thickness (such as the deeper orange-red portion in COL II) was worn very thin, and phenomena such as the subchondral bone layer breaking through to the cartilage layer were observed, indicating severe cartilage damage. The cartilage structure in the PSar group, PSar+GSH group, and UH-PSarPSeMetO-5.0% group was also severely damaged, similar to the PBS group, with COL II almost completely degraded and no obvious repair effect. The repair effect in the UH-PSar-PSeMetO-5.0%+GSH group, UH-PSar-PSeMetO-2.0%+GSH group, and UH-PSarPSeMetO-1.0%+GSH group was significantly better than that in other experimental groups. The cartilage structure was very intact, and the tidal line and other boundaries were complete and continuous without obvious defects. For example, the cartilage structure integrity and COL II content in the UH-PSar-PSeMetO-5.0%+GSH group, the UHPSar-PSeMetO-2.0%+GSH group, and the UH-PSar-PSeMetO-1.0%+GSH group were significantly higher than those in the UH-PSar-PSeMetO-5.0% group, the PSar group, and the PSar+GSH group. Therefore, the combined administration of UH-PSar-PSeMetO+GSH is the most effective treatment, and SeMetO is the key component responsible for this therapeutic effect.

[0175] Based on tissue sections after 4 weeks of treatment, the Mankin histological scoring system systematically evaluated the treatment efficacy in each group. The scores of the PBS group, PSA group, and PSA+GSH group were very similar, indicating that PSA and PSA+GSH had almost no therapeutic effect on OA. Compared with the PBS group, the UH-PSar-PSeMetO-5.0% group had a slightly lower score, but still higher than the UH-PSar-PSeMetO-5.0%+GSH group, UH-PSar-PSeMetO-2.0%+GSH group, and UH-PSar-PSeMetO-1.0%+GSH group. These results indicate that the combined injection of UH-PSar-PSeMetO and GSH is the most effective.

[0176] ​

[0177] Four weeks after injection, the rats were sacrificed, and their hearts, livers, spleens, lungs, kidneys, and lymph nodes were removed. Serum was collected, and the selenium content was detected. ​ The selenium content in various organs of rats was shown 4 weeks after intra-articular injection of selenium-containing polyamino acids according to embodiments of this application.

[0178] The results showed that, 4 weeks after injection, the selenium content in all organs of the rats was comparable to that in the corresponding organs of the uninjected controls. This demonstrates that the selenium-containing polyamino acid of this application exhibits high biocompatibility when used for intra-articular injection.

[0179] The above description is merely a specific embodiment of this disclosure, but the scope of protection of this disclosure is not limited thereto. The scope of protection of this disclosure should be determined by the scope of protection of the claims.

Claims

1. A selenium-containing polyamino acid or a pharmaceutically acceptable salt thereof, said selenium-containing polyamino acid having the structure shown in formula (I): E[-(B) m -(A) n -D] x Formula (I) in, Each time A appears, it is an independent selenium-containing amino acid unit with the structure shown in formula (Ia): in, R1, R2, R3, R4, and R5are each independently at each occurrence selected from the group consisting of H and C 1～6 alkyl, said C 1～6 alkyl, said C 1～6 alkyl, said C 1～6 alkyl, said C 1～6 haloalkyl, phenyl, cyano, nitro, amino, hydroxy, and carboxy, are further substituted with 1 to 3 substituents independently selected from the group consisting of halo, C R6is independently at each occurrence selected from C 1～6 alkyl-S-, and C 1～6 alkyl-S + (O - )-, said C 1～6 alkyl is optionally further substituted with 1 to 3 substituents independently selected from the group consisting of halogen, C 1～6 alkyl, C 1～6 alkoxy, C 1～6 haloalkyl, phenyl, cyano, nitro, amino, hydroxy, and carboxy; Each time B appears, it is independently an oxyethylene unit, a propylene oxide unit, or an amino acid unit selected from the following group: D is selected from H and C 1～6 terminal groups of acyl groups; E is a core portion having x connection sites, wherein the main chain of the core portion comprises 1 to 40 carbon atoms and 1 to 12 heteroatoms independently selected from N, O and S; Each time m appears, it is an integer greater than or equal to 0 and less than or equal to 4,000; each time n appears, it is an integer greater than or equal to 5 and less than or equal to 4,000. x is an integer selected from 1 to 20.

2. The selenium-containing polyamino acid or pharmaceutically acceptable salt thereof of claim 1, wherein, R6is CH3-Se- or CH3-Se + (O - )-.

3. The selenium-containing polyamino acid or pharmaceutically acceptable salt thereof of claim 1, wherein, x is 1 and E is selected from C 1～6 alkylamino and C 6～10 aryl, wherein said C 1～6 alkyl or C 6～10 aryl is optionally substituted with 1 to 3 substituents independently selected from the group consisting of halogen, C 1～6 alkyl, C 1～6 alkoxy, C 1～6 haloalkyl, phenyl, cyano, nitro, amino, hydroxy and carboxy.

4. The selenium-containing polyamino acid or pharmaceutically acceptable salt thereof of claim 1, wherein, x is an integer from 2 to 20, and E is a polyvalent core moiety formed by removing x hydrogen atoms from one or more amino groups selected from the following groups:

5. The selenium-containing polyamino acid or pharmaceutically acceptable salt thereof of claim 1 or 2, wherein, m is 0; and R1, R2, R3, R4 and R5 are all hydrogen.

6. The selenium-containing polyamino acid or pharmaceutically acceptable salt thereof of claim 1 or 2, wherein, Each time m appears, it is an independent integer greater than or equal to 3 and less than or equal to 500; and B is an oxyethylene unit.

7. The selenium-containing polyamino acid or pharmaceutically acceptable salt thereof of claim 6, wherein, x is 1, and E is benzylamino.

8. The selenium-containing polyamino acid or pharmaceutically acceptable salt thereof of claim 1 or 2, wherein, Each occurrence of m is an independent integer greater than or equal to 5 and less than or equal to 2000; and B is a non-seleno-containing amino acid unit derived from sarcosine, lysine, or trifluoroacetyllysine.

9. The selenium-containing polyamino acid or pharmaceutically acceptable salt thereof of claim 8, wherein, x is 1; and E is benzylamino.

10. The selenium-containing polyamino acid or pharmaceutically acceptable salt thereof of claim 8, wherein, 0.01≤m / (m+n)≤0.

99.

11. The selenium-containing amino acid or pharmaceutically acceptable salt thereof of claim 10, wherein, 0.05≤m / (m+n)≤0.

95.

12. The selenium-containing amino acid or pharmaceutically acceptable salt thereof of claim 8, wherein, A and B are arranged in any order in the molecular chain of the selenium-containing polyamino acid.

13. The selenium-containing polyamino acid or pharmaceutically acceptable salt thereof of claim 1 or 2, wherein, The selenium content in the selenium-containing polyamino acid is 0.1% to 30%.

14. A pharmaceutical composition comprising: a selenium-containing polyamino acid or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 13, and a pharmaceutically acceptable carrier.

15. The pharmaceutical composition of claim 14, wherein, The pharmaceutical composition is an injectable aqueous solution.

16. The pharmaceutical composition of claim 15, wherein the injectable aqueous solution comprises PBS as a solvent.

17. The pharmaceutical composition of claim 16, wherein the injectable aqueous solution further comprises GSH as a reducing agent.

18. Use of the selenium-containing polyamino acid or its pharmaceutically acceptable salt as described in any one of claims 1 to 13, or the pharmaceutical composition as described in any one of claims 14 to 17, in the preparation of a medicament for treating diseases selected from osteoarthritis, rheumatoid arthritis, and periodontitis.

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