Capsule protein, multimeric composition thereof, and pharmaceutical composition using the same

Abstract

To solve the problems that, when a drug was housed in a lipocalin-type prostaglandin D synthase with a barrel structure and a disulfide bond was introduced between the α-helix H2 and the E-F loop to confine the drug, the drug was sometimes released from a gap at an opening of the barrel structure.SOLUTION: A capsule protein characterized by substituting alanine for cysteine at an active center of human lipocalin-type prostaglandin D synthase and substituting at least one amino acid in β-strand D with a barrier amino acid inhibits the release of a drug contained therein by a barrier amino acid provided in β-strand D which forms an opening of a barrel structure.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to a capsule protein and its multimeric composition that can be used as a drug delivery system (DDS), and in particular to a capsule protein that can dissolve poorly water-soluble drugs and release the drugs at the affected area after administration, as well as a pharmaceutical composition and processed food that use the same. [Background technology]

[0002] The development of drug delivery systems is a key technology in DDS. Liposomes, microparticles, nanomaterials, and drug-polymer conjugates have been investigated. Among these, polymer micelles have attracted attention as a delivery system advantageous for the delivery of poorly water-soluble drugs. For example, there is a micelle-forming composition consisting of a hydrophobic core surrounded by a hydrophilic shell, where the hydrophilic shell is made of PVP (N-vinyl-2-pyrrolidone) (Patent Document 1).

[0003] Patent Document 2 discloses that capsule proteins modified with lipocalin-type prostaglandin D synthase (hereinafter referred to as "L-PGDS"), a biological product, can be used to dissolve poorly water-soluble drugs in water and administer them to exert their effects. Because L-PGDS-modified capsule proteins are endogenous, they are not antigens and are not toxic to humans, making them a safe and reliable drug delivery vehicle.

[0004] Patent Document 3 discloses a protein in which a capsule protein modified with L-PGDS is tagged with a label that enables it to recognize cells in the affected area. For example, a capsule protein modified with L-PGDS tagged with a label peptide that specifically binds to cancer cells is thought to accumulate in the cancer cells in the affected area, improving the effectiveness of treatment.

[0005] Patent Document 4 discloses a protein in which the 34th and 92nd tryptophans from the N-terminus of an L-PGDS-modified capsule protein are replaced with cysteines to form a cap formed by a disulfide bond that opens and closes in a redox environment. The purpose is to enhance the drug retention capacity by opening and closing the disulfide bond. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 2004-501180 [Patent Document 2] Japanese Patent Application Laid-Open No. 2008-120793 [Patent Document 3] Japanese Patent Application Laid-Open No. 2011-207830 [Patent Document 4] Japanese Patent Application Laid-Open No. 2013-162760 Summary of the Invention [Problem to be solved by the invention]

[0007] Capsule proteins using L-PGDS have the advantage of being able to easily dissolve poorly water-soluble drugs and, because they are biologically produced, are highly safe. However, the barrel structure that holds the drug is shaped like a container without a lid. This poses the problem of the drug being released during transport once it has been incorporated. To solve this problem, Patent Document 4 uses a disulfide bond to fasten the capsule protein using L-PGDS.

[0008] However, it was found that the disulfide bond used to fasten the opening of the capsule protein using L-PGDS was not enough to completely seal the opening of the barrel structure. [Means for solving the problem]

[0009] The present invention has been conceived in view of the above problems, and provides a capsule protein that can prevent unwanted release of a drug by providing an amino acid that acts as a bulky barrier at the opening of the barrel structure.

[0010] More specifically, the capsule protein of the present invention is It is characterized in that the cysteine ​​at the active center of human lipocalin-type prostaglandin D synthase is replaced with alanine, and at least one amino acid in β-strand D is replaced with a barrier amino acid. [Effects of the Invention]

[0011] The capsule protein of the present invention has an amino acid barrier at the opening of the barrel structure composed of the β-strands of mutant L-PGDS, so that the drug inside the barrel structure is less likely to be released during transport, allowing the drug to be delivered efficiently to affected cells.

[0012] Furthermore, by forming the capsule protein of the present invention into a multimeric composition, it is possible to exert an EPR (Enhanced Permeability and Retention) effect, which increases the specificity of cancer cell invasion, enhances the effectiveness of the drug, and suppresses its effects on normal cells, thereby expected to reduce side effects. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows the crystal structure of mutant L-PGDS. [Figure 2] FIG. 1 shows the amino acid sequence of mutant L-PGDS. [Figure 3] FIG. 1 shows a model structure of a D-clip mutant into which a D-clip (disulfide bond) has been introduced. [Figure 4] FIG. 1 shows a model structure of a barrier mutant to which a barrier amino acid has been added. [Figure 5] FIG. 1 is a diagram depicting the concept of multimeric composition. [Figure 6]FIG. 1 shows a model structure of mutant L-PGDS labeled as mutant L-PGDS. [Figure 7] FIG. 11 depicts the model structure of a barriered D-clip mutant labeled as a barriered D-clip mutant. [Figure 8] 1 is a graph showing the retention and release capacity when SN-38 is contained in a capsule protein. [Figure 9] This is a graph showing the retention and release capacity when SN-38 is contained in a capsule protein (barrier-attached mutant). [Figure 10] 1 is a graph showing the retention of dipyridamole when it is contained in a capsule protein (barrier-attached mutant). [Figure 11] 1 is a graph showing the tumor growth inhibitory effect when a capsule protein containing SN-38 was administered to mice bearing human prostate cancer. [Figure 12] 1 is a graph showing the tumor growth inhibitory ability when an octameric composition of capsule protein containing SN-38 was administered to mice bearing human prostate cancer. [Figure 13] 13 is a graph showing changes in body weight of the mice in FIG. 12. DETAILED DESCRIPTION OF THE INVENTION

[0014] The capsule protein according to the present invention will be described below with reference to the drawings and examples. Note that the following description exemplifies one embodiment of the present invention and one example, and the present invention is not limited to the following description. The following description can be modified within the scope of the present invention.

[0015] The capsule protein of the present invention is based on human lipocalin-type prostaglandin D synthase (L-PGDS). Table 1 shows the amino acid sequence of L-PGDS (SEQ ID NO: 1). L-PGDS is composed of 168 amino acids, from the N-terminal alanine to the 168th glutamine.

[0016] [Table 1]

[0017] L-PGDS obtained by transforming E. coli is referred to as "mutant L-PGDS" or simply "mutant." Table 2 shows the amino acid sequence of mutant L-PGDS (SEQ ID NO: 2). For the sake of artificial production, glycine-serine (GS) has been added to the N-terminus, resulting in two more amino acids compared to the sequence shown in Table 1. Hereinafter, when specifying the amino acids of mutant L-PGDS, the sequence will be the L-PGDS sequence with glycine-serine (GS) added to the N-terminus.

[0018] [Table 2]

[0019] In mutant L-PGDS, the cysteine ​​(C) at position 45 from the N-terminus (position 43 in SEQ ID NO: 1) has been substituted with alanine (A) to inactivate the enzyme active site. Furthermore, the cysteine ​​(C) at position 147 (position 145 in SEQ ID NO: 1) has also been substituted with alanine (A) to prevent the mismatching of disulfide bonds. These two positions are enclosed in a box in Table 2 (the same applies to the capsule protein sequences below). This mutant L-PGDS is also referred to as "C45A / C147A."

[0020] Figure 1 shows the crystal structure of mutant L-PGDS. Figure 1(b) shows the structure rotated 90° from Figure 1(a). L-PGDS has the same structure. Figure 2 shows the amino acid sequence (SEQ ID NO: 2) of mutant L-PGDS (C45A / C147A) and the correspondence between the β-strands and α-helices.

[0021] Mutant L-PGDS has eight β-strands, designated A to H, and α-helices, designated H1 to H3. Loops exist between the β-strands, and there are short strand I and short helices H4 and H5.

[0022] β-strands A to H are arranged in a spiral, enclosing a central space. This is called a barrel structure. When mutant L-PGDS holds a drug, it is thought to store the drug within this barrel structure. Also, referring to Figure 1(b), a large opening 10 in the barrel structure is formed between β-strand D and α-helix H2.

[0023] If opening 10 remains open, even if a drug is stored, there is a high risk that it will be released before reaching the intended cell. Therefore, a lid is provided on opening 10 (Patent Document 4). This is achieved by forming a disulfide bond between the loop connecting β-strand E and β-strand F (called the "EF loop") and α-helix H2.

[0024] Referring again to Figure 2, the EF loop consists of four amino acids from the N-terminus of mutant L-PGDS, from proline (P) at position 90 to glycine (G) at position 93. Similarly, the α-helix H2 consists of 10 amino acids from serine (S) at position 32 to alanine (A) at position 41. Disulfide bonds can be obtained by replacing each of these amino acids with cysteine ​​(C). This is called the "disulfide clip" or "D clip."

[0025] For example, Figure 3 shows a model structure of mutant L-PGDS in which the lysine in α-helix H2 (the 38th K from the N-terminus) and the histidine (H) in the EF loop are each replaced with cysteine ​​(C). It can be seen that a portion of the opening 10 is closed by a disulfide clip 12 formed between the cysteines (C). This mutant L-PGDS is called a "D-clip mutant." Patent Document 4 discloses a D-clip mutant.

[0026] However, it was found that the disulfide clip 12 only clips the edge of the opening 10, and the drug housed inside the barrel structure is released from the gap 14 between the disulfide clip 12 and β-strand D. Therefore, in order to block the gap 14 above β-strand D, the present invention provides a barrier amino acid. A barrier amino acid is an amino acid that can reduce the area of ​​the opening 10. By providing a barrier amino acid, it is possible not only to block the gap 14 but also to effectively narrow the opening 10.

[0027] Referring again to Figure 2, β-strand D is a sequence of 11 amino acids from the 68th glutamine (Q) to the 78th proline (P) from the N-terminus of mutant L-PGDS. It is desirable for the barrier amino acid to have a functional group that occupies a wide spatial range, in order to fill the gap 14 as much as possible. Furthermore, it is necessary that the introduction of the barrier amino acid onto β-strand D does not significantly change the crystal structure of mutant L-PGDS, as the barrel structure must be maintained.

[0028] For example, lysine (K), histidine (H), tryptophan (W), tyrosine (Y), phenylalanine (F), etc. can be suitably used as the barrier amino acid. Substitutions do not have to be made at one site. Furthermore, barrier amino acids may be inserted into the amino acid sequence that constitutes the β-strand.

[0029] Figure 4 shows a model structure of mutant L-PGDS in which methionine (M: 74th position from the N-terminus, see Figure 2) on β-strand D has been replaced with tryptophan (W). This is shown as "74W" in the figure. The placement of tryptophan on β-strand D shows that gap 14 is blocked. Mutant L-PGDS with a barrier amino acid like this is called a "barrier mutant."

[0030] A disulfide clip can also be added to the barrier mutant. The mutant L-PGDS, which has both a barrier amino acid and a disulfide clip, is called a "barrier-added D-clip mutant."

[0031] Furthermore, as shown in Patent Document 3, a peptide (label peptide) that recognizes target cells can be added to the N-terminus, C-terminus, or both termini. Furthermore, the peptide can be added not only to the termini but also to overlap the termini. The label peptide is not particularly limited, but for example, a peptide sequence NGR that specifically binds to a membrane protein (CD13) expressed in neovascular endothelial cells can be selected. Alternatively, the internalized-Arg-Gly-Asp (iRGD) motif, which recognizes αvβ3 and αvβ5 integrins, or the Cys-Arg-Gly-Asp-Lys (CRGDK) motif, which recognizes neuropilin 1, may be used.

[0032] Other suitable motifs that can be used include the Lys-Leu-Pro (KLP) motif, which recognizes peritoneal tumors of gastric cancer (Cancer Res, 97, 1075-81, 2006), the Asn-Val-Val-Arg-Gln (NVVRQ) motif, which recognizes metastatic cancer cells (Clin Cancer Res, 14, 5494-502, 2008), and the Phe-Gln-His-Pro-Ser-Phe-Ile (FQHPSFI) motif, which recognizes liver cancer cells (Mol Med, 13, 246-54, 2007).

[0033] A mutant with a barrier attached to a labeled peptide, which is a capsule protein according to the present invention, is called a "labeled barrier mutant." A mutant with a disulfide clip attached is called a "labeled barrier D-clip mutant." For the convenience of producing the capsule protein according to the present invention by genetic recombination, multiple amino acids may be attached to the N-terminus or C-terminus in addition to the GS described above.

[0034] The capsule protein of the present invention can incorporate compounds up to approximately 800 Da in size into its barrel structure. The compounds may be drugs or other compounds. The other compounds may be compounds that serve as nutritional supplements or compounds derived from natural products.

[0035] Furthermore, it is known that the EPR (Enhanced Permeability and Retention) effect of DDS increases the selectivity of entry into cancer cells and prolongs the retention period, allowing for the expected long-term drug effect. Therefore, the barrier mutant, which is the capsule protein of the present invention, may be used as a multimeric composition. To expect the EPR effect, it is recommended that the overall size be 10 nm or greater. Furthermore, a labeled peptide may be added to the multimeric composition.

[0036] When forming a capsule protein multimeric composition, it is preferable to use a multimeric composition in which multiple capsule proteins are linearly linked by linking their C- and N-termini. However, it is more preferable to link the capsule proteins radially. Figure 5 shows a conceptual diagram of the structure of the capsule protein multimeric composition used in the present invention.

[0037] Figure 5(a) shows a conceptual diagram of a multimeric composition of capsule proteins (hereinafter simply referred to as "multimeric composition"). Figure 5(b) is a partially exploded view of multimeric composition 21. Multimeric composition 21 is composed of a tetramer 32 of streptavidin 30 to which a dimer 36 of capsule protein 34, linked by a linker 35, is bound via biotin 38. Therefore, multimeric composition 21 forms an octameric composition of capsule protein 34.

[0038] Mutant L-PGDS or a barrier-attached mutant can be suitably used as the capsule protein 34. Here, the case where a barrier-attached mutant is used will be explained.

[0039] The dissociation constant (Kd) for the binding of biotin 38 and streptavidin 30 is 10 -15M is the strongest known non-covalent interaction between a protein and a ligand. This interaction is formed very rapidly and is not susceptible to pH, temperature, denaturants, or organic solvents. Furthermore, biotin is a small molecule and does not inhibit the functionality of the modified molecule. Therefore, the octamer shown in Figure 5(a) is considered to be very stable.

[0040] 5(c) shows a multimeric composition 22 in which a monomer of capsule protein 34 is bound to a tetramer 32 of streptavidin 30 via biotin 38. This forms a tetramer of capsule protein 34.

[0041] As described below, the octameric composition 21 of capsule protein 34 has a diameter of 10 nm or more, which is larger than that of the tetramer or monomer. Therefore, it can enter cancer cells through the blood vessels in cancer cells, which have larger gaps between endothelial cells than normal cells, but it is less likely to penetrate normal cells. Therefore, it selectively penetrates cancer cells and remains inside, exhibiting the so-called EPR effect.

[0042] The multimeric composition 21 of the present invention (in which capsule protein 34 forms an octamer) has shown effects that are clearly different from those of the capsule protein monomer in the in vivo experiments described below, and is thought to exert an EPR effect.

[0043] Capsule proteins become soluble after incorporating drugs or other compounds. Their function remains unchanged even when they are in the form of a multimeric composition. Therefore, capsule proteins can be suitably used to solubilize poorly soluble drugs and poorly soluble vitamins. For example, SN-38, vitamins A, D, E, and K, thyroid hormones, steroid hormones, isoflavones, and the like can be suitably used. Furthermore, because they can solubilize poorly soluble substances, the hurdle of solubilization can be reduced in drug development.

[0044] The pharmaceutical composition of the present invention, in which a drug is contained in the capsule protein (hereinafter simply referred to as "pharmaceutical composition"), can be provided in liquid form. It can also be provided as a powder by freeze-drying. As shown in Patent Document 3, the effect is maintained even when freeze-dried.

[0045] Therefore, when used for treatment, the pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, or intramuscularly, topically, rectally, transdermally, or nasally). Compositions for oral administration include, for example, tablets, capsules, pills, granules, powders, liquids, suspensions, etc.

[0046] Furthermore, compositions for parenteral administration include, for example, aqueous or oily agents for injection, ointments, creams, lotions, aerosols, suppositories, patches, etc. These preparations are prepared using conventionally known techniques and may contain non-toxic and inactive carriers or excipients that are commonly used in the pharmaceutical field.

[0047] In addition, complexes in which drugs or compounds other than drugs are incorporated into capsule proteins can be provided as processed foods. Examples of processed foods include general processed foods, including luxury foods and health foods such as candy, gum, jelly, biscuits, cookies, rice crackers, bread, noodles, fish and meat paste products, tea, soft drinks, coffee drinks, milk drinks, whey drinks, lactic acid bacteria drinks, yogurt, ice cream, and pudding, as well as health-promoting foods such as foods for specified health uses and foods with nutrient functions defined by the Ministry of Health, Labor, and Welfare's Health Function Food System. Furthermore, processed foods also include nutritional supplements, feed, food additives, and the like. Furthermore, the ability to soluble poorly soluble substances can be utilized for industrial products and industrial materials.

[0048] The processed foods of the present invention can be prepared by adding a capsule protein (complex) containing other compounds to the raw materials of these processed foods. The capsule protein is easily decomposed by heat because the other compounds are enclosed in a protein-containing mutant. Therefore, it is desirable to complete the processed foods of the present invention without adding a heating step after adding the complex. [Example]

[0049] As a sample capsule protein, (1) Mutant L-PGDS (2) Labeled mutant L-PGDS (3) D-clip mutant with barrier (4) Labeled barrier-attached D-clip mutants Four types were produced.

[0050] Mutant L-PGDS is the capsule protein shown in SEQ ID NO: 2, in which the 45th cysteine ​​(C) from the N-terminus has been substituted with alanine (A) and the 147th cysteine ​​(C) has been substituted with alanine (A) ("C45A / C147A"). Mutant L-PGDS has had the 147th cysteine ​​(C) substituted with alanine (A) to inactivate the active site and prevent the formation of incorrect disulfide bonds during the production process.

[0051] The labeled mutant L-PGDS was prepared by adding the iRGD peptide (CRGDKGPDC: SEQ ID NO: 3) to the C-terminus of mutant L-PGDS as a label, which enables tumor accumulation and cell membrane permeation at the same time. However, the iRGD peptide was added so that it overlapped with a portion of the C-terminus of mutant L-PGDS. This was done to eliminate antigenicity in mice in subsequent in vivo experiments. The amino acid sequence of labeled mutant L-PGDS is shown in Table 4 as SEQ ID NO: 4. The labeled peptide portion is indicated by a "■". Figure 6 shows the model structures of mutant L-PGDS and labeled mutant L-PGDS.

[0052] [Table 3]

[0053] [Table 4]

[0054] To create the barrier D-clip mutant, first, methionine (M) on β-strand D was replaced with tryptophan (W) as a barrier amino acid. The methionine is located at the 74th position from the N-terminus of mutant L-PGDS.

[0055] The D-clip mutant with a barrier has an additional D-clip. The D-clip is obtained by substituting the lysine (K) at position 38 from the N-terminus of mutant L-PGDS with a cysteine ​​(C) and the histidine (H) at position 91 with a cysteine ​​(C). The amino acid sequence is shown in Table 5 (SEQ ID NO: 5). In Table 5, the barrier amino acid is indicated by a "▲" and the D-clip (disulfide bond) is indicated by a "★". A disulfide bond is formed between the two cysteines (C) indicated by a "★". Note that the disulfide bond separates in a reducing environment, allowing the D-clip to open and close the opening 10.

[0056] [Table 5]

[0057] The labeled D-clip mutant with a barrier was created by adding an overlapping iRGD peptide (CRGDKGPDC) to the C-terminus of the D-clip mutant (SEQ ID NO: 5). SEQ ID NO: 6 is shown in Table 6. The barrier amino acid is indicated by "▲", and the D-clip (disulfide bond) is indicated by "★". The labeled peptide portion is indicated by "■". Figure 7 shows a model of the D-clip mutant with a barrier and the labeled D-clip mutant with a barrier.

[0058] [Table 6]

[0059] Each capsule protein was expressed as a fusion protein with glutathione S-transferase by transforming Escherichia coli BL21(DE3) using the designed expression plasmids prepared by the megaprimer method.

[0060] The protein-expressing strain was cultured in LB / Amp test tube medium at 37°C for 8 hours with shaking, then subcultured in 2xYT / Amp auto-induction medium and cultured at 37°C for 16 hours with shaking. The resulting cells were disrupted by sonication, and the supernatant was applied to a Glutathione-Sepharose 4B column for affinity chromatography.

[0061] The fusion protein adsorbed on the column was reacted with 165 units of thrombin overnight, and the target protein was eluted and purified.

[0062] Next, these capsule proteins were loaded with SN-38, an abbreviation for the poorly water-soluble anticancer drug 7-ethyl-10-hydroxycamptothecin. SN-38 is known to exhibit high antitumor efficacy at lower doses than irinotecan hydrochloride, the prodrug of SN-38 currently used in clinical trials.

[0063] <Drug release capacity> SN-38 / mutant L-PGDS, SN-38 / barrier-attached D-clip mutant, and SN-38 / labeled D-clip mutant, each adjusted to a SN-38 concentration of 50 μM and a capsule protein concentration of 50 μM, were dialyzed for 72 hours in a 37°C incubator using a dialysis membrane (Size 27 Wako, molecular weight cutoff: 14,000) against 150 mL of PBS as the external solution.

[0064] After the start of dialysis, 500 μL of the external solution was sampled and replaced with the same volume of PBS at 0, 1, 3, 6, 8, 12, 24, 36, 48, 60, and 72 hours. The SN-38 concentration in the sampled external solution was measured using a Hitachi F-7000 spectrofluorometer (excitation wavelength: 365 nm, measurement wavelength: 380-600 nm, measurement temperature: 37°C).

[0065] Next, we evaluated the drug release function of the barrier-attached D-clip mutant and the labeled barrier-attached D-clip mutant in response to a reducing environment by equilibrium dialysis, as described above. Dialysis was performed using PBS and 10 mM DTT / PBS as external solutions. PBS was used as the external solution, representing an oxidizing environment, while DTT was used as the external solution, representing a reducing environment.

[0066] Figure 8 shows the change in SN-38 concentration over time in the dialysis solution plotted against dialysis time. The horizontal axis represents reaction time (hours), and the vertical axis represents SN-38 concentration (nM). Error bars represent the mean ± standard error (n = 3). Abbreviations in the figure include "SN-38 / L-PGDS (●)" for SN-38 / mutant L-PGDS, "SN-38 / Capsule (▲)" for SN-38 / barrier-attached D-clip mutant, and "SN-38 / Cap-sCRGDK (■)" for SN-38 / labeled barrier-attached D-clip mutant. For "SN-38 / Capsule" and "SN-38 / Cap-sCRGDK," "+10 mM DTT (△ and □)" refers to the addition of 10 mM DTT to the respective pharmaceutical composition solutions. The addition of DTT (Dithiothreitol) creates a strongly reducing environment in the solution.

[0067] Referring to Figure 8, when the external solution was PBS, it was revealed that the SN-38 concentration in the external solution of the barrier-equipped D-clip mutant (SN-38 / Capsule) and the labeled barrier-equipped D-clip mutant (SN-38 / Cap-sCRGDK) was lower than that of mutant L-PGDS (SN-38 / L-PGDS) at all times from the start of dialysis, and it was found that the release of SN-38 from the SN-38 / barrier-equipped D-clip mutant and SN-38 / labeled barrier-equipped D-clip mutant was suppressed compared to the release from SN-38 / mutant L-PGDS.

[0068] On the other hand, when the external solution was DTT / PBS, the SN-38 concentration in the external solution increased for the barriered D-clip mutant (SN-38 / Capsule: "△") and the labeled barriered D-clip mutant (SN-38 / Cap-sCRGDK: "□") compared to when the external solution was PBS.

[0069] These results suggest that the barriered D-clip mutant and the labeled barriered D-clip mutant retain SN-38 longer than mutant L-PGDS in an oxidative environment and release SN-38 in response to a reducing environment. Furthermore, there was no difference in the drug release pattern between the barriered D-clip mutant and the labeled barriered D-clip mutant, indicating that the addition of a labeled peptide does not affect drug release control.

[0070] Next, we performed similar experiments on the mutant L-PGDS without the D-clip, a barrier-attached mutant (M74W). The experiment for mutant L-PGDS was a repeat of that shown in Figure 8. The results are shown in Figure 9. Figure 9(a) shows the results for mutant L-PGDS, and Figure 9(b) shows the results for the barrier-attached mutant (M74W). In Figures 9(a) and 9(b), the horizontal axis represents reaction time (hours) and the vertical axis represents SN-38 concentration (μM).

[0071] Referring to Figure 9(a), the SN-38 concentration versus reaction time when the external solution was PBS (black circle "●") was almost the same as in Figure 8, and it can be seen that the experiment in Figure 8 was well reproduced. In the case of mutant L-PGDS, when the external solution was DTT / PBS (white circle "○"), the SN-38 concentration reached 2.0 μM within a short reaction time and appeared to have almost reached a plateau.

[0072] In contrast, referring to Figure 9(b), when the external solution was PBS (black circle ``●''), the SN-38 concentration did not exceed 1.0 μM even over the reaction time, and the release of SN-38 was more suppressed than in the cases of mutant L-PGDS, barrier-attached D-clip mutant, and labeled barrier-attached D-clip mutant shown in Figure 8.

[0073] On the other hand, when the external solution was DTT / PBS (open circle "○"), the SN-38 concentration reached approximately 2.0 μM after 48 hours of dialysis. This value was almost the same as that of the barriered D-clip mutant and the labeled barriered D-clip mutant shown in Figure 8. In other words, the barriered mutant was found to have a superior drug retention capacity than the barriered D-clip mutant and the labeled barriered D-clip mutant. We also confirmed that in a reducing environment, the barriered mutant was able to release almost all of the encapsulated drug, similar to other capsule proteins.

[0074] Next, the drug was changed from SN-38 to dipyridamole, a poorly water-soluble antianginal drug, and the results of a similar experiment are shown in Figure 10. The external solution was PBS. Referring to Figure 10, the horizontal axis represents the reaction time (hours), and the left vertical axis represents the dipyridamole concentration (nM). The right vertical axis represents the left vertical axis converted into the release rate (%). While the dipyridamole concentration increased with reaction time in the mutant L-PGDS, the dipyridamole concentration in the barrier mutant (M74W) only increased to a level close to the detection limit.

[0075] From this, it was found that the mutant with a barrier has excellent ability to retain the encapsulated drug, and for some drugs, it can be retained almost without leakage. It can be considered that it can continue to retain the encapsulated drug until it enters the body and reaches the reducing atmosphere inside the cell, and it can be said that it is highly efficient as a DDS.

[0076] <In-vivo effect> Four-week-old male BALB / C-nu / nu mice (Japan SLC) were housed and acclimated for 1 week with free access to water and food in an animal room maintained at room temperature with a 12-hour light / dark cycle. Then, 100 μL of human prostate cancer cell PC-3 at 5×10 7 cells / mL (PBS: Matrigel = 1:1) was administered subcutaneously to create a prostate cancer model mouse.

[0077] The day when the tumor volume (calculated from the approximate formula: {(major axis)×(minor axis)2} / 2) reached 250 mm 3 was set as the start day of administration (day 零). The mice were randomly classified into each administration group of PBS, SN-38 / mutant L-PGDS (2.0 mg / kg / d), SN-38 / labeled mutant L-PGDS (2.0 mg / kg / d), SN-38 / D clip mutant with a barrier (2.0 mg / kg / d), and SN-38 / labeled D clip mutant with a barrier (2.0 mg / kg / d), and each sample was administered 8 times via the tail vein every other day. Only PBS was administered to the control group.

[0078] The results of the in vivo antitumor experiment are shown in Fig. 11. The horizontal axis is the number of days elapsed from the start day of administration (days), and the vertical axis is the tumor volume (mm 3 ). The abbreviations in the graph are as follows. PBS: Control group SN-38 / L-PGDS: SN-38 / mutant L-PGDS SN-38 / L-PGDS-sCRGDK: SN-38 / labeled mutant L-PGDS SN-38 / Capsule: SN-38 / D clip mutant with a barrier SN-38 / Cap-sCRGDK: labeled barriered D-clip mutant

[0079] In the PBS-treated group, no antitumor effect was observed, and tumor volume continued to increase from the day of treatment.In contrast, significant tumor growth inhibition was observed in the SN-38 / L-PGDS, SN-38 / L-PGDS-sCRGDK, SN-38 / Capsule, and SN-38 / Cap-sCRGDK-treated groups.

[0080] Furthermore, the SN-38 / Capsule and SN-38 / L-PGDS-sCRGDK groups did not show significant tumor growth inhibition compared to the SN-38 / L-PGDS group, indicating that the addition of either the targeting or controlled release function alone does not result in significantly higher antitumor activity than mutant L-PGDS.

[0081] On the other hand, SN-38 / Cap-sCRGDK (a labeled D-clip mutant with a barrier) showed significantly higher antitumor activity than SN-38 / L-PGDS (mutant L-PGDS). These results demonstrate that the synergistic effect of the two functions of drug release control (the barrier amino acid and D-clip) and cancer targeting (the labeled peptide) can significantly suppress tumor growth.

[0082] <Multimer> Next, we will explain how to create a multimeric composition (octameric composition) of capsule proteins. As shown in Figure 5, the octameric composition is composed of a tetramer 32 of streptavidin 30, to which a dimer 36 of capsule protein 34, linked by a linker 35, is bound via biotin 38.

[0083] Therefore, a biotinylated dimer composition in which biotin is bound to a dimer of capsule protein is produced, and then this is combined with a separately produced tetramer of streptavidin to obtain an octamer composition of capsule protein. The linker is encoded by the base sequence shown in Table 7 (SEQ ID NO: 7). Streptavidin is encoded by the base sequence shown in Table 8 (SEQ ID NO: 8).

[0084] [Table 7]

[0085] [Table 8]

[0086] <Construction of dimeric L-PGDS expression vector> The gene sequence of the barriered mutant was amplified by PCR using a forward primer containing a BamHI recognition site and a reverse primer containing an EcoRI recognition site and a linker sequence (GGGGS: SEQ ID NO: 7), followed by agarose gel electrophoresis. The resulting fragment was digested with BamHI and EcoRI to obtain the barriered mutant insert.

[0087] In the barrier mutant, the cysteine ​​(C) at position 45 from the N-terminus is replaced with alanine (A), and the cysteine ​​(C) at position 147 is replaced with alanine (A) ("C45A / C147A"). Furthermore, the methionine (M) on β-strand D is replaced with tryptophan (W) as a barrier amino acid ("M74W"). The methionine is at position 74 from the N-terminus of mutant L-PGDS. The amino acid sequence of the barrier mutant (SEQ ID NO: 9) is shown in Table 9.

[0088] [Table 9]

[0089] A plasmid (pGEX4T-2) was also prepared by treating it with restriction enzymes in the same manner. The barriered mutant insert and the restricted pGEX4T-2 were subjected to agarose gel electrophoresis separately.

[0090] After staining each fragment with ethidium bromide, the DNA was extracted from the excised gel using an Agarose Gel Extraction Kit (Jene Bioscience) and ligated. This procedure inserted the base sequence encoding the barrier mutant and linker into the plasmid pGEX4T-2. This plasmid is called the barrier mutant expression vector.

[0091] The resulting barrier mutant expression vector was used to transform Escherichia coli DH5α(DE3) and inoculated onto LB / Amp plates. Colony direct PCR was performed on the colonies that grew on the plates. Colonies that confirmed the presence of the barrier mutant were inoculated into LB / Amp test tubes (5 mL) and cultured at 37°C for 16 hours. Minipreps were then performed using SV Minipreps (Promega), and the barrier mutant expression vector was purified.

[0092] The resulting barrier mutant expression vector was sequenced to confirm that the base sequences encoding the barrier mutant and the linker had been inserted.

[0093] A barrier mutant insert amplified using primers containing EcoRI and SalI recognition sites was inserted into this barrier mutant expression vector using the same procedure. The E. coli DH5α(DE3) strain was then transformed using the same procedure, cultured, and amplified, and the plasmid was purified. This plasmid contains a base sequence encoding a dimer of the barrier mutant. Therefore, this plasmid is called the dimer barrier mutant expression vector. The resulting dimer barrier mutant expression vector was sequenced to confirm the base sequence.

[0094] <Construction of a modified dimeric L-PGDS expression vector> Next, the 15 amino acid residues (Avitag) that are specifically recognized by biotin ligase (BirA) are TM Annealing was performed using complementary oligo DNAs of SEQ ID NOS: 10 and 11 to encode the Av peptide (hereafter referred to as "Av"). Biotin ligase binds biotin to the lysine residues of the Av peptide. The nucleotide sequences are shown in Tables 10 and 11, respectively. The annealed product was then purified using a purification column, FastGene Gel / PCR Extraction Kit (Nippon Genetics, Tokyo). This nucleotide sequence is referred to as the Av nucleotide sequence.

[0095] [Table 10]

[0096] [Table 11]

[0097] The dimer-barriered mutant expression vector was digested with BamHI (37°C, 3 hours) and subjected to agarose gel electrophoresis. The DNA of the dimer-barriered mutant expression vector was then extracted, and an Av base sequence was inserted into the BamHI cleavage site using the In Fusion® HD Cloning Kit (Clontech). The Av base sequence inserted into the dimer-barriered mutant expression vector is called a modified dimer-barriered mutant expression vector. The resulting vector was sequenced to confirm the insertion of the target sequence.

[0098] <Dimer barrier mutant expression strain> The modified dimer-barrier mutant expression vector was transformed into the E. coli strain AVB101 expressing BirA to obtain a dimer-barrier mutant expression strain. The dimer-barrier mutant expression strain was inoculated into 5 ml of LB / Amp / Chl liquid medium and cultured overnight at 37°C with shaking. It was then subcultured into 1 L of 2xYT / Amp / Chl medium and cultured at 37°C.

[0099] The E. coli strain AVB101 that expresses BirA is an E. coli strain into which an expression vector into which a base sequence encoding BirA has been inserted has been injected.

[0100] <Production of biotinylated dimer-barrier mutants> When the OD600 value of the culture medium of the dimer-barrier mutant expression strain reached 0.6-1.0, IPTG (final concentration 0.1 mM) and biotin (final concentration 50 μM) were added. This procedure resulted in the expression of both the modified dimer-barrier mutant and BirA proteins in the E. coli. BirA then binds biotin to the peptide Av added to the dimer-barrier mutant, resulting in the biotinylation of the dimer-barrier mutant.

[0101] After incubation at 37°C for 6 hours, the culture was centrifuged (8,400 × g, 10 min, 4°C) to collect the bacterial cells, which were then washed with PBS, collected by centrifugation, and disrupted by ultrasound.

[0102] An affinity column containing 15 ml of Glutathione Sepharose 4B (GE Healthcare BioScience, UK) was equilibrated with PBS in an amount five times the column volume that had been passed through a 0.22 μm filter, and the disruption supernatant that had been passed through a 0.22 μm filter was applied.

[0103] After washing with 3x the column volume of 1% Triton X-100 / PBS and 5x the column volume of PBS, 165 units of thrombin (SIGMA) was added and the column was stirred thoroughly and allowed to stand at room temperature for 12 hours or more. The column was eluted with 5x the column volume of PBS and concentrated to 4 ml by repeated centrifugation (8,400 g, 20 min, 4°C) using an Amicon column with a molecular weight cutoff of 10 kDa.

[0104] Next, 5 mM Tris-HCl (pH 8.0) was passed through a 0.22 μm filter and degassed for 10 minutes, and a Superdex 75 16 / 600 (GE Healthcare BioScience, UK) was equilibrated with twice the volume of the same buffer. The concentrate was passed through a 0.22 μm filter and loaded onto the Superdex 75 16 / 600. While monitoring the absorbance at 280 nm UV, the eluate was fractionated in 1.5 ml aliquots at a flow rate of 0.5 ml / min, and the peak fraction corresponding to biotinylated dimer L-PGDS was collected.

[0105] The collected fractions were pooled and dialyzed against 20 mM Na-acetate buffer (pH 5.5), and then concentrated to 4 ml by repeated centrifugation (8,400 g, 20 min, 4°C) using an Amicon with a molecular weight cutoff of 10 kDa.

[0106] The sample was loaded onto a column packed with SP Sepharose Fast Flow (GE Healthcare Bio Science, UK), and cation exchange chromatography was performed using a linear gradient method of 20 mM Na-acetate buffer (pH 5.5) → 1 M NaCl / 20 mM Na-acetate buffer (pH 5.5).

[0107] The eluate was fractionated in 1.5 ml aliquots at a flow rate of 1.0 ml / min while monitoring the absorbance at 280 nm UV. SDS-PAGE analysis was then performed, and the fractions that showed a single band of the biotinylated dimer-barrier mutant were collected.

[0108] <Streptavidin purification> E. coli BL21(DE3) was transformed with a streptavidin expression vector (pET21a-Streptavidin-Alive, Addgene) to obtain a streptavidin-expressing strain. The streptavidin-expressing strain was inoculated into 5 ml of LB / Amp liquid medium and cultured overnight at 37°C with shaking. The strain was then subcultured in 1 L of 2xYT / Amp medium and cultured at 37°C. When the OD600 reached 0.6-1.0, IPTG was added to a final concentration of 0.1 mM to induce expression. After further culturing at 18°C ​​for 24 hours, the culture was centrifuged (8,400 × g, 10 min, 4°C) to collect the bacterial cells.

[0109] The resulting cells were suspended in PBS, and 1 μl of 100 mg / ml Lysozyme was added per gram of cells, followed by stirring on ice. The cells were then ultrasonically disrupted (7 sets of 1 minute on, 2 minutes rest) while stirring on ice, and the resulting solution was centrifuged (4°C, 15,000 rpm) to disrupt the cells, and the supernatant was obtained.

[0110] An affinity column containing 10 ml of Ni Sepharose (GE Healthcare BioScience, UK) was equilibrated with 5 column volumes of 20 mM imidazole / 20 mM Na-phosphate buffer (pH 7.0) passed through a 0.22 μm filter. The supernatant was then applied. The column was washed with 20 mM Na-phosphate buffer (pH 7.0) containing various imidazole concentrations (20, 50, and 100 mM), and eluted with 300 mM imidazole / Na-phosphate buffer (pH 7.0). The column was then concentrated to 4 ml by repeated centrifugation (8,400 g, 20 min, 4°C) using an Amicon column with a 10 kDa molecular weight cutoff.

[0111] Next, PBS (pH 7.4) passed through a 0.22 μm filter was degassed for 10 minutes, and then a Superdex 75 16 / 600 column was equilibrated with twice the volume of the same buffer. The concentrate was passed through a 0.22 μm filter and applied to the Superdex 75 16 / 600. While monitoring the absorbance at 280 nm UV, the eluate was fractionated in 1.5 ml aliquots at a flow rate of 0.5 ml / min, and streptavidin tetramers were isolated by non-reducing SDS-PAGE analysis.

[0112] <Preparation of octamer composition> The purified dimeric barrier mutant (in PBS, pH 7.4) and streptavidin tetramer (in PBS, pH 7.4) were mixed at a molar ratio of 4:1 and left to stand at room temperature for 15 minutes. The mixture was then concentrated to 4 ml by repeated centrifugation (8,400 g, 20 min, 4°C) using an Amicon column with a molecular weight cutoff of 50 kDa.

[0113] Next, PBS (pH 7.4) filtered through a 0.22 μm filter was degassed for 10 minutes, and a Superdex 200 16 / 600 (GE Healthcare BioScience, UK) was equilibrated with twice the volume of the same buffer. The concentrate was passed through a 0.22 μm filter and added to the Superdex 200 16 / 600. While monitoring the absorbance at 280 nm UV, the eluate was fractionated at a flow rate of 0.5 ml / min into 1.5 ml aliquots, and the octamer composition was analyzed by SDS-PAGE.

[0114] Furthermore, a barrier mutant monomer was obtained by directly introducing the barrier mutant expression vector into E. coli before preparing the dimeric barrier mutant expression vector. A labeled barrier mutant monomer was also prepared by adding an iRGD peptide to the barrier mutant. Table 12 shows the amino acid sequence of the labeled barrier mutant (SEQ ID NO: 12). The method for adding the iRGD peptide was the same as for SEQ ID NO: 6.

[0115] [Table 12]

[0116] In addition, by transforming the Escherichia coli strain AVB101 expressing BirA with a modified monomeric mutant expression vector with an Av nucleotide sequence inserted into the mutant expression vector with a barrier, a tetramer composition in which four monomers are bound by streptavidin can also be obtained.

[0117] <Size of the octamer composition> <Size of the octamer composition by DLS>

[0118] <Size of the octamer by SAXS> The size of the obtained octamer composition was measured by small-angle X-ray scattering (SAXS). For comparison, the size of mutant L-PGDS was also measured.

[0119] The SAXS measurement was performed at beamline BL40B2 of SPring-8 (Sayo-gun, Hyogo), a large synchrotron radiation facility. The X-ray wavelength was adjusted to 1.000 Å (angstrom), the camera length was adjusted to 2.193 m, and the experiment was conducted at 25 °C. Each measurement was exposed for 20 - 50 seconds, and the scattered light was detected using PILATUS-2M (RIGAKU, Tokyo).

[0120] To maximize the X-ray scattering, a sample cell with a thickness of 3.0 mm was used, and the cell window was made of a 0.02 mm quartz plate. To avoid measurement errors, the protein sample and the buffer were measured alternately. 25 μL of the sample was placed in the cell for measurement. After measurement, the sample was removed from the cell, and the cell was washed three times with the buffer before the next measurement.

[0121] The scattering patterns of the protein sample and buffer solution recorded two-dimensionally on the detector were converted to one-dimensional data by circular averaging, and the data of the buffer solution were subtracted from the data of the protein sample. The scattering curves in the small-angle region were analyzed using the Guinier approximation for a monodisperse system. The scattering intensity I(S,C), which is a function of the scattering vector S and protein concentration C, is expressed as the scattering intensity at the origin I(0,C) and the radius of gyration R g It can be expressed as equation (1) using (C) (radius of gyration).

[0122]

number

[0123] Here, 2θ represents the scattering angle, and λ represents the X-ray wavelength.

[0124] The radius of gyration R calculated from the Guinier region of the obtained scattering curve g The diameter of the octamer was 6.0±0.69 nm, approximately three times larger than that of the monomer (mutant L-PGDS), and the radius of gyration R g It was revealed that the molecular weight (Mw) was increased by octamerization. The results of various measurements are shown in Table 13. exp ) are the theoretical molecular weights (Mw calc ), it was concluded that the octamerization of the barrier mutant, which is thought to be roughly the same size as the monomer (mutant L-PGDS), had definitely occurred. Since the particle size is well over 10 nm, the octamer composition is expected to accumulate in tumors due to the EPR effect.

[0125] [Table 13]

[0126] <Drug-containing> Next, SN-38 was incorporated into the monomer, the barrier mutant, the labeled barrier mutant, and the octamer composition. SN-38 is an abbreviation for 7-ethyl-10-hydroxycamptothecin, a poorly water-soluble anticancer agent as previously described. It is known to exhibit a high antitumor effect at a low dose compared to irinotecan hydrochloride, a prodrug of SN-38 currently used clinically.

[0127] To the PBS suspension of SN-38 incubated at 37°C, a PBS solution of the monomer (mutant L-PGDS), the barrier mutant, the labeled barrier mutant, or the octamer composition was added so that the final concentration was 1 μM, 0.25 μM, or 0.125 μM, respectively, and stirred at 37°C for 6 hours. After the stirring was completed, free SN-38 was removed by ultrafiltration to prepare a sample in which the drug was contained in the capsule protein.

[0128] <In-vivo effects> Four-week-old male BALB / C-nu / nu mice (Japan SLC) were housed and acclimated for 1 week with free access to water and food in an animal room maintained at room temperature with a 12-hour light / dark cycle. Then, a prostate cancer model mouse was created by subcutaneously administering 100 μL of human prostate cancer cell PC-3 at 5 × 10 7 cells / mL (PBS: Matrigel = 1:1).

[0129] Tumor volume (calculated from the approximate formula: {(major axis) × (minor axis) 2} / 2) reached 250 mm 3 The day when it reached was designated as the start day of administration (day 0), and the mice were randomly classified into each administration group of PBS, the monomer (2.0 mg SN-38 / kg / d), the barrier mutant (2.0 mg SN-38 / kg / d), the labeled barrier mutant (2.0 mg SN-38 / kg / d), and the octamer composition (2.0 mg SN-38 / kg / d). The monomer, the barrier mutant, the labeled barrier mutant, and the octamer composition were each administered via the tail vein 4 times at intervals of 4 days. Only PBS was administered to the control group 4 times at intervals of 4 days.

[0130] Figure 12 shows the results of the in vivo antitumor experiment. The horizontal axis represents the number of days elapsed since day 0 of administration, and the vertical axis represents the tumor volume (mm 3 ) The abbreviations in the graph are as follows: PBS: control group SN-38 / L-PGDS: Monomer (SN-38 / Mutant L-PGDS) SN-38 / M74W:SN-38 / Barrier Mutant SN-38 / M74W-sCRGDK: SN-38 / labeled barrier mutant SN-38 / M74W-octamer: Octamer composition of SN-38 / barrier mutant Compared to the case in Figure 11, none of them have D-clips.

[0131] The labeled barrier mutant had a Cys-Arg-Gly-Asp-Lys (CRGDK) motif added to the C-terminus of the barrier mutant, which recognizes neuropilin 1 (labeled number 12).

[0132] As shown in Figure 12, no antitumor effect was observed in the PBS-treated group, and tumor volume continued to increase from the start of treatment. In contrast, the SN-38 / L-PGDS-treated group exhibited an inhibitory effect on tumor growth. Furthermore, the barrier-containing mutant SN-38 / M74W further inhibited tumor growth.

[0133] On the other hand, SN-38 / M74W-sCRGDK (a labeled mutant with a barrier (no D-clip)) and SN-38 / M74W-octamer (an octamer composition of the barrier mutant) surprisingly showed almost no tumor growth from the start of the experiment. Considering that SN-38 inhibits cancer cell proliferation and does not induce apoptosis, the effectiveness of SN-38 can be fully demonstrated.

[0134] Furthermore, despite being administered four times every four days, tumor growth was completely suppressed for some time even after day 15, when no administration was made. This confirms that the drug remains in the cell tissue and is not excreted by the lymphatic system, etc., allowing the EPR effect to exert its efficacy over a long period of time.

[0135] Figure 13 shows the average body weight of the mice during the experiment in Figure 12. The horizontal axis represents the number of days (days) since day 0 of administration, and the vertical axis represents the weight ratio (%) of the mouse to its body weight at the start of administration. The graph shows the average for each administration group (5 mice). The abbreviations in the graph are the same as in Figure 12. The PBS-administered group, which was the control group in Figure 12, is not included.

[0136] Referring to Figure 13, the barrier mutant (SN-38 / M74W), which was highly effective in suppressing cancer cell proliferation, showed signs of losing less than 80% of its original weight by day 20, and the experiment was discontinued. Considering that the inactivated L-PGDS (SN-38 / L-PGDS) showed weight loss but never fell below 80%, it is thought that the barrier mutant was excellent at retaining SN-38 (the drug) and released SN-38 even into normal cells, resulting in side effects.

[0137] On the other hand, the labeled barrier mutant (SN-38 / M74W-sCRGDK) and the barrier mutant octamer (SN-38 / M74W-octamer), which showed a significant effect in inhibiting cancer cell proliferation, showed only slight weight loss and even a tendency to increase weight after 15 days when SN-38 administration was stopped.

[0138] This suggests that the labeled barrier mutant (SN-38 / M74W-sCRGDK) and the barrier mutant octamer (SN-38 / M74W-octamer) specifically identified cancer cells and released drugs into them.

[0139] Furthermore, by forming them into polymers, the EPR effect can be exerted, making it possible to deliver drugs to any cancer type. This is particularly useful for metastatic cancer, as it allows the drug to be selectively delivered to cancer cells without affecting normal cells, regardless of the location of metastasis. [Industrial Applicability]

[0140] The present invention encapsulates poorly soluble drugs and other compounds, turning them into soluble bodies. After being taken up by cells, the disulfide bond is cleaved in the reducing environment within the cells (the intracellular reduced glutathione concentration is approximately 0.5-10 mM, which is approximately 100-1000 times the extracellular concentration), releasing the encapsulated drug or other compound. Therefore, it can be suitably used as a DDS capsule for poorly soluble compounds. Furthermore, by taking advantage of its ability to soluble poorly soluble substances, it can also be used in industrial products and materials. [Explanation of symbols]

[0141] 10 (barrel structure) opening 12 Disulfide Clip 14 Gap 21 Multimer composition (octamer composition) 22 Multimer composition (tetramer composition) 30 Streptavidin 32 Tetramer 35 Linker 34 Capsule Protein 36 Dimer 38 Biotin

Claims

[Claim 1] A capsule protein characterized in that the cysteine ​​at the active center of human lipocalin-type prostaglandin D synthase is substituted with alanine, and at least one amino acid in β-strand D is substituted with a barrier amino acid.

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