Carrier and medicine

Abstract

Provided is a carrier which includes a structure represented by general formula (1) [in the formula (1), A is a first chain that has a sulfur atom capable of forming a disulfide bond with a sulfur atom in a thiol group in a cysteine residue located at position-34 from the N-terminus of human serum albumin; B is a second chain to be inserted into a hydrophobic group-binding pocket of the human serum albumin; and L is a linker moiety that links the first chain and the second chain to each other].

Description

Carriers and Drugs

[0001] This application claims priority to U.S. Provisional Application No. 63 / 654,081, filed in the United States on May 31, 2024, the contents of which are incorporated herein by reference.

[0002] Conventionally, in order to improve the blood retention of physiologically active substances such as GLP-1, which have a short half-life in blood, a technique has been known in which a drug bound to a carrier is attached to serum albumin present in the blood in the body.

[0003] For example, Patent Document 1 discloses a technology related to a manufacturing method for semaglutide. Semaglutide has a GLP-1 analog and a lipid chain, and the lipid chain enters and interacts with the hydrophobic pocket of serum albumin, allowing it to remain in the blood for approximately one week and exhibit sustained release properties.

[0004] Special table 2019-503369 publication

[0005] However, there is still room for improvement in the blood retention of drugs. The present invention has been made in view of such circumstances, and aims to provide a novel technology that can improve the blood retention of physiologically active substances.

[0006] In order to solve the above problems, one aspect of the present invention includes the following aspects.

[0007] [1] A carrier comprising a structure represented by the following general formula (1): [In the above formula (1), A is a first chain having a sulfur atom capable of forming a disulfide bond with the sulfur atom of the thiol group of the 34th cysteine ​​residue from the N-terminus of human serum albumin, B is a second chain inserted into the hydrophobic group-binding pocket of human serum albumin, and L is a linker moiety connecting the first chain and the second chain.]

[0008] [2] The carrier according to [1], wherein the first chain comprises a structure selected from the group consisting of the following (1-1) to (1-18): [In formula (1-11), R is an alkyl group having 1 or 2 carbon atoms.]

[0009] [3] The carrier according to [1] or [2], wherein the second chain is a chain having 15 to 25 carbon atoms and may contain one or more bonds selected from the group consisting of an ester bond, an ether bond, an amide bond, and a carbonyl bond.

[0010] [4] The carrier according to any one of [1] to [3], wherein the second chain has a structure represented by the following general formula (2): [In formula (2), R1 located at the terminal distal to the linker moiety is a methyl group or a carboxy group, and n1 is an integer of 1 to 20.]

[0011] [5] The carrier according to any one of [1] to [3], wherein the end of the second chain distal to the linker portion is a methyl group or an anionic group.

[0012] [6] The carrier according to [5], wherein the end of the second chain distal to the linker portion is a carboxy group.

[0013] [7] The support according to any one of [1] to [6], wherein the distance between A and B is 5 to 20 Å.

[0014] [8] The support according to any one of [1] to [7], wherein the linker portion has a structure of 5 to 20 carbon atoms and may contain one or more bonds selected from the group consisting of a hydroxy group, an ester bond, an ether bond, a sulfide group, a carbonyl group, an amide bond, and a peptide bond.

[0015] [9] The support according to any one of [1] to [8], which contains a structure represented by the following formula (3): [In formula (3), R1 is a methyl group or a carboxy group, and n1 is an integer from 13 to 18.]

[0016]

[10] A drug comprising the carrier according to any one of [1] to [9] linked to a physiologically active substance having a molecular weight of 4,000,000 or less.

[0017]

[11] The drug according to

[10] , wherein the physiologically active substance is a protein, peptide, antibody, DNA, RNA, siRNA, synthetic nucleic acid, or a small molecule drug with a molecular weight of 1,000 or less.

[0018]

[12] The drug according to

[10] or

[11] , wherein the physiologically active substance is glucagon-like peptide-1 (GLP-1), insulin, GIP (glucose-dependent insulinotropic polypeptide), exendin-3, or exendin-4.

[0019]

[13] The agent according to any one of

[10] to

[12] , further linked to wild-type or mutant human serum albumin.

[0020] According to the present invention, a novel technique can be provided that can improve the retention of a physiologically active substance in blood.

[0021] FIG. 1 is a schematic diagram showing the binding of a drug containing a carrier according to a preferred embodiment of the present invention and a physiologically active substance described below to human serum albumin. FIG. 2 is a diagram showing a method for synthesizing Suyamaglutide. FIG. 3 is a graph showing the MALDI-TOF mass spectrometry spectrum of Suyamaglutide s2. FIG. 4 is a graph showing the MALDI-TOF mass spectrometry spectrum of Suyamaglutide. FIG. 5 is a diagram showing a carrier containing the first and second strands, a carrier containing only the first strand, a carrier containing only the second strand, and fluorescent molecules bound thereto. FIG. 6 is a diagram showing various modified carriers used in in vitro experiments. FIG. 7 is a diagram showing various modified carriers used in in vivo experiments. FIG. 8 is a graph showing the time course of the amount of complex between various modified carriers and human serum albumin. FIG. 9 is a table showing the complex formation rate after mixing human serum albumin and various carriers at 100 μM each and shaking at 37°C for 30 minutes. FIG. 10 is a graph showing the time course of the concentration of modified carriers in the plasma of mice administered with various modified carriers. FIG. 11 is a table showing the half-lives of various modified carriers in mouse blood. FIG. 12 is a graph showing the time course of blood glucose levels when each drug is administered at 10 nmol / kg. FIG. 13 is a graph showing the time course of blood glucose levels when each drug is administered at 100 nmol / kg. FIG. 14 is a graph showing the time course of blood glucose levels for each dose in the native GLP-1 group. FIG. 15 is a graph showing the time course of blood glucose levels for each dose in the Semaglutide group. FIG. 16 is a graph showing the time course of blood glucose levels for each dose in the Suyamaglutide group.

[0022] As used herein, the term "comprise" means that components other than the target component may be included. The term "consist of" means that components other than the target component are not included. As used herein, the term "comprise" encompasses both "consist of" and "consist essentially of" embodiments. The term "consist essentially of" means that components other than the target component are not included in an embodiment that performs a special function (such as an embodiment that completely loses the effect of the invention).

[0023] In addition, in this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​written before and after "to" as the lower and upper limits.

[0024] Hereinafter, a carrier (or carriers) and a drug according to preferred embodiments of the present invention will be described in detail, with reference to the drawings where necessary, although the present invention is not limited to the following embodiments.

[0025] [Support] A support according to a preferred embodiment of the present invention includes a structure represented by the following general formula (1).

[0026]

[0027] [In the above formula (1), A is a first chain having a sulfur atom capable of forming a disulfide bond with the sulfur atom of the thiol group of a cysteine ​​residue (C34) that is the 34th amino acid residue from the N-terminus of human serum albumin (mature peptide; see the amino acid sequence shown in SEQ ID NO: 1), B is a second chain that is inserted into the hydrophobic group-binding pocket of human serum albumin, and L is a linker moiety that connects the first chain and the second chain.]

[0028] Fig. 1 is a schematic diagram showing the binding of a carrier according to a preferred embodiment of the present invention and a drug containing a physiologically active substance described below to human serum albumin. Fig. 1 shows a carrier 10 having a first chain 1 having a disulfide bond as an example of the first chain having a sulfur atom, and a second chain 2, but the carrier of this embodiment is not limited to this.

[0029] 1, when a drug 30 containing a carrier 10 and a physiologically active substance 20 is administered to a human body, the second chain 2 of the carrier 10 is first inserted into a hydrophobic group-binding pocket (hereinafter also referred to as "hydrophobic pocket") 41 of endogenous human serum albumin 40 through hydrophobic interaction and reversibly binds (STEP 1). Specifically, the hydrophobic pocket 41 is pocket 2 of the seven hydrophobic pockets of human serum albumin 40. Pocket 2 is the hydrophobic pocket closest to C34.

[0030] Next, a thiol-disulfide exchange reaction occurs between the thiol group of the cysteine ​​located at the 34th position from the N-terminus of human serum albumin 40 and the disulfide bond of first chain 1. As a result, first chain 1 of carrier 10 is bonded to human serum albumin via a disulfide bond (STEP 2). Therefore, drug 30 containing carrier 10 is firmly bound to human serum albumin 40, and the blood retention of physiologically active substance 20 can be effectively improved.

[0031] In addition, while it takes time for the thiol-disulfide exchange reaction to occur between the disulfide bond of the first chain 1 and human serum albumin 40, the interaction between the second chain 2 and the hydrophobic pocket occurs quickly in the blood, effectively suppressing renal excretion of drug 30 shortly after administration.

[0032] After binding to human serum albumin 40, a thiol exchange reaction occurs between carrier 10 and a compound having a thiol group, such as cysteine, circulating in the blood, and thus the physiologically active substance 20 is gradually released from human serum albumin 40 along with carrier 10 (STEP 3), thereby exerting a physiological function according to its structure.

[0033] (First Chain) As described above, in order to carry out a thiol-disulfide exchange reaction with the cysteine ​​residue, which is the 34th amino acid from the N-terminus of human serum albumin, the first chain of the carrier preferably contains the above-described structure having a disulfide bond containing a sulfur atom.

[0034] For example, the first chain preferably includes, as a structure having a disulfide bond, a structure selected from the group consisting of the following (1-1) to (1-18): In this specification, the wavy line in each formula indicates a bond.

[0035] [R in formula (1-11) is an alkyl group having 1 or 2 carbon atoms.]

[0036] The first chain preferably contains a structure represented by the above formula (1-10) among the above formulas (1-1) to (1-18), and particularly preferably contains a structure represented by the following formula (1-10A).

[0037]

[0038] Furthermore, it is more preferable that the first chain contains a structure represented by the following formula (1-10A1).

[0039]

[0040] (Second Strand) The second strand of the carrier is not particularly limited as long as it has a structure that allows it to interact with and be inserted into the hydrophobic pocket of human serum albumin, but is preferably a chain having 15 to 25 carbon atoms, which may contain one or more bonds selected from the group consisting of ester bonds, ether bonds, amide bonds, and carbonyl bonds. The number of carbon atoms is more preferably 19 to 25, even more preferably 20 to 24, particularly preferably 21 to 23, and most preferably 22.

[0041] The second chain of the carrier preferably has a structure represented by the following general formula (2).

[0042] [In formula (2), R1 located at the terminal distal to the linker moiety is a methyl group or a carboxy group, and n1 is an integer of 1 to 20.]

[0043] In the above formula (2), n1 represents a —CH group located between R1 and the carbon atom to which the oxygen atom is bonded via a double bond. ２ The above R1 may be a methyl group, but is preferably a carboxy group from the viewpoint of increasing blood retention.

[0044] In the above formula (2), n1 is preferably an integer of 10 to 18, more preferably an integer of 12 to 16, even more preferably an integer of 13 to 15, and particularly preferably 14.

[0045] Furthermore, the end of the second chain distal to the linker portion is preferably a methyl group or an anionic group from the viewpoint of enhancing hydrophobic interaction with the hydrophobic pocket, regardless of whether the second chain has the structure represented by formula (2) above.

[0046] The type of the anionic group is not particularly limited, but may be, for example, a carboxy group, a sulfonic acid group, or a phosphate group, and among these, the anionic group is preferably a carboxy group.

[0047] The second chain of the carrier more preferably has a structure represented by the following general formula (2-1).

[0048] [In formula (2-1), R1 located at the terminal distal to the linker moiety is a methyl group or a carboxy group, and n1a is an integer of 13 to 15.]

[0049] In the above formula (2-1), n1a may be 14, and R1 may be a carboxy group.

[0050] The second chain of the carrier particularly preferably has a structure represented by the following general formula (2-1A).

[0051] [In formula (2), R1 located at the terminal distal to the linker moiety is a methyl group or a carboxy group, and n1a is an integer of 13 to 15.]

[0052] In the above formula (2-1a), n1a may be 14, and R1 may be a carboxy group.

[0053] For the purpose of inserting the second chain into the hydrophobic pocket of human serum albumin and forming a disulfide bond between the first chain and C34 of human serum albumin, the distance between A and B, more specifically, the distance between the atom of the first chain bound to the linker moiety and the atom of the second chain bound to the linker moiety, is preferably 10 to 100 Å, more preferably 10 to 50 Å, even more preferably 15 to 30 Å, and may be 5 to 20 Å. For example, when a carrier binds to pocket 5, if the pocket and C34 are linked by the linker moiety, it is thought that the linkage will overlap with the FcRn-binding region of human serum albumin, inhibiting the binding of human serum albumin to FcRn. To avoid occlusion of the region between pocket 5 and C34 by the carrier, it is thought that the length of the linking portion between the first chain and the second chain is designed to match the distance between pocket 2 and C34.

[0054] The structure of the linker is not particularly limited as long as it is long enough to allow the second chain to be inserted into the hydrophobic pocket of human serum albumin and to form a disulfide bond between the first chain and C34 of human serum albumin, but may be, for example, a structure having 5 to 20 carbon atoms which may contain one or more bonds selected from the group consisting of a hydroxy group, an ester bond, an ether bond, a sulfide group, a carbonyl group, an amide bond, and a peptide bond. The number of carbon atoms may be 7 to 17, 10 to 14, 11 to 13, or 12.

[0055] The linker portion may be a hydroxy group, an ester bond, an ether bond, a sulfide group, a carbonyl group, an amide bond, a peptide bond, or an oxyethylene unit (-CH ２ -CH ２ The number of carbon atoms may be 7 to 17, 10 to 14, 11 to 13, or 12.

[0056] The linker portion is formed by an amide bond and an oxyethylene unit (-CH ２-CH ２ The number of carbon atoms may be 7 to 17, 10 to 14, 11 to 13, or 12.

[0057] The linker portion may have a structure represented by the following formula (4):

[0058]

[0059] The carrier of this embodiment containing the linker portion described above in detail may contain a structure represented by the following formula (3).

[0060] [In formula (3), R1 is a methyl group or a carboxy group, and n1 is an integer from 13 to 18.]

[0061] In the formula (3), R1 is preferably a carboxy group, and n1 is more preferably an integer of 13 to 15, and even more preferably 14.

[0062] The support having the structure represented by the above formula (3) may contain a structure represented by the following formula (3A).

[0063]

[0064] The carrier of this embodiment may also include a structure represented by the following general formula (3A-1). The carrier of this embodiment may also have a structure (excluding R) represented by reference numeral 100 in FIG. 5, which will be described later. In this case, the terminal methyl group at the end of the string of 14 methylene groups in FIG. 5 may be a carboxy group or another anionic group. When the carrier has the structure represented by reference numeral 100 in FIG. 5, (excluding R), R is a physiologically active substance, which will be described later, and the drug, which will be described later, may have the structure represented by reference numeral 100, which includes a physiologically active substance represented by R.

[0065] [In formula (3A-1), R1 is a methyl group or a carboxy group.]

[0066] The support having the structure represented by the above formula (3A-1) may contain a structure represented by the following formula (3A-1A):

[0067]

[0068] The carrier of this embodiment can improve the blood retention of a physiologically active substance, which will be described in detail later, and extend its blood half-life, thereby reducing the frequency of administration or ingestion of the physiologically active substance and easing the burden on patients.

[0069] [Drug] A drug according to a preferred embodiment of the present invention is a drug (in other words, a pharmaceutical composition) in which the carrier described in detail in the above embodiment is linked to a physiologically active substance having a molecular weight of 4,000,000 or less. That is, the drug of this embodiment includes the above-mentioned carrier and a physiologically active substance having a molecular weight of 4,000,000 or less linked to the carrier. The drug of this embodiment may be a drug in which the carrier and the physiologically active substance are synthesized separately and then linked, or may be a drug consisting of a compound in which the carrier portion and the physiologically active substance portion are synthesized consecutively.

[0070] The physiologically active substance is not particularly limited as long as it induces physiological activity in the human body, and may be, for example, a protein, peptide, antibody, DNA, RNA, siRNA, synthetic nucleic acid, or a small molecule drug with a molecular weight of 1,000 or less. The DNA and RNA may be a nucleic acid drug such as an antisense oligonucleotide.

[0071] The physiologically active substance may also be glucagon-like peptide-1 (GLP-1), insulin, GIP (glucose-dependent insulinotropic polypeptide), exendin-3, or exendin-4. GLP-1 may also be a modified GLP-1 agonist. An example of the GLP-1 agonist is a peptide comprising the amino acid sequence shown in SEQ ID NO: 2.

[0072] An example of a drug (in other words, a small molecule drug, pharmaceutical composition, or ligand) in which a GLP-1 agonist comprising the amino acid sequence shown in SEQ ID NO: 2 is linked to the above-mentioned carrier (specifically, a carrier having a structure represented by the above formula (3A-1A)) is a compound represented by the following formula (5):

[0073] [In formula (5), Aib is a 2-aminoisobutyric acid residue, which is an unnatural amino acid. AEEA means 2-[2-(2-aminoethoxy)ethoxy]acetic acid. The leading H indicates the N-terminus of the peptide moiety. Lys(pal) indicates a structure in which the carboxyl group in the palmitoyl group is amide-bonded to the ε-amino group of lysine.]

[0074] Placing an unnatural amino acid at the above position can confer resistance to cleavage in the human body. Furthermore, the present inventors have found that conjugating palmitic acid to the ε-amino group of lysine to form a second chain exhibits higher binding efficiency to human serum albumin than conjugating myristic acid (C13) or stearic acid (C17).

[0075] In the formula (5), the bond indicated by the wavy line in the formula (3A-1A) in the carrier is bound to a cysteine ​​residue located at the C-terminus of the GLP-1 agonist, but the binding position of the carrier to the GLP-1 agonist is not particularly limited. For example, the carrier may be bound to a lysine residue in the GLP-1 agonist represented by the formula (5). By binding the carrier to the lysine residue, inhibition of the affinity of the drug for the GLP-1 receptor can be suppressed.

[0076] Furthermore, a drug in which a GLP-1 agonist containing the amino acid sequence shown in SEQ ID NO: 2 is linked to a carrier may have a structure represented by the following formula (6): In the following formula (6), the carrier is bound to a lysine residue of the GLP-1 agonist represented by the above formula (5). [In formula (6), Aib is a 2-aminoisobutyric acid residue, which is an unnatural amino acid. The "H-" at the beginning indicates the N-terminus of the peptide moiety, and the "-OH" at the opposite end indicates the C-terminus.]

[0077] Furthermore, the drug of this embodiment may be linked (bound) to wild-type or mutant human serum albumin. In this case, the wild-type or mutant human serum albumin may be linked to the above-mentioned physiologically active substance such as a GLP-1 agonist via the above-mentioned carrier.

[0078] The amino acid sequence of the mutant human serum albumin is not particularly limited, but may be, for example, an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

[0079] Herein, the sequence identity of a subject amino acid sequence to a reference amino acid sequence (the amino acid sequence shown in SEQ ID NO: 1) can be determined, for example, as follows: First, the reference amino acid sequence and the subject amino acid sequence are aligned. Each amino acid sequence may contain gaps to maximize sequence identity. Next, the number of matching amino acids in the reference amino acid sequence and the subject amino acid sequence is calculated, and the sequence identity can be calculated according to the following formula (i):

[0080] Sequence identity (%) = number of matching amino acids / total number of amino acids in the target amino acid sequence × 100 (i)

[0081] [Pharmaceutical Composition] A pharmaceutical composition according to another preferred embodiment of the present invention comprises a drug as described in detail in the above embodiment of [Drug] and a pharmaceutically acceptable carrier. The pharmaceutical composition of this embodiment contains the drug as an active ingredient.

[0082] The pharmaceutical composition of this embodiment may be a drug or a quasi-drug, for example, as defined in the Japanese "Act on Ensuring Quality, Efficacy and Safety of Pharmaceuticals, Medical Devices, etc. (PMD Act)" or may be a quasi-drug.

[0083] The term "pharmaceutically acceptable carrier" refers to a carrier that does not inhibit the physiological activity of the active ingredient, the aforementioned drug, and is not substantially toxic to the subject to which it is administered. "Not substantially toxic" means that the ingredient is not toxic to the subject to which it is administered at a dose normally used. Pharmaceutically acceptable carriers include any known pharmaceutically acceptable ingredient that is typically considered an inactive ingredient. Pharmaceutically acceptable carriers include, but are not limited to, solvents, diluents, vehicles, excipients, glidants, binders, granulating agents, dispersing agents, suspending agents, wetting agents, lubricants, disintegrants, solubilizers, stabilizers, emulsifiers, fillers, etc. Pharmaceutically acceptable carriers may be used alone or in combination of two or more.

[0084] The pharmaceutical composition may contain other ingredients in addition to the above-mentioned components. The other ingredients are not particularly limited, and those commonly used in the pharmaceutical field can be used without particular limitation. Examples of the other ingredients include pharmaceutical additives other than those mentioned above. Examples of pharmaceutical additives include, but are not limited to, preservatives (e.g., antioxidants), chelating agents, flavoring agents, sweeteners, thickeners, buffers, coloring agents, etc.

[0085] The pharmaceutical composition may contain active ingredients other than the above-mentioned drugs, such as, but not limited to, other blood glucose lowering agents, digestive absorption enhancers, anti-inflammatory agents, antipyretics, analgesics, etc.

[0086] The dosage form of the pharmaceutical composition is not particularly limited and can be any dosage form commonly used for pharmaceutical preparations. The pharmaceutical composition of this embodiment may be an oral or subcutaneous formulation, or other formulations, with oral or subcutaneous formulations being preferred. Oral formulations include, for example, tablets, coated tablets, pills, powders, granules, capsules, syrups, fine granules, liquids, drops, and emulsions. Pharmaceutical compositions in these dosage forms can be formulated according to standard methods (e.g., methods described in the Japanese Pharmacopoeia). The same applies to the above-mentioned drugs. Note that when wild-type or mutant human serum albumin is bound to a drug described in detail in the above [Drug] embodiment, the pharmaceutical composition containing the drug is an injection and is administered intravenously.

[0087] The pharmaceutical composition can be administered in a therapeutically effective amount of the above-mentioned drug. "Therapeutically effective amount" means the amount of drug effective for treating or preventing the target disease. For example, a therapeutically effective amount for diabetes may be an amount effective for lowering blood glucose levels. The therapeutically effective amount may be determined appropriately depending on the patient's symptoms, weight, age, sex, etc., as well as the dosage form and administration method of the pharmaceutical composition, etc.

[0088] The pharmaceutical composition may be administered in a single dose or multiple doses. In the case of multiple doses, the administration interval may be appropriately determined depending on the patient's symptoms, body weight, age, sex, etc., as well as the dosage form and administration method of the pharmaceutical composition. The administration interval may be, for example, once every 10 days, once every two weeks, once every three weeks, once a month, once every few months, etc. The pharmaceutical composition is used to treat diabetes. The subject to which the pharmaceutical composition is administered is a human.

[0089] [Other Embodiments] In another embodiment, the present invention provides a method for treating diabetes, comprising administering a drug to a patient in need of treatment. The drug comprises a carrier as described in detail in the [Carrier] embodiment and a physiologically active substance. The physiologically active substance is GLP-1, a GLP-1 agonist, insulin, GIP, exendin-3, or exendin-4. The drug is the same as the drug described in detail in the [Drug] embodiment.

[0090] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the invention described in the claims, and it goes without saying that these modifications are also included within the scope of the present invention.

[0091] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0092] Experimental Example 1 In this experiment, Suyamaglutide, a drug containing the above-mentioned carrier and a GLP-1 agonist, was synthesized. Figure 2 illustrates the synthesis method for Suyamaglutide. In Figure 2, the circle symbol represents a solid-phase resin. First, Suyamaglutide s1 was synthesized on Rink amide-MBHA resin (0.68 mmol / g, 0.15 mmol scale) using an automated synthesizer (CSBio II) by Fmoc solid-phase synthesis. Each amino acid (1 mmol, 6.7 equivalents) was condensed in DMF using HBTU and Oxyma Pure (6.7 equivalents each) with DIEA (13.5 equivalents). The Fmoc group was removed with a 20% solution of piperidine in DMF. Washing was performed with DMF.

[0093] After the above synthesis, TFA / EDT / TIS / H ２ The resin was removed using 2 mL of a 94:2.5:1:2.5 (v / v) mixture of TFA and PEG-1000. The resin was then washed three times with 2 mL of pure TFA. 37 mL of diethyl ether was added, the mixture was stirred, and the mixture was centrifuged at 3500 × g for 5 minutes at 4 °C. The supernatant was then removed. This procedure was repeated three times. 8 mL of crude peptide was obtained.

[0094] The resulting crude peptide (Suyamaglutide s2) was purified by reversed-phase HPLC (C18 column, Inertsil ODS-3 5 μm (20 mm × 250 mm)). The mobile phase was H 2 O containing 0.1% TFA. ２ The target fraction was collected using O (A) and MeCN (B) under equal volume elution conditions at a flow rate of 8 mL / min, and Suyamaglutide s2 was purified by lyophilization.

[0095] Thereafter, the obtained Suyamaglutide s2 was dissolved in MeOH (0.5 mM, 2.2 mg / mL), mixed with a DPDS solution of the same concentration at a molar ratio of 1:1, and shaken at room temperature for 1 hour to introduce a disulfide bond.

[0096] The resulting sample was purified by reverse-phase HPLC. The HPLC was performed using the above-mentioned C18 column and a Hitachi LaChrom Elite system at a flow rate of 8 mL / min. Solvent A (water containing 0.1% (v / v) formic acid) and solvent B (MeCN containing 0.1% (v / v) formic acid) were used, and the concentration of solvent B was linearly increased from 30% to 60% over 30 minutes under gradient conditions. Fractions containing the target Suyamaglutide (Suyamaglutide s3 in Figure 2) were collected and lyophilized.

[0097] The thus obtained Suyamaglutide s2 and Suyamaglutide were subjected to MALDI-TOF mass spectrometry in the positive ion mode. CHCA (10 mg / mL) was selected as the matrix and mixed with the sample. Figure 3 is a graph showing the MALDI-TOF mass spectrum of Suyamaglutide s2. Figure 4 is a graph showing the MALDI-TOF mass spectrum of Suyamaglutide. In Figure 4, additional peaks are observed due to the cleavage of disulfide bonds.

[0098] [Experimental Example 2] In this experiment, the difference in the amount of binding to human serum albumin was confirmed between a carrier containing the first and second chains, a carrier containing only the first chain, and a carrier containing only the second chain.

[0099] Figure 5 is a diagram showing a carrier 100 containing the first and second strands, a carrier 200 containing only the first strand, and a carrier 300 containing only the second strand, with fluorescent molecules a or b bound to them. Figure 6 is a diagram showing various modified carriers used in in vitro experiments. Figure 7 is a diagram showing various modified carriers used in in vivo experiments. In Figure 5, fluorescent molecule a is carboxyfluorescein used in in vitro experiments, and fluorescent molecule b is Cy7 used in in vivo experiments. In the following, a modified carrier in which fluorescent molecule a is bound to carrier 100 will be referred to as modified carrier 100a, a modified carrier in which fluorescent molecule b is bound to carrier 100 will be referred to as modified carrier 100b, a modified carrier in which fluorescent molecule a is bound to carrier 200 will be referred to as modified carrier 200a, a modified carrier in which fluorescent molecule b is bound to carrier 200 will be referred to as modified carrier 200b, a modified carrier in which fluorescent molecule a is bound to carrier 300 will be referred to as modified carrier 300a, and a modified carrier in which fluorescent molecule b is bound to carrier 300 will be referred to as modified carrier 300b.

[0100] (In vitro experiment) Carboxyfluorescein-modified carriers 100a-300a (100 μM) were incubated with fatty acid-free human serum albumin (500 μM) in phosphate-buffered saline (pH 7.4) at 37°C for 1, 5, 10, 30, or 60 minutes to evaluate the rate of disulfide bond formation. During the reaction of each modified carrier with human serum albumin, the disulfide exchange reaction was stopped at predetermined time intervals by adding an excess amount of N-ethylmaleimide (NEM). The resulting disulfide-bonded complexes were isolated by reversed-phase high-performance chromatography (RP-HPLC). The complexes were treated with tris(2-carboxyethyl)phosphine (TCEP) to cleave the disulfide bond, and the released carrier was quantified by RP-HPLC.

[0101] Figure 8 is a graph showing the time course of the amount of complex between various modified carriers and human serum albumin. As shown in Figure 8, approximately 36 μM of modified carrier 100a bound to human serum albumin in the first minute (modification rate: 36%). In contrast, the amount of modified carrier 200a was approximately 19 μM, meaning that modified carrier 100a formed 1.7 times more complex with human serum albumin than modified carrier 200a. The reason why modified carrier 100a has a faster disulfide bond formation rate than modified carrier 200a is due to the presence of the second chain.

[0102] This suggests that the carrier having the second chain accelerates the formation of disulfide bonds with C34 due to the rapid initial binding. During the 60-minute reaction time, modified carrier 100a always showed a higher amount of complex than modified carrier 200a.

[0103] Figure 9 is a table showing the complex formation rate after mixing human serum albumin and various carriers at 100 μM and shaking at 37°C for 30 minutes. In Figure 9, N.D. indicates that no detection occurred. As shown in Figure 9, the modified carrier having the first and second chains had the highest binding rate (complex formation rate with human serum albumin) at about 30%. Note that about 70% of C34 of human serum albumin is oxidized to SO ２ , etc., it was revealed that substantially 100% of the human serum albumin was bound to the modified carrier 100a.

[0104] (In vivo experiment) A single dose of Cy7-modified carrier (100b, 200b, or 300b, 0.171 mM, 100 μL / mouse, 10% DMSO + saline) was intravenously administered to C57BL / 6N male mice (9 weeks old, n=3), and 40 μL of blood was collected over a period of 0 to 24 hours after administration. The fluorescence intensity in the plasma (excitation wavelength Ex: 750 nm, emission wavelength Em: 773 nm) was measured using a plate reader, and the concentration of each modified carrier was quantified.

[0105] Figure 10 is a graph showing the time course of modified carrier concentration in the plasma of mice administered various modified carriers. Figure 11 is a table showing the half-life of various modified carriers in mouse blood. In Figure 11, kel represents the clearance rate (drug elimination rate constant). As shown in Figure 10, modified carrier 100b having a first chain and a second chain remained at the highest concentration. In addition, modified carriers 100b and 300b having a second chain (alkyl) had high initial concentrations. This suggests that the initial concentration increased due to interaction with the hydrophobic pocket. In contrast, modified carrier 200b without a second chain was suggested to undergo a significant amount of renal excretion.

[0106] In addition, it was suggested that the modified carriers 100b and 200b having the first chain (disulfide bond) have good retention in blood.

[0107] [Experimental Example 3] In this experiment, the effect of Suyamaglutide on glucose tolerance was evaluated by an oral glucose tolerance test (OGTT). (Test animals) Five-week-old male C57BL / 6J mice were purchased and all were kept in a pathogen-free environment at the Fukuoka University Animal Center. Water and standard feed were available ad libitum during breeding. All animal experiments were conducted with the approval of the Fukuoka University Animal Experiment Ethics Committee. (Drugs used) The following compounds were used as GLP-1 receptor agonists: native GLP-1 Semaglutide Suyamaglutide (synthesized in Experimental Example 1)

[0108] Each drug was dissolved in DMSO, diluted with PBS, and administered intraperitoneally at a dose of 10 nmol / kg or 100 nmol / kg. A vehicle was administered to the control group in the same manner.

[0109] (OGTT) Seven-week-old mice were fasted for 16 hours, and then the GLP-1 receptor agonist (or vehicle) was administered intraperitoneally 15 minutes before the first glucose solution administration. At 0 and 135 minutes, 2 g / kg of glucose solution was administered orally. Blood was then collected from the tail vein and serum glucose concentrations were measured. Based on the obtained blood glucose data, the area under the blood glucose curve (AUC) for each group was calculated using the trapezoidal method. Each group contained 3-4 mice (n = 3-4).

[0110] (Statistical analysis) Data for each experimental group are shown as mean ± standard error. Comparisons between two groups were performed using Student's t test, and comparisons between three or more groups were performed using one-way ANOVA with Fisher's PLSD test. A P value of <0.05 was considered to indicate a statistically significant difference.

[0111] (Comparative evaluation between groups) Figure 12 is a graph showing the time course of blood glucose levels when each drug was administered at 10 nmol / kg. In the following figures, "i.p." indicates the timing of intraperitoneal administration of the GLP-1 receptor agonist, and "Glu" indicates the timing of oral administration of the glucose solution. As shown in Figure 12, the native GLP-1 group showed a blood glucose lowering effect starting 15 minutes after the first glucose load, but the blood glucose lowering effect following the second glucose load disappeared, and the group showed high blood glucose levels, similar to the vehicle group.

[0112] The Semaglutide group showed a blood glucose lowering effect 30 minutes after glucose loading, and blood glucose levels had fallen to baseline 120 minutes after glucose loading. After the second glucose load, the Semaglutide group showed a longer-lasting blood glucose lowering effect compared to the vehicle and native GLP-1 groups.

[0113] The Suyamaglutide group showed a blood glucose lowering effect starting 60 minutes after glucose loading, and blood glucose levels had fallen to baseline 120 minutes after glucose loading. After the second glucose load, the Suyamaglutide group showed a long-lasting blood glucose lowering effect equivalent to that of the Semaglutide group, compared to the vehicle and native GLP-1 groups.

[0114] Figure 13 is a graph showing the time course of blood glucose levels when each drug was administered at 100 nmol / kg. As shown in Figure 13, the native GLP-1 group showed a blood glucose lowering effect starting 15 minutes after the first glucose load, but the blood glucose lowering effect after the second glucose load disappeared, and blood glucose levels remained high, similar to the vehicle group.

[0115] The Semaglutide group showed a blood glucose lowering effect starting 15 minutes after glucose loading, and blood glucose levels had fallen to baseline 120 minutes after glucose loading. After the second glucose load, the Semaglutide group showed a longer-lasting blood glucose lowering effect compared to the vehicle and native GLP-1 groups.

[0116] The Suyamaglutide group showed a blood glucose lowering effect starting 15 minutes after glucose loading, and blood glucose levels had returned to baseline 120 minutes after glucose loading. After the second glucose load, the Suyamaglutide group showed a long-lasting blood glucose lowering effect equivalent to that of the Semaglutide group, compared to the vehicle and native GLP-1 groups.

[0117] (Evaluation of dose-dependent effect) Figure 14 is a graph showing the change in blood glucose level over time for each dose in the native GLP-1 group. As shown in Figure 14, in the native GLP-1 group, a blood glucose lowering effect was observed starting 15 minutes after the first glucose load at 10 nmol / kg, and a blood glucose lowering effect was observed dose-dependently at 100 nmol / kg. The blood glucose lowering effect following the second glucose load disappeared, and high blood glucose levels were similarly observed even at 100 nmol / kg administration.

[0118] Figure 15 is a graph showing the change in blood glucose levels over time for each dose in the Semaglutide group. As shown in Figure 15, the Semaglutide group showed a blood glucose lowering effect 30 minutes after glucose loading at 10 nmol / kg, and 15 minutes after glucose loading at 100 nmol / kg, demonstrating dose-dependence. After the second glucose load, both 10 nmol / kg and 100 nmol / kg showed equivalent blood glucose lowering effects compared to the vehicle group.

[0119] 16 is a graph showing the change in blood glucose levels over time for each dose in the Suyamaglutide group. As shown in FIG. 16, the Suyamaglutide group showed a blood glucose lowering effect starting 60 minutes after glucose loading at 10 nmol / kg, and starting 15 minutes after glucose loading at 100 nmol / kg, demonstrating dose-dependence. After the second glucose load, both 10 nmol / kg and 100 nmol / kg showed a dose-dependent blood glucose lowering effect compared to the vehicle group.

[0120] (Comparative evaluation of AUC during OGTT) The area under the curve (AUC) during OGTT was evaluated. In the native GLP-1 group, AUC tended to decrease compared to the vehicle group at both 10 nmol / kg and 100 nmol / kg, but no significant difference was observed.

[0121] At 10 nmol / kg, the semaglutide group showed a tendency to decrease compared to the vehicle group, but no significant difference was observed.On the other hand, at 100 nmol / kg, the semaglutide group showed a significant decrease compared to the vehicle group.

[0122] In the Suyamaglutide group, at 10 nmol / kg, there was a tendency for blood pressure to decrease compared to the vehicle group, but no significant difference was observed. On the other hand, in the Suyamaglutide group, at 100 nmol / kg, there was a significant decrease compared to the vehicle group. There was no significant difference between the Semaglutide group (100 nmol / kg) and the Suyamaglutide group (100 nmol / kg), which were significantly decreased compared to the vehicle group, and they were equivalent.

[0123] These results demonstrate that a single dose of Suyamaglutide in a long-duration OGTT had a longer-lasting blood glucose lowering effect than vehicle and native GLP-1, and was equivalent to that of Semaglutide. Although there were concerns that Suyamaglutide, whose C-terminus is chemically modified, may have a weaker effect on the GLP-1 receptor, it demonstrated a non-inferior blood glucose improving effect to Semaglutide, confirming its pharmacological activity in vivo.

[0124] The present invention provides a novel technique that can improve the blood retention of a physiologically active substance, and is therefore industrially applicable.

[0125] 1 First strand 2 Second strand 10 Carrier 20 Physiologically active substance 40 Human serum albumin 41 Hydrophobic group binding pocket (hydrophobic pocket) 100 Carrier 100a to 300a Carboxyfluorescein-modified carrier 100b to 300b Cy7-modified carrier 200 Carrier 300 Carrier a, b Fluorescent molecule

Claims

1. A carrier comprising a structure represented by the following general formula (1): [In the above formula (1), A is a first chain having a sulfur atom capable of forming a disulfide bond with the sulfur atom of the thiol group of the 34th cysteine ​​residue from the N-terminus of human serum albumin, B is a second chain inserted into the hydrophobic group-binding pocket of human serum albumin, and L is a linker moiety connecting the first chain and the second chain.] 2. The carrier according to claim 1, wherein the first chain comprises a structure selected from the group consisting of (1-1) to (1-18) below. [In formula (1-11), R is an alkyl group having 1 or 2 carbon atoms.] 3. The carrier according to claim 1, wherein the second chain is a chain having 15 to 25 carbon atoms and optionally containing one or more bonds selected from the group consisting of an ester bond, an ether bond, an amide bond, and a carbonyl bond.

4. The carrier according to claim 1, wherein the second chain has a structure represented by the following general formula (2): [In formula (2), R1 located at the terminal distal to the linker moiety is a methyl group or a carboxy group, and n1 is an integer of 1 to 20.] 5. The carrier according to claim 1, wherein the end of the second chain distal from the linker portion is a methyl group or an anionic group.

6. The carrier according to claim 5, wherein the terminus of the second strand distal to the linker portion is a carboxy group.

7. The carrier according to claim 1, wherein the distance between A and B is 5 to 20 Å.

8. The carrier according to claim 1, wherein the linker portion has a structure of 5 to 20 carbon atoms and may contain one or more bonds selected from the group consisting of a hydroxy group, an ester bond, an ether bond, a sulfide group, a carbonyl group, an amide bond, and a peptide bond.

9. The carrier according to claim 1, which comprises a structure represented by the following formula (3): [In formula (3), R1 is a methyl group or a carboxy group, and n1 is an integer from 13 to 18.] 10. A drug comprising the carrier according to any one of claims 1 to 9 linked to a physiologically active substance having a molecular weight of 4,000,000 or less.

11. The drug according to claim 10, wherein the physiologically active substance is a protein, peptide, antibody, DNA, RNA, siRNA, synthetic nucleic acid, or small molecule drug with a molecular weight of 1,000 or less.

12. The drug according to claim 10, wherein the physiologically active substance is glucagon-like peptide-1 (GLP-1), insulin, GIP (glucose-dependent insulinotropic polypeptide), exendin-3, or exendin-4.

13. The agent according to claim 10, further comprising wild-type or mutant human serum albumin linked thereto.

Images