CN111848778B - Teriparatide analogues - Google Patents

Teriparatide analogues Download PDF

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CN111848778B
CN111848778B CN202010355587.3A CN202010355587A CN111848778B CN 111848778 B CN111848778 B CN 111848778B CN 202010355587 A CN202010355587 A CN 202010355587A CN 111848778 B CN111848778 B CN 111848778B
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teriparatide
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glu
pth
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CN111848778A (en
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冯军
张喜全
阮思达
陆伟根
东圆珍
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Shanghai Duomirui Biological Technology Co ltd
Shanghai Institute of Pharmaceutical Industry
Chia Tai Tianqing Pharmaceutical Group Co Ltd
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Shanghai Institute of Pharmaceutical Industry
Chia Tai Tianqing Pharmaceutical Group Co Ltd
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    • 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
    • C07K14/575—Hormones
    • C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
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    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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Abstract

The invention belongs to the field of medicines, and particularly relates to a teriparatide analogue. The invention obtains a new class of teriparatide analogues through modification and/or amino acid replacement at 13, 26 and/or 27 of the N-terminal of teriparatide, has enhanced cell membrane penetration capability, and can be used for oral administration.

Description

Teriparatide analogues
Technical Field
The invention belongs to the field of medicines, and particularly relates to a teriparatide analogue.
Background
At present, the incidence rate of osteoporosis in the elderly is second only to diabetes and senile dementia, and the third place of senile diseases is jumped, so that the development of medicaments for treating osteoporosis is also a research hot spot. The drugs for treating osteoporosis can be mainly classified into the following three types: 1. drugs that promote bone mineralization, such as vitamin D and calcium preparations, etc.; 2. drugs that inhibit bone resorption, such as bisphosphonates, diumab, estrogens, calcitonin, and the like; 3. drugs for promoting bone formation, mainly parathyroid hormone (PTH) and its analogues, calcium receptor antagonists, anti-sclerostin monoclonal antibodies, etc. Parathyroid hormone has been the drug of choice for the treatment of osteoporosis in postmenopausal women.
Parathyroid hormone is a polypeptide composed of 84 amino acids synthesized, stored and secreted by parathyroid epithelial cells, and has the main physiological functions of promoting bone formation, mobilizing blood calcium into bones and promoting the reabsorption of calcium by renal tubules and gastrointestinal tracts, thereby achieving the purpose of treating osteoporosis. Parathyroid hormone PTH (1-84) is rapidly metabolized into an N-terminal PTH (1-34) fragment when entering the human body, and the fragment has complete physiological activity and can be combined with a tissue corresponding specific receptor so as to exert biological effects. Since PTH (1-34) has the same physiological activity as parathyroid hormone, it has also been developed as a drug for the treatment of osteoporosis.
Teriparatide (trademark Forteo) is a recombinant PTH (1-34) developed by Eli Lilly company in the united states, FDA approved for its marketing in 2002, and marketed in china in 2013 (futaiao), and has the amino acid sequence H-Ser-Val-Ser-Glu-Ile-gin-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Leu-gin-Asp-His-Asn-Phe-OH, which is approved for the treatment of post-menopausal osteoporosis, early-or hypogonadal male osteoporosis patients, and of persistent, systemic glucocorticoid-related osteoporosis patients.
The currently marketed forms of teriparatide are injections, which are administered intermittently at low doses and require one subcutaneous injection per day, thus causing great pain to the patient. The clinical compliance of patients can be greatly improved if the oral administration of teriparatide can be realized.
However, for oral administration of polypeptides, the bioavailability in vivo is low, mainly because: (1) The complex enzyme environment of the gastrointestinal tract causes the polypeptide to be easily degraded in the gastrointestinal tract; (2) has a large molecular weight and is not easy to pass through the mucous layer; (3) Lacking lipid solubility, poor permeability makes it difficult to penetrate small intestine epithelial cells into the systemic circulation. The polypeptide is modified by lipid acylation, so that the lipophilicity of the polypeptide can be increased, and the polypeptide can enter the systemic circulation through gastrointestinal cells. At the same time, there are receptors on the cell surface of the gastrointestinal tract, and chemical modification of the polypeptide with ligands that bind specifically to the receptor is performed to facilitate penetration of the polypeptide into the cell, such as cholic acid (sodium-dependent cholic acid transporter ASBT) and biotin (sodium-dependent biotin receptor). In US9993430B2, the lipophilicity of the polypeptide is increased by performing the modification of the lipid acylation on GLP-1, and the oral bioavailability of the GLP-1 polypeptide is improved by the combined action of the polypeptide and a penetration enhancer SNAC; cho et al/Lancet 2 (2012) reports an oral insulin IN-105, which is an oral administration mode of insulin by modifying a PEG-fatty acid amphipathic side chain on a beta chain Lys29 to increase the lipophilicity of insulin; in US20110014247, 5-CNAC is added as a permeation enhancer to form a compound with polypeptide in a non-covalent bond, so that the lipophilicity of the polypeptide is increased to improve the permeation effect of the polypeptide in the gastrointestinal tract; in US8962015, biotin is used as a target head to assist in penetrating the drug-carrying liposome into a single cell layer, so that the bioavailability of the oral polypeptide is improved. The enhancement of cellular penetration of polypeptides by increasing lipid solubility has been widely used in the field of oral administration of polypeptides, however, chemical modifications may result in conformational changes in the polypeptide, resulting in loss of polypeptide activity. Thus, in the modification of polypeptides, it is also critical to maintain the activity of the polypeptide.
Disclosure of Invention
The present patent application relates to teriparatide analogues which are modified and/or amino acid replaced at positions 13, 26 and/or 27 of the N-terminal teriparatide.
In one aspect, the present application provides teriparatide analogs that are modified at one, two or three lysines at positions 13, 26 and/or 27 of the N-terminus of teriparatide.
In some embodiments, the modification is an acylation modification.
In some embodiments, the modification site is located at one, two or three of the three lysines at positions 13, 26 and 27 of the N-terminus of teriparatide.
In some embodiments, the modification site is located at one, two or three of the three free epsilon-amino groups distributed over three lysines at positions 13, 26 and 27 of the N-terminus of teriparatide.
In some embodiments, the fatty chain structure for the acylated modified teriparatide is of the general formula: HOOC- (AEEA) m -(Xaa) n -R 1 Wherein Xaa is one or more of D-alanine (D-Ala), beta-alanine (beta-Ala), 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), 2-aminobutyric acid (Abu), arginine (Arg), aspartic acid (Asp), asparagine (Asn), cysteine (Cys), D-glutamic acid (D-Glu), gamma-glutamic acid (gamma-Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), proline (Pro), phenylalanine (Phe), serine (Ser), tyrosine (Tyr), threonine (Thr), tryptophan (Trp), valine (Val), methionine (Met) or default. m is a number from 0,1,2,3,4, 5. n is one number of 0,1,2, 3. R is R 1 Is aliphatic straight chain, branched chain C 6 -C 20 Acyl groups of (2), deoxycholic acid, biotin or deletion.
In some embodiments, m is a number from 0,1,2,3,4,5, preferably from 0 to 3, more preferably 2; n is a number 0,1,2,3, preferably 0 or 1, more preferably 1. When n is 1, (Xaa) n Is one of D-alanine (D-Ala), beta-alanine (beta-Ala), 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), 2-aminobutyric acid (Abu), arginine (Arg), aspartic acid (Asp), asparagine (Asn), cysteine (Cys), D-glutamic acid (D-Glu), gamma-glutamic acid (gamma-Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), proline (Pro), phenylalanine (Phe), serine (Ser), tyrosine (Tyr), threonine (Thr), tryptophan (Trp), valine (Val), methionine (Met); preferably 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), D-alanine (D-Ala), beta-alanine (beta-Ala), aspartic acid (Asp), cysteine (Cys), gamma-glutamic acid (gamma-Glu), glycine (Gly), proline (Pro), phenylalanine (Phe); more preferably, it is gamma-glutamic acid (gamma-Glu), proline (Pro). R is R 1 Selected from heptanoyl, methylheptanoyl, octanoyl, and methyloctanoyl Acyl, nonenoyl, methylnonenoyl, decanoyl, methyldecanoyl, lauroyl, myristoyl, palmitoyl, octadecanoyl, 17-carboxyheptadecanoyl, 15-carboxypentadecanoyl, 13-carboxytridecanoyl, 11-carboxyundecanoyl, deoxycholic acid and biotin; preferably lauroyl, myristoyl, palmitoyl, stearoyl, 17-carboxyheptadecanoyl, deoxycholic acid, biotin; more preferably an octadecanoyl group, a 17-carboxyheptadecanoyl group.
The present application provides a class of teriparatide lipidated derivatives having the following structure (polypeptide sequence in order from N-terminal to C-terminal):
when two or three lysine modifications exist at positions 13, 26 or 27, T in the modification group is the same structure, wherein the chemical structural formula T may be represented as:
wherein m is a number from 0,1,2,3,4,5, n is a number from 0,1,2,3, p is an integer from 6 to 20, R 2 is-H or-COOH; xaa is one or more of D-alanine (D-Ala), beta-alanine (beta-Ala), 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), 2-aminobutyric acid (Abu), arginine (Arg), aspartic acid (Asp), asparagine (Asn), cysteine (Cys), D-glutamic acid (D-Glu), gamma-glutamic acid (gamma-Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), proline (Pro), phenylalanine (Phe), serine (Ser), tyrosine (Tyr), threonine (Thr), tryptophan (Trp), valine (Val), methionine (Met) Several or default.
More specifically, the side chain structure may be represented by the following structural formula:
wherein m is selected from integers from 0 to 5, preferably 0 to 2, more preferably 2; xaa 1 Selected from D-alanine (D-Ala), beta-alanine (beta-Ala), 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), 2-aminobutyric acid (Abu), arginine (Arg), aspartic acid (Asp), asparagine (Asn), cysteine (Cys), D-glutamic acid (D-Glu), gamma-glutamic acid (gamma-Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), proline (Pro), phenylalanine (Phe), serine (Ser), tyrosine (Tyr), threonine (Thr), tryptophan (Trp), valine (Val), methionine (Met); preferably 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), D-alanine (D-Ala), beta-alanine (beta-Ala), aspartic acid (Asp), cysteine (Cys), gamma-glutamic acid (gamma-Glu), glycine (Gly), proline (Pro), phenylalanine (Phe); more preferably, it is gamma-glutamic acid (gamma-Glu), proline (Pro).
In another aspect, the present application provides a class of teriparatide analogues with substitutions of lysine at position 13, position 26 or position 27 of the N-terminus of teriparatide, said analogues having the general formula:
H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-X 1 -His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-X 2 -X 3 -Leu-Gln-Asp-Val-His-Asn-Phe-OH
Wherein X is 1 ，X 2 ，X 3 Not simultaneously Lys, and X 1 ，X 2 ，X 3 At least one of which is Lys; in some embodiments, X 1 ，X 2 ，X 3 Any one or any two of which is D-alanine (D-Ala), beta-alanine (beta-Ala), 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), 2-aminobutyric acid (Abu), arginine (Arg), aspartic acid (Asp), asparagine (Asn), cysteine (Cys), D-glutamic acid (D-Glu), gamma-glutamic acid (gamma-Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), proline (Pro), phenylalanine (Phe), serine (Ser), tyrosine (Tyr), threonine (Thr), tryptophan (Trp), valine (Val), methionine (Met), preferably D-alanine (D-Ala), beta-alanine (beta-Ala), 2-aminoisobutyric acid (Aib), arginine (Arg), cysteine (Cys), glycine (Gly), more preferably arginine (Arg).
In another aspect, the present application provides acylated modifications of a class of teriparatide analogs having substitutions of lysines at positions 13, 26 and/or 27 of the N-terminus of teriparatide, said analogs having the general formula:
H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-X 1 -His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-X 2 -X 3 -Leu-Gln-Asp-Val-His-Asn-Phe-OH
wherein X is 1 ，X 2 ，X 3 Not simultaneously Lys, and X 1 ，X 2 ，X 3 At least one of which is Lys; in some embodiments, X 1 ，X 2 ，X 3 Any one or any two of the amino acids are D-alanine (D-Ala), beta-alanine (beta-Ala), 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), 2-aminobutyric acid (Abu), arginine (Arg), aspartic acid (Asp), asparagine (Asn), cysteine (Cys), D-glutamic acid (D-Glu), gamma-glutamic acid (gamma-Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), proline (Pro), phenylalanine (Phe), serine (Ser), tyrosine (Tyr), threonine (Thr), tryptophan (Trp), valine (Val), methionine (Met); preferably D-alanine (D-Ala), beta-alanine (beta-Ala), 2-aminoisobutyric acid (Aib), arginine (Arg), cysteine (Cys), glycine (Gly), more preferably arginine (Arg);
wherein the acylation modification site is located at X 1 ，X 2 Or X 3 Lys of the above, further, the acylation site is located at X 1 ，X 2 Or X 3 Lys on the free epsilon-amino group.
In some embodiments, the fatty chain structure for the acylation modification is of the formula: HOOC- (AEEA) m -(Xaa) n -R 1 Wherein Xaa is one or more of D-alanine (D-Ala), beta-alanine (beta-Ala), 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), 2-aminobutyric acid (Abu), arginine (Arg), aspartic acid (Asp), asparagine (Asn), cysteine (Cys), D-glutamic acid (D-Glu), gamma-glutamic acid (gamma-Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), proline (Pro), phenylalanine (Phe), serine (Ser), tyrosine (Tyr), threonine (Thr), tryptophan (Trp), valine (Val), methionine (Met) or default. m is a number from 0,1,2,3,4, 5. n is one number of 0,1,2, 3. R is R 1 Is aliphatic straight chain, branched chain C 6 -C 20 Acyl groups of (2), deoxycholic acid, biotin or deletion.
In some embodiments, m is a number from 0,1,2,3,4,5, preferably from 0 to 3, more preferably 2; n is a number 0,1,2,3, preferably 0 or 1, more preferably 1. When n is 1, (Xaa) n Is one of D-alanine (D-Ala), beta-alanine (beta-Ala), 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), 2-aminobutyric acid (Abu), arginine (Arg), aspartic acid (Asp), asparagine (Asn), cysteine (Cys), D-glutamic acid (D-Glu), gamma-glutamic acid (gamma-Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), proline (Pro), phenylalanine (Phe), serine (Ser), tyrosine (Tyr), threonine (Thr), tryptophan (Trp), valine (Val), methionine (Met); preferably 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), D-alanine (D-Ala), beta-alanine (beta-Ala), aspartic acid (Asp), cysteine (Cys), gamma-glutamic acid (gamma-Glu), glycine (Gly), proline (Pro), phenylalanine (Phe); more, thePreferably, it is gamma-glutamic acid (gamma-Glu), proline (Pro). R is R 1 Selected from heptanoyl, methylheptanoyl, octanoyl, methyloctanoyl, nonanoyl, methylnonanoyl, decanoyl, methyldecanoyl, lauroyl, myristoyl, palmitoyl, octadecanoyl, 17-carboxyheptadecanoyl, 15-carboxypentadecanoyl, 13-carboxytridecanoyl, 11-carboxyundecanoyl, deoxycholic acid and biotin. Preferably lauroyl, myristoyl, palmitoyl, stearoyl, 17-carboxyheptadecanoyl, deoxycholic acid, biotin. More preferably an octadecanoyl group, a 17-carboxyheptadecanoyl group.
In another aspect, the present application provides an acylated modification of a teriparatide analog, the modification being of the general formula (the polypeptide sequence being in order from the N-terminus to the C-terminus):
X 2 26 ,X 3 27 ,Lys 13 -PTH(1-34)
or (b)
X 1 13 ,X 3 27 ,Lys 26 -PTH(1-34)
Or (b)
X 1 13 ,X 2 26 ,Lys 27 -PTH(1-34)
Wherein X is 1 Selected from D-Ala, beta-Ala, GABA, aib, abu, arg, asp, asn, cys, D-Glu, gamma-Glu, gln, gly, his, ile, leu, pro, phe, ser, tyr, thr, trp, val, met, preferably beta-Ala, GABA, aib, abu, arg, cys, more preferably Arg; x is X 2 Selected from D-Ala, beta-Ala, GABA, aib, abu, arg, asp, asn, cys, D-Glu, gamma-Glu, gln, gly, his, ile, leu, pro, phe, ser, tyr, thr, trp, val, met, preferably beta-Ala, GABA, aib, abu, arg, cys, more preferably Arg; x is X 3 Selected from D-Ala, beta-Ala, GABA, aib, abu, arg, asp, asn, cys, D-Glu, gamma-Glu, gln, gly, his, ile, leu, pro, phe, ser, tyr, thr, trp, val, met, preferably beta-Ala, GABA, aib, abu, arg, cys, more preferably Arg;
wherein, the chemical structural general formula T can be expressed as:
wherein m is a number from 0,1,2,3,4,5, n is a number from 0,1,2,3, p is an integer from 6 to 20, R 2 is-H or-COOH; xaa is one or more of D-alanine (D-Ala), beta-alanine (beta-Ala), 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), 2-aminobutyric acid (Abu), arginine (Arg), aspartic acid (Asp), asparagine (Asn), cysteine (Cys), D-glutamic acid (D-Glu), gamma-glutamic acid (gamma-Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), proline (Pro), phenylalanine (Phe), serine (Ser), tyrosine (Tyr), threonine (Thr), tryptophan (Trp), valine (Val), methionine (Met) or default.
More specifically, the side chain structure T may be represented by the following structural formula:
/>
wherein m is selected from 0 to 5, preferably 0 to 2, more preferably 2; xaa 1 Selected from D-alanine (D-Ala), beta-alanine (beta-Ala), 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), 2-aminobutyric acid (Abu), argininAcid (Arg), aspartic acid (Asp), asparagine (Asn), cysteine (Cys), D-glutamic acid (D-Glu), gamma-glutamic acid (gamma-Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), proline (Pro), phenylalanine (Phe), serine (Ser), tyrosine (Tyr), threonine (Thr), tryptophan (Trp), valine (Val), methionine (Met); preferably 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib) D-alanine (D-Ala), beta-alanine (beta-Ala), aspartic acid (Asp), cysteine (Cys) gamma-glutamic acid (gamma-Glu), glycine (Gly), proline (Pro), phenylalanine (Phe); more preferably, it is gamma-glutamic acid (gamma-Glu), proline (Pro).
In some embodiments, the present application provides acylated modifications of a class of teriparatide analogs selected from the group consisting of compounds having the following structure (polypeptide sequences in order from N-terminus to C-terminus):
1.N-ε 27 -PTH(1-34)-AEEA-AEEA-Pro-C 17 -COOH：
2.N-ε 13 -PTH(1-34)-AEEA-AEEA-α-Glu-C 16 ：
3.N-ε 26 -PTH-γ-Glu-C 12 ：
4.N-ε 27 -PTH (1-34) -AEEa-deoxycholic acid:
5.N-ε 13 -(Arg 26 ，Gly 27 )PTH(1-34)-γ-Glu-C 16 ：
6.N-ε 27 -(Arg 13 ，Arg 26 )PTH(1-34)-AEEA-Gly-AEEA-Gly-γ-Glu-C 17 -COOH：
7.N-ε 26 -(Cys 13 ，Ala 27 )PTH(1-34)-Phe-AEEA-AEEA-AEEA-C 12 ：
8.N-ε 26 -(Arg 13 ，Arg 27 )PTH(1-34)-AEEA-AEEA-C 11 -biotin:
9.N-ε 27 -(Arg 13 ，Arg 26 )PTH(1-34)-AEEA-AEEA-C 17 -COOH：
10.N-ε 27 -(Arg 13 ，Arg 26 )PTH(1-34)-AEEA-Glu-C 17 -COOH：
11.N-ε 27 -(Arg 13 ，Arg 26 )PTH(1-34)-AEEA-AEEA-Ala-C 17 -COOH：
12.N-ε 27 -(Arg 13 ，Arg 26 )PTH(1-34)-AEEA-AEEA-C 16 ：
in the application, the in-vitro biological activity of the teriparatide analogue is not greatly changed compared with that of the teriparatide, the modification of a side chain does not influence the biological activity of the polypeptide, the polarity is obviously reduced, and in reverse chromatography, the elution proportion of the analogue acetonitrile phase is increased from 40% to 55% -60% of PTH, which shows that the polarity is reduced after the acylation of the lipid, thereby being beneficial to realizing the transmembrane transport of the polypeptide.
The cell permeability of the teriparatide analogue in the application is obviously improved, and in an in vitro Caco-2 cell transmembrane transport experiment, the PTH derivative has stronger capacity of penetrating through a monolayer cell layer than that of a PTH reference substance, and the apparent permeability coefficient P of the PTH derivative app The value is 1.89-4.03 times that of PTH standard, which shows that the modification of the lipid acylation can effectively enhance the cell membrane penetration capacity.
In the application, only one lysine is remained in the sequence of the teriparatide by replacing the amino acid of the teriparatide, so that only one modification site for lipid acylation is remained, the purification process is simplified, and the purity of the product is improved.
The present application provides a pharmaceutically acceptable salt of the aforementioned teriparatide analogue or use thereof.
The polypeptide related to the application of the invention can be used for treating osteoporosis.
The teriparatide analogue can be used as an effective pharmaceutical ingredient of an oral preparation; can also be used as the effective medicine component of injection medicine, such as intravenous injection, subcutaneous injection, intramuscular injection, etc.; can also be used as effective pharmaceutical ingredient for topical application
The teriparatide analogues of the present application may be formulated into pharmaceutically effective dosage units by existing pharmaceutical techniques, and the effective dosage units may be in the form of oral, pharmaceutical tablets, capsules or liquids.
The teriparatide analogue component can be prepared into an aqueous preparation, wherein the moisture is not less than 50%.
In some embodiments, the oral formulation may be in the form of tablets, troches, pills, capsules (e.g., hard, soft, enteric, micro-capsules), elixirs, granules, syrups, granules, emulsions, suspensions, solutions, dispersions, and sustained release formulations for oral or non-oral administration, wherein tablets containing various excipients (e.g., calcium carbonate, calcium phosphate, etc.) may also be formulated as disintegrating formulations.
In some embodiments, the pharmaceutical composition may be released in a controlled manner, including slow release or rapid release, and the controlled release dosage of the relevant pharmaceutical composition may be achieved by known pharmaceutical techniques.
The present application provides a pharmaceutical composition comprising a polypeptide of the present application or a pharmaceutically acceptable salt thereof. The pharmaceutical compositions of the present application further comprise a pharmaceutically acceptable excipient, diluent or carrier.
In some embodiments, the teriparatide analog of the present application is included in the pharmaceutical compositions described herein in an amount of at least about 0.1mg, or at least about 0.2mg, or at least about 0.3mg, or at least about 0.4mg, or at least about 0.6mg, or at least about 0.8mg, or at least about 1mg, or at least about 1.5mg, or at least about 2mg, or at least about 2.5mg, or at least about 3mg, or at least about 5mg, or at least about 7mg, or at least about 10mg, or at least about 12mg, or at least about 15mg, or at least about 20mg, or at least about 30mg, or at least about 50mg, or at least about 70mg, or at least about 100mg.
In some embodiments, the amount of teriparatide analog of the present application in the pharmaceutical compositions described herein is in the range of 2.5 to 99.4% by weight. In some embodiments, the amount of teriparatide analog of the present application in the pharmaceutical composition described herein is in the range of 2.5 to 10 wt%, or in the range of 8 to 15 wt%, or in the range of 10 to 20 wt%, or in the range of 15 to 30 wt%, or in the range of 20 to 40 wt%, or in the range of 30 to 50 wt%, or in the range of 40 to 60 wt%, or in the range of 50 to 70 wt%.
The teriparatide analogues of the present application, when used in the treatment of osteoporosis, in some embodiments, are administered orally at least 100 μg; in some embodiments, at least 200 μg is administered orally; in some embodiments, at least 500 μg is administered orally; in some embodiments, at least 100 μg is administered orally; in some embodiments, at least 100 μg is administered orally. The teriparatide analogs of the present application, when used in the treatment of osteoporosis, in some embodiments, are administered orally in an amount of 20mg or less; in some embodiments, the dosage is 10mg or less for oral administration; in some embodiments, the dosage is 5mg or less for oral administration; in some embodiments, 3mg or less is administered orally; in some embodiments, 2000 μg or less is administered orally; in some embodiments, 1000 μg or less is administered orally. The teriparatide analogues of the present application, when used in the treatment of osteoporosis, in some embodiments, are administered orally at 200 μg to 20mg; in some embodiments, 200 μg to 10mg is administered orally; in some embodiments, 200 μg to 5mg are administered orally; in some embodiments, 200 μg to 3000 μg is administered orally; in some embodiments, 200 μg to 2000 μg is administered orally; in some embodiments, 500 μg to 1000 μg is administered orally; in some embodiments, 750 μg is administered orally.
The teriparatide analogues of the present application, or a pharmaceutical composition thereof, when used in the treatment of osteoporosis, are administered orally 1-3 times per day according to any of the various embodiments described herein; in some embodiments, oral administration according to any of the various embodiments described herein is performed 1 or 2 times per day; in some embodiments, oral administration according to any of the various embodiments described herein is performed 1 time per day.
The scheme of this application still includes:
1. a teriparatide analogue, characterized in that the analogue is modified and/or amino acid replaced at positions 13, 26 and/or 27 of the N-terminal teriparatide.
2. The teriparatide analogue according to claim 1, which is acylated modified at positions 13, 26 and/or 27 of the N-terminal of teriparatide.
3. Teriparatide analogue according to any of the preceding claims, characterized in that the acylation modification site is located at one, two or three of the lysines at position 13, 26 or 27 of the N-terminal of teriparatide.
4. Teriparatide analogues according to any of the preceding claims, characterized in that the fatty chain structure for the acylation modification has the general formula: HOOC- (AEEA) m -(Xaa) n -R 1 Wherein Xaa is one or more of D-alanine, beta-alanine, 4-aminobutyric acid, 2-aminoisobutyric acid, 2-aminobutyric acid, arginine, aspartic acid, asparagine, cysteine, D-glutamic acid, gamma-glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, proline, phenylalanine, serine, tyrosine, threonine, tryptophan, valine and methionine or the default; m is a number from 0,1,2,3,4, 5; n is one number of 0,1,2, 3; r is R 1 Selected from aliphatic straight chain, branched chain C 6 -C 20 Acyl groups of (c), deoxycholic acid, biotin or deletion.
5. Teriparatide analogue according to any of the preceding claims, characterized in that m is a number from 0,1,2,3,4,5, preferably 0-3, more preferably 2.
6. Teriparatide analogues according to any of the preceding claims, characterized in that n is a number 0,1,2,3, preferably 0 or 1, more preferably 1.
7. Teriparatide analogues according to any of the preceding claims, characterized in that said n is 1, said (Xaa) n Selected from 4-aminobutyric acid, 2-aminoisobutyric acid, D-alanine, beta-alanine, aspartic acid, cysteine, gamma-glutamic acid, glycine, proline, phenylalanine, preferably gamma-glutamic acid or proline.
8. Teriparatide analogues as claimed in any of the foregoing, characterized in that R 1 Selected from heptanoyl, methylheptanoyl, octanoyl,Methyl octanoyl, nonanoyl, methyl nonanoyl, decanoyl, methyl decanoyl, lauroyl, myristoyl, palmitoyl, octadecanoyl, 17-carboxyheptadecanoyl, 15-carboxypentadecanoyl, 13-carboxytridecanoyl, 11-carboxyundecanoyl, deoxycholic acid and biotin, preferably lauroyl, myristoyl, palmitoyl, octadecanoyl, 17-carboxyheptadecanoyl, deoxycholic acid, biotin, more preferably octadecanoyl, 17-carboxyheptadecanoyl.
9. Teriparatide analogue according to any one of the preceding claims, characterized in that the analogue has a structure as described in one of the following:
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wherein, the chemical structural general formula T can be expressed as:
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when two or three lysine modifications are present at positions 13, 26 or 27, T in the modification group is of the same structure, wherein m is a number from 0,1,2,3,4,5, n is a number from 0,1,2,3, p is an integer from 6 to 20, R 2 is-H or-COOH.
10. Teriparatide analogue according to any one of the preceding claims, characterized in that T may be represented by the following structure:
/>
wherein m is selected from integers from 0 to 5, preferably 0 to 2, more preferably 2; xaa 1 Selected from the group consisting of D-alanine, beta-alanine, 4-aminobutyric acid, 2-aminoisobutyric acid, 2-aminobutyric acid, arginine, aspartic acid, asparagine, cysteine, D-glutamic acid, gamma-glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, proline, phenylalanine, serine, tyrosine, threonine, tryptophan, valine, methionine; preferably 4-aminobutyric acid, 2-aminoisobutyric acid, D-alanine, beta-alanine, aspartic acid, cysteine, gamma-glutamic acid, glycine, proline, phenylalanine; more preferably gamma-glutamic acid or proline.
11. A teriparatide analogue according to any one of the preceding claims, which analogue has a substitution of a lysine at position 13, 26 or 27 of the N-terminus of teriparatide, the analogue having the general formula:
H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-X 1 -His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-X 2 -X 3 -Leu-Gln-Asp-Val-His-Asn-Phe-OH, wherein X 1 ，X 2 ，X 3 Not simultaneously Lys, and X 1 ，X 2 ，X 3 At least one of which is Lys.
12. Teriparatide analogues according to any of the preceding claims, characterized in that said X 1 ，X 2 ，X 3 Any one or any two of the amino acids are one or two of D-alanine, beta-alanine, 4-aminobutyric acid, 2-aminoisobutyric acid, arginine, aspartic acid, asparagine, cysteine, D-glutamic acid, gamma-glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, proline, phenylalanine, serine, tyrosine, threonine, tryptophan, valine and methionine; preferably D-alanine, beta-alanine, 2-aminoisobutyric acid, arginineCysteine, glycine; more preferably arginine.
13. The teriparatide analogue of any one of the preceding claims, characterized in that the teriparatide analogue has an acylation modification, the acylation modification site being located at X 1 ，X 2 Or X 3 Lys on.
14. Teriparatide analogues according to any of the preceding claims, characterized in that the acylation modification is a linked fatty chain structure of the general formula: HOOC- (AEEA) m -(Xaa) n -R 1 Wherein Xaa is one or more of D-alanine, beta-alanine, 4-aminobutyric acid, 2-aminoisobutyric acid, 2-aminobutyric acid, arginine, aspartic acid, asparagine, cysteine, D-glutamic acid, gamma-glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, proline, phenylalanine, serine, tyrosine, threonine, tryptophan, valine and methionine or the default; m is a number from 0,1,2,3,4, 5; n is one number of 0,1,2, 3; r is R 1 Selected from aliphatic straight chain, branched chain C 6 -C 20 Acyl groups of (c), deoxycholic acid, biotin or deletion.
15. Teriparatide analogue according to any of the preceding claims, characterized in that m is a number from 0,1,2,3,4,5, preferably 0-3, more preferably 2.
16. Teriparatide analogue according to any one of the preceding claims, characterized in that n is a number 0,1,2,3, preferably 0 or 1, more preferably 1.
17. The teriparatide analogue of any one of the preceding claims, characterized in that n is 1, xaa n Selected from 4-aminobutyric acid, 2-aminoisobutyric acid, D-alanine, beta-alanine, aspartic acid, cysteine, gamma-glutamic acid, glycine, proline, phenylalanine, preferably gamma-glutamic acid or proline.
18. Teriparatide analogues as claimed in any of the foregoing, characterized in that R 1 Selected from the group consisting of heptanoyl, methylheptanoyl, octanoyl, methyloctanoyl, nonanoyl, methylnonanoyl, decanoyl, methyldecanoyl, lauroyl, myristoyl,Palmitoyl, octadecanoyl, 17-carboxyheptadecanoyl, 15-carboxypentadecanoyl, 13-carboxytridecanoyl, 11-carboxyundecanoyl, deoxycholic acid and biotin, preferably lauroyl, myristoyl, palmitoyl, octadecanoyl, 17-carboxyheptadecanoyl, deoxycholic acid, biotin, more preferably octadecanoyl, 17-carboxyheptadecanoyl.
19. Teriparatide analogue according to any one of the preceding claims, characterized in that the analogue has the structure:
wherein X is 1 Selected from D-Ala, beta-Ala, GABA, aib, abu, arg, asp, asn, cys, D-Glu, gamma-Glu, gln, gly, his, ile, leu, pro, phe, ser, tyr, thr, trp, val, met, preferably beta-Ala, GABA, aib, abu, arg, cys, more preferably Arg; x is X 2 Selected from D-Ala, beta-Ala, GABA, aib, abu, arg, asp, asn, cys, D-Glu, gamma-Glu, gln, gly, his, ile, leu, pro, phe, ser, tyr, thr, trp, val, met, preferably beta-Ala, GABA, aib, abu, arg, cys, more preferably Arg; x is X 3 Selected from D-Ala, beta-Ala, GABA, aib, abu, arg, asp, asn, cys, D-Glu, gamma-Glu, gln, gly, his, ile, leu, pro, phe, ser, tyr, thr, trp, val, met, preferably beta-Ala, GABA, aib, abu, arg, cys, more preferably Arg;
wherein, the chemical structural general formula T can be expressed as:
wherein m is a number from 0,1,2,3,4,5, n is a number from 0,1,2,3, p is an integer from 6 to 20,R 2 is-H or-COOH.
20. The teriparatide analogue of any one of the preceding claims, wherein T may be of the structure:
/>
wherein m is selected from integers from 0 to 5, preferably 0 to 2, more preferably 2; xaa 1 Selected from the group consisting of D-alanine, beta-alanine, 4-aminobutyric acid, 2-aminoisobutyric acid, 2-aminobutyric acid, arginine, aspartic acid, asparagine, cysteine, D-glutamic acid, gamma-glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, proline, phenylalanine, serine, tyrosine, threonine, tryptophan, valine, methionine, preferably 4-aminobutyric acid, 2-aminoisobutyric acid, D-alanine, beta-alanine, aspartic acid, cysteine, gamma-glutamic acid, glycine, proline, phenylalanine, more preferably gamma-glutamic acid or proline.
21. Teriparatide analogue according to any one of the preceding claims, characterized in that the analogue is selected from compounds having one of the following structures:
1).
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22. a pharmaceutical composition comprising a teriparatide analogue of any one of claims or a pharmaceutically acceptable salt thereof.
23. The pharmaceutical composition of any of the preceding claims, further comprising a pharmaceutically acceptable carrier and/or excipient.
24. Pharmaceutical composition according to any of the preceding claims, characterized in that it is an injectable or an oral formulation.
25. Pharmaceutical composition according to any one of the preceding claims, characterized in that the oral formulation may be in the form of tablets, troches, pills, capsules, elixirs, granules, syrups, granules, emulsions, suspensions, solutions, dispersions and sustained release formulations for oral or non-oral administration.
26. Use of a pharmaceutical composition according to any of the preceding claims for the preparation of a medicament for the treatment of osteoporosis.
Definition of the definition
The following terms used in this application have the following meanings, unless otherwise indicated. A particular term, unless otherwise defined, shall not be construed as being ambiguous or otherwise unclear, but shall be construed in accordance with the ordinary meaning in the art. When trade names are presented herein, it is intended to refer to their corresponding commercial products or active ingredients thereof.
The term "optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, ethyl "optionally" substituted with halogen means that ethyl may be unsubstituted (CH 2 CH 3 ) Monosubstituted (e.g. CH 2 CH 2 F) Polysubstituted (e.g. CHFCH 2 F、CH 2 CHF 2 Etc.) or fully substituted (CF) 2 CF 3 ). It will be appreciated by those skilled in the art that for any group comprising one or more substituents, no substitution or pattern of substitution is introduced that is sterically impossible and/or synthetic.
Herein, a textC used m-n Meaning that the moiety has m to n carbon atoms. For example, "carbon 3-10 Cycloalkyl "means that the cycloalkyl has 3 to 10 carbon atoms. "carbon 0-6 Alkylene "means that the alkylene has from 0 to 6 carbon atoms, and when the alkylene has 0 carbon atoms, the group is a bond.
As used herein (AEEA) m Or (Xaa) n Refers to AEEA or Xaa groups having m or n linkages in the moiety.
Numerical ranges herein refer to individual integers within a given range. For example "C 1-6 By "is meant that the group may have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.
When any variable (e.g., R) occurs more than once in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if one group is substituted with 2R's, then each R has an independent option.
The term "substituted" means that any one of the subsequent hydrogen atoms on a particular atom is substituted with a substituent, provided that the valence of the particular atom is normal and the substituted compound is stable. When the substituent is oxo (i.e., =o), meaning that two hydrogen atoms are substituted, oxo does not occur on the aromatic group.
PTH, unless otherwise specified, in the present embodiment refers to PTH (1-34) or an analog having PTH (1-34) as a parent nucleus.
AEEA in the present embodiment refers to 8-amino-3, 6-dioctanoic acid.
The term "pharmaceutically acceptable" is intended to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
As pharmaceutically acceptable salts, for example, metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like can be mentioned. Non-limiting examples of metal salts include, but are not limited to, salts of alkali metals, such as sodium, potassium, and the like; salts of alkaline earth metals, such as calcium salts, magnesium salts, barium salts, and the like; aluminum salts, and the like. Non-limiting examples of salts with organic bases include, but are not limited to, salts with trimethylamine, triethylamine, pyridine, picoline, 2, 6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and the like. Non-limiting examples of salts formed with inorganic acids include, but are not limited to, salts formed with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like. Non-limiting examples of salts formed with organic acids include, but are not limited to, salts formed with formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, malic acid, maleic acid, tartaric acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like. Non-limiting examples of salts with basic amino acids include, but are not limited to, salts with arginine, lysine, ornithine and the like. Non-limiting examples of salts formed with acidic amino acids include, but are not limited to, salts formed with aspartic acid, glutamic acid, and the like.
The term "pharmaceutical ingredient" refers to a formulation of one or more compounds of the present application or salts thereof with excipients, diluents, or carriers commonly accepted in the art for delivering biologically active compounds to an organism (e.g., a human). The purpose of the pharmaceutical composition is to facilitate administration of the compounds of the present application to an organism.
The term "pharmaceutically acceptable excipient, diluent, or carrier" refers to those excipients, diluents, or carriers that do not significantly stimulate the organism and do not impair the biological activity or performance of the active compound. Suitable carriers, diluents and excipients are well known to those skilled in the art and include materials such as carbohydrates, waxes, water soluble and/or water swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water and the like.
The present application also includes the same meaning as those described herein, but where one or more atoms are replaced by an atom of a different atomic weight or mass number than that commonly found in natureA potentiometric labeled compound of the present application. Examples of isotopes that can be incorporated into compounds of the present application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as, respectively 2 H、 3 H、 11 C、 13 C、 14 C、 13 N、 15 N、 15 O、 17 O、 18 O、 31 P、 32 P、 35 S、 18 F、 123 I、 125 I and 36 cl, and the like.
Certain isotopically-labeled compounds of the present application (e.g., with 3 H is H 14 C-labeled) can be used in compound and/or substrate tissue distribution analysis. Tritiated (i.e., 3H) and carbon-14 (i.e., 14C) isotopes are particularly preferred for their ease of preparation and detectability. Furthermore, substitution with heavier isotopes such as deuterium (i.e., 2H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and therefore may be preferred in certain circumstances. Positron emitting isotopes such as 15O, 13N, 11C and 18F can be used in Positron Emission Tomography (PET) studies to determine substrate occupancy. Isotopically-labeled compounds of the present application can generally be prepared by following procedures analogous to those disclosed in the schemes and/or examples below by substituting an isotopically-labeled reagent for an non-isotopically-labeled reagent.
The compounds of the present application may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent alternatives well known to those skilled in the art, preferred embodiments including but not limited to the examples of the present application.
The chemical reactions of the embodiments of the present application are accomplished in a suitable solvent that is suitable for the chemical changes of the present application and the reagents and materials needed. In order to obtain the compounds of the present application, modifications or choices of synthesis steps or reaction schemes based on the existing embodiments are sometimes required by those skilled in the art.
The following examples are merely representative of one aspect of the present invention and are not limiting of the inventive subject matter.
Description of the drawings:
fig. 1: shows teriparatide analogs prior to acylation modification with a 13,26,27 amino acid substitution to PTH (1-34): (Arg) 26 ，Gly 27 )Lys 13 -PTH，(Cys 13 ，Ala 27 )Lys 26 -PTH (Arg) 13 ，Arg 26 )Lys 27 -in vitro activity EC50 value of PTH
Specific embodiments:
EXAMPLE 1N- ε 27 -PTH(1-34)-AEEA-AEEA-Pro-C 17 -COOH
(1) Materials and reagents
2-CTC resin, substitution value 1.15mmol/g.
The amino acid is: fmoc-Pro-OH, fmoc-AEEA-OH, mono-tert-butyl octadecanedioate
The synthesis reagent comprises the following steps: HOBt, DIC, DMF, DCM piperidine, DIEA.
(2) Instrument for measuring and controlling the intensity of light
CS-BIO type polypeptide synthesizer, waters600 semi-preparative high performance liquid chromatograph, beckman centrifuge, BUCHI rotary evaporator.
(3) Operating procedure
a. Solid phase chemical synthesis of polypeptides
Weighing 1.00g of 2-CTC resin, placing the resin into a reactor of a polypeptide synthesizer, adding 10mLDCM, soaking for 1h, weighing 2-3 times of Fmoc-AEEAc-OH and 4-6 times of DIEA, adding the mixture into the reactor after dissolving the mixture, putting the mixture into the reactor for reaction at room temperature for two hours, namely, coupling the first amino acid to the resin, washing the resin for 6 times by DCM, and determining the substitution value (SD) of the resin at the moment; then 10ml of 20% PIP/DMF solution is added, the mixture is mixed for 30min to remove amino protecting groups, the resin is washed for 6 times by DCM, the second amino acid is coupled, three times of Fmoc-AEEAc-OH, HOBt and DIC are weighed, 10ml of mixed solvent of DMF/DCM (1:1) is added for dissolution, the reaction is carried out, the reaction temperature is room temperature, the progress of the reaction is monitored by ninhydrin reaction, the colorless is monitored, and the resin is washed for 6 times by DCM. And then, the coupling reaction of proline and octadecanedioic acid can be continued according to the coupling method, and the cycle is performed until all amino acid coupling is completed.
b. Cleavage and precipitation
Adding a cracking reagent according to the proportion of 5mL of the cracking reagent to 1g of resin, wherein the reagent proportion is TFE, DCM=1:4 (V: V), stirring at room temperature for reacting for 1 hour, filtering, steaming filtrate at 40 ℃ until the filtrate is dry, adding 10mL of LDCM into a rotary steaming bottle, steaming the filtrate again until the filtrate is dry, repeating for 2-3 times, finally adding 3mL of LDCM to dissolve polypeptide, adding 40mL of glacial ethyl ether, placing the mixture in a refrigerator at-20 ℃ for 20min, centrifuging, drying in vacuum, and weighing crude peptide.
c. Liquid phase reaction for preparing teriparatide derivative
The dried crude peptide was weighed about 0.1mmol, HOSU about 0.095mmol, and DIC 15. Mu.L was pipetted, and about 5mL of tetrahydrofuran was added and reacted at room temperature for 1-2 hours, and THF was removed by rotary evaporation to give a yellow oil. TFA H is added 2 And (3) performing tert-butyl ester removal protection in a mixed solvent of O=90:10, reacting for 1 hour at room temperature, pouring in glacial diethyl ether for precipitation, centrifuging to obtain a solid, washing with diethyl ether three times, drying under reduced pressure to obtain solid powder, weighing, and dissolving with DMF to obtain a side chain activated ester solution.
0.5mmol of teriparatide is dissolved in 0.1mol/L triethylamine solution, and a side chain activated ester solution is slowly added dropwise into the solution, wherein the feeding ratio is 1:1.2, stirring at room temperature for 10 minutes, and stopping the reaction by adjusting the pH to 8.0 with 0.1mol/L hydrochloric acid.
The crude product was purified by semi-preparative RP-HPLC.
1 purification
Chromatographic column: nano Micro C18 column (10 mm. Times.250 mm,10 μm)
Flow rate: 5ml/min
Detection wavelength: 215nm
Mobile phase: phase A: 1% HAC/water solution
And B phase: 1% HAC/acetonitrile
Gradient elution procedure is as in table 3:
TABLE 3 gradient elution table
Analysis of the collected product by Agilent 1260HPLC
Chromatographic column: YMC-pack ODS-AQ C18 analytical column (4.6 mm. Times.250 mm,5 μm)
Flow rate: 1ml/min
Detection wavelength: 215nm
Mobile phase: phase A: 0.1% TFA/water
And B phase: 0.1% TFA/acetonitrile
Gradient elution procedure is as in table 4:
TABLE 4 gradient elution Table
Collecting target components with purity of more than 90%, removing acetonitrile by rotary evaporation, and vacuum freeze drying. Molecular weight confirmation by ESI-MS, M/Z= 1201.62 [ M+4H ] + Consistent with theoretical molecular weight.
EXAMPLE 2N- ε 13 -PTH(1-34)-AEEA-AEEA-α-Glu-C 16
The preparation was carried out as described in example 1, with molecular weight confirmation via ESI-MS, M/Z= 1194.6 [ M+4H ] + Is consistent with the theoretical molecular weight.
Example 3: n-epsilon 26 -PTH(1-34)-γ-Glu-C 12
The preparation process is as followsMolecular weight confirmation was performed by ESI-MS as described in example 1, M/Z= 1108.2 [ M+4H ] + Is consistent with the theoretical molecular weight.
Example 4: n-epsilon 27 -PTH (1-34) -AEEA-AEEA-deoxycholic acid
The preparation was carried out as described in example 1, with molecular weight confirmation via ESI-MS, M/Z= 1196.5 [ M+4H ] + Is consistent with the theoretical molecular weight.
Example 5: n-epsilon 13 -(Arg 26 ，Gly 27 )PTH-γ-Glu-C 16
(1) Materials and reagents
2-CTC resin, substitution value 1.15mmol/g.
Teriparatide analogues (Arg) 26 ，Gly 27 ) PTH is obtained by engineering bacteria expression or chemical synthesis method, M/Z= 1019.2 (M+4H) +
The amino acid is: fmoc-gamma-Glu-Otbu, palmitic acid
The synthesis reagent comprises the following steps: HOBt, DIC, DMF, DCM piperidine, DIEA.
(2) Instrument for measuring and controlling the intensity of light
CS-BIO type polypeptide synthesizer, waters600 semi-preparative high performance liquid chromatograph, beckman centrifuge, and bushi rotary evaporator.
(3) Operating procedure
a. Solid phase chemical synthesis of polypeptides
Weighing 1.00g of 2-CTC resin, placing the resin in a reactor of a polypeptide synthesizer, adding 10mLDCM, soaking for 1h, weighing 2-3 times of Fmoc-gamma-Glu-OH and 4-6 times of DIEA, adding the mixture into the reactor for dissolution, putting the mixture into the reactor for reaction at room temperature (preferably 25 ℃ and above, or else prolonging the reaction time), reacting for two hours, namely, coupling the first amino acid to the resin, washing the resin for 6 times by DCM, and determining the substitution value (SD) of the resin at the moment; then 10ml of 20% PIP/DMF solution is added, the mixture is mixed for 30min to remove amino protecting groups, the resin is washed for 6 times by DCM, the second amino acid is coupled, three times of palmitic acid, hoBT and DIC are weighed, 10ml of mixed solvent of DMF/DCM (1:1) are added together for dissolution, the reaction is carried out, the reaction temperature is room temperature, the progress of the reaction is monitored by ninhydrin reaction, the colorless is monitored to be the completion of the reaction, and the resin is washed for 6 times by DCM.
b. Cleavage and precipitation
After the synthesis of the polypeptide is completed, the wet weight is weighed. Adding a cracking reagent according to the proportion of 5mL of the cracking reagent to 1g of resin, wherein the reagent proportion is TFE (TFE) DCM=1:4 (V: V), stirring at room temperature for reaction for 1 hour, filtering to a 25mL rotary steaming bottle, rotary steaming at 40 ℃ until no liquid exists, adding 10mL of LDCM into the rotary steaming bottle, rotary steaming again until no liquid exists, repeating for 2-3 times, finally adding 3mL of LDCM, dissolving the cleaved polypeptide, transferring the solution to a 50mL centrifuge tube, adding 40mL of diethyl ether, placing in a refrigerator at-20 ℃ for 20min, centrifuging, vacuum drying, and weighing the crude peptide.
c. Liquid phase reaction for preparing teriparatide derivative
The dried crude peptide was weighed to about 0.1mmol, HOSU to about 0.095mmol, and DIC to 15. Mu.L was pipetted, and about 5mL of tetrahydrofuran was added to dissolve in a 5mLEP tube, reacted at room temperature for 1-2 hours, then the reaction liquid was transferred to a 10mL rotary evaporator, THF was removed by rotary evaporation, then about 5mLDMF was added to the rotary evaporator, and the reaction product in the rotary evaporator was dissolved to obtain an activated ester of a side chain.
Then 0.5mmol of teriparatide analogue is dissolved in 0.1mol/L triethylamine solution, and the side chain activated ester solution is slowly added dropwise into the solution, wherein the feeding ratio is 1:1.2, stirring at room temperature for 10 minutes, after which the reaction was stopped by adjusting the pH to 8.0 with 0.1N hydrochloric acid.
The crude product was purified by semi-preparative RP-HPLC.
1 purification
Chromatographic column: nano Micro C18 column (10 mm. Times.250 mm,10 μm)
Flow rate: 5ml/min
Detection wavelength: 215nm
Mobile phase: phase A: 1% HAC/water solution
And B phase: 1% HAC/acetonitrile
Gradient elution procedure is as in table 3:
TABLE 3 gradient elution table
Analysis of the collected product by Agilent 1260HPLC
Chromatographic column: YMC-pack ODS-AQ C18 analytical column (4.6 mm. Times.250 mm,5 μm)
Flow rate: 1ml/min
Detection wavelength: 215nm
Mobile phase: phase A: 0.1% TFA/water
And B phase: 0.1% TFA/acetonitrile
Gradient elution procedure is as in table 4:
TABLE 4 gradient elution Table
Collecting target component with purity higher than 90%, rotary evaporating under low pressure, and freeze drying. Molecular weight confirmation by ESI-MS, M/Z= 1111.1 [ M+4H ] + Is consistent with the theoretical molecular weight.
Example 6: n-epsilon 27 -(Arg 13 ，Arg 26 )PTH(1-34)-AEEA-Gly-AEEA-Gly-Glu-C 17 -COOH
(Arg 13 ，Arg 26 )Lys 27 -PTH (1-34) analogues obtained by engineering bacterial expression or chemical synthesis, M/z= 1044.3 [ m+4h ] +
The procedure for the preparation of the analogue derivatives was as described in example 4, with molecular weight confirmation via ESI-MS, M/z= 1251.8 [ m+4h ] + Is consistent with the theoretical molecular weight.
Example 7: n-epsilon 26 -(Cys 13 ，Ala 27 )PTH(1-34)-Phe-AEEA-AEEA-AEEA-C12
(Cys 13 ，Ala 27 )Lys 26 -PTH (1-34) analogues obtained by engineering bacterial expression or chemical synthesis, M/z= 1009.7 [ m+4h ] +
The preparation of the derivatives was carried out as described in example 4, with molecular weight confirmation via ESI-MS, M/z= 1200.7 [ m+4h ] + Is consistent with the theoretical molecular weight.
Example 8: n-epsilon 26 -(Arg 13 ，Arg 27 )PTH(1-34)-AEEA-AEEA-C 11 -biotin
(Arg 13 ，Arg 27 )Lys 26 -PTH (1-34) analogues obtained by engineering bacterial expression or chemical synthesis, M/z= 1044.5 [ m+4h ] +
The preparation of the derivatives was carried out as described in example 4, with molecular weight confirmation via ESI-MS, M/z= 1218.2 [ m+4h ] + Is consistent with the theoretical molecular weight.
Example 9: n-epsilon 27 -(Arg 13 ，Arg 26 )PTH(1-34)-AEEA-AEEA-C 17 -COOH
(Arg 13 ，Arg 26 )Lys 27 -PTH (1-34) analogues obtained by engineering bacterial expression or chemical synthesis methods M/z= 1044.5 [ m+4h ] +
Process for the preparation of derivativesMolecular weight confirmation was performed by ESI-MS as described in example 4, M/z= 1189.8 [ m+4h ] + Is consistent with the theoretical molecular weight.
Example 10: n-epsilon 27 -(Arg 13 ，Arg 26 )PTH(1-34)-AEEA-Glu-C 17 -COOH
(Arg 13 ，Arg 26 )Lys 27 Preparation of the-PTH (1-34) analogs using engineering bacterial expression or chemical synthesis to obtain M/z= 1044.5 [ m+4h ] derivatives the molecular weight was confirmed by ESI-MS using the procedure described in example 4, M/z= 1186.7 [ m+4h ] + Is consistent with the theoretical molecular weight.
Example 11: n-epsilon 27 -(Arg 13 ，Arg 26 )PTH(1-34)-AEEA-AEEA-Ala-C 17 -COOH
(Arg 13 ，Arg 26 )Lys 27 -PTH (1-34) analogues obtained by engineering bacterial expression or chemical synthesis methods M/z= 1044.5 [ m+4h ] + The preparation of the derivatives was carried out as described in example 4, with molecular weight confirmation via ESI-MS, M/z= 1208.5 [ m+4h ] + Is consistent with the theoretical molecular weight.
Example 12: n-epsilon 27 -(Arg 13 ，Arg 26 )PTH(1-34)-AEEA-AEEA-C 16
(Arg 13 ，Arg 26 )Lys 27 -PTH (1-34) analogues obtained by engineering bacterial expression or chemical synthesis methods M/z= 1044.5 [ m+4h ] + The procedure for the preparation of the derivatives is as described in example 4Molecular weight confirmation by ESI-MS, M/Z= 1175.3 [ M+4H ] + Is consistent with the theoretical molecular weight.
Example 13: detection of in vitro Activity EC50
CHO-K1 cells are adopted, teriparatide acetate is used as a reference substance, and the method for detecting the bioactivity of the teriparatide acetate is adopted according to the United states pharmacopoeia. In vitro cell activity assays were performed on PTH (1-34) and derivatives of PTH (1-34). Teriparatide is able to bind specifically to the cell surface PTH receptor (PTH 1R), leading to the activation of adenylate cyclase and thus the production of cyclic adenosine monophosphate (cAMP), which stimulates cellular metabolic pathways. The cAMP formed in these cells in dependence on PTH1-34 concentration was quantitatively analyzed using time resolved fluorescence technique (TR-FRET) (the detection kit is LANCE UltracAMP Kit from Perkinelmer). And analyzing experimental data by Prism5 software, taking the concentration of a sample as an X axis, the corresponding fluorescence ratio as a Y axis, fitting by selecting a four-parameter equation, and drawing a dose response curve of the active reference substance and the test substance to obtain an EC50 value.
Example 14: research on transmembrane transport of derivatives
Caco-2 cells were grown at 5X10 5 Individual/cm 2 The cells were densely inoculated on the Apical side of a Millicell-CM insert cell culture dish (Transwell plate number 3401 from Corning Co.) and the liquid was changed every day, and after the third week, the transmembrane resistance was measured, and when the resistance was more than 500. Omega. CM 2 Can be used for transportation experiments. In the transport experiments on Caco-2 monolayer cells, teriparatide acetate (self-made in laboratory) was used as a control. 0.3ml of PTH (1-34) or PTH (1-34) derivative solution (100. Mu.g/ml) was added to the donor pool (AP end), 0.7mg Hank's buffer was added to the acceptor pool (BP end), 0.1ml of solution was withdrawn from the acceptor pool at various time points (0,20,45,60 and 90 minutes), and an equal amount was replenishedBlank Hank's solution, using ELISA kit to determine the concentration, and plotting the concentration versus time using apparent epithelial cell permeability coefficient P app Formula calculation
A is the permeate membrane area, here the donor cell membrane area (cm 2 )，C 0 Is the initial sample concentration (ng/ml) of the donor cell, dQ/dt is the diffusion flux (ng/s).
Teriparatide has a large polarity, has poor cell penetration capability, and cannot realize transcellular transport. Surprisingly, however, the modified teriparatide derivatives (examples 1 and 3) and the teriparatide analogue derivatives (examples 5 and 7) are P app The value is improved by 1.8-4 times, and according to the results of the example 4 and the example 8, the permeability of an epithelial cell membrane is obviously improved after the deoxycholic acid and the biotin are modified, so that the transmembrane transport capacity of the polypeptide is enhanced, and a compound basis is effectively provided for the polypeptide molecules to cross the epithelial cell layer of the gastrointestinal tract.
In accordance with the present disclosure, while the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention.
The disclosures of all documents cited herein are hereby incorporated by reference to the extent that they provide exemplary, procedural and other details supplementary to those set forth herein.

Claims (6)

1. Teriparatide analogues characterized in that said analogues are selected from compounds of one of the following structures:
1).
2).
3).
4).
5).
6).
7).
8).
9).
10).
11).
12).
2. a pharmaceutical composition comprising a teriparatide analogue according to any one of claims 1 or a pharmaceutically acceptable salt thereof.
3. The pharmaceutical composition of claim 2, further comprising a pharmaceutically acceptable carrier and/or excipient.
4. A pharmaceutical composition according to claim 3, characterized in that it is an injectable or an oral formulation.
5. The pharmaceutical composition according to claim 4, wherein the oral formulation is in the form of tablets, pills, capsules, elixirs, syrups, granules, emulsions, suspensions, dispersions and sustained release formulations for oral or non-oral administration.
6. Use of a pharmaceutical composition according to claim 2 for the preparation of a medicament for the treatment of osteoporosis.
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CN102731643A (en) * 2012-06-26 2012-10-17 深圳翰宇药业股份有限公司 Method for preparing polypeptide used for treating osteoporosis
CN103467595A (en) * 2013-09-06 2013-12-25 深圳翰宇药业股份有限公司 Method for preparing teriparatide
CN104017064A (en) * 2014-06-13 2014-09-03 杭州诺泰制药技术有限公司 Method for preparing teriparatide
CN104910269A (en) * 2015-06-02 2015-09-16 成都圣诺生物科技股份有限公司 Method for synthesizing teriparatide
CN107501408A (en) * 2017-09-22 2017-12-22 扬子江药业集团四川海蓉药业有限公司 A kind of preparation method of Teriparatide

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CN102731643A (en) * 2012-06-26 2012-10-17 深圳翰宇药业股份有限公司 Method for preparing polypeptide used for treating osteoporosis
CN103467595A (en) * 2013-09-06 2013-12-25 深圳翰宇药业股份有限公司 Method for preparing teriparatide
CN104017064A (en) * 2014-06-13 2014-09-03 杭州诺泰制药技术有限公司 Method for preparing teriparatide
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