Method for determining the effect of carrier peptides on the binding of therapeutic proteins to cell membranes

Abstract

This application relates to a method for measuring the effect of a carrier peptide on the binding of therapeutic proteins, particularly botulinum toxin type A 150 kD, to cells. The carrier peptide and its use are also described.

Description

[Technical Field]

[0001] Claim of priority This international application claims priority based on U.S. Provisional Application No. 63 / 584,901, filed on 25 September 2023, which is incorporated herein by reference in its entirety.

[0002] Sequence List The sequence listing, created on September 25, 2024, and submitted with this application as an XML file named “13720.0038P1.xml” with a size of 21 kilobytes, is incorporated herein by reference in accordance with 37 §152e(5) of the Code of Federal Regulations.

[0003] This invention relates to a method for measuring the effect of a carrier peptide on the binding of therapeutic proteins, particularly botulinum toxin type A 150 kD, to cells. The carrier peptide and its use are also described. [Background technology]

[0004] Subcutaneously administered therapeutic proteins may need to be further taken up into cells to achieve their desired therapeutic effect. Peptide carriers are excipients that can promote the uptake of therapeutic proteins into cells. Assays measuring the effect of peptide carriers on the uptake of therapeutic proteins into cells may be useful. [Overview of the project] [Problems that the invention aims to solve]

[0005] This disclosure relates to a method for determining the effect of carrier peptides on the binding of therapeutic proteins to cell membranes. [Means for solving the problem]

[0006] One aspect of the present disclosure is a method for measuring the effect of a carrier peptide on the binding of a therapeutic protein to a cell membrane, comprising the steps of: preparing cells in a cell culture maintained in a container; adding a composition comprising a labeled therapeutic protein and a peptide carrier to the cell culture; incubating the cell culture with the composition to enable the peptide and the labeled therapeutic protein to bind to the cells in the cell culture; washing away excess labeled therapeutic protein and excess peptide carrier from the container; and measuring the amount of labeled therapeutic protein bound to the cells in the cell culture.

[0007] Another aspect of the present disclosure is a method for measuring the effect of a carrier peptide on the binding of a therapeutic protein to a cell membrane, comprising the steps of: preparing cells in a cell culture maintained in a container; adding a peptide carrier to the cell culture; incubating the cell culture with the peptide carrier in the container; adding a labeled therapeutic protein to the cell culture; incubating the cell culture with the labeled therapeutic protein to enable the labeled therapeutic protein to bind to cells in the cell culture; washing out excess labeled therapeutic protein and excess peptide carrier from the container; and measuring the amount of labeled therapeutic protein bound to cells in the cell culture.

[0008] Other embodiments include carrier peptides, botulinum toxin compositions containing them, and the use of them. [Brief explanation of the drawing]

[0009] [Figure 1] A representative graph showing the effect of peptide backbone type on BoNT / A binding to the cell membrane. [Figure 2] A representative graph showing the effect of cell-permeable peptide type and arrangement on the amount of BoNT / A binding to the cell membrane. [Figure 3]A representative graph showing the effect of peptide backbone length on the binding of BoNT / A to the cell membrane. [Modes for carrying out the invention]

[0010] This disclosure relates to a method for determining the effect of carrier peptides on the binding of therapeutic proteins to cell membranes.

[0011] In one embodiment, a method for measuring the effect of a carrier peptide on the binding of a therapeutic protein to a cell membrane includes the steps of: preparing cells in a cell culture maintained in a container such as a well; adding a peptide carrier to the cell culture; incubating the cell culture with the peptide carrier in the container; optionally removing excess peptide from the cell culture after incubation with the peptide; adding a labeled therapeutic protein to the cell culture; incubating the cell culture with the labeled therapeutic protein to enable the labeled therapeutic protein to bind to cells in the cell culture; washing away excess labeled therapeutic protein from the cell culture; and measuring the amount of labeled therapeutic protein bound to cells in the cell culture.

[0012] In another embodiment, a method for measuring the effect of a carrier peptide on the binding of a therapeutic protein to a cell membrane includes the steps of: preparing cells in a cell culture maintained in a container; adding a composition comprising a labeled therapeutic protein and a peptide carrier to the cell culture; incubating the cell culture with the composition to allow the peptide and the labeled therapeutic protein to bind to the cells in the cell culture; washing away excess labeled therapeutic protein from the cells; and measuring the amount of labeled therapeutic protein bound to the cells in the cell culture.

[0013] The therapeutic protein can be any protein, particularly one intended to be administered by subcutaneous injection or applied topically (including to mucous membranes). In embodiments, the therapeutic protein is botulinum toxin or botulinum toxin type A complex or 150 kd of type A botulinum toxin. Other types of botulinum toxin, including types B, C, D, E, F, G, and H (either in complex or as the neurotoxin itself), are also intended.

[0014] The label can be any compound that can conjugate to the protein and be detected by spectroscopy, such as UV / Vis, IR, or fluorescence. In embodiments, the label is peroxidase or horseradish peroxidase. In the case of peroxides, UV / VIS absorbance is used to measure the amount of labeled protein in the container.

[0015] This method may include a step of washing away excess labeled therapeutic protein from the container. The washing step may include flushing the container once, twice, three times, four times, five times, six times, seven times, or eight times with 0.5–2% v / v or about 1% v / v of a nonionic surfactant (e.g., polysorbate, e.g., polysorbate 20 or 80) in phosphate-buffered saline (PCST is a common solution having this combination that can be used for washing).

[0016] The cells in the cell culture can be any cells that are suitable for culture, are target cells for therapy, or have properties similar to such cells. In embodiments, the cells in the cell culture can be nerve cells, such as neuroblastoma cells. In embodiments, the cells are cell lines selected from SiMa, Neuro-2a, NT2N, GT1-7, GN11, ND7 / 23, SH-Sy5Y, PC12, and HT-22.

[0017] The peptide carrier is configured to bind to the therapeutic protein non-covalently. Depending on the properties of the therapeutic protein, the peptide carrier can be either positively charged or negatively charged.

[0018] Generally, a positively charged support (also called a “positively charged skeleton”) is typically a chain of atoms in which groups within the chain possess a positive charge at physiological pH, or in which positively charged groups are bonded to side chains extending from the skeleton. In certain preferred embodiments, the positively charged skeleton is a cationic peptide. As used herein, the term “peptide” refers to an amino acid sequence but does not imply the number of amino acid residues in the amino acid sequence. Thus, the term “peptide” can also encompass polypeptides and proteins. In certain preferred embodiments, the positively charged skeleton itself has no distinct enzymatic activity or therapeutic biological activity. In certain embodiments, the skeleton is a linear hydrocarbon skeleton, which in some embodiments is interrupted by heteroatoms selected from nitrogen, oxygen, sulfur, silicon, and phosphorus. The majority of atoms in the skeleton chain are usually carbon. In addition, the skeleton is often a polymer of repeating units (e.g., amino acids, poly(ethyleneoxy), poly(propyleneamine), and polyalkyleneimines), but may also be a heteropolymer. In one group of embodiments, the positively charged skeleton is a polypropyleneamine in which multiple amine nitrogen atoms exist as positively charged (tetrasubstituted) ammonium groups. In another embodiment, the positively charged skeleton is a non-peptidyl polymer, which may be a heteropolymer or homopolymer, such as a polyalkylene imine, such as polyethyleneimine or polypropyleneimine, and may have a molecular weight of about 10,000 to about 2,500,000, preferably about 100,000 to about 1,800,000, and most preferably about 500,000 to about 1,400,000. In another group of embodiments, the skeleton is bonded to multiple side chain portions containing positively charged groups (e.g., ammonium groups, pyridinium groups, phosphonium groups, sulfonium groups, guanidinium groups, or amidinium groups). In this group of embodiments, the side chain portions may be arranged along the skeleton at fixed or variable intervals. In addition, the lengths of the side chains may be similar or different.For example, in one group of embodiments, the side chain can be a linear or branched hydrocarbon chain having 1 to 20 carbon atoms, and terminates at the distal end (the side away from the backbone) with one of the above positively charged groups. The binding between the positively charged carrier and the botulinum toxin is via non-covalent interactions, non-limiting examples of which include ionic interactions, hydrogen bonds, van der Waals forces, or combinations thereof. Examples of positively charged backbones and efficiency groups for use in the present invention are described in U.S. Patent Nos. 8,623,811 and 9,211,248, which are hereby incorporated by reference in their entirety.

[0019] In one group of embodiments, the positively charged backbone is a polypeptide having a plurality of positively charged side chain groups (e.g., lysine, arginine, ornithine, homoarginine, and the like). Preferably, the polypeptide has a molecular weight of from about 100 to about 1,500,000, more preferably from about 500 to about 1,200,000, and most preferably from about 1000 to about 1,000,000. Those skilled in the art will appreciate that when amino acids are used in this part of the present invention, the side chains may have D- or L-form (R configuration or S configuration) at the center of the linkage. In certain preferred embodiments, the polypeptide has a molecular weight of from about 500 to about 5000, more preferably from 1000 to about 4000, more preferably from 2000 to about 3000. In other preferred embodiments, the polypeptide comprises 10 to 20 amino acids, or 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids, preferably polylysine.

[0020] Alternatively, the backbone may comprise amino acid analogs and / or synthetic amino acids. The backbone can also be an analog of a polypeptide such as a peptoid. See, for example, Kessler, Angew. Chem. Int. Ed. Engl. 32:543 (1993), Zuckermann et al. Chemtracts-Macromol. Chem. 4:80 (1992), and Simon et al. Proc. Nat'l. Acad. Sci. USA 89:9367 (1992). Briefly, peptoids are polyglycines in which the side chains are bonded to the backbone nitrogen atoms rather than the alpha-carbon atoms. As noted above, the side chain moieties typically terminate in a positively charged group, forming a positively charged backbone component. The synthesis of peptoids is described, for example, in U.S. Pat. No. 5,877,278, which is incorporated herein by reference in its entirety. As the term is used herein, a positively charged backbone having a peptoid backbone structure is considered "non-peptidic" because it is not composed of amino acids with naturally occurring side chains at the alpha-carbon positions.

[0021] For example, the amide bond of a peptide can be an ester bond, thioamide (--CSNH--), reverse thioamide (--NHCS--), aminomethylene (--NHCH2--) or reverse methyleneamino (--CH2NH--) group, ketomethylene (--COCH2--) group, phosphinate (--PO2RCH2--), phosphoamide and phosphoamide ester (--PO2RNH--), reverse peptide (--NHCO--), trans-alkene (--CR=CH--), fluoroalkene (--CF=CH--), dimethylene (--CH2CH2) Various other skeletons can be used with steric or electronic mimics of polypeptides in which the skeleton is replaced by malonate and / or gem-diamino-alkyl subunits, for example, Fletcher et al. ((1998) Chem. Rev. 98:763). Many of the aforementioned substitutions result in polymer skeletons that are nearly isovalent in electron distribution compared to skeletons formed from α-amino acids.

[0022] In each of the above skeletons, a positively charged side chain group can be added. For example, in the sulfonamide-linked skeletons (--SO2NH-- and --NHSO2--), the side chain group can be bonded to the nitrogen atom. Similarly, in the hydroxyethylene (--CH(OH)CH2--) bond, the side chain group can be bonded to the hydroxy substituent. Those skilled in the art can easily adapt the chemical properties of other bonds using standard synthetic methods to obtain positively charged side chain groups.

[0023] In one embodiment, the positively charged backbone is a polypeptide having a protein transfer domain (also known as an efficiency group). As used herein, the efficiency group or protein transfer domain is any active substance that has the effect of promoting the translocation of the positively charged backbone through a tissue or cell membrane. Non-limiting examples of the protein transfer domain or efficiency group include -(gly) n1 -(arg) n2 Examples include (SEQ ID NO: 5), HIV-TAT or a fragment thereof, or the protein transduction domain (PTD) or a fragment thereof of Antennapedia, where the subscript n1 is an integer from 0 to 20, more preferably from 0 to 8, and even more preferably from 2 to 5, and the subscript n2 is an odd integer independently from about 5 to about 25, more preferably from about 7 to about 17, and most preferably from about 7 to about 13. In some embodiments, the HIV-TAT fragment does not contain the cysteine-rich region of the HIV-TAT molecule to minimize problems associated with disulfide aggregation. Preferably, the fragments of the protein transduction domains of HIV-TAT and Antennapedia retain the protein transduction activity of the whole protein. Even more preferably, the HIV-TAT fragment has an amino acid sequence (gly) p -RGRDDRRQRRR-(gly) q (Sequence ID 1), (gly) p -YGRKKRRQRRR-(gly) q (Sequence ID 2) or (gly) p -RKKRRQRRR-(gly) qhaving (SEQ ID NO: 3), wherein the subscripts p and q are each independently an integer of 0 to 20, or p and q are each independently an integer of 1 in an embodiment. In another embodiment, the fragment or efficiency moiety is attached to the backbone via the C-terminus or N-terminus of the amino acid sequence of the fragment or efficiency moiety. In certain preferred embodiments, p is 1 and q is zero, or p is zero and q is 1. Preferred HIV-TAT fragments are those in which the subscripts p and q are each independently an integer of 0 to 8, more preferably 0 to 5. In another preferred embodiment, the positively charged side chain or branching group is the Antennapedia (Antp) protein transduction domain (PTD), or a fragment thereof that retains activity. These are known in the art, for example, based on Console et al., J. Biol. Chem. 278:35109 (2003), and non-limiting examples of Antennapedia PTD contemplated by the present invention are the PTD having the amino acid sequence SGRQIKIWFQNRRMKWKKC (SEQ ID NO: 6). In other embodiments, the positively charged carrier has the amino acid sequence RKKRRQRRR-G-(K) for use in the compositions and methods of the present invention 15 -G-RKKRRQRRR (SEQ ID NO: 4), or a positively charged peptide having the amino acid sequence YGRKKRRQRRR-G-(K) 15 -G-YGRKKRRQRRR (SEQ ID NO: 7), or a positively charged peptide having the amino acid sequence RGRDDRRQRRR-G-(K) 15 -G-RGRDDRRQRRR (SEQ ID NO: 8).

[0024] Preferably, the positively charged carrier comprises the side chain positively charged protein transduction domain or the positively charged efficiency moiety in an amount of at least about 0.01% by percentage of the total weight of the carrier, preferably from about 0.01 to about 50 weight percent, more preferably from about 0.05 to about 45 weight percent, and most preferably from about 0.1 to about 30% by weight. The formula -(gly) n1 -(arg) n2For a positively charged protein transfer domain having (SEQ ID NO: 5), the preferred range is approximately 0.1 to approximately 25%.

[0025] In another embodiment, the backbone is polylysine, and the positively charged protein transfer domain is attached to the amino group of the lysine side chain, or to the C-terminus or N-terminus. In some preferred embodiments, the polylysine may have molecular weights of at least 100D, 200D, 300D, 400D, 500D, 600D, 700D, 800D, 900D, 1000D, 1500D, 2000D, 2500D, 3000D, 3500D, 4000D, 4500D, 5000D, 5500D, or 6000D, and molecular weights of less than about 2,000,000D, less than 1,000,000D, less than 500,000D, less than 250,000D, less than 100,000D, less than 75,000D, less than 50,000D, and less than 25,000D. Within the range of 100 to 2,000,000 D, the lower and / or upper limits of the range can be increased or decreased by 100 each, and the resulting subranges are specific embodiments of the invention. In some exemplary embodiments, the polylysine has a molecular weight of about 1,000 to about 1,500,000 D, about 2,000 to about 800,000 D, or about 3,000 to about 200,000 D. In other exemplary embodiments, the polylysine has a molecular weight of about 100 to about 10,000 D, about 500 to about 5,000 D, about 1,000 to about 4,000 D, about 1,500 to about 3,500 D, or about 2,000 to about 3,000 D. Preferably, the polylysine polypeptide has 10 to 20 lysine (SEQ ID NO: 9), more preferably 15 lysine. In some embodiments, the polylysine intended by the present invention may be any of the commercially available polylysines (Sigma Chemical Company, St. Louis, Mo., USA), such as polylysine having more than 70,000 MW, polylysine having 70,000 to 150,000 MW, polylysine having 150,000 to 300,000 MW, and polylysine having more than 300,000 MW. The selection of an appropriate polylysine depends on the remaining components of the composition and should be sufficient if it imparts a net positive charge to the composition as a whole, preferably with a length of 1 to 4 times the combined length of the negatively charged components.Preferred positively charged protein transfer domains or efficient groups include, for example, -gly-gly-gly-arg-arg-arg-arg-arg-arg-arg(-Gly3Arg7(SEQ ID NO: 10)) or HIV-TAT.

[0026] In another preferred embodiment, the positively charged skeleton is a polyalkyleneimine, non-limiting examples of which include polyethyleneimine, polypropyleneimine, and polybutyleneimine. In certain embodiments, the polyalkyleneimine has a molecular weight of at least 100D, 200D, 300D, 400D, 500D, 600D, 700D, 800D, 900D, 1000D, 1500D, 2000D, 2500D, 3000D, 3500D, 4000D, 4500D, 5000D, 5500D, or 6000D, as well as molecular weights less than approximately 2,000,000D, less than 1,000,000D, less than 500,000D, less than 250,000D, less than 100,000D, less than 75,000D, less than 50,000D, and less than 25,000D. Within the range of 100 to 2,000,000 D, the lower and / or upper limits of the range can be increased or decreased by 100 each, and the resulting subranges are specific embodiments of the present invention.

[0027] In other embodiments of the present invention, the carrier is a relatively short polylysine or polyethyleneimine (PEI) skeleton (which may be linear or branched) having positively charged branched groups. While we do not wish to be bound by theory, such carriers are considered useful for minimizing the uncontrolled aggregation of the skeleton and botulinum toxin in the therapeutic composition, which significantly reduces transport efficiency. When the carrier is a relatively short linear polylysine or PEI skeleton, the skeleton will have a molecular weight of less than 75,000 D, more preferably less than 30,000 D, and most preferably less than 25,000 D. However, when the carrier is a relatively short branched polylysine or PEI skeleton, the skeleton will have a molecular weight of less than 60,000 D, more preferably less than 55,000 D, and most preferably less than 50,000 D.

[0028] Positively charged carriers are listed in Table 1 of the Examples. Pharmaceutical compositions containing carrier peptides are also intended. Such embodiments include any carrier peptide from Table 1 and botulinum toxin, for example, botulinum toxin type A or 150 kd of type A botulinum toxin. Such compositions are intended, but compositions having other types of toxins, with or without their natural accessory proteins, are also intended. Further embodiments are intended to provide a method for reducing the appearance of fine wrinkles or creases, or for treating migraines, cervical dystonia, or upper limb spasticity, comprising the step of administering an effective amount of such a pharmaceutical composition. [Examples] [Examples]

[0029] Cell-based carrier peptide enhancement assay To determine the effect of carrier peptides on BoNT / A binding to cells, cell-based assays were performed. SiMa cells pre-coated with one of the peptides listed in Table 1 were placed in 96-well black clear-bottom plates treated with CellBind and fed into 7.5 × 10°F growth medium (RPMI-1640 + 10% FBS). 4 Cells were seeded in wells and allowed to condense for 2 days. Horseradish peroxidase (HRP) was conjugated to 150 kDa BoNT / A using the Mix-n-Stain HRP antibody labeling kit (Biotium, Fremont CA, USA) according to the manufacturer's protocol. Cells were then incubated with 100 μL of 150 kDa BoNT / A labeled with quantified HRP (3.3 nM) at 37°C for 1 hour. The plates were then washed five times again with PBST, followed by the addition of 100 μL of TMB substrate and incubation for 10–15 minutes. The reaction was stopped by adding 50 μL of 1 M sulfuric acid solution, and absorbance was read at 450 nm using an M5 Spectramax plate reader.

[0030] [Table 1]

[0031] The effect of the backbone was tested by comparing the relative amount of BoNT / A binding between PTD-K15-PTD and PTD-R15-PTD. The results are shown in Figure 1. The effects of different cell-penetrating peptides (PTD vs. PTD4) and the number of cell-penetrating peptides on the polylysine backbone were tested, and the results are shown in Figure 2. The effect of backbone length was tested by comparing the effects of PTD-K5-PTD, PTD-K10-PTD, and PTD-K15-PTD. The results are shown in Figure 3. This test showed that in all of these tests, RTP004 showed the highest percentage change in binding compared to the absence of the peptide.

Claims

1. A method for measuring the effect of carrier peptides on the binding of therapeutic proteins to cell membranes, A step of preparing a cell culture containing cells to be maintained in a container; A step of adding a peptide carrier to the cell culture; The step of incubating the cell culture in the container together with the peptide carrier; A step of adding a labeled therapeutic protein to the cell culture; The step of incubating the cell culture with the labeled therapeutic protein to enable the labeled therapeutic protein to bind to the cells in the cell culture; A step of washing the cell culture to remove excess labeled therapeutic proteins and excess peptide carriers; and A step of measuring the amount of labeled therapeutic protein bound to the cells in the cell culture; The method, including the method described above.

2. A method for measuring the effect of carrier peptides on the binding of therapeutic proteins to cell membranes, A step of preparing a cell culture containing cells to be maintained in a container; A step of adding a composition comprising a labeled therapeutic protein and a peptide carrier to the cell culture; A step of incubating the cell culture with the composition to enable the peptide and the labeled therapeutic protein to bind to the cells in the cell culture; A step of washing the cells for excess labeled therapeutic proteins and excess peptide carriers; A step of measuring the amount of labeled therapeutic protein bound to the cells in the cell culture; The method, including the method described above.

3. The method according to claim 1 or 2, wherein the labeled therapeutic protein is botulinum toxin, botulinum toxin type A complex, or 150 kd of botulinum toxin type A.

4. The method according to any one of claims 1 to 3, wherein the label of the labeled therapeutic protein is peroxidase or horseradish peroxidase.

5. The method according to any one of claims 1 to 4, wherein the step of measuring the amount of labeled therapeutic protein includes the step of measuring the amount of UV / VIS absorbance of the label in a cell culture.

6. The method according to any one of claims 1 to 5, wherein the step of washing excess labeled therapeutic protein from the wells comprises flushing the wells once, twice, three times, four times, five times, six times, seven times, or eight times with phosphate-buffered saline in an amount of nonionic surfactant of 0.5 to 2% v / v or about 1% v / v, or with PBST.

7. The method according to any one of claims 1 to 6, wherein the cell culture comprises neuroblastoma cells, neuroblastoma SiMa cells, or neuroblastoma N2a cells.

8. The method according to any one of claims 1 to 7, wherein the peptide carrier is positively charged.

9. The method according to claim 8, wherein the peptide carrier contains polylysine.

10. The method according to claim 8 or 9, wherein the peptide carrier comprises one or more cell-penetrating peptides.

11. The method according to any one of claims 1 to 7 and 10, wherein the peptide carrier is negatively charged.

12. The method according to claim 1, further comprising the step of removing excess peptide from a cell culture after incubation with the peptide.

13. A carrier peptide as described in any of SEQ ID NOs: 11 to 18.

14. A pharmaceutical composition comprising the carrier peptide and botulinum toxin as described in claim 13.

15. The composition according to claim 14, wherein the botulinum toxin is a botulinum toxin type A complex or 150 kd type A botulinum toxin.

16. A method for reducing the appearance of fine wrinkles or creases, or for treating migraine, cervical dystonia, or upper limb spasticity, comprising the step of administering an effective amount of the pharmaceutical composition according to claim 14 or 15.

17. The method according to any one of claims 1 to 12, wherein the cells are nerve cells, for example, cells from a cell line selected from SiMa, Neuro-2a, NT2N, GT1-7, GN11, ND7 / 23, SH-Sy5Y, PC12, and HT-22.

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