WO2019178759A1 - 促进局部肌肉增长、减缓或防止局部肌肉萎缩的组合物及其用途 - Google Patents

促进局部肌肉增长、减缓或防止局部肌肉萎缩的组合物及其用途 Download PDF

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WO2019178759A1
WO2019178759A1 PCT/CN2018/079716 CN2018079716W WO2019178759A1 WO 2019178759 A1 WO2019178759 A1 WO 2019178759A1 CN 2018079716 W CN2018079716 W CN 2018079716W WO 2019178759 A1 WO2019178759 A1 WO 2019178759A1
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composition
polypeptide
muscle atrophy
local muscle
muscle
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PCT/CN2018/079716
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English (en)
French (fr)
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洪诚孝
詹勋锦
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维多利亚生物医学控股股份有限公司
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Priority to CN201880090991.3A priority Critical patent/CN111902157B/zh
Priority to JP2020550596A priority patent/JP7183291B2/ja
Priority to US16/981,391 priority patent/US11672845B2/en
Priority to PCT/CN2018/079716 priority patent/WO2019178759A1/zh
Priority to EP18910803.8A priority patent/EP3769779B1/en
Publication of WO2019178759A1 publication Critical patent/WO2019178759A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1841—Transforming growth factor [TGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/06—Anabolic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/085—Staphylococcus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin

Definitions

  • the present invention relates to a composition, and more particularly to a composition for promoting local muscle growth; the invention further provides for the use of a composition, in particular a medicament for the manufacture of a medicament for promoting local muscle growth.
  • the invention further relates to a composition, in particular to a composition for slowing or preventing local muscle atrophy; the invention further provides for the use of a composition, in particular for the manufacture of a slowing or preventing local muscle atrophy.
  • the use of the drug is referred to a composition, and more particularly to a composition for promoting local muscle growth; the invention further provides for the use of a composition, in particular a medicament for the manufacture of a medicament for promoting local muscle growth.
  • the invention further relates to a composition, in particular to a composition for slowing or preventing local muscle atrophy; the invention further provides for the use of a composition, in particular for the manufacture of a slowing or preventing local muscle atrophy.
  • the use of the drug is
  • Myostatin is highly similar to other members of the transforming growth factor ⁇ (TGF- ⁇ ) family in terms of its sequence.
  • the gene structure of myostatin consists of three parts: (1) the N-terminal hydrophobic domain as a signal for protein secretion release; (2) a highly conserved protein cleavage site RXRR; and (3) cysteine-rich (cysteine) C-terminal active domain.
  • TGF- ⁇ transforming growth factor ⁇
  • the existing research proposes a monoclonal antibody JA16 having high specificity for myostatin (Whittemore et al., 2003, Biochemical and Biophysical Research Communications 300: 965-971), and finds binding by analyzing the binding position of myostatin.
  • the position is located at the C-terminal 15 amino acids DFGLDCDEHSTESRC of mouse myostatin, and it is known that the C-terminal domain is an antigenic fragment.
  • Taiwanese invention patent I540968 discloses the fusion of a muscle doubling fragment (myostatin) with a Pseudomonas aeruginosa exotoxin region Ia fragment to form a polypeptide fragment or antibody, wherein the Pseudomonas aeruginosa exotoxin fragment is effective for enhancing an immune response; however, It is known to the skilled person that Pseudomonas aeruginosa may produce an inclusion body during the production of bacteria, so that additional urea (urea) is required to extract the fragments or antibodies to destroy the cells, resulting in urea residues, and refolding. Fragments may affect protein function; more importantly, the effects disclosed in this patent are still systemic responses, and therefore, the prior art myostatin antibodies still have room for improvement.
  • myostatin muscle doubling fragment
  • Pseudomonas aeruginosa exotoxin region Ia fragment to form a polypeptide fragment
  • enterotoxin secreted by Staphylococcus aureus can be divided into several types, such as A, B, C1, C2, D, E and F, among which Staphylococcus aureus
  • the toxins of the toxins (Staphylococcal enterotoxin) SEA, SEB, SEC1 and SEC2) are similar in molecular weight and structurally similar, so they all cause a systemic immune response and have the same clinical symptoms, such as fever. Large side effects such as elevated blood pressure.
  • the invention relates to a composition for promoting local muscle growth, delaying local muscle atrophy or preventing local muscle atrophy, comprising: a first polypeptide which is similar to that shown in SEQ ID NO: a sequence of at least 90% or more; and a second polypeptide comprising from 1 to 10 repeat units of the sequence set forth in SEQ ID NO: 14.
  • the second polypeptide is a linear array epitope (LAE) of tandem repeated units, wherein the second polypeptide is as shown in SEQ ID NO: 14. Sequence 1 to 10 repeating units.
  • LAE linear array epitope
  • the second polypeptide is a linear array of repeating antigens of a tandem repeat unit, wherein the second polypeptide comprises 6 repeat units of the sequence set forth in SEQ ID NO: 14.
  • the first polypeptide has T or L corresponding to position 7 in SEQ ID NO: 8, has G or E at position 9, has a Y or V at position 13, and has a H or Y at position 105. .
  • the first polypeptide is selected from the group consisting of SEQ ID Nos: 4, 5, 6, 7, 8, 9, 10, 11 and 12.
  • the composition further comprises: a linker between the first polypeptide and the second polypeptide; wherein the sequence of the composition is as shown in SEQ ID NO: .
  • first polypeptide and the second polypeptide may be selected from the following:
  • the invention in another aspect, relates to a composition for promoting local muscle growth, delaying local muscle atrophy or preventing local muscle atrophy comprising: a polypeptide of S. aureus enterotoxin; and a myostatin polypeptide.
  • polypeptide of S. aureus enterotoxin is selected from the group consisting of Staphylococcus aureus enterotoxins A, B, C1, C2, D, E, F, G and H.
  • SEC2 has a molecular weight of 27 kDa and contains 239 amino acids.
  • the SEC2 transcribed by ⁇ is a 266 amino acid protein with a molecular weight of 30 kDa; the SEC2 polypeptide is cleaved at alanine 27 to produce a mature toxin containing 239 amino acids with a molecular weight of 27 kDa.
  • the N-terminal polypeptide sequencing determines the position of the cleavage of the message polypeptide in SEC2 and confirms the N-terminus of the mature toxin, thereby demonstrating that SEC2 is effective in enhancing the immune response elicited. More preferably, the polypeptide sequence of the S.
  • aureus enterotoxin C2 sequence has at least 90% sequence identity to the sequence set forth in SEQ ID NO: 8, for example, corresponding to SEQ ID NO: The sequence shown is selected from the group consisting of having a T or L at position 7, a G or E at position 9, a Y or V at position 13, and a H or Y at position 105.
  • the myostatin polypeptide comprises, but is not limited to, growth differentiation factor 8, GDF8, follistatin or activin receptor type- 2B, ACTR-IIB).
  • the myostatin polypeptide is selected from the group consisting of a growth differentiation factor 8 such as the sequence shown in SEQ ID NO: 13, a follistatin such as the sequence shown in SEQ ID NO: 15, and a second type activin receptor.
  • a growth differentiation factor 8 such as the sequence shown in SEQ ID NO: 13
  • a follistatin such as the sequence shown in SEQ ID NO: 15
  • a second type activin receptor a group consisting of the sequences shown in SEQ ID NO: 16.
  • the epitope polypeptide of myostatin is a linear array epitope (LAE) of tandem repeated units.
  • LAE linear array epitope
  • the invention is directed to a pharmaceutical composition that promotes local muscle growth, delays local muscle atrophy, or prevents local muscle atrophy, comprising a composition as previously described and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes, but is not limited to, water, alcohols, glycols, hydrocarbons [such as petroleum jelly and white).
  • adjuvant includes, but is not limited to, alum precipitate, Freund's complete adjuvant, Freund's incomplete adjuvant, and monophosphoryloxy.
  • alum precipitate As used herein, “adjuvant” includes, but is not limited to, alum precipitate, Freund's complete adjuvant, Freund's incomplete adjuvant, and monophosphoryloxy.
  • compositions of the present invention may exist in a variety of forms. These forms include, but are not limited to, liquid, semi-solid, and solid pharmaceutical forms, including, but not limited to, dispersions or suspensions; semi-solids and solids including, but not limited to, tablets, pills, powders, liposomes, and suppositories .
  • the preferred form depends on the intended mode of administration and therapeutic application. More preferably, the pharmaceutical composition is in the form of an orally or infusible solution.
  • the invention relates to a nucleic acid that promotes local muscle growth, delays local muscle atrophy, or prevents local muscle atrophy, which encodes a composition consisting of the amino acid sequence set forth in SEQ ID NO:17.
  • the invention relates to the use of a composition as described above for the manufacture of a medicament for promoting local muscle growth by administering a drug to the recipient at an effective dose to localize Muscles achieve growth.
  • the aforementioned composition is used to achieve localized muscle growth in mammals, including administration of a fusion protein of myostatin fragment and SEC2, thereby obtaining specific immune cells against myostatin.
  • the immune cells of the present invention can be introduced into other animals by the epitope of the myostatin fragment, and purified or introduced into the mammal by immunizing the cells or their epitopes, so that the mammal itself can increase muscle growth.
  • the immune cells of the present invention may be a plurality of B lymphocytes or T cell strains; preferably, the aforementioned immune cells are regulatory T cells.
  • the invention relates to the use of a composition as hereinbefore described, which is for use in the manufacture of a medicament for preventing local muscle atrophy, which is to administer the medicament to the recipient at an effective dose to slow or Prevents the effect of local muscle atrophy.
  • the composition of the composition is applied to the leg muscles on the side of the damaged nerve so that the muscles of the lateral leg can maintain muscle size to avoid nerve damage.
  • the composition of the composition is applied to the leg muscles on the side of the blocked nerve so that the muscles of the lateral leg can maintain muscle size to avoid nerve blockage.
  • compositions and uses are suitable for use in animals.
  • it is suitable for vertebrates; more preferably, the myostatin of human, pig, cow, sheep, dog and poultry, waterfowl and other animals has been selected, and its amino acid sequence is highly conserved. Therefore, it can be assumed that the myostatin of the aforementioned animal has the same function; more preferably, the aforementioned mammal belongs to a human, a pig, a cow, a sheep or a dog.
  • compositions of the present invention are useful in a variety of conditions of muscle wasting, which may be caused by drugs including, but not limited to, by the use of glucocorticoids such as cortisol, dexamethasone, and cresitol. (betamethasone), prednisone, methylprednisolone or prednisolone.
  • Muscle atrophy can also be denervated by neurological trauma or by degenerative, neuronal necrosis Caused, metabolic or inflammatory neuropathy, such as Guillian-Barre syndrome, peripheral neuropathy, or exposure to environmental toxins.
  • muscle atrophy can be caused by muscle diseases including, but not limited to, myotonic dystrophy, congenital myopathies, familial periodic paralysis (FPP), metabolic muscle disease ( Metabolic myopathies; caused by liver glycogen or lipid storage diseases, dermatomyositis, polymyositis, inclusion body myositis (IBM), myositis (myositis) Ossificans) or rhabdomyolysis (rhabdomyolysis).
  • muscle diseases including, but not limited to, myotonic dystrophy, congenital myopathies, familial periodic paralysis (FPP), metabolic muscle disease ( Metabolic myopathies; caused by liver glycogen or lipid storage diseases, dermatomyositis, polymyositis, inclusion body myositis (IBM), myositis (myositis) Ossificans) or rhabdomyolysis (rhabdomyolysis).
  • Muscle atrophy can also be caused by diseases including, but not limited to, motor neuron diseases (MND), spinal muscular atophy (SMA), amyotrophic lateral sclerosis (amyotrophic lateral sclerosis). ), juvenile spinal muscular atrophy (also known as SMA-III), myasthenia Gravis (MG), paralysis due to stroke or spinal cord injury, bone fixation due to trauma, prolonged bed rest, Autonomic inactivity, non-autonomous inactivity, metabolic stress or undernutrition, cancer, AIDS, fasting, thyroid disease, diabetes, central core disease (CCD), burns, chronic obstructive pulmonary disease Muscle atrophy caused by liver disease (such as fibrosis, cirrhosis), sepsis, renal failure, congestive heart failure, aging, space navigation, or spending a period of time in a zero-gravity environment.
  • MND motor neuron diseases
  • SMA spinal muscular atophy
  • amyotrophic lateral sclerosis amyotrophic lateral sclerosis
  • epitope refers to a fragment capable of eliciting an immune response to produce an antigen in a protein antigen, which can be observed by structure prediction or by selecting a protein fragment to observe an immune animal. immune response.
  • the term "effective dose” refers to an amount effective to achieve local muscle growth or to slow or prevent local muscle atrophy at dose and for the time required; as exemplified by the present invention
  • the dose effective to promote local muscle growth can be known by promoting the local muscle growth test (Example 1); the dose effective to slow or prevent local muscle atrophy can be slowed or prevented by local muscle atrophy test (Examples 2, 3) And learned.
  • the epitope is a small polypeptide fragment, if the small polypeptide fragment is directly immunized in an animal, the immune response may be less than ideal.
  • a linear array epitope (LAE) comprising a tandem repeat unit is constructed to improve the immune response.
  • bacterial toxins can be used to aid antigen delivery by using toxins that eliminate toxin activity as a transport system to enhance the overall immune effect with the properties of the toxin.
  • the linear array of repeat epitopes of the epitope of the C-terminus of the myostatin of the present invention is fused to S. aureus enterotoxin C2 (SEC2).
  • a host cell comprising the above nucleic acid is produced.
  • examples include E. coli, insect cells, plant cells, yeast cells, and mammalian cells.
  • the nucleic acid molecule can be used to express a polypeptide or fusion protein described herein, which can be operably linked to a multiple cloning site (MCS) of a suitable vector to produce the polypeptide or fusion protein.
  • MCS multiple cloning site
  • Vector embodiments include plasmids.
  • the vector comprises a promoter, an enhancer, a multiple cloning site, etc., and after the nucleic acid molecule is ligated to a multiple cloning site of a suitable vector, the expression vector can be introduced into a host cell to produce a polypeptide as described herein or Fusion protein.
  • the host cell includes, but is not limited to, Escherichia coli, B. pertussis, Bacillus, African green monkey kidney cells, Haemophilus, fungi or yeast.
  • An advantage of the present invention is that it is a breakthrough in the prior art by the composition comprising the polypeptide of the S. aureus enterotoxin C2 sequence and the antigen-determining polypeptide of the myostatin C-terminus to achieve local muscle growth.
  • the composition is administered to the leg muscles on the side of the nerve injury or nerve block, and it is unexpectedly found that the side leg muscles can maintain muscle size, which will contribute to clinical encounter with nerve damage or When the nerve is blocked, the patient's local muscle atrophy can be effectively avoided.
  • Fig. 1 is a line graph showing the ratio of muscle cross-sectional area of a low-dose experimental group and a low-dose control group of the present invention. Among them, the low-dose experimental group was indicated by a broken line indicating ⁇ and the low-dose control group was indicated by a broken line indicating ⁇ .
  • Fig. 2 is a line diagram showing the ratio of the muscle cross-sectional area of the dose test group and the medium dose control group in the present invention.
  • the middle dose experimental group is indicated by a broken line indicating ⁇
  • the middle dose control group is indicated by a broken line indicating ⁇ .
  • Figure 3 is a line graph showing the ratio of the muscle cross-sectional area of the high-dose experimental group of the present invention compared with the high-dose control group.
  • the high-dose experimental group was indicated by a broken line indicating ⁇
  • the high-dose control group was indicated by a broken line indicating ⁇ .
  • Figure 4 is a longitudinal sectional view and a cross-sectional scan of the computerized leg of the mouse in the hind leg of the present invention, each cross-sectional scan is a cross section of the cross-sectional line of the corresponding longitudinal section scan;
  • (A) is a low dose in Figure 4.
  • the longitudinal section scan of the control group the area calculated by the muscle volume between the upper and lower dashed lines;
  • (B) is the longitudinal section scan of the middle dose control group in Fig. 4, and the area calculated by the muscle volume between the upper and lower dashed lines;
  • C is a longitudinal section scan of the high-dose control group, and the area calculated by the muscle volume between the upper and lower dotted lines;
  • D in Fig.
  • FIG. 4 is a cross-sectional scan of the low-dose control group;
  • Figure 4 (F) is a cross-sectional scan of the high-dose control group;
  • Figure 4 (G) is a longitudinal section scan of the low-dose experimental group, with muscle volume between the upper and lower dashed lines The calculated area;
  • (H) in Fig. 4 is a longitudinal section scan of the middle dose experimental group, and the area between the upper and lower dotted lines is the area calculated by the muscle volume;
  • (I) in Fig. 4 is the longitudinal section scan of the high dose experimental group, up and down The area between the dotted lines calculated for the muscle volume;
  • in Figure 4 (J) is the low dose experimental group Cross-sectional scans;
  • FIG. 4 (K) is a cross-sectional scan dose experimental group;
  • FIG. 4 (L) is a cross-sectional view of a high dose experiment scanning group.
  • Figure 5 is a bar graph of the hind leg volume ratio of the low dose control group and the low dose experimental group of the present invention.
  • A.U. indicates an Arbitrary Unit (arbitrary unit).
  • Figure 6 is a bar graph of the hind leg volume ratio of the mouse in the dose control group and the medium dose experimental group in the present invention.
  • A.U. indicates an Arbitrary Unit (arbitrary unit).
  • Figure 7 is a bar graph of the hind leg volume ratio of the high dose control group and the high dose experimental group of the present invention.
  • A.U. indicates an Arbitrary Unit (arbitrary unit).
  • Figure 8 is a staining diagram of hematoxylin-eosin (H-E) tissue staining of the calf muscle fibers of the high-dose control mice of the present invention.
  • Figure 9 is a staining diagram of the H-E tissue of the calf muscle fibers of the high-dose experimental group of the present invention.
  • Figure 10 is a diagram showing immunohistochemical staining of myogenic myofibrillar fibers against myostatin in the mouse of the present invention; wherein (A) to (C) in Fig. 10 are a control group; Figs. 10(D) to (F) are respectively 50. Nack (ng) (low dose), 500 ng (medium dose) and 5000 ng (high dose) experimental group.
  • Figure 11 is a line graph showing the change in body weight of the low, medium and high dose experimental group mice of the present invention after administration of the composition.
  • the low-dose experimental group is indicated by a broken line indicating ⁇
  • the medium-dose experimental group is indicated by a broken line indicating ⁇
  • the high-dose experimental group is indicated by a broken line indicating ⁇ .
  • Figure 12 is a bar graph showing the ratio of muscle volume after 1000 ng of the composition of the present invention was administered to the experimental group and the experimental group of the local muscles after sciatic nerve destruction; wherein the left leg was subjected to sciatic nerve destruction surgery, and the right leg did not undergo sciatic nerve destruction. surgery.
  • Figure 13 is a bar graph of the muscle ratio obtained by dividing the left leg muscle volume ratio by the right leg muscle volume ratio between the control group and the experimental group of Figure 12 .
  • Figure 14 is a bar graph showing the ratio of muscle volume of the composition of the present invention to 1000 ng and 5000 ng of the composition of the present invention after the local muscles of the control group, the experimental group A and the experimental group B were subjected to sciatic nerve truncation; Sciatic nerve destruction surgery, the right leg did not undergo sciatic nerve destruction surgery.
  • Figure 15 is a bar graph of the ratio of muscles obtained by dividing the left leg muscle volume ratio by the right leg muscle volume ratio of the control group, the experimental group A, and the experimental group B of Figure 14 .
  • the fusion protein used in this embodiment is a vector expressed by the pET expression system of Escherichia coli (E. coli); preferably, pET-28a is used.
  • E. coli Escherichia coli
  • pET-28a pET-28a
  • the first polypeptide "SEC2m" is a S.
  • aureus enterotoxin C2 having a point mutation the nucleic acid sequence is as shown in SEQ ID NO: 1, and the protein sequence is as SEQ ID NO: 8 (mutation point is in place) Point 7 has L, site 9 has E, site 13 has V and site 105 has Y); the second polypeptide "Myo epitope” shows antigenic determinant of myostatin, which is myogenesis inhibition C-terminal 15 amino acids (as shown in SEQ ID NO: 14; this sequence is highly conserved, so multiple species have this sequence) with 6 repeats, the nucleic acid sequence of a single fragment is SEQ ID NO :2 is shown.
  • the gene sequence located in the multiple cloning site (MCS) of the pET vector is sequenced from the N-terminus to "SEC2m", linker and "Myo epitope", as shown in SEQ ID NO: 3 .
  • a 35 L fermentation culture program was established to culture E. coli BL21 (DE3) strain containing the SEQ ID NO: 3 pET vector in a 50 L fermentor. 4 tubes of 5 mL strains were cultured overnight at 37 ° C with LB/Ampicillin medium, and each strain was inoculated into 0.2 liters of LB/ampicillin medium for 1 liter, and continuously cultured at 37 ° C to OD 600. Is 0.3. Thereafter, it was added to 35 ⁇ L of the medium for culture, and the OD 600 was measured every two hours to monitor the change of the growth curve, and the appropriate time point was further selected according to the growth curve, and the final concentration of 0.1 mM of isopropyl group was added.
  • ⁇ -D-thiogalactopyranoside IPTG induced Escherichia coli to express the fusion protein highly, and the culture was continued at 37 ° C for 3 hours with shaking, and the strain was recovered by centrifugation.
  • the expression of the fusion protein was determined by SDS-PAGE electrophoresis and Western blotting to determine the optimal 35 L fermentation conditions.
  • the fusion polypeptide expressed by Escherichia coli BL21 (DE3) is subjected to extraction and isolation of the fusion polypeptide after cell lysis, and finally a composition (as shown in SEQ ID NO: 17) is obtained;
  • the extraction and separation are conventional techniques and will not be described here.
  • mice used in the animal experiments of the present example were "C57BL/6" strains of 8 weeks old (12 months old) mother rats, and a control group and an experimental group of 9 mice, the experimental time was 6 months. From the time the mice grew to 12 months of age, high lipid diets were used and they were bred with drinking water containing high fructose syrup. Feed and water are changed every two days to avoid deterioration. The feed was stored at -20 ° C, and the high fructose syrup was stored at 4 ° C; the body weight was measured every two weeks. After 6 months of feeding, the experimental animals began intramuscular injection of the composition once a week.
  • composition obtained in Preparation Example 1 (diluted into a different concentration by physiological saline) was injected into the muscles of the left hind leg of the mice into different doses of the experimental group, and the right lower calf was injected with saline (saline).
  • the experimental groupings are shown in Table 1 below.
  • the collection of experimental data is measured weekly by a vernier scale, measuring the long diameter (a) and short diameter (b) and body weight of the muscle, and the measurement position is the position of the injected drug.
  • the calculated value is calculated as the ellipse area: "a ⁇ b ⁇ 3.14", and the approximate cross-sectional area of the muscle is calculated.
  • the calculation range is shown in Fig. 4 (A), 4 (B), 4 (C), 4 (G), 4 ( H), 4 (I)
  • the area between the upper and lower dotted lines is the muscle volume calculation for comparison between the experimental groups.
  • each experimental group was compared with the control group, and the cross-sectional area of the calf injected with the high-dose composition was increased by 8.19% compared with the high-dose control group.
  • the cross-sectional area of the calf of the dosage composition increased by 5.5% compared to the medium dose control group, while the cross-sectional area of the low-dose composition increased by 5.67% compared to the low-dose control group.
  • each cross-sectional scan is a cross-section of the cross-sectional line of the corresponding longitudinal section scan.
  • Figure 4 (A), Figure 4 (B), Figure 4 (C), Figure 4 (G), Figure 4 (H), Figure 4 (I) between the dotted lines for the calculation of muscle volume Range the muscle volume is obtained by integrating each cross-sectional area. After 19 weeks of administration, the experiment was terminated, and the mice were sacrificed and their calves were fixed, fixed with 10% formalin, and subjected to computed tomography.
  • the low-dose experimental group increased by 4.6% compared with the low-dose control group
  • the medium-dose experimental group increased by 8.5% compared with the middle-dose control group. It increased by 19.2% in the high dose control group. Since the test is based on the muscle of the left hind leg of the same mouse as the experimental group, and the right posterior leg is the control group, the results show that the injection of the composition of the present invention into the left calf only causes the calf muscle to grow on the side. On the other side (right side), the muscles of the lower leg did not grow, so that the composition of the present invention produced muscle growth only by the local application, and did not cause a reaction of systemic muscle enlargement.
  • FIG. 8 is the posterior leg muscle fiber of the high-dose control group
  • FIG. 9 is the calf muscle fiber of the high-dose experimental group, and the muscle fiber of the high-dose experimental group is known. Significantly increased compared to the control group.
  • FIG. 10 (A), FIG. 10 (B), and FIG. 10 (C) different doses of the control group (left posterior calf) myostatin can be significantly stained; (D), (E) in Fig. 10, and (F) in Fig. 10, the expression of myostatin was significantly inhibited in the low dose, medium dose, or high dose experimental group (right hind leg).
  • the right calf (ie, the control group) of the same mouse had a high concentration of myostatin, and the right calf muscle did not become significantly larger; but the left of the same mouse
  • the myostatin of the calf (ie, the experimental group) is inhibited by the composition of the present invention, so that the myostatin concentration is low, and the left calf muscle is enlarged, that is, the composition of the present invention has a local muscle mass. Effect (not affected by systemic blood circulation).
  • Body weight was measured at week 19, as shown in Figure 11, and there was no significant difference in body weight between the groups after administration of the composition in either the low, medium or high dose groups. Therefore, in this test, the topical application of the composition only increases the muscle at the site of the application site, and does not cause systemic muscle enlargement.
  • Example 2 Test to slow or prevent local muscle atrophy caused by nerve damage
  • the sciatic nerve destruction surgery is to apply the anesthetic (abdominal anesthesia) to the mice, remove the body hair from the knee to the buttocks of the mouse, fix the mouse legs and disinfect the surgical site with alcohol cotton, find the thigh femur position, open near the buttocks.
  • a parallel femoral incision, after peeling off the muscle layer, the sciatic nerve parallel to the femur can be seen, the sciatic nerve is picked up, the sciatic nerve of the mouse is damaged by special tools, the sciatic nerve is returned to the original position, and the mouse is observed daily after suturing the skin. Wound healing, gait changes, and overall status are designed to mimic the state of nerve damage.
  • the right thigh muscle volume as the baseline ratio 1.
  • the left thigh muscle volume ratio is the left thigh muscle volume divided by the right thigh muscle volume (as shown in the control group in Figure 12), showing that the control group mice have left leg muscles compared to the right leg. Muscle showed atrophy; another group of mice had a left thigh muscle volume of about 1888 mm 3 and a right thigh muscle volume of about 1705 mm 3 .
  • the right thigh muscle volume was used as a reference ratio and the left thigh muscle volume ratio was The left thigh muscle volume is divided by the right thigh muscle volume (as shown in the experimental group in Fig. 12).
  • the left leg of the mouse undergoes sciatic nerve destruction surgery, the left leg muscles are not atrophied due to the administration of the composition of the present invention. .
  • the left thigh/right thigh muscle volume ratio of the control group was about 0.86 (less than 1 means atrophy); the left thigh/right thigh muscle volume ratio of the experimental group was about 1.11, so the experimental group compared
  • the control group exhibits a phenomenon of maintaining a muscle volume by administering the composition of the present invention.
  • the composition of the present invention by administering the composition of the present invention, local muscle atrophy or even the maintenance of the original muscle volume can be reduced or relieved.
  • Example 3 a test to slow or prevent local muscle atrophy caused by nerve truncation
  • the composition of the preparation of the present invention 5000 ng was administered by intramuscular injection for 14 days, and finally the degree of muscle atrophy of the mouse was observed to evaluate the effect of the composition of the present invention against muscle atrophy (the right leg of each group of mice above) Neither sciatic nerve truncation was performed.
  • the sciatic nerve truncation procedure is similar to that of Example 2, except that the sciatic nerve is directly truncated after being picked up.
  • the left thigh muscle volume ratio is the left thigh muscle volume divided by the right thigh muscle volume (as shown in the control group in Figure 14), indicating that the left leg muscle of the control group is atrophied compared to the right leg muscle, and the right leg muscle is not.
  • experimental group A mouse left thigh muscle volume is about 1289mm 3
  • right thigh muscle volume is about 1394mm 3
  • the right leg has not undergone sciatic nerve destruction surgery, so the right thigh muscle volume as a reference ratio 1, left thigh muscle
  • the volume ratio is the left thigh muscle volume divided by the right thigh muscle volume (as shown in experimental group A in Figure 14).
  • the left leg muscle atrophy is significantly slowed down; the experimental group B mouse left thigh muscle volume is about 1958mm 3 , the right thigh muscle volume is about 1869mm 3 , because the right leg has not undergone sciatic nerve destruction surgery, so the right thigh muscle volume as a reference ratio 1, left
  • the thigh muscle volume ratio is the left thigh muscle volume divided by the right thigh muscle volume (as shown in experimental group B of Figure 14), although the left leg of the mouse undergoes sciatic nerve destruction surgery, the combination of the present invention is applied to the left leg muscle. 5000ng, no shrinkage.
  • the left thigh/right thigh muscle volume ratio of the control group was about 0.87, showing atrophic state (less than 1 means that the atrophy state was present); the experimental group A's left thigh/right thigh muscle volume ratio was about 0.92 experimental group. A showed a slowing of muscle atrophy compared with the control group; the left thigh/right thigh muscle volume ratio of experimental group B was about 1.04, and experimental group B could maintain muscle volume even compared with the control group.
  • the local muscle atrophy can be reduced or relieved, and the present embodiment 3 can maintain the original muscle volume.

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Abstract

一种促进局部肌肉增长或减缓或防止局部肌肉萎缩的组合物，其包含金黄色葡萄球菌肠毒素C2区域的多肽，以及肌生成抑制素多肽。藉由所述的组合物，克服现有技术仅能全身性肌肉增长的缺陷，以达成促进局部肌肉增长、减缓或防止局部肌肉萎缩的效果。

Description

促进局部肌肉增长、减缓或防止局部肌肉萎缩的组合物及其用途 技术领域
本发明是关于一种组合物，特别是指一种促进局部肌肉增长的组合物；本发明另提供一种组合物的用途，特别是指一种用于制造促进局部肌肉增长的药物的用途。本发明另关于一种组合物，特别是指一种减缓或防止局部肌肉萎缩的组合物；本发明另提供一种组合物的用途，特别是指一种用于制造减缓或防止局部肌肉萎缩的药物的用途。
背景技术
肌生成抑制素(myostatin)就其序列而言，与转化生长因子(transforming growth factor β，TGF-β)家族其他成员高度相似。肌生成抑制素的基因结构包括有三部分：(1)作为蛋白质分泌释放信号的N端疏水结构域；(2)高度保守的蛋白质切割位置RXRR；以及(3)富含半胱氨酸(cysteine)的C端活性结构域。许多研究报告指出，脊椎动物中肌生成抑制素的氨基酸序列在C端活性结构域具有高度的保守性。现有研究提出对肌生成抑制素具有高特异性的单株抗体JA16(Whittemore et al.,2003,Biochemical and Biophysical Research Communications300:965-971)，藉由分析肌生成抑制素的结合位置，发现结合位置位在小鼠肌生成抑制素C端15个氨基酸DFGLDCDEHSTESRC，从而可知C端结构域为抗原片段(antigenic fragment)。
然而，目前施予肌生成抑制素的抗体皆会产生全身性的反应，如Camporez等人于2016年的文献指出，局部注射抗肌生成抑制素抗体后，使年老的小鼠全身性的肌肉增长，进而使全身重量增加。此外，针对肌生成抑制剂ACE-031(myostatin inhibitor)于2010年的临床试验结果显示虽可用于增加全身肌肉及加强肌肉力量，但受试者分别出现自发性出血(spontaneous bleeding)、流鼻血(nosebleeds)、皮肤微血管扩张(small expansion of blood vesselsin skins)或头痛(headaches)等副作用，因此该临床试验不得不因负面现象(negative phenomena)而于2011年中止试验；尤其抗体会引起受体免疫系统的全身性作用，诸如过敏反应、寒颤、腹泻、恶心呕吐、皮肤瘙痒等症状。此外，如H.N.Peiris于2012年的文献[Placenta 33(2012 902-907)]指出，在“双重肌肉”基因型(double-muscling)的牛种中，产犊(calving)和生育(fertility)都存在困难；然而在myostatin基因缺乏的小鼠(Mstn -/-)却是可生育的(fertile)；由此推测，肌肉生长抑制素可能与妊娠期促成胎盘及其功能有关。
中国台湾发明专利I540968揭露含有肌肉倍增片段(myostatin)与绿脓杆菌外毒素区域Ia 片段融合形成多肽片段或抗体，其中绿脓杆菌外毒素片段的目的在于有效增强诱导免疫反应；然而，就所属领域技术人员所知，绿脓杆菌在细菌生产过程中可能会产生内涵体(inclusion body)，因此萃取片段或抗体时需要额外添加尿素(urea)去破坏细胞而导致尿素残留，以及重新折迭(refolding)片段而可能影响蛋白质功能；更重要的是，该篇专利所揭露的效果仍是全身性的反应，因此，现有技术的肌生成抑制素抗体仍有改善的空间。
另外，目前研究显示，金黄色葡萄球菌(Staphylococcus aureus)分泌的肠外蛋白(enterotoxin)可被分为A、B、C1、C2、D、E和F等几种类型，其中金黄色葡萄球菌肠毒素(Staphylococcal enterotoxin)SEA、SEB、SEC1及SEC2)的肠毒素蛋白分子量都相近似且在结构上有较高的类似性，故皆会引起全身性的免疫反应并具有相同的临床症状，如发烧、血压升高等较大的副作用。
有鉴于此，如何发展出只影响局部肌肉而不引起全身性反应的药剂，实为各相关业者积极研究开发的目标。
发明内容
为了克服现有技术的缺点，本发明的目的在于提供一种组合物，以达成局部肌肉增长的效果。
在一个态样中，本发明是关于一种促进局部肌肉增长、延缓局部肌肉萎缩或防止局部肌肉萎缩的组合物，其包含：第一多肽，其是如SEQ ID NO：8所示具有相似度至少90％以上的序列；以及第二多肽，其包含如SEQ ID NO：14所示序列1至10个重复单元(repeat units)。
较佳的，所述的第二多肽为纵列重复单元(tandem repeated units)的线性排列重复抗原(linear array epitope，LAE)，其中所述第二多肽为如SEQ ID NO：14所示序列1至10个重复单元。
更佳的，所述的第二多肽为纵列重复单元的线性排列重复抗原，其中所述第二多肽包含如SEQ ID NO：14所示序列6个重复单元。
较佳的，所述的第一多肽在SEQ ID NO：8对应于位点7具有T或L、位点9具有G或E、位点13具有Y或V及位点105具有H或Y。
较佳的，所述的第一多肽选自由如SEQ ID NO：4、5、6、7、8、9、10、11及12所组成的群组。
较佳的，所述的组合物，其还包含：连接子，其是介于所述第一多肽以及第二多肽之间；其中所述组合物的序列如SEQ ID NO：17所示。
在一个较佳的制备例中，所述第一多肽与第二多肽可选自以下例示：
Figure PCTCN2018079716-appb-000001
在另一个态样中，本发明是关于一种促进局部肌肉增长、延缓局部肌肉萎缩或防止局部肌肉萎缩的组合物，其包含：金黄色葡萄球菌肠毒素的多肽；以及肌生成抑制素多肽。
较佳的，所述的金黄色葡萄球菌肠毒素的多肽选自于由金黄色葡萄球菌肠毒素A、B、C1、C2、D、E、F、G及H所组成的群组。
SEC2具有27kDa的分子量，含有239个氨基酸。甫经转录的SEC2为266个氨基酸的蛋白质，分子量计为30kDa；于丙氨酸27处切断SEC2多肽可产生成熟毒素，其含有239个氨基酸，分子量计为27kDa。藉由N端多肽定序可确定SEC2中的讯息多肽切割位置并确认成熟毒素的N端，藉此可证明SEC2能够有效地增强所引发的免疫反应。更佳的，所述的金黄色葡萄球菌肠毒素C2序列的多肽序列与SEQ ID NO：8中所示序列具有至少90％以上的序列一致性，举例而言，对应于SEQ ID NO：8所示序列中选自由位点7具有T或L、位点9具有G或E、位点13具有Y或V及位点105具有H或Y所组成的群组。
较佳的，所述的肌生成抑制素多肽包含、但不限于生长分化因子8(growth differentiation factor 8，GDF8)、滤泡抑素(follistatin)或第二型活化素受体(activin receptor type-2B，ACTR-IIB)。
更佳的，所述的肌生成抑制素多肽选自由生长分化因子8如SEQ ID NO：13所示序列、滤泡抑素如SEQ ID NO：15所示序列及第二型活化素受体如SEQ ID NO：16所示序列所组成的群组。
较佳的，所述的肌生成抑制素的抗原决定位多肽为纵列重复单元(tandem repeated  units)的线性排列重复抗原(linear array epitope，LAE)。
在另一态样中，本发明是关于一种促进局部肌肉增长、延缓局部肌肉萎缩或防止局部肌肉萎缩的医药组成物，其包含如前所述的组合物以及药学上可接受的载剂。
在本文中，所述的“药学上可接受的载剂”包含，但不限于水、醇(alcohols)、甘醇(glycol)、碳氢化合物(hydrocarbons)[诸如石油胶(petroleum jelly)以及白凡士林(white petrolatum)]、蜡(wax)[诸如石蜡(paraffin)以及黄蜡(yellow wax)]、保存剂(preserving agents)、抗氧化剂(antioxidants)、溶剂(solvent)、乳化剂(emulsifier)、悬浮剂(suspending agent)、分解剂(decomposer)、黏结剂(binding agent)、赋形剂(excipient)、安定剂(stabilizing agent)、螯合剂(chelating agent)、稀释剂(diluent)、胶凝剂(gelling agent)、防腐剂(preservative)、润滑剂(lubricant)、吸收增强剂(absorption enhancers)、活性剂(active agents)、保湿剂(humectants)、气味吸收剂(odor absorbers)、香料(fragrances)、pH调整剂(pH adjusting agents)、闭塞剂(occlusive agents)、软化剂(emollients)、增稠剂(thickeners)、助溶剂(solubilizing agents)、渗透增强剂(penetration enhancers)、抗刺激剂(anti-irritants)、着色剂(colorants)、推进剂(propellants)表面活性剂(surfactant)、佐剂(adjuvant)，及其他类似或适用本发明的载剂。
在本文中，所述的“佐剂”包括，但不限于，明矾沉淀、弗朗氏完全佐剂(Freund’s complete adjuvant)、弗朗式不完全佐剂(Freund’s incomplete adjuvant)、及单磷氧基-脂质A/海藻糖dicorynomycolate佐剂。
本发明所述的医药组成物可以多种形式存在。这些形式包括，但不限于液体、半固体及固体药剂形式，其中液体包括，但不限于分散液或悬浮液；半固体及固体包括，但不限于锭剂、丸剂、粉剂、脂质粒及栓剂。较佳的形式取决于预期的投药模式及治疗应用。更佳的，所述的医药组合物呈可口服或可输注溶液形式。
在另一态样中，本发明是关于一种促进局部肌肉增长、延缓局部肌肉萎缩或防止局部肌肉萎缩的核酸，其编码由SEQ ID NO：17中所示的氨基酸序列组成的组合物。
在另一态样中，本发明是关于一种如前所述的组合物的用途，其是用于制造促进局部肌肉增长的药物，其是将药物以有效剂量施予受体局部以使局部肌肉达到增长的效果。
在本发明的一个较佳实施例中，使用前述组合物以达成促进哺乳类局部肌肉增长，包括给予肌生成抑制素片段和SEC2的融合蛋白，从而获得抗肌生成抑制素的特异性免疫细胞。本发明的免疫细胞可以由肌生成抑制素片段的抗原决定位引入其他动物中，并经过纯化或藉由将免疫细胞或其抗原决定位引入哺乳动物体内，以使哺乳动物身体本身能 够增加肌肉生长。本发明的免疫细胞可为多株B淋巴球或T细胞殖株；较佳的是，前述免疫细胞是调节型T细胞。
在另一态样中，本发明是关于一种如前所述的组合物的用途，其是用于制造防止局部肌肉萎缩的药物，其是将药物以有效剂量施予受体局部以减缓或防止使局部肌肉萎缩的效果。在一实施例中，是使一侧腿神经受损后，施予前述组合物于被受损神经一侧的腿部肌肉，以使该侧腿部肌肉可维持肌肉大小避免因神经受损而造成肌肉萎缩的现象。在一实施例中，是使一侧腿神经阻断后，施予前述组合物于被阻断神经一侧的腿部肌肉，以使该侧腿部肌肉可维持肌肉大小避免因神经阻断而造成肌肉萎缩的现象。
前述组合物及用途适用于动物。较佳的是，适用于脊椎动物；更佳的是，人、猪、牛、羊、犬和家禽、水禽等动物的肌生成抑制素已获选殖，且其氨基酸序列具有高度的保守性，因此可假定前述动物的肌生成抑制素具有相同的功能；更佳的是，前述哺乳动物属于人、猪、牛、羊或犬。
本发明所述的组合物适用于各种肌肉萎缩症状，肌肉萎缩可由药物引起，其包括，但不限于由使用糖皮质激素(诸如皮质醇(cortisol)、地塞米松(dexamethasone)、倍皮质醇(betamethasone)、普赖松(prednisone)、甲基培尼皮质醇(methylprednisolone)或去氢皮质醇(prednisolone)治疗引起。肌肉萎缩亦可由神经创伤造成去神经化或由退化性、由神经坏死所引起、代谢性或发炎性神经病变，例如格巴二氏症候群(Guillian-Barre syndrome)、周边神经病变，或暴露于环境毒素引起。
此外，肌肉萎缩另可由肌肉疾病所引起，包括，但不限于肌肉强直症(myotonic dystrophy)、先天性肌病(congenital myopathies)、家族周期性麻痹(familial periodic paralysis，FPP)、代谢性肌肉病(metabolic myopathies；诸如由肝糖或脂质储存疾病所致)、皮肌炎(dermatomyositis)、多发性肌炎(polymyositis)、包涵体肌炎(inclusion body myositis，IBM)、骨化性肌炎(myositis ossificans)或横纹肌溶解症(rhabdomyolysis)所致。
肌肉萎缩亦可由以下疾病引起，包括，但不限于成人运动神经元疾病(motorneuron diseases，MND)、脊髓性肌肉萎缩症(spinal muscular atophy，SMA)、肌萎缩性脊髓侧索硬化症(amyotrophic lateral sclerosis)、少年型脊髓性肌萎缩症(juvenile spinal muscular atrophy；又称SMA-Ⅲ)、肌无力症(myasthenia Gravis，MG)、因中风或脊髓损伤的瘫痪、因创伤的骨骼固定化、长期卧床、自主不活动、非自主不活动、代谢压力或营养不足、癌症、爱滋病(AIDS)、禁食、甲状腺病症、糖尿病、肌中央轴空病(central core disease，CCD)、烧烫伤、慢性阻塞性肺病、肝病(诸如纤维化、硬化)、败血症、肾衰竭、充血性心脏衰竭、衰老、太空航行或在零重力环境中度过一段时间所引起的肌肉萎缩。
另一方面，在本文中，用语“抗原决定位(epitope)”是指能够引发免疫反应以产生蛋白质抗原中的抗原的片段，其可以藉由结构预测或藉由选择蛋白片段来观察免疫动物的免疫反应。
另一方面，在本文中，用语“有效剂量”是指在剂量上及对于所需要的时间而言对达成所要促进局部肌肉增长或减缓或防止局部肌肉萎缩有效的量；其如本发明所例示的，有效促进局部肌肉增长的剂量可通过促进局部肌肉增长试验(实施例1)而得知；有效减缓或防止局部肌肉萎缩的剂量可通过减缓或防止局部肌肉萎缩试验(实施例2、3)而得知。
由于所述抗原决定位是小型多肽片段，如果以该小型多肽片段在动物中直接进行免疫，则免疫反应可能未尽理想。较佳的是，建构含有纵列重复单元的线性排列重复抗原(linear array epitope，LAE)，以改善免疫反应。此外，可以使用细菌毒素来帮助抗原的传递，藉由使用消除毒素活性的毒素作为运输系统，从而以毒素的性质来加强整体免疫效果。在一态样中，本发明的肌生成抑制素C端的抗原决定位的线性排列重复抗原与金黄色葡萄球菌肠毒素C2(SEC2)融合。
一种宿主细胞包含上述核酸是可被生产的。实施例包括大肠杆菌、昆虫细胞、植物细胞、酵母菌细胞及哺乳动物细胞。该核酸分子可以用于表达本说明书所述的多肽或融合蛋白，操作上可将该核酸分子连接至合适的载体的多克隆位点(multiple cloning site，MCS)以产生所述的多肽或融合蛋白。
载体实施例包含质粒。该载体较佳的是包括启动子、增强子、多克隆位点等，核酸分子连接至合适的载体的多克隆位点后，该表达载体可以被导入宿主细胞以产生本说明书所述的多肽或融合蛋白。所述宿主细胞包括，但不限于大肠杆菌、百日咳杆菌、芽孢杆菌、非洲绿猴肾脏细胞、嗜血杆菌、真菌或酵母。
本发明的优点在于藉由包含金黄色葡萄球菌肠毒素C2序列的多肽以及肌生成抑制素C端的抗原决定位多肽的组合物，以达成促进局部肌肉增长的效果，是突破现有技术的困难且未有前例的。此外，所述的组合物施予前述组合物于神经损伤或神经阻断一侧的腿部肌肉，意外地发现该侧腿部肌肉可维持肌肉大小，将有助于临床上遇到神经损伤或神经阻断时可有效地避免病人局部肌肉萎缩的现象。
附图说明
以下附图仅旨在于对本发明做示意性说明和解释，并不限定本发明的范围。其中：
图1是本发明低剂量实验组与低剂量对照组对比的肌肉截面积比率折线图。其中，低 剂量实验组以标示●的折线表示、低剂量对照组以标示■的折线表示。
图2是本发明中剂量实验组与中剂量对照组对比的肌肉截面积比率折线图。其中，中剂量实验组以标示●的折线表示、中剂量对照组以标示■的折线表示。
图3是本发明高剂量实验组与高剂量对照组对比的肌肉截面积比率折线图。其中，高剂量实验组以标示●的折线表示、高剂量对照组以标示■的折线表示。
图4是本发明小鼠后腿电脑断层纵截面扫描图及横截面扫描图，各横截面扫描图是相应纵截面扫描图的剖面线标示处的横截面；图4中(A)是低剂量对照组的纵截面扫描图，上下虚线之间为肌肉体积计算的区域；图4中(B)是中剂量对照组的纵截面扫描图，上下虚线之间为肌肉体积计算的区域；图4中(C)是高剂量对照组的纵截面扫描图，上下虚线之间为肌肉体积计算的区域；图4中(D)是低剂量对照组的横截面扫描图；图4中(E)是中剂量对照组的横截面扫描图；图4中(F)是高剂量对照组的横截面扫描图；图4中(G)是低剂量实验组的纵截面扫描图，上下虚线之间为肌肉体积计算的区域；图4中(H)是中剂量实验组的纵截面扫描图，上下虚线之间为肌肉体积计算的区域；图4中(I)是高剂量实验组的纵截面扫描图，上下虚线之间为肌肉体积计算的区域；图4中(J)是低剂量实验组的横截面扫描图；图4中(K)是中剂量实验组的横截面扫描图；图4中(L)是高剂量实验组的横截面扫描图。
图5是本发明低剂量对照组及低剂量实验组的小鼠后腿体积比率的柱状图；A.U.表示Arbitrary Unit(任意单位)。
图6是本发明中剂量对照组及中剂量实验组的小鼠后腿体积比率的柱状图；A.U.表示Arbitrary Unit(任意单位)。
图7是本发明高剂量对照组及高剂量实验组的小鼠后腿体积比率的柱状图；A.U.表示Arbitrary Unit(任意单位)。
图8是本发明高剂量对照组小鼠后小腿肌丝纤维苏木素-伊红染色(hematoxylin-eosin，H-E)组织组织染色图。
图9是本发明高剂量实验组小鼠后小腿肌丝纤维H-E组织染色图。
图10是本发明小鼠后腿肌丝纤维针对肌生成抑制素的免疫组织染色图；其中图10中(A)至(C)为对照组；图10(D)至(F)分别为50纳克(ng)(低剂量)、500ng(中剂量)与5000ng(高剂量)实验组。
图11是本发明低、中、高剂量实验组小鼠在施予组合物后的体重变化折线图。其中，低剂量实验组以标示●的折线表示、中剂量实验组以标示■的折线表示、高剂量实验组以标示▲的折线表示。
图12是控制组与实验组小鼠局部肌肉历经坐骨神经破坏手术后，实验组施予本发明组合物1000ng后观察肌肉体积比率的柱状图；其中左腿历经坐骨神经破坏手术，右腿没有历经坐骨神经破坏手术。
图13是图12的控制组与实验组各别以左腿肌肉体积比除以右腿肌肉体积比获得的肌肉比率的柱状图。
图14是控制组、实验组A与实验组B小鼠局部肌肉历经坐骨神经截断手术后，实验组A、B分别施予本发明组合物1000ng、5000ng观察肌肉体积比率的柱状图；其中左腿历经坐骨神经破坏手术，右腿没有历经坐骨神经破坏手术。
图15是图14的控制组、实验组A、与实验组B各别以左腿肌肉体积比除以右腿肌肉体积比获得之肌肉比率的柱状图。
具体实施方式
以下配合附图及本发明的较佳实施例，进一步阐述本发明为达成预定发明目的所采取的技术手段。
制备例1、制备含SEC2片段与肌生成抑制素片段的组合物
本实施例所采用的融合蛋白是采用大肠杆菌(E.coli)的pET表达系统表达的载体；较佳的，是选用pET-28a。其中第一多肽“SEC2m”所示是金黄色葡萄球菌肠毒素C2具有点突变(point mutation)，核酸序列如SEQ ID NO：1所示、蛋白质序列如SEQ ID NO：8(突变点于位点7具有L、位点9具有E、位点13具有V及位点105具有Y)；第二多肽“Myo epitope”所示则为肌生成抑制素的抗原决定位，其是肌生成抑制素C端15个氨基酸(如序列SEQ ID NO：14所示；该序列具有高度的保守性，因此多个物种皆具有此序列)具有6个重复片段，其单一片段的核酸序列如SEQ ID NO：2所示。位于pET载体多克隆位点(multiple cloning site，MCS)内的基因序列从N端依序为“SEC2m”、连接子(linker)与“Myo epitope”，如序核酸列SEQ ID NO：3所示。
建立35L发酵培养程序以在50L发酵罐中培养含有SEQ ID NO：3pET载体的大肠杆菌BL21(DE3)菌株。4管5mL菌株在37℃下用LB/氨芐青霉素(Ampicillin)培养基培养过夜，各菌株分别接种到0.2升的LB/氨芐青霉素培养基中，共1升，于37℃持续摇动培养至OD 600为0.3。其后加入至35μL培养基中进行培养，并以每两小时取样测定OD 600为以监测生长曲线的变化，并根据生长曲线进一步选择合适的时间点，加入最终浓度为0.1mM的异丙基-β-D-硫代半乳糖苷(Isopropyl β-D-1-thiogalactopyranoside，IPTG)诱导大肠杆菌以高度表达融合蛋白，于37℃持续摇动培养3小时，离心回收菌株。藉由SDS-PAGE电泳和 西方墨点法测定融合蛋白的表达，以确定最佳35L发酵条件。大致上，是将大肠杆菌BL21(DE3)表达的融合多肽在细胞裂解后，进行该融合多肽的萃取与分离，最终获得一组合物(如SEQ ID NO：17所示)；由于所述表达蛋白的萃取与分离属于习知技术，在此不再赘述。
实施例1、促进局部肌肉增长试验
本实施例的动物实验所采用的小鼠为“C57BL/6”品系8周龄(12个月龄)的母鼠，对照组和实验组共9只小鼠，实验时间为6个月。自小鼠成长至12个月龄时，开始采用高脂质饲料，并使用含高果糖浆的饮水饲育。饲料及饮水，每两天更换一次，以避免变质。饲料保存于-20℃，高果糖浆则保存于4℃；每两周量测一次体重。实验动物经6个月的喂食后，开始每周以肌肉内注射该组合物一次。取自制备例1的组合物(以生理食盐水将组合物稀释成不同浓度)分别注射于小鼠左后小腿的肌肉成为不同剂量的实验组，右后小腿则为注射生理食盐水(saline)；其实验分组如下表1所示。
表1、动物实验分组与组合物的施予方式
Figure PCTCN2018079716-appb-000002
实验数据的收集，是每周以游标量尺测量一次、量测肌肉的长径(a)及短径(b)和体重，量测位置为注射药物的位置。将所得数值以椭圆面积计算公式︰「a×b×3.14」，算得肌肉概约截面积，计算范围如图4(A)、4(B)、4(C)、4(G)、4(H)、4(I)上下虚线之间为肌肉体积计算的区域，以进行各实验组间的比较。
(1)比较实验组(左后小腿)及对照组(右后小腿)注射组合物后的肌肉截面积
请参阅图1至图3所示，于第19周时将各实验组与其对照组相比，其中注射高剂量组合物的小腿横截面积相较于高剂量对照组增加了8.19％，接受中剂量组合物的小腿横截面积相较于中剂量对照组增加5.5％，而接受低剂量组合物的横截面积相较于低剂量对照组也增加5.67％。
(2)比较实验组(左后小腿)及对照组(右后小腿)注射组合物后的肌肉体积
请参阅图4，小鼠后腿电脑断层纵截面扫描图及横截面扫描图，各横截面扫描图是相应纵截面扫描图的剖面线标示处的横截面。其中图4中(A)、图4中(B)、图4中(C)、图4中(G)、图4中(H)、图4中(I)虚线之间为计算肌肉体积的范围，将各截面积以积分获得肌肉 体积。投药至19周，结束实验，将小鼠牺牲并取其两只小腿，以10％福马林固定，进行电脑断层扫描。
请参阅图5至图7所示，肌肉体积中，低剂量实验组相较于低剂量对照组增加4.6％，中剂量实验组相较于中剂量对照组增加8.5％，高剂量实验组相较于高剂量对照组增加19.2％。由于本试验是以同一小鼠的左后小腿的肌肉为实验组，右后小腿则为对照组，因此结果显示，将本发明的组合物注射于左侧小腿只会造成该侧小腿肌肉增长，另一侧(右侧)小腿的肌肉并无增长，故本发明所述的组合物经由局部施予后仅于该局部产生肌肉增长的效果，并无产生全身肌肉变大的反应。
(3)测量实验组(左后小腿)及对照组(右后小腿)的注射组合物后肌丝纤维粗细情形
于第19周牺牲后并经电脑断层扫描后，将实验小鼠的小腿骨取出，肌肉部分则进行石蜡包埋及切片，并以H-E组织染色比较肌丝纤维粗细。请参阅图8及图9所示，图8为高剂量对照组小鼠后小腿肌丝纤维、图9为高剂量实验组小鼠后小腿肌丝纤维，可知高剂量实验组小鼠肌丝纤维较对照组显著增大。
(4)测量实验组(左后小腿)及对照组(右后小腿)的注射组合物后肌生成抑制素含量分布
于第19周牺牲后并经电脑断层扫描后，将实验小鼠的小腿骨取出，肌肉部分则进行石蜡包埋及切片，并以肌生成抑制素抗体进行免疫组织化学染色(immunohistochemistry，IHC)观察肌生成抑制素含量分布。请参阅图10所示，图10中(A)、图10中(B)、图10中(C)不同剂量的对照组(左后小腿)肌生成抑制素可明显被染色；反观图10中(D)、图10中(E)、图10中(F)，无论是低剂量、中剂量、或高剂量实验组(右后小腿)肌生成抑制素的表达明显被抑制。也就是说，无论低剂量、中剂量或是高剂量，同一只老鼠的右小腿(即对照组)的肌生成抑制素浓度高，右小腿肌肉并无明显变大；但同一只小鼠的左小腿(即实验组)的肌生成抑制素被本发明的组合物所抑制，故肌生成抑制素浓度低，左小腿肌肉变大，即本发明所述的组合物具有能够使局部肌肉变大的效果(不受全身血液循环影响)。
(5)比较注射低剂量、中剂量、或高剂量的组合物后体重变化
于第19周时量测体重，请参阅图11所示，无论是低剂量、中剂量、或高剂量实验组在施予组合物之后，各组间体重并没影显著差异。因此，本试验藉由局部施予组合物只会在施予部位的局部增大肌肉，并不会造成全身性肌肉增大。
实施例2、减缓或防止局部肌肉因神经损伤导致萎缩的试验
准备10只ICR小鼠(八周龄，购自于乐斯科生技股份有限公司)以正常饲料喂食1周至2周后，分成2组(每组各5只)，于实验第1天进行坐骨神经破坏手术使各组小鼠的左腿坐骨 神经造成神经损伤(各组小鼠的右腿皆不进行坐骨神经破坏手术)，并于第28天牺牲，其中各组分别为：控制组(左腿进行坐骨神经破坏手术，但不给药)，实验组(左腿进行坐骨神经破坏手术，且每只小鼠于第1、3、7、14天以肌肉内注射施予本发明制备例1所述的组合物1000ng，最后观察小鼠肌肉萎缩程度来评估本发明的组合物对于减缓或防止肌肉萎缩的效果。
坐骨神经破坏手术是对小鼠施予麻醉剂(腹腔麻醉)后，剔除小鼠手术侧膝盖至臀部的体毛，固定小鼠腿部并以酒精棉消毒手术部位，找到大腿股骨位置，于靠近臀部处开一道平行股骨的切口，剥离肌肉层后可看见平行于股骨的坐骨神经，将坐骨神经挑起，以特殊工具损伤小鼠的坐骨神经后，将其坐骨神经放回原本位置，缝合皮肤后每日观察小鼠的伤口愈合情况、步态变化及整体状态，其目的为模拟神经损伤的状态。
结果显示，于第28天测量其中一只控制组小鼠的左大腿肌肉体积约为1373立方毫米(mm 3)、右大腿肌肉体积约为1595mm 3，由于右腿未进行坐骨神经破坏手术，故将右大腿肌肉体积做为基准比率1、左大腿肌肉体积比率则为左大腿肌肉体积除以右大腿肌肉体积(如图12控制组所示)，显示控制组小鼠左腿肌肉相较于右腿肌肉呈现萎缩现象；另测量其中一只实验组小鼠左大腿肌肉体积约为1888mm 3、右大腿肌肉体积约为1705mm 3，将右大腿肌肉体积做为基准比率1、左大腿肌肉体积比率则为左大腿肌肉体积除以右大腿肌肉体积(如图12实验组所示)，尽管小鼠左腿进行坐骨神经破坏手术但因施予本发明所述的组合物，故显示左腿肌肉并无萎缩现象。
如图13所示，控制组的左大腿/右大腿肌肉体积比率约为0.86(小于1即表示呈现萎缩状态)；实验组的左大腿/右大腿肌肉体积比率约为1.11，故实验组相较于控制组因施予本发明所述的组合物而呈现维持肌肉量体积的现象。由此可见，于神经损伤的情况下，经由施予本发明所述的组合物可减少或舒缓局部肌肉萎缩、甚至维持原本肌肉体积的现象。
实施例3、减缓或防止局部肌肉因神经截断导致萎缩的试验
准备10只ICR小鼠(八周龄，购自于乐斯科生技股份有限公司)以正常饲料喂食1周至2周后，分成2组(每组各5只)，于实验第1天进行坐骨神经截断手术，并于第28天牺牲，其中各组分别为：控制组(左腿进行坐骨神经截断手术，但不给药)，实验组A(左腿进行坐骨神经截断手术，且每只小鼠于第1、3、7、14天以肌肉内注射施予本发明制备例1所述的组合物1000ng，实验组B(左腿进行坐骨神经截断手术，且每只小鼠于第1、3、7、14天以肌肉内注射施予本发明制备例1所述的组合物5000ng，最后观察小鼠肌肉萎缩程度来评估本发明的组合物对于抗肌肉萎缩的效果(以上各组小鼠的右腿皆不进行坐骨神经截 断手术)。坐骨神经截断手术方式大略同实施例2，差异在于将坐骨神经挑起后直接截断。
结果显示，于第28天测量控制组小鼠左大腿肌肉体积约为1375mm 3、右大腿肌肉体积约为1560mm 3，由于右腿未进行坐骨神经破坏手术，故将右大腿肌肉体积做为基准比率1、左大腿肌肉体积比率则为左大腿肌肉体积除以右大腿肌肉体积(如图14控制组所示)，显示控制组小鼠左腿肌肉相较于右腿肌肉呈现萎缩，右腿肌肉亦无萎缩现象；实验组A小鼠左大腿肌肉体积约为1289mm 3、右大腿肌肉体积约为1394mm 3，由于右腿未进行坐骨神经破坏手术，故将右大腿肌肉体积做为基准比率1、左大腿肌肉体积比率则为左大腿肌肉体积除以右大腿肌肉体积(如图14实验组A所示)，在坐骨神经截断手术后，左腿肌肉萎缩现象显著减缓；实验组B小鼠左大腿肌肉体积约为1958mm 3、右大腿肌肉体积约为1869mm 3，由于右腿未进行坐骨神经破坏手术，故将右大腿肌肉体积做为基准比率1、左大腿肌肉体积比率则为左大腿肌肉体积除以右大腿肌肉体积(如图14实验组B所示)，尽管小鼠左腿历经坐骨神经破坏手术，但因左腿肌肉施予本发明所述的组合物5000ng，并无产生萎缩现象。
如图15所示，控制组的左大腿/右大腿肌肉体积比率约为0.87呈现萎缩状态(小于1即表示呈现萎缩状态)；实验组A的左大腿/右大腿肌肉体积比率约为0.92实验组A相较于控制组呈现减缓肌肉萎缩；实验组B的左大腿/右大腿肌肉体积比率约为1.04，实验组B相较于控制组甚至可以维持肌肉体积。由此可见，于坐骨神经截断的情况下，经由施予本发明所述的组合物，除了如实施例2可减少或舒缓局部肌肉萎缩、本实施例3甚至可维持原本肌肉体积的现象。
以上所述仅是本发明的较佳实施例而已，并非对本发明做任何形式上的限制，虽然本发明已以较佳实施例揭露如上，然而并非用以限定本发明，任何熟悉本领域的技术人员，在不脱离本发明技术方案的范围内，当可利用上述揭示的技术内容作出些许更动或修饰为等同变化的等效实施例，但凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰，均仍属于本发明技术方案的范围内。

Claims (12)

  1. 一种促进局部肌肉增长、延缓局部肌肉萎缩或防止局部肌肉萎缩的组合物，其包含：
    第一多肽，其是如SEQ ID NO：8所示具有相似度至少90％以上的序列；以及
    第二多肽，其包含如SEQ ID NO：14所示序列1至10个重复单元。
  2. 根据权利要求1所述的组合物，其中所述第二多肽为纵列重复单元的线性排列重复抗原，其中所述第二多肽为如SEQ ID NO：14所示序列1至10个重复单元。
  3. 根据权利要求1所述的组合物，其中所述第一多肽在SEQ ID NO：8对应于位点7具有T或L、位点9具有G或E、位点13具有Y或V及位点105具有H或Y。
  4. 根据权利要求1所述的组合物，其中所述第一多肽选自由如SEQ ID NO：4、5、6、7、8、9、10、11及12所组成的群组。
  5. 根据权利要求1至4中任一项所述的组合物，其还包含：
    连接子，其是介于所述第一多肽以及第二多肽之间；其中所述组合物的序列如SEQ ID NO：17所示。
  6. 一种促进局部肌肉增长、延缓局部肌肉萎缩或防止局部肌肉萎缩的核酸，其编码由SEQ ID NO：17中所示的氨基酸序列组成的组合物。
  7. 一种促进局部肌肉增长、延缓局部肌肉萎缩或防止局部肌肉萎缩的医药组成物，其包含如权利要求1至5中任一项所述的组合物以及药学上可接受的载剂。
  8. 一种如权利要求1至5中任一项所述的组合物用于制造促进局部肌肉增长的药物的用途。
  9. 一种如权利要求1至5任一项所述的组合物用于制造减缓或防止局部肌肉萎缩的药物的用途。
  10. 根据权利要求9所述的用途，其中，所述组合物是用于制造适用于药物引起的肌肉萎缩、神经创伤引起的肌肉萎缩、神经坏死所引起的肌肉萎缩、自体免疫反应引起的肌肉萎缩、环境毒素引起的肌肉萎缩或外力引起的肌肉萎缩。
  11. 一种促进局部肌肉增长、延缓局部肌肉萎缩或防止局部肌肉萎缩的组合物，其包含：
    金黄色葡萄球菌肠毒素的多肽；以及，
    肌生成抑制多肽。
  12. 根据权利要求11所述的组合物，其中所述肌生成抑制多肽选自于由生长分化因子8、滤泡抑素及第二型活化素受体所组成的群组。
PCT/CN2018/079716 2018-03-21 2018-03-21 促进局部肌肉增长、减缓或防止局部肌肉萎缩的组合物及其用途 WO2019178759A1 (zh)

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