# Patent

## Title
Glycosylated polypeptide and drug composition containing said polypeptide

## Abstract

[Problem] To provide a glycosylated polypeptide having affinity to somatostatin receptors and, compared to somatostatins, having improved in-blood stability. [Solution] The glycosylated polypeptide is characterized by at least two amino acids in a somatostatin or an analogue thereof being replaced by glycosylated amino acids.

## Background
STATEMENT OF PRIORITY
This application is a 35 U.S.C. 0.371 national phase application of International Application Serial No. PCT/JP2012/072383, filed Sep. 3, 2012, which claims the benefit, under 35 U.S.C. §119 (a) of Japanese Patent Application No. 2011-192203, filed Sep. 4, 2011, the entire contents of each of which are incorporated by reference herein.
STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING
A Sequence Listing in ASCII text format, submitted under 37 C.F.R. §1.821, entitled 9778-9TS_ST25v2.txt, 99,959 bytes in size, generated on Sep. 11, 2015 and filed via EFS-Web, is provided in lieu of a paper copy. This Sequence Listing is hereby incorporated by reference into the specification for its disclosures.
TECHNICAL FIELD
The present invention relates to a glycosylated polypeptide and a pharmaceutical composition comprising said polypeptide.
BACKGROUND ART
Somatostatin is a cyclic peptide present in both the central nervous system and the surrounding tissue. Somatostatin was first isolated from mammalian hypothalamus, and was identified from anterior pituitary gland as an important inhibitor of growth hormone secretion. This peptide is widely distributed in e.g. the hypothalamus, the pancreas, and the gastrointestinal tract, and its action is exerted via binding to a somatostatin receptor. In addition, somatostatin is known for its secretory suppression of growth hormone (GH) and thyroid-stimulating hormone (TSH) in the pituitary gland, as well as secretion suppression of various hormones such as gastrin, selectin, cholecystokinin (CCK), and VIP (Vasoactive Intestinal Polypeptide) in the gastrointestinal tract, and glucagon and insulin in the pancreas. It is also known to have an action to suppress gastrointestinal motility.
Natural somatostatin having the structural formula: Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys (SEQ ID NO. 1) (also known as somatotropin release inhibiting factor (SRIF)) was first isolated by Guillemin and coworkers. This somatostatin exerts its effect by interacting with a family of receptors. Somatostatin receptor (SSTR) has 1 to 5 subtypes (SSTR1-SSTR5), and among these, SSTR2 is known to be distributed in each tissues of the GH-secreting human pituitary gland adenoma, the central nervous system, the anterior pituitary gland, the retina, the adrenal medulla, the stomach, the duodenal mucosa, the small intestines, and the colon, as well as in the glucagon-secreting A-cell of the pancreatic islet. Each of these receptors is also known to be expressed in various tumors. For example, it has been reported that SSTR1 and SSTR5 are expressed in a functional pituitary adenoma, SSTR2 as well as SSTR1 and SSTR3 are expressed in a gastrointestinal tumor, SSTR3 is expressed in a pheochromocytoma, SSTR1 and SSTR5 are expressed in a prostate cancer, and SSTR5 is expressed in a colorectal cancer (Non-Patent Literature 1). Moreover, SSTR4 is reported in regards to its function as a receptor having an antagonistically modulating action and its possibility of being essential in the treatment of a glaucoma-related disease (Non-Patent Literature 2). As such, somatostatin and analogs thereof are potentially useful therapeutic drugs for somatostatin-related diseases or various types of tumors.
Meanwhile, because naturally-occurring somatostatin has a short half-life in blood of 2-3 minutes, it shows two undesirable properties of having low bioavailability and short duration of action, and thus its use or application as a therapeutic is limited. For this reason, various somatostatin analogs have been developed in order to find a somatostatin analog superior in any one of efficacy, biostability, duration of action, or selectivity considering the release suppression of growth hormone, insulin, or glucagon.
Octreotide (Patent Literatures 1 and 2) is reported as the first approved somatostatin analog that can be clinically utilized, and this octreotide is known to have affinity towards somatostatin receptors SSTR2, SSTR3, and SSTR5.
Octreotide has been developed as a cyclic peptide consisting of eight amino acids which has a sequence of four amino acids (Phe-Trp-Lys-Thr) (SEQ ID NO. 170) that is an important portion for showing the biological activity of somatostatin, Cys that forms a disulfide (S—S) bond at the two terminals of the sequence, and further D-Phe and Thr(ol) outside of the Cys at the two terminals. This octreotide can render persistence of action by improving the half-life in blood by its amino acid sequence, as well as has a higher selectivity towards growth hormone (GH) than somatostatin which enables it to have a strong action.
Such somatostatin analogs including octreotide can be used for treating patients who have a hormone-secreting and hormone-dependent tumor. Currently, symptoms related to metastatic carcinoid tumor which is a tumor of the neuroendocrine system (flushing, diarrhea, cardiac valve disease, and abdominal pain) and symptoms related to vasoactive intestinal peptide (VIP)-secreting adenoma (watery diarrhea) are treated with octreotide.
For example, in a carcinoid and VIP-producing tumor, octreotide inhibits both secretion and action of its active factor. Accordingly, in a VIP-producing tumor characterized in profusely-secreting diarrhea, a somatostatin analog can reduce its diarrhea by secretory inhibition of VIP as well as by directly influencing intestinal secretion.
On the other hand, however, many neuroendocrine tumors are reported to have resistance to somatostatin analogs such as octreotide (Non-Patent Literature 3). Moreover, although octreotide is used in the treatment of acromegaly, it is reported to have no effect on approximately one third of acromegaly patients. Further, it is reported that in majority of carcinoid tumor patients, octreotide exerts its effect only during initial administration, and tachyphylaxis is caused when the duration of administration is prolonged. Further, it is reported that octreotide does not show any effect on suppression of adrenocorticotropic hormone (ACTH) production in early Cushing's disease patients.
In light of the problems above, development of a somatostatin analog that binds to multiple receptor subtypes with high affinity like that of a natural somatostatin is desired for a tumor expressing multiple somatostatin receptors, and it is suggested that a somatostatin analog having such affinity towards somatostatin receptors may possibly also have effect on patients who were therapeutically ineffective with or patients who have resistance to past somatostatin analogs (Non-Patent Literature 4).
Accordingly, development of a somatostatin analog having a structure similar to a naturally-occurring somatostatin, similarly having affinity towards somatostatin receptors, and having extended half-life in blood compared to somatostatin has been desired.
Meanwhile, it has been becoming clear that sugar chains are responsible for various roles in vivo, and a method for adding a sugar chain to octreotide in order to extend the half-life in blood has also been proposed (such as Patent Literature 3).
However, research is delayed due to the complexity or diversity of its structure, and it cannot be said that the type of sugar chain or the position for adding a sugar chain is always optimized. A glycosylated polypeptide that has overcome the problems of past somatostatin analogs has not been reported.
CITATION LIST
Patent Literatures


    [Patent Literature 1]U.S. Pat. No. 4,310,518
    [Patent Literature 2] U.S. Pat. No. 4,235,886
    [Patent Literature 3] Japanese Published Unexamined Patent Application Publication No. Hei 03 (1991)-014599


Non-Patent Literatures


    [Non-Patent Literature 1] Current Opinion in Pharmacology, 2004, Vol. 4, pp. 608-613
    [Non-Patent Literature 2]J. Med. Chem. 2003, Vol. 46, pp. 5587-5596
    [Non-Patent Literature 3] Mol. Endocrinol., 2010, Vol. 24 (1), pp. 240-249
    [Non-Patent Literature 4] Molecular and Cellular Endocrinology, Vol. 286, 2008, pp. 69-74


SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
The object of the present invention is to provide a glycosylated polypeptide having affinity towards somatostatin receptors and improved stability in blood compared to somatostatin.
Means for Solving the Problems
As a result of repeated research to solve the above problems, the present inventors found a glycosylated polypeptide which maintains affinity towards somatostatin receptors and has improved stability in blood.
In other words, the present invention relates to a glycosylated polypeptide selected from the group consisting of: (A) SRIF14 consisting of the amino acid sequence represented by SEQ ID NO. 1; (B) a polypeptide having one or a few amino acids deleted, substituted, or added from/to SRIF14 consisting of the amino acid sequence represented by SEQ ID NO. 1; (C) a SRIF14 analog; (D) a polypeptide having 80% or more homology with SRIF14 consisting of the amino acid sequence represented by SEQ ID NO. 1; (E) a polypeptide further comprising N amino acids (wherein N is an integer from 1 or more to 20 or less) at the N-terminal side of (A)-(D); and (F) a polypeptide further comprising M amino acids (wherein M is an integer from 1 or more to 6 or less) at the C-terminal side of (A)-(D); characterized in that at least two amino acids are substituted with glycosylated amino acids, and the polypeptide has affinity towards somatostatin receptors.
Here, one embodiment of the glycosylated polypeptide of the present invention is characterized in that at least one of the amino acids substituted with said glycosylated amino acid is the amino acid corresponding to position 19 of SRIF14.
Moreover, in one embodiment of the glycosylated polypeptide of the present invention, said glycosylated polypeptide is characterized in that at least two amino acids are substituted with glycosylated amino acids in said polypeptide (E), and at least one of the amino acids substituted with said glycosylated amino acid is present at any of said N amino acids at the N-terminal side of said polypeptide (E).
Moreover, in one embodiment of the glycosylated polypeptide of the present invention, said glycosylated polypeptide is characterized in that at least two amino acids are substituted with glycosylated amino acids in said polypeptide (F), and at least one of the amino acids substituted with said glycosylated amino acid is present at any of said M amino acids at the C-terminal side of said polypeptide (F).
Moreover, in one embodiment of the glycosylated polypeptide of the present invention, said glycosylated polypeptide is characterized in that at least two amino acids are substituted with glycosylated amino acids in said polypeptide (E), and further the sequence of said N amino acids added onto the N-terminal side is represented by X—Y—, wherein X means a sequence of any L amino acids (wherein L is an integer from 1 or more to 6 or less), and Y is a sequence selected from the group consisting of: (1) Lys, (2) Arg-Lys, (3) Glu-Arg-Lys, (4) Arg-Glu-Arg-Lys (SEQ ID NO. 171), (5) Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 172), (6) Ala-Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 173), (7) Met-Ala-Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 174), (8) Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 175), (9) Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 176), (10) Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 177), (11) Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 178), (12) Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 179), (13) Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 180), (14) Ser-Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 181), and (15) a sequence having one or a few amino acids deleted, substituted, or added from/to the above sequences (2)-(14).
Moreover, one embodiment of the glycosylated polypeptide of the present invention is characterized in that at least one of the amino acids substituted with said glycosylated amino acid is present in any of L amino acids which means X in X—Y-representing the sequence of said N amino acids added onto the N-terminal side of said polypeptide (E).
Moreover, one embodiment of the glycosylated polypeptide of the present invention is characterized in that the N amino acids added onto the N-terminal side are linked to said N-terminal side via a linker.
Moreover, in another embodiment of the glycosylated polypeptide of the present invention, a polypeptide selected from the group consisting of: (A) SRIF28 consisting of the amino acid sequence represented by SEQ ID NO. 2; (B) a polypeptide having one or a few amino acids deleted, substituted, or added from/to SRIF28 consisting of the amino acid sequence represented by SEQ ID NO. 2; (C) a SRIF28 analog; (D) a polypeptide having 80% or more homology with SRIF28 consisting of the amino acid sequence represented by SEQ ID NO. 2; (E) a polypeptide further comprising J amino acids (wherein J is an integer from 1 or more to 15 or less) at the N-terminal side of (A)-(D); and (F) a polypeptide further comprising K amino acids (wherein K is an integer from 1 or more to 6 or less) at the C-terminal side of (A)-(D); is characterized in that at least two amino acids are substituted with glycosylated amino acids, and the polypeptide has affinity towards somatostatin receptors.
Moreover, one embodiment of the glycosylated polypeptide of the present invention is characterized in that at least one of the amino acids substituted with said glycosylated amino acid is at least one amino acid selected from the group consisting of the amino acid corresponding to position 1, the amino acid corresponding to position 5, the amino acid corresponding to position 9, the amino acid corresponding to position 12, the amino acid corresponding to position 13, the amino acid corresponding to position 14, and the amino acid corresponding to position 19 of SRIF28.
Moreover, in one embodiment of the glycosylated polypeptide of the present invention, said glycosylated polypeptide is characterized in that at least two amino acids are substituted with glycosylated amino acids in said polypeptide (E), wherein at least one of the amino acids substituted with said glycosylated amino acid is present in said J amino acids at the N-terminal side of said polypeptide (E).
Moreover, in one embodiment of the glycosylated polypeptide of the present invention, said glycosylated polypeptide is characterized in that at least two amino acids are substituted with glycosylated amino acids in said polypeptide (F), and at least one of the amino acids substituted with said glycosylated amino acid is present in said K amino acids of the C-terminal side of said polypeptide (F).
Moreover, in one embodiment of the glycosylated polypeptide of the present invention, said glycosylated polypeptide is characterized in that it has increased stability in blood compared to SRIF28.
Moreover, in one embodiment of the glycosylated polypeptide of the present invention, said glycosylated polypeptide is characterized in that it has increased half-life in blood by 10-fold or more compared to SRIF28.
Moreover, one embodiment of the glycosylated polypeptide of the present invention is characterized in that said affinity towards somatostatin receptors has affinity towards at least two or more receptors selected from the group consisting of SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5.
Moreover, in one embodiment of the glycosylated polypeptide of the present invention, said glycosylated polypeptide is characterized in that it has affinity towards any one of at least SSTR1 and SSTR4.
Moreover, in one embodiment of the glycosylated polypeptide of the present invention, said glycosylated polypeptide is characterized in that it has affinity towards both SSTR1 and SSTR4.
Moreover, in one embodiment of the glycosylated polypeptide of the present invention, said glycosylated polypeptide is characterized in that it has affinity towards all of SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5.
Moreover, one embodiment of the glycosylated polypeptide of the present invention is characterized in that each of said glycosylated amino acids is glycosylated Asn or glycosylated Cys.
Moreover, one embodiment of the glycosylated polypeptide of the present invention is characterized in that in each of said glycosylated amino acids, the sugar chain and the amino acid are linked without a linker.
Moreover, one embodiment of the glycosylated polypeptide of the present invention is characterized in that in each of said glycosylated amino acids, the sugar chain consists of 4 or more sugars.
Moreover, one embodiment of the glycosylated polypeptide of the present invention is characterized in that in each of said glycosylated amino acids, the sugar chain is a biantennary complex-type sugar chain, a triantennary complex-type sugar chain, or a tetraantennary complex-type sugar chain.
Moreover, one embodiment of the glycosylated polypeptide of the present invention is characterized in that in each of said glycosylated amino acids, the sugar chain is a biantennary complex-type sugar chain.
Moreover, one embodiment of the glycosylated polypeptide of the present invention is characterized in that said biantennary complex-type sugar chain is a sugar chain selected from the group consisting of a disialo sugar chain, a monosialo sugar chain, an asialo sugar chain, a diGlcNAc sugar chain, and a dimannose sugar chain.
Moreover, one embodiment of the glycosylated polypeptide of the present invention is characterized in that in each of said glycosylated amino acids, the sugar chain is a sugar chain represented by the following formula:




[wherein R1 and R2 are identical or different and are:





and Ac is an acetyl group].

Moreover, one embodiment of the glycosylated polypeptide of the present invention is characterized in that said sugar chain has at least one sialic acid at the non-reducing terminal, and the carboxy group of said sialic acid is modified by an alkylamino group, a benzyl group, an amino group, or an aminoethylamino group, having 1-30 carbons.
Moreover, in one embodiment of the glycosylated polypeptide of the present invention, said glycosylated polypeptide is characterized in that it has multiple glycosylated amino acids, wherein the sugar chains on each of said glycosylated amino acids are all identical.
Moreover, in one embodiment of the glycosylated polypeptide of the present invention, said glycosylated polypeptide is characterized in that it has Cys corresponding to Cys at position 17 and Cys at position 28 of SRIF28, and further these Cys are bound by a disulfide bond with each other.
Moreover, one embodiment of the glycosylated polypeptide of the present invention is characterized in that the C-terminal of said glycosylated polypeptide is amidated.
Moreover, one embodiment of the glycosylated polypeptide of the present invention is characterized in that an azido group is introduced at the N-terminal of said glycosylated polypeptide.
Moreover, one embodiment of the glycosylated polypeptide of the present invention is characterized in that it is labeled.
Moreover, another aspect of the present invention relates to a pharmaceutical composition characterized in that it comprises (I) the glycosylated polypeptide described above and/or a pharmaceutically acceptable salt thereof, and (II) a pharmaceutically acceptable carrier.
Moreover, one embodiment of the pharmaceutical composition of the present invention is characterized in that the sugar chains in said glycosylated polypeptide are substantially uniform.
Moreover, one embodiment of the pharmaceutical composition of the present invention is characterized in that it is employed for treatment or prevention of a somatostatin-related disease.
Moreover, one embodiment of the pharmaceutical composition of the present invention is characterized in that said somatostatin-related disease is at least one disease selected from the group consisting of acromegaly, gigantism, Alzheimer's disease and other forms of dementia, cancer, hormone-producing tumor, endocrine tumor, carcinoid, VIPoma, insulinoma, glucagonoma, Cushing's disease, hormone secretion defect, diabetes and complications thereof, pains, arthritis, diarrhea, gastric ulcer, inflammatory bowel disease, irritable bowel syndrome, gastrointestinal obstruction, ileus, postoperative restenosis, radiation damage, eye disease, dry eye, glaucoma, interstitial keratitis, iritis, cataract, and conjunctivitis.
Moreover, another aspect of the present invention relates to a method for treating or preventing a somatostatin-related disease, characterized in administering an effective amount of the glycosylated polypeptide described above.
Moreover, one embodiment of the treatment or prophylactic method of the present invention is characterized in that said somatostatin-related disease is at least one disease selected from the group consisting of acromegaly, gigantism, Alzheimer's disease and other forms of dementia, cancer, hormone-producing tumor, endocrine tumor, carcinoid, VIPoma, insulinoma, glucagonoma, Cushing's disease, hormone secretion defect, diabetes and complications thereof, pains, arthritis, diarrhea, gastric ulcer, inflammatory bowel disease, irritable bowel syndrome, gastrointestinal obstruction, ileus, postoperative restenosis, radiation damage, eye disease, dry eye, glaucoma, interstitial keratitis, iritis, cataract, and conjunctivitis.
Effects of the Invention
The glycosylated polypeptide of the present invention has affinity towards somatostatin receptors, and has significantly improved stability in blood compared to somatostatin by having at least two sugar chains in the polypeptide. Moreover, the glycosylated polypeptide of the present invention can be employed for treating a somatostatin-related disease by virtue of having the above characteristics.
Moreover, since the sugar chain added onto the glycosylated somatostatin of the present invention is easily degraded in vivo, no drug-induced suffering is caused to a living body by accumulation thereof.
Moreover, a part or all of the sugar chains added onto the glycosylated somatostatin of the present invention is a sugar chain present in the living body of e.g. mammals and birds including humans or a modified version thereof, and the possibility of showing side effects or antigenicity when administered in vivo is low. There is less concern for e.g. an allergic reactions or antibody production to occur and thereby losing drug effect.
Further, since many of the sugar chains employed herein are relatively short, those having uniform structure can be obtained without going through complex manufacturing steps.
Accordingly, a pharmaceutical grade high quality glycosylated polypeptide having somatostatin activity can be stably obtained in a large scale.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1D show each compound name of the glycosylated polypeptides of an embodiment of the present invention and the amino acid sequence information of the glycosylated polypeptide having said compound name. Moreover, FIG. 1E is a graph showing the IC50 value in regards to cAMP production suppressing action (agonist activity) when somatostatin receptor expression cells were treated with SRIF14, SRIF28, and the glycosylated polypeptides of an embodiment of the present invention.
FIG. 2 is graphs showing the plasma concentration transition of each polypeptide when SRIF28 and S1C(disialo)-SRIF28 were administered intravenously or subcutaneously to rats. In FIG. 2, the graph on the left is a graph showing the plasma concentration transition of each polypeptide when administered intravenously, and the graph on the right is a graph showing the plasma concentration transition of each polypeptide when administered subcutaneously.
FIG. 3 is graphs showing the plasma concentration transition when SRIF28, S1C(disialo)-SRIF28, N5C(disialo)-SRIF28, and S1C(disialo).N5C(disialo)-SRIF28 were administered intravenously and subcutaneously to rats. In FIG. 3, the graph on the left is a graph showing the plasma concentration transition of each polypeptide when administered intravenously, and the graph on the right is a graph showing the plasma concentration transition of each polypeptide when administered subcutaneously.
FIG. 4 is graphs showing the plasma concentration transition when SRIF28, S1C(disialo)-SRIF28, S1C(disialo).R13C(disialo)-SRIF28, and S1C(disialo).N5C(disialo).A9C(disialo)-SRIF28 were administered intravenously and subcutaneously to rats. In FIG. 4, the graph on the left is a graph showing the plasma concentration transition of each polypeptide when administered intravenously, and the graph on the right is a graph showing the plasma concentration transition of each polypeptide when administered subcutaneously.
FIG. 5 is graphs showing the plasma concentration transition when S1C(disialo)-SRIF28, S1C(disialo).N5C(disialo).A9C(disialo)-SRIF28, 3C(disialo)-SRIF28, 5C(disialo)-SRIF28, and 10C(disialo)-SRIF28 were administered intravenously and subcutaneously to rats. In FIG. 5, the graph on the left is a graph showing the plasma concentration transition of each polypeptide when administered intravenously, and the graph on the right is a graph showing the plasma concentration transition of the polypeptides when administered subcutaneously.
FIG. 6 is graphs showing the plasma concentration transition when S1C(disialo)-SRIF28, N5C(disialo)-SRIF28, A9C(disialo)-SRIF28, S1C(disialo) N5C(disialo)-SRIF28, N5C(disialo) A9C(disialo)-SRIF28, and S1C(disialo) N5C(disialo) A9C(disialo)-SRIF28 were administered intravenously and subcutaneously to rats. In FIG. 6, the graph on the left is a graph showing the plasma concentration transition of each polypeptide when administered intravenously, and the graph on the right is a graph showing the plasma concentration transition of each polypeptide when administered subcutaneously.
FIG. 7 is graphs showing the plasma concentration transition when S1C(disialo)-SRIF28, S1-2C(disialo)-SRIF28, and S1-3C(disialo)-SRIF28 were administered intravenously and subcutaneously to rats. In FIG. 7, the graph on the left is a graph showing the plasma concentration transition of each polypeptide when administered intravenously, and the graph on the right is a graph showing the plasma concentration transition of each polypeptide when administered subcutaneously.
FIG. 8 is graphs showing the plasma concentration transition when S1C(disialo)-SRIF28, S1C(disialo(amide))-SRIF28, and S1C(disialo(aminoethylamide))-SRIF28 were administered intravenously and subcutaneously to rats. In FIG. 8, the graph on the left is a graph showing the plasma concentration transition of each polypeptide when administered intravenously, and the graph on the right is a graph showing the plasma concentration transition of each polypeptide when administered subcutaneously.
FIG. 9 is graphs showing the plasma concentration transition when S1C(disialo)-SRIF28, S1C(disialo)-D-Trp22-SRIF28, and S1C(disialo(Bn))-SRIF28 were administered intravenously and subcutaneously to rats. In FIG. 9, the graph on the left is a graph showing the plasma concentration transition of each polypeptide when administered intravenously, and the graph on the right is a graph showing the plasma concentration transition of each polypeptide when administered subcutaneously.
FIG. 10 is graphs showing the plasma concentration transition when S1C(disialo)-SRIF28, R13C(disialo)-SRIF28, and K14C(disialo)-SRIF28 were administered intravenously and subcutaneously to rats. In FIG. 10, the graph on the left is a graph showing the plasma concentration transition of each polypeptide when administered intravenously, and the graph on the right is a graph showing the plasma concentration transition of each polypeptide when administered subcutaneously.
FIG. 11 is graphs showing the plasma concentration transition when S1C(disialo)-SRIF28, E12C(disialo)-SRIF28, N19C(disialo)-SRIF28, 29C(disialo)-SRIF28, S1C(monosialo)-SRIF28, and S1C(asialo)-SRIF28 were administered intravenously and subcutaneously to rats. In FIG. 11, the graph on the left is a graph showing the plasma concentration transition of each polypeptide when administered intravenously, and the graph on the right is a graph showing the plasma concentration transition of each polypeptide when administered subcutaneously.
FIG. 12 is graphs showing the plasma concentration transition when S1C(disialo)-SRIF28, K14C(disialo)-SRIF28, and C(disialo)-SRIF14 were administered intravenously and subcutaneously to rats. In FIG. 12, the graph on the left is a graph showing the plasma concentration transition of each polypeptide when administered intravenously, and the graph on the right is a graph showing the plasma concentration transition of each polypeptide when administered subcutaneously.
FIG. 13 is graphs showing the plasma concentration transition when C(disialo)-SRIF14, C(disialo)-C12 linker-SRIF14, and C(disialo)-PEG linker-SRIF14 were administered intravenously and subcutaneously to rats. In FIG. 13, the graph on the left is a graph showing the plasma concentration transition of each polypeptide when administered intravenously, and the graph on the right is a graph showing the plasma concentration transition of each polypeptide when administered subcutaneously.
FIG. 14 is graphs showing the plasma concentration transition when SRIF28, S1C(disialo)-SRIF28, S1C(asialo)-SRIF28, S1C(diGlcNAc)-SRIF28, S1C(diMan)-SRIF28, and S1C(GlcNAc)-SRIF28 were administered intravenously and subcutaneously to rats. In FIG. 14, the graph on the left is a graph showing the plasma concentration transition of each polypeptide when administered intravenously, and the graph on the right is a graph showing the plasma concentration transition of each polypeptide when administered subcutaneously.
FIG. 15 is graphs showing the plasma concentration transition when S1C(disialo)-SRIF28, S1C(trisialo)-SRIF28, S1C(tetrasialo)-SRIF28, S1-2C(disialo)-SRIF28, and S1C(Asn(disialo))-SRIF28 were administered intravenously and subcutaneously to rats. In FIG. 15, the graph on the left is a graph showing the plasma concentration transition of each polypeptide when administered intravenously, and the graph on the right is a graph showing the plasma concentration transition of each polypeptide when administered subcutaneously.
FIG. 16 is graphs showing the plasma concentration transition when SRIF28, S1C(disialo)-SRIF28, S1-2C(disialo)-SRIF28, S1-3C(disialo)-SRIF28, and S1-4C(disialo)-SRIF28 were administered intravenously and subcutaneously to rats. In FIG. 16, the graph on the left is a graph showing the plasma concentration transition of each polypeptide when administered intravenously, and the graph on the right is a graph showing the plasma concentration transition of each polypeptide when administered subcutaneously.
FIG. 17 is graphs showing the plasma concentration transition when SRIF14, C(disialo)-SRIF14, C(disialo)-K-SRIF14, C(disialo)-R—K-SRIF14, 2C(disialo)-R—K-SRIF14, and 3C(disialo)-R—K-SRIF14 were administered intravenously and subcutaneously to rats. In FIG. 17, the graph on the left is a graph showing the plasma concentration transition of each polypeptide when administered intravenously, and the graph on the right is a graph showing the plasma concentration transition of each polypeptide when administered subcutaneously.
FIG. 18 is graphs showing the plasma concentration transition when S1C(asialo)-SRIF28, S1-2C(asialo)-SRIF28, and S1-3C(asialo)-SRIF28 were administered intravenously and subcutaneously to rats. In FIG. 18, the graph on the left is a graph showing the plasma concentration transition of each polypeptide when administered intravenously, and the graph on the right is a graph showing the plasma concentration transition of each polypeptide when administered subcutaneously.
FIG. 19 is graphs showing the plasma concentration transition when S1C(disialo)-SRIF28, S1-2C(disialo)-SRIF28, S1-2C(asialo)-SRIF28, S1-2C(disialo(amide))-SRIF28, S1-2C(disialo(Bn))-SRIF28, and C(disialo(aminoethylamide))-S1C(disialo)-SRIF28 were administered intravenously and subcutaneously to rats. In FIG. 19, the graph on the left is a graph showing the plasma concentration transition of each polypeptide when administered intravenously, and the graph on the right is a graph showing the plasma concentration transition of each polypeptide when administered subcutaneously.
FIG. 20 is a graph showing the result of plasma stability test employing rat plasma for the glycosylated polypeptides of an embodiment of the present invention.



DESCRIPTION OF EMBODIMENTS
A “Somatostatin” herein refers to SRIF14 consisting of a sequence of 14 amino acids or SRIF28 consisting of a sequence of 28 amino acids.
In the present specification, the N-terminal Ser in the SRIF28 amino acid sequence will be set to as position 1, and the C-terminal Cys as position 28. SRIF14 is in perfect match with the amino acid sequence of positions 15 to 28 in the SRIF28 amino acid sequence. Note that position 15 of the SRIF28 amino acid sequence is Ala, and the N-terminal Ala in the SRIF14 amino acid sequence (SEQ ID NO. 1) will be set as position 15 in correspondence with position 15 of SRIF28. SRIF14 and SRIF28 have a disulfide bond at Cys at position 17 and Cys at position 28.
SRIF14 has the amino acid sequence below (SEQ ID NO. 1). In the amino acid sequence below, 15 in “Ala15” means Ala at position 15.
Ala15-Gly16-Cys17-Lys18-Asn19-Phe20-Phe21-Trp22-Lys23-Thr24-Phe25-Thr26-Ser27-Cys28 (SEQ ID NO. 1)
SRIF28 has the amino acid sequence below (SEQ ID NO. 2). Ser1-Ala2-Asn3-Ser4-Asn5-Pro6-Ala7-Met8-Ala9-Pro10-Arg11-Glu12-Arg13-Lys14-Ala15-Gly16-Cys17-Lys18-Asn19-Phe20-Phe21-Trp22-Lys23-Thr24-Phe25-Thr26-Ser27-Cys28 (SEQ ID NO. 2)
An “amino acid” herein is employed in its broadest meaning, and includes not only natural amino acids but also non-natural amino acids such as amino acid variants and derivatives. Those skilled in the art will recognize in light of this broad definition that examples of amino acids herein include, e.g., natural proteinogenic L-amino acids; D-amino acids; chemically modified amino acids such as amino acid variants and derivatives; natural non-proteinogenic amino acids such as norleucine, 3-alanine, and ornithine; and chemically synthesized compounds having properties well-known in the art characteristic of amino acids. Examples of non-natural amino acids include an α-methylamino acid (such as α-methylalanine), a D-amino acid, a histidine-like amino acid (such as 2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine, and α-methyl-histidine), an amino acid having excess methylenes on the side chain (“homo” amino acid), and an amino acid in which the carboxylic functional group amino acid in the side chain is substituted with a sulfonate group (such as cysteic acid). Some of the somatostatin analogs having affinity towards somatostatin receptors are known to comprise a non-natural amino acid. In a preferred aspect, the amino acid contained in the compound of the present invention consists only of natural amino acids.
As used herein, when one of the amino acids or a few amino acids are said to be deleted, substituted, or added, the number of amino acids substituted etc. is not particularly limited as long as affinity towards somatostatin receptors is retained, but is 1-9, preferably 1-5, and more preferably approximately 1-3 amino acids, or 20% or less and preferably 10% or less of the entire length. The amino acid to be substituted or added may be a natural amino acid, a non-natural amino acid, or an amino acid analog, preferably a natural amino acid. Examples of somatostatin peptides having one of the amino acids or a few amino acids deleted, substituted, or added include, e.g., a somatostatin peptide having Trp at position 22 substituted with a D-form Trp (D-Trp), Asn at position 19 deleted (J. Med. Chem., 2001, 44, 2238-2246), Phe at position 25 substituted with Tyr, and Met at position 8 substituted with Leu (Endocrinology, 1982, 10:1049-1051).
A “SRIF14 or SRIF28 analog” herein includes a polypeptide structurally similar to somatostatin and/or a polypeptide having an overlapping structure with somatostatin, e.g. a polypeptide having one of the amino acids or a few amino acids of somatostatin conservatively substituted, a modified somatostatin, a somatostatin fragment having affinity towards somatostatin receptors, and an elongated somatostatin having affinity towards somatostatin receptors.
“Having one of the amino acids or a few amino acids conservatively substituted” herein refers to an amino acid substitution in which the hydrophylicity and/or hydrophobicity index are similar between the original amino acid and the amino acid to be substituted, and wherein apparent reduction or dissipation of affinity towards somatostatin receptors before and after such substitution is not caused.
A “modified somatostatin” herein is a modified version of somatostatin including a naturally-occurring variant of somatostatin or an artificially modified compound of somatostatin. Examples of such modifications include, e.g., alkylation, acylation (such as acetylation), amidation, carboxylation, ester formation, disulfide bond formation, glycosylation, lipidation, phosphorylation, hydroxylation, and binding of a labeling component of one or more amino acid residues of somatostatin.
A “somatostatin fragment having affinity towards somatostatin receptors” herein is a peptide having one or more amino acids deleted from the N- and/or C-terminals of somatostatin which maintains affinity towards somatostatin receptors.
An “elongated somatostatin having affinity towards somatostatin receptors” herein is a peptide having one or more amino acids added to the N- and/or C-terminals of SRIF28 or SRIF14 which maintains affinity towards somatostatin receptors.
The glycosylated polypeptide of the present invention comprises a glycosylated polypeptide which is a polypeptide consisting of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO. 1; a polypeptide consisting of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO. 2; or a polypeptide having an amino acid further added to the N- or C-terminal of these polypeptides; wherein at least one amino acid is substituted with a glycosylated amino acid, and the polypeptide has affinity towards somatostatin receptors.
A polypeptide having homology with SEQ ID NO. 1 or 2 can have preferably 80% or more, 85% or more, 90% or more, and 95% or more homology, as long as it has affinity towards somatostatin receptors.
The glycosylated polypeptide of the present invention also comprises a polypeptide having an amino acid further added to the N- and/or C-terminals of SRIF14 or SRIF28 as described above.
In the glycosylated polypeptide of the present invention, the amino acid further added to the N-terminal of SRIF14 is not particularly limited as long as it maintains affinity towards somatostatin receptors. For example, 1 or more to 20 or less amino acids can be added.
Here, the amino acid further added to the N-terminal of SRIF14 can be represented by X—Y—. Y is an amino acid that binds directly to the N-terminal amino acid of SRIF14 polypeptide, and Y consists of any of amino acid sequences (1)-(15) below:
(1) Lys,
(2) Arg-Lys,
(3) Glu-Arg-Lys,
(4) Arg-Glu-Arg-Lys (SEQ ID NO. 171),
(5) Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 172),
(6) Ala-Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 173),
(7) Met-Ala-Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 174),
(8) Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 175),
(9) Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 176),
(10) Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 177),
(11) Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 178),
(12) Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 179),
(13) Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 180),
(14) Ser-Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 181), and
(15) a sequence having one or a few amino acids deleted, substituted, or added from/to the above sequences (2)-(14).
Moreover, X is any amino acid from 1 or more to 6 or less, and shows any 1, 2, 3, 4, 5, or 6 amino acids. Preferably, X is a glycosylated amino acid, and more preferably glycosylated Asn or glycosylated Cys.
In the glycosylated polypeptide of the present invention, the amino acid further added to the N-terminal of SRIF28 is not particularly limited as long as it maintains affinity towards somatostatin receptors. For example, any amino acids from 1 or more to 15 or less can be added, and any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids can be added. The amino acid further added to the N-terminal of SRIF28 is preferably a glycosylated amino acid, and more preferably glycosylated Asn or glycosylated Cys. Moreover, all of the any 1 or more to 15 or less amino acids can be glycosylated amino acids.
In the glycosylated polypeptide of the present invention, the amino acid further added to the C-terminal of SRIF14 or SRIF28 is not particularly limited as long as it maintains affinity towards somatostatin receptors. For example, any amino acids from 1 or more to 6 or less can be added, and any 1, 2, 3, 4, 5, or 6 amino acids can be added. The amino acid further added to the C-terminal of SRIF14 or SRIF28 is preferably a glycosylated amino acid, and more preferably glycosylated Asn or glycosylated Cys. Moreover, all of the any 1 or more to 6 or less amino acids can be glycosylated amino acids.
A “peptide having one or a few amino acids further added to the N-terminal side of somatostatin (position 1 of SRIF28 or position 15 of SRIF14)” herein refers to, in the case of SRIR28, those having any amino acid or glycosylated amino acid further added to the N-terminal Ser at position 1. Moreover, in the case of SRIF14, it refers to those having any amino acid or glycosylated amino acid further added to the N-terminal Ala at position 15.
Similarly, a “peptide having one or a few amino acids further added to the C-terminal side (position 28 of SRIF28 or SRIF14)” refers to those having any amino acid or glycosylated amino acid further added to Cys at position 28 of SRIF28 or SRIF14.
An amino acid can also be further added to the glycosylated polypeptide of the present invention at its N- or C-terminal via a linker. Examples of such linkers can include, e.g., an alkyl chain or a polyethylene glycol chain having amino and carboxy groups at the two terminals so that it can form a peptide bond with the amino acid. Examples of such linkers can include, e.g., —NH—(CH2)n—CO— (wherein n is an integer and is not limited as long as it does not inhibit the linker function of interest, but is preferably an integer from 1-15) or —NH—(CH2CH2O)m—CH2CH2—CO— (wherein m is an integer and is not limited as long as it does not inhibit the linker function of interest, but is preferably an integer from 1-7). More specifically, examples can include —NH—(CH2)11—CO— (C12 linker) or —NH— (CH2CH2O)3—CH2CH2—CO— (PEG linker). Moreover, the amino acid added to the glycosylated polypeptide via a linker is not particularly limited, but is preferably a glycosylated amino acid. Examples of a glycosylated amino acid can include glycosylated Asn or glycosylated Cys.
Moreover, in one embodiment of the present invention, an azido group can also be introduced into the glycosylated polypeptide at its N-terminal. Azidation of the N-terminal of the glycosylated polypeptide is preferred because it will allow various molecules to be selectively introduced by utilizing azide-alkyne [3+2]cycloaddition reaction or Staudinger reaction. The method for azidating the glycosylated polypeptide is not particularly limited, but it can be obtained by e.g. condensing the N-terminal of a glycopeptide synthesized on a resin in solid phase synthesis and an azido-substituted fatty acid with a condensation agent. Azido-substituted fatty acids can include 4-azidobutanoic acid, 5-azidopentanoic acid, and 6-azidohexanoic acid.
Moreover, in one embodiment of the present invention, a labeling compound can also be added to the glycosylated polypeptide at its N-terminal. Examples of the labeling compound used herein can include, but is not limited to, e.g. biotin, fluorescent dyes, and metal ion chelators. The labeling compound can be directly bound to the N-terminal of the glycopeptide, or it can also be bound to the N-terminal of the glycopeptide via a linker. For example, by adding biotin as the labeling compound to the N-terminal of the glycopeptide, strong binding with avidin can be utilized to enable application as research reagents, clinical test agents, or missile therapy.
The addition of a labeling compound to the glycosylated polypeptide of the present invention can be performed by a conventional method well-known to those skilled in the art. For example, the N-terminal of the glycosylated polypeptide on the resin in solid phase synthesis and a labeling compound can be condensed with a condensation agent.
The “glycosylated polypeptide” of the present invention is characterized in that at least two amino acids are substituted by glycosylated amino acids.
A “glycosylated polypeptide” herein includes e.g. a polypeptide having at least two amino acids of somatostatin substituted with glycosylated amino acids and a polypeptide having at least two amino acids substituted with glycosylated amino acids in the above somatostatin analog, each of which is included in the glycosylated polypeptide of the present invention even if one of the amino acids or a few amino acids other than the glycosylated amino acid is further deleted, substituted, or added. A peptide having at least two amino acids substituted with glycosylated amino acids in a peptide in which the C-terminal thereof is amidated (such as SRIF14NH2 having the amino acid sequence of Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-NH2 (SEQ ID NO. 3) or SRIF28NH2 having the amino acid sequence of Ser-Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-NH2 (SEQ ID NO. 4) is also included in the glycosylated polypeptide of the present invention.
Further, a salt of these peptides is also included in glycosylated polypeptides.
A salt as used herein may be any of acid addition salts or base addition salts. Acids generally employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, and phosphoric acid, as well as organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carboxylic acid, succinic acid, citric acid, benzoic acid, and acetic acid. Base addition salts include salts derived from an inorganic base such as ammonium hydroxide or alkali or alkaline earth metal hydroxides, carbonates, and bicarbonates. In particular, pharmaceutically acceptable salts are preferred.
A “glycosylated (sugar chain attached) amino acid” herein is an amino acid having a sugar chain bound thereto, and the sugar chain and the amino acid may be bound via a linker. The binding site of the sugar chain and the amino acid is not particularly restricted, but the amino acid is preferably bound to the reducing terminal of the sugar chain.
The type of amino acid to which the sugar chain binds is not particularly limited, and any of natural amino acids and non-natural amino acids can be employed. With respect to the glycosylated amino acids having identical or similar structure as those present as glycopeptides (glycoproteins) in vivo, the glycosylated amino acid is preferably glycosylated Asn, like N-linked sugar chain as well as glycosylated Ser and glycosylated Thr, and glycosylated Asn, like O-linked sugar chain is particularly preferred.
Moreover, when the sugar chain and the amino acid are bound via a linker, with respect to easy binding with the linker, the amino acid of the glycosylated amino acid is preferably an amino acid having two or more carboxy groups in a molecule such as aspartic acid and glutamic acid, an amino acid having two or more amino groups in a molecule such as lysine, arginine, histidine, and tryptophan, an amino acid having a hydroxyl group in the molecule such as serine, threonine, and tyrosine, an amino acid having a thiol group in the molecule such as cysteine, and an amino acid having an amide group in the molecule such as asparagine and glutamine.
In particular, with respect to reactivity, aspartic acid, glutamic acid, lysine, arginine, serine, threonine, cysteine, asparagine, and glutamine are preferred.
In any glycosylated polypeptide of the present invention, it is thought that if the sugar chain structure, the structure other than the sugar chain, the number of addition sites for the sugar chain, and the number of sugar chains added are identical, there is no major difference in the half-life in blood of the glycosylated polypeptide of the present invention whether the glycosylated amino acid is glycosylated Asn (without a linker) or glycosylated Cys (with a linker).
When the sugar chain and the amino acid are bound via a linker, linkers employed in the art can be widely used, examples of which can include, e.g., —NH—(CO)—(CH2)a—CH2—(wherein a is an integer and is not limited as long as it does not inhibit the linker function of interest, but is preferably an integer from 0-4), C1-10 polymethylene, —CH2—R— (wherein R is a group produced by having one hydrogen atom detached from a group selected from the group consisting of an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, a substituted alkynyl, an aryl, a substituted aryl, a carbocyclic group, a substituted carbocyclic group, a heterocyclic group, and a substituted heterocyclic group), and —(CO)—(CH2)a—(CO)— (wherein a is an integer and is not limited as long as it does not inhibit the linker function of interest, but is preferably an integer from 0-4).
In the glycosylated amino acid, when the sugar chain and the amino acid on the somatostatin backbone are bound via a linker, it is preferred that the linker also comprises an amino acid at the sugar chain terminal. The type of amino acid is not particularly limited, but a preferred example can include Asn.
The manufacturing method of the glycosylated polypeptide of the present invention is not to be limited in any way by a description therefor (such as the description a “glycosylated polypeptide having an amino acid substituted with a glycosylated amino acid”), and a glycosylated polypeptide manufactured with any of methods A or B described below is included in the “glycosylated polypeptide having an amino acid substituted with a glycosylated amino acid”. Moreover, for example, a glycosylated polypeptide in which a sugar chain without any amino acid bound thereto is bound directly or via a linker to an amino acid on a peptide; a glycosylated polypeptide in which a sugar or a sugar chain is further added to the sugar chain added in the glycosylated polypeptide in order to elongate the already added sugar chain; a glycosylated polypeptide in which one or a few amino acids are bound to the amino and/or carboxy group of a glycosylated amino acid, and further linked to one or more somatostatin fragments; and a glycosylated polypeptide in which a sugar chain having an amino acid bound thereto is bound via a linker to an amino acid on a peptide are also included in the glycosylated polypeptide of the present invention, as long as the final structure matches.
In the glycosylated polypeptide of the present invention, the number of amino acids to be substituted with a glycosylated amino acid may be appropriately adjusted by e.g. bioactivity such as stability in blood or secretory suppression of growth hormone etc., the number of amino acids present in the final glycosylated polypeptide, or the molecular weight of the glycosylated polypeptide before and after glycosylation. For example, the glycosylated polypeptide of the present invention can have 2-15 amino acids in its amino acid sequence substituted to glycosylated amino acids. With respect to stability in blood, as long as the desired activity is obtained, it is preferred to substitute 2 or more, e.g. it is preferred to substitute 2-10, and more preferably to substitute 2-3 amino acids. In general, in a glycosylated polypeptide having one amino acid of somatostatin substituted with a glycosylated amino acid, stability in blood will increase when one or more of amino acids other than the glycosylated amino acid are further substituted by glycosylated amino acids. On the other hand, although affinity towards somatostatin receptors will tend to decrease, the stability in blood of the glycosylated polypeptide will increase, and it is therefore possible to compensate for or increase the decreased somatostatin activity.
Moreover, when multiple sugar chains are present in a glycosylated polypeptide, the each sugar chains can be added to consecutive amino acids, or can be added to amino acids with intervals in the amino acid sequence of the glycosylated polypeptide. Placing the sugar chains densely is preferred because there is no rapid increase in plasma concentration.
When the sugar chains are placed densely, for example, approximately 2-15 consecutive glycosylated amino acids can be added to the N-terminal of SRIF14 or SRIF28. Moreover, with respect to bioavailability, the preferred glycosylation position is adding multiple sugar chains with intervals rather than adding densely to consecutive amino acids.
In the glycosylated polypeptide of the present invention, the site for substituting an amino acid with a glycosylated amino acid can be appropriately adjusted with respect to having affinity towards at least one somatostatin receptor, and preferably, it can be appropriately adjusted with respect to having affinity towards multiple somatostatin receptors.
In one aspect of the present invention, the site for substituting an amino acid with a glycosylated amino acid can include, with respect to having affinity towards multiple receptors in SSTR1-SSTR5 among affinity of the glycosylated polypeptide towards somatostatin receptors, e.g., the amino acid corresponding to position 19 of SRIF14, the amino acids added at the 1st, 2nd, 3rd, 6th, 10th, and 14th positions from the N-terminal side among the amino acids further added to the N-terminal side of SRIF14, and the amino acids added at the 1st and 2nd positions from the C-terminal side among the amino acids further added to the C-terminal side of SRIF14.
Preferably, it can include the amino acids added at the 3rd, 6th, 10th, and 14th positions from the N-terminal side among the amino acids further added to the N-terminal side of SRIF14.
Moreover, similarly with respect to having affinity towards multiple receptors in SSTR1-SSTR5, it can include the amino acids corresponding to positions 1, 5, 9, 12, 13, 14, and 19 of SRIF28, and the amino acids added at the 1st and 2nd positions from the C-terminal side among the amino acids further added to the C-terminal of SRIF28. Preferably, it can include the amino acids corresponding to positions 1, 5, 9, and 12 of SRIF28.
Moreover, particularly, examples of substitution of two or more amino acids of the glycosylated polypeptide of the present invention with glycosylated amino acids can include, with respect to the glycosylated polypeptide having affinity towards multiple somatostatin receptors, e.g., substitutions of the amino acids added at the 10th and 14th positions from the N-terminal side among the amino acids further added to the N-terminal side of SRIF14; the amino acids added at the 6th and 10th positions from the N-terminal side among the amino acids further added to the N-terminal side of SRIF14; the amino acids added at the 2nd and 14th positions from the N-terminal side among the amino acids further added to the N-terminal side of SRIF14; the amino acids added at the 14th and 15th positions from the N-terminal side among the amino acids further added to the N-terminal side of SRIF14; the amino acids added at the 14th, 15th, and 16th positions from the N-terminal side among the amino acids further added to the N-terminal side of SRIF14; and the amino acids added at the 6th, 10th, and 14th positions from the N-terminal side among the amino acids further added to the N-terminal side of SRIF14.
Preferably, it can include, e.g., substitutions of the amino acids added at the 10th and 14th positions from the N-terminal side among the amino acids further added to the N-terminal side of SRIF14; the amino acids added at the 6th and 10th positions from the N-terminal side among the amino acids further added to the N-terminal side of SRIF14; the amino acids added at the 14th and 15th positions from the N-terminal side among the amino acids further added to the N-terminal side of SRIF14; the amino acids added at the 14th, 15th, and 16th positions from the N-terminal side among the amino acids further added to the N-terminal side of SRIF14; and the amino acids added at the 6th, 10th, and 14th positions from the N-terminal side among the amino acids further added to the N-terminal side of SRIF14.
Moreover, similarly, examples of substitution of two or more amino acids with glycosylated amino acids can include, with respect to the glycosylated polypeptide having affinity towards multiple somatostatin receptors, e.g., substitutions of the amino acids corresponding to positions 1 and 5; the amino acids corresponding to positions 5 and 9; the amino acids corresponding to positions 1 and 13; the amino acid corresponding to position 1 and the amino acid added at the 1st position from the N-terminal side among the amino acids further added to the N-terminal side of position 1; the amino acid corresponding to position 1 and the amino acids added at the 1st and 2nd positions from the N-terminal side among the amino acids further added to the N-terminal side of position 1; and the amino acids corresponding to positions 1, 5, and 9; preferably, substitutions of the amino acids corresponding to positions 1 and 5; substitutions of the amino acids corresponding to positions 5 and 9; substitution of the amino acid corresponding to position 1 and the amino acid added at the 1st position among the amino acids further added to the N-terminal side of position 1; the amino acid corresponding to position 1 and the amino acids added at the 1st and 2nd positions from the N-terminal side among the amino acids further added to the N-terminal side of position 1; and substitutions of the amino acids corresponding to positions 1, 5, and 9 of SRIF28.
In one aspect of the present invention, the site for substituting an amino acid with a glycosylated amino acid can include, with respect to the stability in blood of the glycosylated polypeptide, e.g., the amino acid corresponding to position 19 of SRIF14, the amino acids added at the 1st, 2nd, 3rd, 6th, 10th, 14th positions from the N-terminal side among the amino acids further added to the N-terminal side of SRIF14, and the amino acids added at the 1st and 2nd positions from the C-terminal side among the amino acids further added to the C-terminal side of SRIF14. Preferably, it can include the amino acid corresponding to position 19 of SRIF14, the amino acids added at the 1st and 2nd positions from the N-terminal side among the amino acids further added to the N-terminal side of SRIF14, and the amino acids added at the 1st and 2nd positions from the C-terminal side among the amino acids further added to the C-terminal side of SRIF14. More preferably, it is the amino acid corresponding to position 19 of SRIF14, the amino acid added at the 1st position from the N-terminal side among the amino acids further added to the N-terminal side of SRIF14, and the amino acid added at the 1st position from the C-terminal side among the amino acids further added to the C-terminal side of SRIF14.
Moreover, similarly with respect to the stability in blood of the glycosylated polypeptide, it is e.g. one or more amino acids selected from the group consisting of the amino acids corresponding to positions 1, 5, 9, 12, 13, 14, and 19 of SRIF28 and the amino acids added at the 1st and 2nd positions from the C-terminal side among the amino acids further added to the C-terminal side of SRIF28, preferably one or more amino acids selected from the group consisting of the amino acids corresponding to positions 13, 14, and 19 of SRIF28 and the amino acids added at the 1st and 2nd positions from the C-terminal side among the amino acids further added to the C-terminal side of SRIF28, and particularly preferably one or more amino acids selected from the group consisting of the amino acids corresponding to positions 14 and 19 of SRIF28 and the amino acid added at the 1st position from the C-terminal side among the amino acids further added to the C-terminal side of SRIF28. In particular, substitution of an amino acid at a distal site from the N-terminal side of SRIF28 is also preferred.
Here, an “amino acid corresponding to a particular amino acid” refers to an amino acid at the same position corresponding to the amino acid sequence of SRIF14 or SRIF28, as long as there is no addition or deletion etc. of an amino acid in the glycosylated polypeptide. Moreover, if an addition or deletion of an amino acid is present in the amino acid sequence of a glycosylated polypeptide, it refers to the amino acid at the position that takes into account the shift on the amino acid sequence by the addition or deletion of an amino acid. For example, in a glycosylated polypeptide having the sequence Ser1-Ala2-Asn3-Ser4-(SEQ ID NO:182) at positions 1 to 4, when one amino acid (Trp) is added between the amino acids at positions 2 and 3 (Ser-Ala-Trp-Asn-Ser-) (SEQ ID NO:183), the amino acid corresponding to the amino acid at position 3 (Asn) refers to the amino acid (Asn) in the glycosylated polypeptide which has been shifted one to the C-terminal side by the insertion of Trp.
In one aspect of the present invention, the amino acid substituted with an glycosylated amino acid is preferably one or more amino acids selected from an amino acid other than the amino acids corresponding to positions 17, 21, 22, 23, and 28 of SRIF28, and in particular, it is more preferably one or more amino acids selected from an amino acid other than the amino acids corresponding to positions 17, 22, 23, and 28 of SRIF28.
In one aspect of the present invention, when two or more amino acids are substituted with glycosylated amino acids, any combination above can be employed for the site for substituting an amino acid with a glycosylated amino acid but is not limited thereto. For example, a combination of one site being selected from the above preferred sites and other sites being selected from any site of SRIF14 or SRIF28; or a combination of one site being selected from the above preferred sites and other sites being selected from any of one or a few amino acids further added to the N-terminal (position 1 of SRIF28 or position 15 of SRIF14) or C-terminal of somatostatin are also included in one preferred aspect of the present invention. Moreover, in a glycosylated polypeptide, it is more preferred that the two or more amino acids are substituted with glycosylated amino acids at the amino acids specified above. Moreover, it is further preferred that all the glycosylated amino acids included in the glycosylated polypeptide are substituted with glycosylated amino acids at the amino acids specified above.
Among the glycosylated polypeptides of the present invention, the combination of the two substituted amino acids in a glycosylated polypeptide having two amino acids substituted with glycosylated amino acids can be, e.g., two amino acids selected from the group consisting of the amino acid corresponding to position 19 of SRIF14, the 1st, 2nd, 3rd, 6th, 10th, and 14th amino acids from the N-terminal side among the amino acids further added to the N-terminal side of SRIF14, and the 1st and 2nd amino acids from the C-terminal side among the amino acids further added to the C-terminal side of SRIF14. Moreover, similarly, it can include two amino acids selected from the group consisting of the amino acids corresponding to positions 1, 5, 9, 12, 13, 14, and 19 of SRIF28, as well as the 1st amino acid further added to the C-terminal of SRIF28 and the 2nd amino acid further added to the C-terminal of SRIF28.
Similarly, substitution positions for a glycosylated polypeptide having three amino acids substituted with glycosylated amino acids or a glycosylated polypeptide having four or more amino acids substituted with glycosylated amino acids can include a combination of three or four or more amino acids selected from the group consisting of the amino acids specified above. However, the glycosylated polypeptide of the present invention is not limited to the combinations listed above, and comprises other combinations as long as it has affinity towards somatostatin receptors and improved stability in blood compared to a naturally-occurring somatostatin.
In one aspect of the present invention, the site where a deletion, substitution, or addition of an amino acid other than glycosylated amino acids occurs is preferably e.g. one or more amino acids selected from an amino acid other than the amino acids corresponding to positions 17, 22, 23, and 28 of SRIF28.
A “sugar chain” herein refers to a compound made from a string of one or more unit sugars (monosaccharides and/or derivatives thereof). When there is a string of two or more unit sugars, each unit sugar is bound with each other by a dehydration condensation with a glycoside bond in between. Such sugar chains include, but are not limited to e.g. a wide range such as monosaccharides and polysaccharides contained in vivo (glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), sialic acid, and conjugates and derivatives thereof), as well as a sugar chain degraded or derived from conjugated biomolecules such as degraded polysaccharides, glycoproteins, proteoglycans, glycosaminoglycans, and glycolipids. The sugar chain may be linear or branched.
Moreover, a “sugar chain” herein also includes a sugar chain derivative, and examples of sugar chain derivatives include, but are not limited to, a sugar chain wherein the sugar constituting the sugar chain is e.g. a sugar having a carboxy group (such as aldonic acid in which C-position 1 is oxidized to become a carboxylic acid (such as D-gluconic acid which is oxidized D-glucose) and uronic acid in which the terminal C atom has become a carboxylic acid (D-glucuronic acid which is oxidized D-glucose)), a sugar having an amino group or an amino group derivative (such as acetylated amino group) (such as N-acetyl-D-glucosamine and N-acetyl-D-galactosamine), a sugar having both amino and carboxy groups (such as N-acetylneuraminic acid (sialic acid) and N-acetylmuramic acid), a deoxylated sugar (such as 2-deoxy-D-ribose), a sulfated sugar comprising a sulfate group, and a phosphorylated sugar comprising a phosphate group.
A preferred sugar chain herein is a sugar chain that increases the stability in blood of somatostatin and will not dissipate affinity towards somatostatin receptors when added to somatostatin (when substituted with an amino acid of somatostatin in the form of a glycosylated amino acid). In an aspect of the present invention, a preferred sugar chain is a sugar chain that can maintain affinity towards multiple receptors of somatostatin, and further preferably a sugar chain that can maintain affinity towards all the receptors of somatostatin when added to somatostatin (when substituted with an amino acid of somatostatin in the form of a glycosylated amino acid).
The sugar chain in the glycosylated polypeptide of the present invention is not particularly limited, and may be a sugar chain that exists as a glycoconjugate in vivo (such as a glycopeptide (or a glycoprotein), a proteoglycan, and a glycolipid), or it may be a sugar chain that does not exist as a glycoconjugate in vivo.
A sugar chain that exists as a glycoconjugate in vivo is preferred with respect to the fact that the glycosylated polypeptide of the present invention is administered in vivo. Examples of such sugar chains include N- or O-linked sugar chains which are sugar chains bound to a peptide (or a protein) in vivo as a glycopeptide (or a glycoprotein). An N-linked sugar chain is preferably employed. N-linked sugar chains can include, e.g., a high-mannose form, a complex form, or a hybrid form, particularly preferably a complex form.
Examples of preferred complex-type sugar chains used herein include, e.g., a sugar chain represented by the following general formula:




[wherein R1 and R2 are identical or different and are:





and Ac is an acetyl group].

In the glycosylated polypeptide of the present invention, the sugar chain may be bound to the somatostatin peptide with a method other than O- and N-linking, even if it is a sugar chain that exists as a glycoconjugate in vivo. For example, as described above, those in which the sugar chain is bound to Cys or Lys via a linker are also included in the glycosylated polypeptide of the present invention.
In one aspect of the present invention, it is preferred that the sugar chain in the glycosylated polypeptide of the present invention is a sugar chain consisting of 4 or more, for example, 5 or more, 7 or more, in particular 9 or more, or 11 or more sugars.
In one preferred aspect of the present invention, the sugar chain in the glycosylated polypeptide of the present invention is a sugar chain consisting of 5-11, 9-11, or 11 sugars.
In one preferred aspect of the present invention, the sugar chain in the glycosylated polypeptide of the present invention is a biantennary complex-type sugar chain. A complex-type sugar chain is characterized in that it comprises two or more types of monosaccharides, and has the basic structure shown below and a lactosamine structure shown by Galβ1-4GlcNAc.




A biantennary complex-type sugar chain refers to those having a monoantennary sugar chain consisting of 0-3 sugars bound to each of the two mannoses at the end of the basic structure. Examples of biantennary complex-type sugar chains are preferably e.g. a disialo sugar chain as shown below:




a monosialo sugar chain:





an asialo sugar chain:





a diGlcNAc sugar chain:





and a dimannose sugar chain:





and more preferably a disialo sugar chain.

Moreover, the complex-type sugar chain of the present invention includes not only the above biantennary complex-type sugar chains, but also a triantennary complex-type sugar chain (triple-branched complex-type sugar chain) and a tetraantennary complex-type sugar chain (quadruple-branched complex-type sugar chain). For example, triantennary and tetraantennary complex-type sugar chains can include a trisialo sugar chain represented by the structural formula below:




and a tetrasialo sugar chain represented by the structural formula below:





Moreover, triantennary and tetraantennary complex-type sugar chains can also include a sugar chain having one or more sugars deleted from the non-reducing terminal of these trisialo or tetrasialo sugar chains.

Further, the complex-type sugar chain of the present invention includes those with a fucose added. Complex-type sugar chains with a fucose added can include fucose-containing complex-type sugar chains represented by the structural formula below:







Moreover, a sugar chain having one or more sugars deleted from the non-reducing terminal of these fucose-containing complex-type sugar chains can also be included.

Moreover, a “disialo sugar chain,” a “biantennary complex-type sugar chain,” a “monosialo sugar chain,” an “asialo sugar chain,” a “diGlcNAc sugar chain,” a “dimannose sugar chain,” a “triantennary complex-type sugar chain,” a “tetraantennary complex-type sugar chain,” and a “fucose-containing complex-type sugar chain” herein include not only those shown in the above chemical formulae, but also those with a binding mode different from examples shown in the chemical formulae, and such sugar chains are also preferably employed as a sugar chain of the present invention. Examples of such sugar chains include, e.g., a disialo or monosialo sugar chain in which sialic acid and galactose are bound with an (α2→3) bond.
Moreover, when the sugar chain is one having a sialic acid at the non-reducing terminal of the sugar chain, a sugar chain having the carboxy group of the sialic acid modified can also be employed. The modification of the carboxy group of the sialic acid is preferably a group capable of dissipating the negative charge of the carboxy group or converting it into positive charge, examples of which can include, e.g., an alkylamino group, a benzyl group, an amino group, and an aminoethylamino group, having 1-30 carbons. The introduction of these modifying groups will dissipate the negative charge of the carboxyl group of the sialic acid (such as benzyl or amino group) or convert it into positive charge (such as aminoethylamino group), and it is thus possible to contemplate blood clearance or control of body distribution of glycosylated polypeptide.
Moreover, the high-mannose sugar chain employed herein is a sugar chain having 2 or more mannoses further bound to the basic structure of the complex-type sugar chain described above. Because high-mannose sugar chains are bulky, stability in blood may become higher by binding a high-mannose sugar chain to the peptide. A sugar chain comprising 5-9 mannoses such as a mammalian high-mannose sugar chain is preferred, but it may be a sugar chain comprising more mannoses such as a yeast high-mannose sugar chain. Examples of high-mannose sugar chains preferably employed herein can include, e.g., high-mannose-5 (M-5):




and high-mannose-9 (M-9):





Preferred sugar chains herein can include, e.g., the sugar chains described below which are a sugar chain having an identical structure (a sugar chain in which the type of constituent sugar and the binding mode thereof are identical) with a sugar chain that exists in a human body as a glycoprotein bound to a protein (such as a sugar chain described in “FEBS LETTERS Vol. 50, No. 3, February 1975”), or a sugar chain having one or more sugars deleted from the non-reducing terminal of the same.





































In one preferred aspect of the present invention, the sugar chain structure of the glycosylated amino acids in the glycosylated polypeptide of the present invention can be substantially identical, or they may have different sugar chain structures. When the sugar chain structure in the glycosylated polypeptide is substantially identical, they are preferably e.g. 90% or more identical or 99% or more identical, and it is most preferable that the sugar chain structure is completely identical. As used herein, the sugar chain structure in the glycopeptide is identical refers to the fact that in a glycosylated polypeptide having two or more sugar chains added, the type of sugar constituting the sugar chain, the binding order, and the binding mode are identical in the glycopeptide when said sugar chains are compared with each other. Moreover, in one preferred aspect of the present invention, it is preferred that the sugar chains of the glycosylated polypeptide of the present invention are uniform. As used herein, the sugar chains in the glycosylated polypeptide are uniform refers to the fact that the glycosylation sites in the peptide, the type of each sugar constituting the sugar chain, the binding order, and the binding mode between sugars are identical between glycopeptides when sugar chains are compared between glycosylated polypeptides, and that at least 90% or more, preferably 95% or more, and more preferably 99% or more of the sugar chain structure is uniform. In particular, a composition etc. comprising a glycopeptide in which the sugar chains are uniform between glycopeptides has a constant quality, and is preferred particularly in fields such as pharmaceuticals manufacture or assays. The proportion of the uniform sugar chain can be measured for example by a method employing e.g. HPLC, capillary electrophoresis, NMR, and mass spectrometry.
The preferred glycosylated polypeptides herein are the glycosylated polypeptides (SEQ ID NOs. 21-26, 29, 30, 33, 34, 88, 89, 125, 127, 129, 148, 149, 157, 164, and 169) manufactured in Examples 20-25, 28, 29, 32-35, 49-51, 63-64, and 66-68 described below. In other words, in the following SRIF28 amino acid sequence:
Ser1-Ala2-Asn3-Ser4-Asn5-Pro6-Ala7-Mete-Ala9-Pro10-Arg11-Glu12-Arg13-Lys14-Ala15-Gly16-Cys17-Lys18-Asn19-Phe20-Phe21-Trp22-Lys23-Thr24-Phe25-Thr26-Ser27-Cys28 (SEQ ID NO. 2),
(a1) a glycosylated polypeptide having disialo sugar chain attached Cys substituted for Ser at position 1, and having one disialo sugar chain attached Cys further added at the N-terminal side (Example 20) (SEQ ID NO. 21);
(a2) a glycosylated polypeptide having disialo sugar chain attached Cys substituted for Ser at position 1 and Asn at position 5 (Example 21) (SEQ ID NO. 22);
(a3) a glycosylated polypeptide having disialo sugar chain attached Cys substituted for Ser at position 1 and Arg at position 13 (Example 22) (SEQ ID NO. 23);
(a4) a glycosylated polypeptide having disialo sugar chain attached Cys substituted for Asn at position 5 and Ala at position 9 (Example 23) (SEQ ID NO. 24);
(a5) a glycosylated polypeptide having disialo sugar chain attached Cys substituted for Ser at position 1, and having two disialo sugar chain attached Cys further added at the N-terminal side (Example 24) (SEQ ID NO. 25);
(a6) a glycosylated polypeptide having disialo sugar chain attached Cys substituted for Ser at position 1, Asn at position 5, and Ala at position 9 (Example 25) (SEQ ID NO. 26);
(a7) a glycosylated polypeptide having asialo sugar chain attached Cys substituted for Ser at position 1, and having one asialo sugar chain attached Cys further added at the N-terminal side (Example 28) (SEQ ID NO. 29);
(a8) a glycosylated polypeptide having asialo sugar chain attached Cys substituted for Ser at position 1, and having two asialo sugar chain attached Cys further added at the N-terminal side (Example 29) (SEQ ID NO. 30);
(a9) a glycosylated polypeptide having disialo sugar chain attached Cys substituted for Ser at position 1, and having GlcNAc-added Cys substituted for Asn at position 19 (Example 32) (SEQ ID NO. 33);
(a10) a glycosylated polypeptide having disialo sugar chain attached Cys substituted for Ser at position 1, and having dimannose glycosylated Cys substituted for Asn at position 19 (Example 33) (SEQ ID NO. 34);
(a11) a glycosylated polypeptide having disialo sugar chain attached Cys substituted for Ser at position 1, and having four disialo sugar chain attached Cys further added at the N-terminal side (Example 34) (SEQ ID NO. 88);
(a12) a glycosylated polypeptide having disialo sugar chain attached Cys substituted for Ser at position 1, and having nine disialo sugar chain attached Cys further added at the N-terminal side (Example 35) (SEQ ID NO. 89);
(a13) a glycosylated polypeptide having disialo sugar chain attached Cys substituted for Ser at position 1 and Glu at position 12 (Example 49) (SEQ ID NO. 125);
(a14) a glycosylated polypeptide having disialo sugar chain attached Cys substituted for Ser at position 1 in which the carboxy group of the sialic acid on the sugar chain is converted into carboxylic amide, and having one disialo sugar chain attached Cys further added at the N-terminal side in which the carboxy group of the sialic acid on the sugar chain is converted into carboxylic amide (Example 63) (SEQ ID NO. 148);
(a15) a glycosylated polypeptide having disialo sugar chain attached Cys substituted for Ser at position 1 in which the carboxy group of the sialic acid on the sugar chain is protected by a benzyl group, and having one disialo sugar chain attached Cys further added at the N-terminal side in which the carboxy group of the sialic acid on the sugar chain is protected by a benzyl group (Example 64) (SEQ ID NO. 149);
(a16) a glycosylated polypeptide having disialo sugar chain attached Asn substituted for Ser at position 1, and having dimannose glycosylated Cys substituted for Asn at position 19 (Example 66) (SEQ ID NO. 157);
(a17) a glycosylated polypeptide having disialo sugar chain attached Cys substituted for Ser at position 1, and having one disialo sugar chain attached Cys further added at the N-terminal side in which the carboxy group of the sialic acid on the sugar chain has formed an amide bond with one end of 1,2-ethylenediamine (Example 67) (SEQ ID NO. 164); and
(a18) a glycosylated polypeptide having disialo sugar chain attached Cys substituted for Ser at position 1, and having three disialo sugar chain attached Cys further added at the N-terminal side (Example 68) (SEQ ID NO. 169) are preferred.
Moreover, in the SRIF14 amino acid sequence:
Ala15-Gly16-Cys17-Lys18-Asn19-Phe20-Phe21-Trp22-Lys23-Thr24-Phe25-Thr26-Ser27-Cys28 (SEQ ID NO. 1),
(a19) a glycosylated polypeptide having disialo sugar chain attached Cys-disialo sugar chain attached Cys-Arg-Lys-further added at the N-terminal side of Ala at position 15 (Example 50) (SEQ ID NO. 127); and
(a20) a glycosylated polypeptide having disialo sugar chain attached Cys-disialo sugar chain attached Cys-disialo sugar chain attached Cys-Arg-Lys- further added at the N-terminal side of Ala at position 15 (Example 51) (SEQ ID NO. 129) are preferred.
The glycosylated polypeptide of the present invention can be manufactured by integrating a glycosylation step into a peptide synthesis method well-known to those skilled in the art. A method utilizing an enzyme represented by transglutaminase can also be employed for glycosylation, but since there are problems in this case such as the need for a large amount of the sugar chain to be added, the complication of purification after the final step, and the restriction for glycosylation position and the sugar chain capable of addition, it cannot be said to be a practical method for large scale manufacturing such as pharmaceuticals manufacture though it is possible to employ for synthesis of a small amount such as for assays.
As specific examples of simple manufacturing methods of the glycosylated polypeptide of the present invention which are stable manufacturing methods of a glycosylated polypeptide having uniform sugar chain structure, a method for manufacturing a glycosylated polypeptide by using glycosylated Asn as the glycosylated amino acid and applying a well-known peptide synthesis method such as solid and liquid phase synthesis (method A), and a method for manufacturing a glycosylated polypeptide by manufacturing a peptide in which any amino acid of somatostatin is substituted with Cys according to a well-known peptide synthesis method, and then adding a sugar chain to Cys by chemical synthesis (method B) will be exemplified below. Those skilled in the art is able to manufacture various glycosylated polypeptides by referring to these manufacturing methods, and the glycosylated polypeptide obtained and the manufacturing method thereof are extremely useful especially in the field of pharmaceuticals manufacture.
Moreover, these methods A and B can be performed in a combination of two or more. If it is a synthesis of a small amount such as for assays, it is also possible to further combine the above method employing a sugar chain elongation reaction by a transferase. Method A is described in International Publication No. 2004/005330 (US 2005222382 (A1)) and method B is described in International Publication No. 2005/010053 (US 2007060543 (A1)), the disclosures of which are incorporated herein by reference in their entirety. Moreover, the manufacture of sugar chains having uniform sugar chain structure employed in methods A and B are described in e.g. International Publication No. 03/008431 (US 2004181054 (A1)), International Publication No. 2004/058984 (US 2006228784 (A1)), International Publication No. 2004/058824 (US 2006009421 (A1)), International Publication No. 2004/070046 (US 2006205039 (A1)), and International Publication No. 2007/011055, the disclosures of which are incorporated herein by reference in their entirety.
Method for Manufacturing Glycosylated Polypeptide (Method A)
The glycosylated polypeptide can be manufactured by for example a solid phase synthesis employing glycosylated Asn, the outline of which is shown below.
(1) The carboxy group of an amino acid having the amino group nitrogen protected with a lipophilic protecting group is bound to a resin. In this case, since the amino group nitrogen of the amino acid is protected with a lipophilic protecting group, self-condensation between amino acids is prevented, and the resin reacts with the amino acid to cause binding.
(2) The lipophilic protecting group of the reactant obtained is detached to form a free amino group.
(3) This free amino group and the carboxy group of any amino acid having the amino group nitrogen protected with a lipophilic protecting group are subjected to amidation reaction.
(4) The above lipophilic protecting group is detached to form a free amino group.
(5) By repeating the above steps (3) and (4) for once or more, a peptide in which any number of any amino acids are linked and having a resin bound at one end and a free amino group at the other end is obtained.
(6) Finally, by cleaving the resin with an acid, a peptide having the desired amino acid sequence can be obtained.
In (1), if glycosylated Asn having the amino group nitrogen protected with a lipophilic protecting group is employed instead of the amino acid having the amino group nitrogen protected with a lipophilic protecting group, and the carboxy group of said asparagine portion is reacted with the hydroxyl group of the resin, a peptide having a glycosylated Asn at the C-terminal can be obtained.
Moreover, after (2), or after repeating (3) and (4) for any number of times that is once or more, if glycosylated Asn having the amino group nitrogen protected with a lipophilic protecting group is employed instead of the amino acid having the amino group nitrogen protected with a lipophilic protecting group in (3), a sugar chain can be added at any position.
As such, by employing glycosylated Asn having the amino group nitrogen protected with a lipophilic protecting group instead of the amino acid having the amino group nitrogen protected with a lipophilic protecting group for twice or more times in any of steps (1) and (3), a peptide having a sugar chain added at any two or more positions can be obtained.
After binding the glycosylated amino acid, if the lipophilic protecting group is detached to form a free amino group, and step (6) is performed immediately thereafter, a peptide having a glycosylated Asn at the N-terminal can be obtained.
The resin may be a resin generally used in solid phase synthesis, and for example, 2-chlorotrityl chloride resin (from Merck) functionalized with chlorine, Amino-PEGA resin (from Merck) functionalized with an amino group, NovaSyn TGT alcohol resin (from Merck), Wang resin (from Merck), or HMPA-PEGA resin (from Merck) etc. having a hydroxyl group can be employed. Moreover, a linker may exist between the Amino-PEGA resin and the amino acid, and examples of such linkers can include, e.g., 4-hydroxymethylphenoxyacetic acid (HMPA) and 4-(4-hydroxymethyl-3-methoxyphenoxy)-butylacetic acid (HMPB). H-Cys(Trt)-Trityl NovaPEG resin (from Merck) etc. in which the C-terminal amino acid is bound to the resin in advance can also be employed.
Moreover, when the C-terminal is to be amidated, it is preferred to employ e.g. Rink-Amide-PEGA resin (from Merck) functionalized with an amino group. By cleaving this resin and the peptide with an acid, the C-terminal amino acid of the peptide can be amidated.
In the binding between the resin and the amino acid having the amino group nitrogen protected with a lipophilic protecting group, for example, in order to use a resin having a hydroxyl group or a resin functionalized with chlorine, the carboxy group of the amino acid is bound to the resin via an ester bond. Moreover, if a resin functionalized with an amino group is to be used, the carboxy group of the amino acid is bound to the resin via an amide bond.
The 2-chlorotrityl chloride resin is preferred in that it can prevent the racemization of the terminal Cys when elongating the peptide chain in solid phase synthesis.
Any amino acid can be employed as the amino acid, and examples can include the natural amino acids serine (Ser), asparagine (Asn), valine (Val), leucine (Leu), isoleucine (Ile), alanine (Ala), tyrosine (Tyr), glycine (Gly), lysine (Lys), arginine (Arg), histidine (His), aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), threonine (Thr), cysteine (Cys), methionine (Met), phenylalanine (Phe), tryptophan (Trp), and proline (Pro).
Moreover, the D-form of the above natural amino acid can also be used.
Examples of lipophilic protecting groups can include, e.g., carbonate- or amide-based protecting groups such as a 9-fluorenylmethoxycarbonyl (Fmoc) group, a t-butyloxycarbonyl (Boc) group, a benzyl group, an allyl group, an allyloxycarbonyl group, and an acetyl group. When introducing a lipophilic protecting group into an amino acid, e.g. when introducing an Fmoc group, introduction can be carried out by adding 9-fluorenylmethyl-N-succinimidyl carbonate and sodium hydrogen carbonate and allowing reaction. The reaction may be performed at 0-50° C., preferably at room temperature for approximately about 1-5 hours.
As the amino acid protected with a lipophilic protecting group, those commercially available can also be used. Examples can include Fmoc-Ser-OH, Fmoc-Asn-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Tyr-OH, Fmoc-Gly-OH, Fmoc-Lys-OH, Fmoc-Arg-OH, Fmoc-His-OH, Fmoc-Asp-OH, Fmoc-Glu-OH, Fmoc-Gln-OH, Fmoc-Thr-OH, Fmoc-Cys-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Trp-OH, and Fmoc-Pro-OH.
Moreover, an amino acid protected with a lipophilic protecting group wherein the protecting group is introduced into the side chain can include, e.g., Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Cys (StBu)-OH, Fmoc-Cys (tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, and Fmoc-Tyr(tBu)-OH.
Moreover, if it is desired to add a linker in the amino acid sequence of the glycosylated polypeptide, the linker can be inserted at a preferred position by using a linker protected with a lipophilic protecting group instead of the above amino acid protected with a lipophilic protecting group in the process of solid phase synthesis.
When employing a 2-chlorotrityl chloride resin, esterification can be performed with a base such as diisopropylethylamine (DIPEA), triethylamine, pyridine, and 2,4,6-collidine. Moreover, when employing a resin having a hydroxyl group, e.g. a well-known dehydration condensation agent such as 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole (MSNT), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIC) can be employed as the esterification catalyst. The proportion of the amino acid and the dehydration condensation agent used is 1 part by weight of the former to generally 1-10 parts by weight, preferably 2-5 parts by weight of the latter.
The esterification reaction is preferably performed by for example placing the resin in a solid phase column, washing this resin with a solvent, and then adding the amino acid solution. Examples of washing solvents can include, e.g., dimethylformamide (DMF), 2-propanol, and dichloromethane. Examples of solvents for dissolving the amino acid can include, e.g., dimethyl sulfoxide (DMSO), DMF, and dichloromethane. The esterification reaction may be performed at 0-50° C., preferably at room temperature for approximately about 10 minutes-30 hours, preferably for approximately 15 minutes-24 hours.
It is also preferred to acetylate and cap the unreacted hydroxyl groups on the solid phase at this time with e.g. acetic anhydride.
The detachment of the lipophilic protecting group can be performed by for example treatment with a base. Examples of bases can include, e.g., piperidine and morpholine. In such a case, it is preferred that this is performed in the presence of a solvent. Examples of solvents can include, e.g., DMSO, DMF, and methanol.
The amidation reaction of the free amino group with the carboxy group of any amino acid having the amino group nitrogen protected with a lipophilic protecting group is preferably performed in the presence of an activator and a solvent.
Examples of activators can include, e.g., dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSC/HCl), diphenylphosphorylazide (DPPA), carbonyldiimidazole (CDI), diethylcyanophosphonate (DEPC), benzotriazol-1-yloxy-trispyrrolidinophosphonium (DIPCI), benzotriazol-1-yloxy-trispyrrolidinophosphonium hexafluorophosphate (PyBOP), 1-hydroxybenzotriazole (HOBt), hydroxysuccinimide (HOSu), dimethylaminopyridine (DMAP), 1-hydroxy-7-azabenzotriazole (HOAt), hydroxyphthalimide (HOPht), pentafluorophenol (Pfp-OH), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 1-[bis (dimethylamino)methylene]-5-chloro-1H-benzotriazolium 3-oxide hexafluorophosphate (HCTU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphonate (HATU), O-benzotriazol-1-yl-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), and 3,4-dihydro-3-hydrodi-4-oxa-1,2,3-benzotriazine (Dhbt).
The amount of the activator used is preferably 1-20 equivalents, preferably 1-10 equivalents, and further preferably 1-5 equivalents to any amino acid having the amino group nitrogen protected with a lipophilic protecting group.
Examples of solvents can include, e.g., DMSO, DMF, and dichloromethane. The reaction may be performed at 0-50° C., preferably at room temperature for approximately about 10-30 hours, preferably for approximately 15 minutes-24 hours. The detachment of the lipophilic protecting group can be performed similarly to the above.
Treatment with an acid is preferred in order to cleave the peptide chain from the resin. Examples of acids can include, e.g., trifluoroacetic acid (TFA) and hydrogen fluoride (HF).
In this way, a glycosylated polypeptide having the desired position substituted with glycosylated Asn can be obtained. Moreover, the glycosylated polypeptide purified as such allows formation of a disulfide bond between deprotected Cys as described below.
In one embodiment of the present invention, when sialic acid is contained in the non-reducing terminal on the sugar chain of glycosylated Asn employed in solid phase synthesis, the sialic acid is prevented from being cleaved by the acid treatment. It is therefore preferred that the carboxy group of said sialic acid is protected by a protecting group. Examples of protecting groups can include, e.g., a benzyl group, an allyl group, and a diphenylmethyl group. The method for introduction and detachment of the protecting group can be performed by a well-known method. Moreover, the detachment of the protecting group is preferably performed after the glycosylated polypeptide manufactured by solid phase synthesis is cleaved from the resin. After cleaving from the resin, if the glycosylated polypeptide is to be cyclized by allowing formation of a disulfide bond in the glycosylated polypeptide, the detachment of the protecting group may be before or after the step of forming a disulfide bond.
Method for Manufacturing Glycosylated Polypeptide (Method B)
The glycosylated polypeptide can also be manufactured by a method of first synthesizing a peptide chain, and then adding a sugar chain to the synthesized peptide chain. Specifically, a peptide comprising Cys at the position to be glycosylated is manufactured by e.g. a solid phase synthesis method, a liquid phase synthesis method, a method of synthesis by a cell, and a method of separating and extracting those that naturally exist. Here, Cys that will not be glycosylated such as Cys at a position planned to form a disulfide bond are protected with for example an acetamidomethyl (Acm) group. Moreover, if Cys that will neither be glycosylated nor be used in the formation of a disulfide bond is to be introduced into the glycosylated polypeptide, Cys can be introduced by protecting the Cys with a protecting group during the glycosylation and disulfide bond formation steps, and then deprotecting the same. Such protecting groups can include, e.g., tert-butyl (tBu) or 4-methoxybenzyl.
Moreover, when a different sugar chain is to be added to Cys in the glycosylated polypeptide, the different sugar chain can be introduced by first unprotecting the Cys where the sugar chain is to be introduced, and then protecting the Cys where a different sugar chain is to be introduced with e.g. StBu. Specifically, when synthesizing a peptide by e.g. solid phase synthesis, Cys where the first sugar chain is to be introduced is unprotected, and Cys where the second sugar chain is to be introduced is made into Cys having a protecting group with e.g. Fmoc-Cys(StBu)-OH. Then, the sugar chain is introduced to the unprotected Cys while retaining the protecting group such as StBu. Next, the StBu group etc. is deprotected to introduce a different sugar chain to the Cys that became unprotected. Note that Cys where the first sugar chain is to be introduced and Cys where the second sugar chain is to be introduced can be one or more Cys.
In the deprotection of the StBu group, the deprotection can be performed by reaction with a reductant such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP), dithiothreitol (DTT), and tributylphosphine. The above reaction may be performed generally at 0-80° C., preferably at 5-60° C., and further preferably at 10-35° C. The reaction time is preferably approximately 30 minutes-5 hours in general. After completion of the reaction, it may be purified with a well-known method (such as high performance liquid column chromatography (HPLC)) as appropriate.
When introducing different sugar chains, it is preferred that the introduction starts from sugar chains more stable against the reduction condition in the deprotection step of Cys or the acidic condition in the purification step such as HPLC. In particular, when a sialic acid-containing sugar chain is to be introduced, it is preferred that a sugar chain that does not have a sialic acid or a sugar chain having fewer sialic acid residues is introduced first.
Moreover, if it is desired to add a linker in the amino acid sequence of the glycosylated polypeptide, for example, the linker can be inserted at a preferred position of the synthesized polypeptide by using a linker protected with a lipophilic protecting group instead of an amino acid protected with a lipophilic protecting group in the process of solid phase synthesis.
Next, by reacting a haloacetylated complex-type sugar chain derivative with the peptide comprising an unprotected Cys obtained above, the sugar chain is reacted with the thiol group of the unprotected Cys and bound to the peptide. The above reaction may be performed in a phosphate buffer, a Tris-hydrochloride buffer, a citrate buffer, or a mixed solution thereof, generally at 0-80° C., preferably at 10-60° C., and further preferably at 15-35° C. The reaction time is generally 10 minutes-24 hours, and preferably approximately 30 minutes-5 hours in general. After completion of the reaction, it may be purified with a well-known method (such as HPLC) as appropriate.
A haloacetylated complex-type sugar chain derivative is for example a compound wherein the hydroxyl group bound to the carbon at position 1 of a complex-type asparagine-linked sugar chain is substituted with —NH—(CH2)a—(CO)—CH2X (wherein X is a halogen atom, and a is an integer and is not limited as long as it does not inhibit the linker function of interest, but is preferably an integer from 0-4).
Specifically, a haloacetylated complex-type sugar chain derivative and a Cys-containing peptide are reacted in a phosphate buffer at room temperature. After completion of the reaction, a glycosylated polypeptide substituted with a glycosylated Cys can be obtained by purification with HPLC.
Moreover, the reaction can also be performed in a mixed solution of an organic solvent such as DMSO, DMF, methanol, and acetonitrile with the above buffer. Here, the organic solvent can be added to the above buffer at a proportion in the range of 0-99% (v/v). For a peptide comprising an unprotected Cys with low solubility to the buffer, addition of such an organic solvent can improve the solubility to the reaction solution and is thus preferred.
Alternatively, the reaction can also be performed in an organic solvent such as DMSO, DMF, methanol, and acetonitrile or a mixed solution thereof. In this case, it is preferably performed in the presence of a base. Examples of bases can include, e.g., DIPEA, triethylamine, pyridine, and 2,4,6-collidine. Moreover, the reaction can also be performed in a mixed solution of guanidine hydrochloride or urea added to the buffer solution. Guanidine hydrochloride or urea can be added to the above buffer so that the final concentration will be 1 M-8 M. The solubility of a peptide with low solubility in the buffer can also be improved by addition of guanidine hydrochloride or urea and is thus preferred.
Further, in order to prevent the peptide comprising an unprotected Cys from forming a dimer via a disulfide bond, tris(2-carboxyethyl)phosphine hydrochloride (TCEP) or dithiothreitol (DTT) can also be added to the buffer for reaction. TCEP or DTT can be added to the buffer so that the final concentration will be 10 μM-10 mM.
Moreover, after binding the sugar chain to the Cys of interest, the protecting group of Cys protected with Acm etc. is deprotected. When the protecting group is an Acm group, it can be deprotected by subjecting to reaction with e.g. iodine, mercury acetate (II), silver nitrate (I), or silver(I) acetate in water, methanol, acetic acid, or a mixed solution thereof.
The above reaction may be performed generally at 0-80° C., preferably at 5-60° C., and further preferably at 10-35° C. The reaction time is preferably approximately 5 minutes-24 hours in general. After completion of the reaction, it may be treated with DTT or hydrochloric acid etc., and then purified with a well-known method (such as HPLC) as appropriate.
In this way, a glycosylated polypeptide having the desired position substituted with glycosylated Cys can be obtained. Moreover, the glycosylated polypeptide purified as such allows formation of a disulfide bond between deprotected Cys as described below.
Moreover, when manufacturing a glycosylated polypeptide having multiple sialic acid-containing sugar chains such as disialo or monosialo sugar chain in the peptide sequence, a sialic acid-containing sugar chain in which the carboxy group of the sialic acid on the sugar chain to be introduced is protected by e.g. a benzyl (Bn) group, an allyl group, a diphenylmethyl group, and a phenacyl group can be employed.
When a sugar chain having the carboxy group of the sialic acid protected is introduced, a step of deprotecting the sialic acid protecting group can be carried out after a step of forming a disulfide bond in the glycosylated polypeptide described below.
Accordingly, by protecting the carboxy group of the sialic acid with a benzyl group etc., a separation and purification step by e.g. HPLC in the manufacturing step will be facilitated. Moreover, the protection of the carboxy group of the sialic acid will also enable the prevention of detachment of an acid-labile sialic acid.
The protection reaction of the carboxy group of the sialic acid on the sugar chain can be performed by a method well-known to those skilled in the art. Moreover, in a glycosylated polypeptide in which a disulfide bond was formed, the protecting group of the carboxy group of the sialic acid can be deprotected by hydrolysis under basic conditions. The above reaction may be performed generally at 0-50° C., preferably at 0-40° C., and further preferably at 0-30° C. The reaction time is preferably approximately 5 minutes-5 hours in general. After completion of the reaction, it may be neutralized with a weak acid such as phosphoric or acetic acid, and then purified with a well-known method (such as HPLC) as appropriate.
Moreover, in the glycosylated polypeptide prepared by the above methods A and B, a disulfide bond between Cys can be formed with a method well-known to those skilled in the art employing e.g. air and/or oxygen, iodine, DMSO, a mixture of oxidized and reduced glutathione, potassium ferricyanide, Ellman's reagent (5,5′-dithiobis(2-nitrobenzoic acid)), thallium(III) trifluoroacetate, and alkyltrichlorosilane sulfoxide.
When forming a disulfide bond between Cys-Cys, Cys in the glycosylated polypeptide which desirably do not form a disulfide bond is to be protected with a protecting group. As such protecting groups, a protecting group which is stable under oxidizing conditions such as Acm, tBu, 4-methoxybenzyl, and 4-methylbenzyl can be employed.
Moreover, in method B, it is also possible to perform the formation of disulfide bond before the introduction of the sugar chain. However, if a protecting group is introduced to Cys that is to be subject to disulfide bonding, the deprotection step will come before the disulfide bond formation step.
(Activity)
The glycosylated polypeptide of the present invention has affinity towards somatostatin receptors. Having “affinity towards somatostatin receptors” herein means having affinity towards at least one of somatostatin receptors SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5.
The glycosylated polypeptide of the present invention preferably has affinity towards two or more somatostatin receptors, more preferably has affinity towards three or more receptors, further preferably has affinity towards four or more receptors, and most preferably has affinity towards all five receptors SSTR1-SSTR5 similarly to a natural somatostatin (SRIF28 and SRIF14). In particular, it is preferred that it has affinity towards any one of at least SSTR1 and SSTR4 and affinity towards other SSTRs.
As such, the glycosylated polypeptide of the present invention has somatostatin activity (agonist activity) and antagonist activity towards a somatostatin receptor by having affinity towards a somatostatin receptor.
For example, affinity towards each somatostatin receptor can be measured with e.g. competitive binding experiment in vitro.
In the measurement of affinity by competitive binding experiment, the affinity of the test substance (such as 50% inhibitory concentration: IC50 value or binding inhibition constant: Ki value) can be measured by competitively binding a labeled ligand and the test substance to the receptor and seeking the release of the labeled ligand when the test substance is administered.
For example, somatostatin activity (agonist activity) can be evaluated by a cAMP production suppression test in vitro employing somatostatin receptor expression cells as shown in Examples 69-3 and 69-4.
The cAMP production suppression test can be evaluated by treating somatostatin receptor expression cells with the glycosylated polypeptide or the control compounds SRIF14 or SRIF28, measuring the amount of cAMP accumulated in the cell after culturing for a certain amount of time, and comparing with the control compound. The amount of cAMP can be measured by a well-known method such as the enzyme immunoassay method (EIA).
For example, somatostatin activity (agonist activity) can be evaluated by a GH production suppression test in vivo as shown in Examples 89 and 90.
The GH production suppression test can be carried out for example as follows. The glycosylated polypeptide is subcutaneously administered to non-fasted rats. Rats are administered a GH release enhancer under general anesthesia, and then blood is collected in approximately 5 minutes. The collected blood is the plasma sample, and GH concentration is measured by a well-known method such as the enzyme immunoassay method (EIA). Moreover, as a control, plasma samples are obtained from rats which were administered with saline that does not contain glycosylated polypeptide, and somatostatin activity can be evaluated by comparing the GH concentrations measured.
In the glycosylated polypeptide of the present invention, even if some extent of attenuation in affinity towards each receptor is triggered by glycosylating its structure, the half-life in blood is extended and as a result, somatostatin activity equivalent to a naturally-occurring somatostatin that is not glycosylated (hereinafter may be referred to as “non-glycosylated somatostatin”) can be maintained, and it can preferably have increased somatostatin activity. In light of this extension of half-life in blood, the ratio between the Ki value of the glycosylated polypeptide of the present invention against each receptor and the Ki value of unglycosylated SRIF14 is preferably in the range of 1000:1-0.3:1, more preferably in the range of 100:1-0.3:1, and further preferably in the range of 10:1-0.3:1, as measured in e.g. the method described in Example 69-1.
The stability in blood of the glycosylated polypeptide of the present invention is preferably equivalent to a naturally-occurring somatostatin (non-glycosylated somatostatin) or more. Stability in blood can be measured with a method well-known to those skilled in the art, and can be decided with e.g. stability in plasma, half-life in blood, and AUC (drug plasma concentration—area under the time curve) as indicators. Moreover, decrease in renal or hepatic clearance also contributes to an increase in stability in blood.
The glycosylated polypeptide of the present invention has increased half-life in blood compared to an non-glycosylated SRIF28, and its half-life is increased by 4-folds or more, preferably 10-fold or more, more preferably 20-folds or more, and further preferably 30-folds or more compared to SRIF28, as measured in e.g. the experimental method shown in Example 70.
Moreover, the glycosylated polypeptide of the present invention has preferably 4-folds or more, more preferably 10-fold or more, and further preferably 20-folds or more stability in blood compared to SRIF28, as measured in e.g. the experimental method shown in Example 88.
(Pharmaceutical Composition)
Next, a pharmaceutical composition containing the glycosylated polypeptide of the present invention as the active ingredient will be described.
The pharmaceutical composition containing the glycosylated polypeptide of the present invention as the active ingredient is effective for the treatment or prevention of somatostatin-related diseases. As described above, various actions are known for somatostatin, and diseases related to these actions also vary. For example, examples of somatostatin-related diseases include, e.g., acromegaly, gigantism, Alzheimer's disease and other forms of dementia, cancer, hormone-producing tumor, endocrine tumor (such as carcinoid, VIPoma, insulinoma, and glucagonoma), Cushing's disease, hormone secretion defect, diabetes and complications thereof, pains, arthritis, diarrhea, gastric ulcer, inflammatory bowel disease, irritable bowel syndrome, gastrointestinal obstruction, ileus, postoperative restenosis, radiation damage, and eye disease (such as dry eye, glaucoma, interstitial keratitis, iritis, cataract, and conjunctivitis). The pharmaceutical composition containing the glycosylated polypeptide of the present invention as the active ingredient is effective for the treatment or prevention of the above diseases, in particular, acromegaly, dementia, cancer, hormone-producing tumor, endocrine tumor, Cushing's disease, hormone secretion defect, diabetes complication, diarrhea, gastrointestinal obstruction, ileus, radiation damage, eye disease, various tumors or gut-associated disease, and gastrointestinal symptoms accompanying excess hormone production.
The above pharmaceutical composition is one formulated into the form of an ordinary pharmaceutical composition with a diluent or an excipient such as a generally used filler, expander, binder, moisturizer, disintegrant, surfactant, and lubricant.
Examples of such pharmaceutical compositions include, e.g., tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, suppositories, inhalants, ophthalmic solutions, and injections.
The amount of the glycosylated polypeptide of the present invention contained in the pharmaceutical composition is not particularly limited and can be selected as appropriate from a broad range, but in general 1-70% by weight of the glycosylated polypeptide of the present invention is preferably contained in the pharmaceutical composition.
The pharmaceutical composition containing the glycosylated polypeptide of the present invention as the active ingredient can either further contain other active ingredients, or it can be employed in combination with a pharmaceutical composition containing other active ingredients. Moreover, the pharmaceutical composition containing the glycosylated polypeptide of the present invention as the active ingredient can either comprise the glycosylated polypeptide as a pharmaceutically acceptable salt, or further contain one or more different glycosylated polypeptides of the present invention as active ingredients. Moreover, it can also be employed in combination with the pharmaceutical composition containing one or more different glycosylated polypeptides of the present invention as the active ingredient. Moreover, Examples of other ingredients that can be contained in the pharmaceutical composition can include a pharmaceutically acceptable carrier known to those skilled in the art.
Moreover, a treatment using the glycosylated polypeptide of the present invention may include e.g. radiation therapy, and it is also useful in scintigraphy for measuring the distribution of cells and tissues expressing any of SSTR1-SSTR5 throughout the body. The use of extracorporeal imaging by radiation scanning or magnetic resonance will enable in vivo semiquantitative detection.
A radiolabeled glycosylated polypeptide is useful for therapeutic treatment of a malignant tumor expressing any of SSTR1-SSTR5, for example in a human body in a tissue that does not comprise a substantial amount of SSTR1-SSTR5 in a healthy state. Moreover, the labeled glycosylated polypeptide can be administered for scintigraphy, or as a composition containing an effective amount to suppress a tumor. Examples of such labels are different isotopes such as iodine (123I, 125I, and 13I), indium (111In), carbon (11C), fluorine (18F), technetium (99mTc), and yttrium (90Y), or a fluorescent label.
The labeling can be carried out by a method well-known to those skilled in the art, and the label can be contained in the glycosylated polypeptide, bound directly thereto, or bound to an appropriate compound and then bound to the glycosylated polypeptide. For example, in iodization of tyrosine, the labeling can be performed by e.g. a method employing chloramine-T etc.
The method for administering the pharmaceutical composition according to the present invention is not particularly restricted, and it is administered with a method according to various drug formulations, age, sex, and the disease condition of the patient, and other conditions. The method of administration for tablets, pills, liquids, suspensions, emulsions, granules, and capsules include e.g. oral administration. Moreover, in case of injections, it can be intravenously, intramuscularly, intradermally, subcutaneously, or intraperitoneally administered alone or mixed with an ordinary fluid replacement such as glucose or amino acid. In case of suppositories, it is intrarectally administered. In case of ophthalmic solutions, it is applied to an eye tissue such as the conjunctival sac. In case of inhalants, it is applied to the bronchial tube or the lung.
The administration dose of the above pharmaceutical composition may be selected as appropriate according to usage, age, sex, and the disease extent of the patient, and other conditions, and for example can be an administration dose that will be 0.1-900 nmol, preferably 1-100 nmol, and more preferably 1-10 nmol of the glycosylated polypeptide of the present invention per 1 kg of body weight.
The administration frequency of the above pharmaceutical composition may be selected as appropriate according to usage, age, sex, and the disease extent of the patient, and other conditions, and 3 times/day, twice/day, once/day, and further at a less frequent administration frequency according to stability in blood thereof (such as once/week and once/month) may be selected. Preferably, the administration frequency of the above pharmaceutical composition is once or less per day.
The sugar chain added to the glycosylated polypeptide of the present invention is easily degraded by the metabolic system in the body. Moreover, in one aspect of the present invention, said sugar chain has a structure that exists as bound to a glycopeptide (or a glycoprotein) in vivo. Accordingly, a pharmaceutical composition comprising the glycosylated polypeptide of the present invention and said peptide as active ingredients has advantages such as not showing side effects or antigenicity when administered in vivo and less concern for losing drug effect by allergic reactions or antibody production.
Further, the glycosylated polypeptide of the present invention can be stably and easily supplied in large amounts, and it is extremely useful with respect to providing pharmaceuticals having stable and high quality.
Moreover, the present invention also provides a method for treating or preventing a somatostatin-related disease, characterized in administering an effective amount of the glycosylated polypeptide of the present invention.
The terms used herein are to be employed to describe particular embodiments, and do not intend to limit the invention.
Moreover, the term “comprising” as used herein, unless the content clearly indicates to be understood otherwise, intends the presence of the described items (such as components, steps, elements, and numbers), and does not exclude the presence of other items (such as components, steps, elements, and numbers).
Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meanings as those broadly recognized by those skilled in the art of the technology to which the present invention belongs. The terms used herein, unless explicitly defined otherwise, are to be construed as having consistent meanings with the meanings herein and related technical fields, and shall not be construed as having idealized or excessively formal meanings.
The embodiments of the present invention may be described referring to schematic diagrams. In case of schematic diagrams, they may be exaggerated in presentation in order to allow clear description.
Terms such as first and second are employed to express various elements, and it should be recognized that these elements are not to be limited by these terms. These terms are employed solely for the purpose of discriminating one element from another, and it is for example possible to describe a first element as a second element, and similarly, to describe a second element as a first element without departing from the scope of the present invention.
The present invention will now be described in further detail referring to Examples. However, the present invention can be embodied by various aspects, and shall not be construed as being limited to the Examples described herein.
EXAMPLES
The notation system of the glycosylated polypeptides herein will be described below.
For example, S1C(disialo) N5C(disialo)-SRIF28 shows that Ser at position 1 (S1) and Asn at position 5 of polypeptide SRIF28 are both substituted by disialo sugar chain attached Cys (C(disialo)).
Moreover, S1C(disialo)-D-Trp22-SRIF28 shows that Ser at position 1 (S1) of polypeptide SRIF28 is substituted by disialo sugar chain attached Cys (C(disialo)), and further Trp at position 22 is substituted with D-Trp.
Moreover, C(disialo)-R—K-SRIF14 shows that glycosylated Cys-Arg-Lys- is added at the N-terminal side of SRIF14.
Moreover, 29C(disialo)-SRIF28 or 30C(disialo)-SRIF28 shows peptides having one disialo sugar chain attached Cys further added to Cys at position 28 which is the C-terminal of SRIF28 (29C(disialo)-SRIF28), or having —W-disialo sugar chain attached Cys (W is any amino acid that binds to Cys at position 28) further added to Cys at position 28 which is the C-terminal of SRIF28 (30C(disialo)-SRIF28).
Moreover, S1-2C(disialo)-SRIF28 shows that Ser at position 1 present at the N-terminal of polypeptide SRIF28 is substituted with two consecutive disialo sugar chain attached Cys.
In the meantime, “disialo” means a disialo sugar chain, “monosialo” means a monosialo sugar chain, “asialo” means an asialo sugar chain, “diGlcNAc” means a diGlcNAc sugar chain, “GlcNAc” means an N-acetylglucosamine, “diMan” means a dimannose sugar chain, “trisialo” means a trisialo sugar chain, and “tetrasialo” means a tetrasialo sugar chain.
Moreover, (disialo(aminoethylamide)) means that the carboxy group of the sialic acid of the disialo sugar chain is modified by an aminoethylamino group. “Bn,” “amide,” or “hexadecylamide” instead of “aminoethylamide” each mean that the carboxy group of the sialic acid on the sugar chain is protected by a benzyl group, an amino group, hexadecylamino group.
Example 1
Synthesis of S1C(disialo)-SRIF28
1-1 Glycosylation Reaction of Thiol
Peptide 1 (SEQ ID NO. 38) represented by the following formula (1) (from APC, Inc.) (60.6 mg, 18.3 μmol) and compound a represented by the following formula (a) (bromoacetamidated oligosaccharide: from Otsuka Chemical Co., Ltd.) (85.8 mg, 36.6 μmol, 2.0 equivalents to peptide 1) were dissolved in 33 mM phosphate buffer (pH 7.4, 5.5 mL), and reacted at room temperature for 30 minutes.




The reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous acetic acid (AcOH), B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=90:10→75:25, 15 minutes, linear gradient elution] to obtain glycopeptide 2 (SEQ ID NO. 39) represented by the following formula (2) (60.5 mg, 10.9 μmol, yield 59%).




ESI-MS: (m/z) calcd for C229H358N50O102S4: [M+3H]3+ 1858.3, [M+4H]4+ 1394.0, [M+5H]5+ 1115.4. found 1858.1, 1393.8, 1115.2.
1-2 Deprotection of Acm Group
To glycopeptide 2 obtained in the method described in the above 1-1 (51.2 mg, 9.19 μmol) was added an aqueous solution (3.7 mL) of silver(I) acetate (18.8 mg, 113 μmol), and reacted at room temperature for 40 minutes. DTT (43.6 mg, 282 μmol) dissolved in 200 mM Tris-HCl buffer (pH 7.4, 3.7 mL) and 100 mM ascorbic acid aqueous solution (0.92 mL) were added, and this was promptly filtered with a filter. The filtrate was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=90:10→75:25, 15 minutes, linear gradient elution] to obtain glycopeptide 3 (SEQ ID NO. 40) represented by the following formula (3) (29.2 mg, 5.38 μmol, yield 58%).




ESI-MS: (m/z) calcd for C223H348N48O100S4: [M+3H]3+ 1810.9, [M+4H]4+ 1358.4, [M+5H]5+ 1086.9, [M+6H]6+ 906.0. found 1810.7, 1358.3, 1086.6, 905.7.
1-3 Formation of Disulfide Bond
Glycopeptide 3 obtained in the method described in the above 1-2 (29.2 mg, 5.38 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO (1/1, v/v, 10.8 mL), and reacted at room temperature for 2 days. The reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=77:23→64:36, 17 minutes, linear gradient elution] to obtain a fraction containing compound (S1C(disialo)-SRIF28) represented by the following formula (4) (SEQ ID NO. 5).




This fraction was further purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=90:10→75:25, 15 minutes, linear gradient elution] to obtain S1C(disialo)-SRIF28 (17.2 mg, 3.17 μmol, yield 59%).
ESI-MS: (m/z) calcd for C223H346N48O100S4: [M+3H]3+ 1810.2, [M+4H]4+ 1357.9, [M+5H]5+ 1086.5. found 1810.0, 1357.5, 1086.4.
Example 2
Synthesis of N5C(disialo)-SRIF28
A compound represented by the following formula (6) (N5C(disialo)-SRIF28) (SEQ ID NO. 6) was synthesized similarly to Example 1, except that a compound represented by the following formula (5) (peptide 5) (SEQ ID NO. 41) was employed instead of peptide 1.




Example 3
Synthesis of A9C(disialo)-SRIF28
A compound represented by the following formula (8) (A9C(disialo)-SRIF28) (SEQ ID NO. 7) was synthesized similarly to Example 1, except that a compound represented by the following formula (7) (peptide 7) (SEQ ID NO. 42) was employed instead of peptide 1.




Example 4
Synthesis of E12C(disialo)-SRIF28
A compound represented by the following formula (10) (E12C(disialo)-SRIF28) (SEQ ID NO. 8) was synthesized similarly to Example 1, except that a compound represented by the following formula (9) (peptide 9) (SEQ ID NO. 43) was employed instead of peptide 1.




Example 5
Synthesis of R13C(disialo)-SRIF28
A compound represented by the following formula (12) (R13C(disialo)-SRIF28) (SEQ ID NO. 9) was synthesized similarly to Example 1, except that a compound represented by the following formula (11) (peptide 11) (SEQ ID NO. 44) was employed instead of peptide 1.




Example 6
Synthesis of K14C(disialo)-SRIF28
A compound represented by the following formula (14) (K14C(disialo)-SRIF28) (SEQ ID NO. 10) was synthesized similarly to Example 1, except that a compound represented by the following formula (13) (peptide 13) (SEQ ID NO. 45) was employed instead of peptide 1.




Example 7
Synthesis of A15C(disialo)-SRIF28
A compound represented by the following formula (16) (A15C(disialo)-SRIF28) (SEQ ID NO. 11) was synthesized similarly to Example 1, except that a compound represented by the following formula (15) (peptide 15) (SEQ ID NO. 46) was employed instead of peptide 1.




Example 8
Synthesis of G16C(disialo)-SRIF28
A compound represented by the following formula (18) (G16C(disialo)-SRIF28) (SEQ ID NO. 12) was synthesized similarly to Example 1, except that a compound represented by the following formula (17) (peptide 17) (SEQ ID NO. 47) was employed instead of peptide 1.




Example 9
Synthesis of K18C(disialo)-SRIF28
A compound represented by the following formula (20) (K18C(disialo)-SRIF28) (SEQ ID NO. 13) was synthesized similarly to Example 1, except that a compound represented by the following formula (19) (peptide 19) (SEQ ID NO. 48) was employed instead of peptide 1.




Example 10
Synthesis of N19C(disialo)-SRIF28
A compound represented by the following formula (22) (N19C(disialo)-SRIF28) (SEQ ID NO. 14) was synthesized similarly to Example 1, except that a compound represented by the following formula (21) (peptide 21) (SEQ ID NO. 49) was employed instead of peptide 1.




Example 11
Synthesis of F21C(disialo)-SRIF28
A compound represented by the following formula (24) (F21C(disialo)-SRIF28) (SEQ ID NO. 15) was synthesized similarly to Example 1, except that a compound represented by the following formula (23) (peptide 23) (SEQ ID NO. 50) was employed instead of peptide 1.




Example 12
Synthesis of T26C(disialo)-SRIF28
A compound represented by the following formula (26) (T26C(disialo)-SRIF28) (SEQ ID NO. 16) was synthesized similarly to Example 1, except that a compound represented by the following formula (25) (peptide 25) (SEQ ID NO. 51) was employed instead of peptide 1.




Example 13
Synthesis of 29C(disialo)-SRIF28
A compound represented by the following formula (28) (29C(disialo)-SRIF28) (SEQ ID NO. 17) was synthesized similarly to Example 1, except that a compound represented by the following formula (27) (peptide 27) (SEQ ID NO. 52) was employed instead of peptide 1.




Example 14
Synthesis of 30C(disialo)-SRIF28
A compound represented by the following formula (30) (30C(disialo)-SRIF28) (SEQ ID NO. 18) was synthesized similarly to Example 1, except that a compound represented by the following formula (29) (peptide 29) (SEQ ID NO. 53) was employed instead of peptide 1.




Example 15
Synthesis of S1C(disialo)-D-Trp22-SRIF28
A compound represented by the following formula (32) (S1C(disialo)-D-Trp22-SRIF28) (SEQ ID NO. 19) was synthesized similarly to Example 1, except that a compound represented by the following formula (31) (peptide 31) (SEQ ID NO. 54) was employed instead of peptide 1.




Example 16
Synthesis of A9C(disialo)-D-Trp22-SRIF28
A compound represented by the following formula (34) (A9C(disialo)-D-Trp22-SRIF28) (SEQ ID NO. 20) was synthesized similarly to Example 1, except that a compound represented by the following formula (33) (peptide 33) (SEQ ID NO. 55) was employed instead of peptide 1.




Example 17
Synthesis of C(disialo)-SRIF14
A compound represented by the following formula (36) (C(disialo)-SRIF14) (SEQ ID NO. 35) was synthesized similarly to Example 1, except that a compound represented by the following formula (35) (peptide 35) (SEQ ID NO. 56) was employed instead of peptide 1.




Example 18
Synthesis of C(disialo)-R—K-SRIF14
A compound represented by the following formula (38) (C(disialo)-R—K-SRIF14) (SEQ ID NO. 36) was synthesized similarly to Example 1, except that a compound represented by the following formula (37) (peptide 37) (SEQ ID NO. 57) was employed instead of peptide 1.




Example 19
Synthesis of C(disialo)-C12 linker-SRIF14
19-1 Synthesis of Peptide
2-chlorotrityl chloride resin (100 μmol) was taken in a column for solid phase synthesis, and after washing with DMF and dichloromethane, a solution of Fmoc-Cys(Acm)-OH (49.7 mg, 120 μmol) and DIPEA (104.5 μL, 600 μmol) in dichloromethane (3.0 mL) was added, and this was shaken for 1 hour. After washing with dichloromethane and DMF, the Fmoc protecting group was removed by treating with 20% piperidine in DMF. After washing with DMF, in a peptide solid phase synthesis method with Fmoc strategy employing a PreludeTM peptide synthesizer, a protected peptide 39 (SEQ ID NO. 58) represented by the following formula (39) was synthesized in a state bound to the resin. The condensation reaction was performed in DMF using HCTU as the condensation agent.




The Fmoc protecting group was removed by treating with 20% piperidine in DMF. After washing with DMF and dichloromethane, employing HCTU as the condensation agent, Fmoc-12-aminododecanoic acid and Fmoc-Cys(Trt)-OH were condensed in sequence. After condensation, the Fmoc protecting group was removed by treating with 20% piperidine in DMF. After washing with DMF and dichloromethane, TFA:water:triisopropylsilane:ethanedithiol (=90:2.5:5:2.5) was added, and this was shaken for 3 hours at room temperature. This leads to the detachment of the protecting group of the amino acid side chain (other than the Acm group), as well as cleaving between the peptide and the resin. The resin was filtered off, cold diethyl ether was added to the filtrate, and crude peptide was obtained as precipitate. A part of the crude peptide was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=60:40→36.2:63.8, 20 minutes, linear gradient elution] to obtain a compound represented by the following formula (40) (peptide 40) (SEQ ID NO. 59) (11.2 mg).




ESI-MS: (m/z) calcd for C97H144N12O23S3: [M+2H]2+ 1042.3, [M+3H]3+ 695.2. found 1042.0, 695.0.
19-2 Glycosylation Reaction of Thiol
Peptide 40 obtained in the method described in the above 19-1 (6.8 mg, 3.3 μmol) and compound a represented by the above formula (a) (19.1 mg, 8.15 μmol) were dissolved in 0.2 M phosphate buffer (pH 7.4, 0.96 mL) containing 7 M guanidine hydrochloride and 330 μM TCEP, and reacted at room temperature for 2 hours. After confirming the disappearance of raw materials with HPLC, the reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=80:20→50:50, 25 minutes, linear gradient elution] to obtain a compound represented by the following formula (41) (glycopeptide 41) (SEQ ID NO. 60) (7.1 mg, 1.6 μmol, yield 50%).




ESI-MS: (m/z) calcd for C183H283N29O85S3: [M+3H]3+ 1449.5, [M+4H]4+ 1087.4, [M+5H]5+ 870.1. found 1449.3, 1087.2, 870.0.
19-3 Deprotection of Acm Group
To glycopeptide 41 obtained in the method described in the above 19-2 (10.3 mg, 2.37 μmol) was added an aqueous solution (0.95 mL) of silver(I) acetate (9.7 mg, 58 μmol), and reacted at room temperature for 30 minutes. DTT (22.3 mg, 145 μmol) dissolved in 100 mM phosphate buffer (pH 7.4, 0.95 mL) and 100 mM ascorbic acid aqueous solution (0.95 mL) were added, and this was promptly filtered with a filter. The filtrate was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=80:20→50:50, 25 minutes, linear gradient elution] to obtain glycopeptide 42 represented by the following formula 42 (SEQ ID NO. 61) (5.8 mg, 1.4 μmol, yield 59%).




ESI-MS: (m/z) calcd for C177H273N273O83S3: [M+3H]3+ 1402.1, [M+4H]4+ 1051.8, [M+5H]5+ 841.7. found 1401.9, 1051.7, 841.5.
19-4 Formation of Disulfide Bond
Glycopeptide 42 obtained in the method described in the above 19-3 (5.8 mg, 1.4 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO (1/1, v/v, 3.5 mL), and reacted at room temperature for 30 hours. The reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=70:30→50:50%, 25 minutes, linear gradient elution] to obtain a fraction containing compound (C(disialo)-C12 linker-SRIF14) represented by the following formula 43 (SEQ ID NO. 37).




This fraction was further purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=80:20→50:50, 25 minutes, linear gradient elution] to obtain C(disialo)-C12 linker-SRIF14 (3.6 mg, 0.86 μmol, yield 61%).
ESI-MS: (m/z) calcd for C177H271N27O83S3: [M+3H]3+ 1401.5, [M+4H]4+ 1051.3, [M+5H]5+ 841.3. found 1401.2, 1051.2, 841.1.
Example 20
Synthesis of S1-2C(disialo)-SRIF28
20-1 Synthesis of Peptide
2-chlorotrityl chloride resin (100 μmol) was taken in a column for solid phase synthesis, and after washing with DMF and dichloromethane, a solution of Fmoc-Cys(Acm)-OH (49.7 mg, 120 μmol) and DIPEA (104.5 μL, 600 μmol) in dichloromethane (3.0 mL) was added, and this was shaken for 1 hour. After washing with dichloromethane and DMF, the Fmoc protecting group was removed by treating with 20% piperidine in DMF. After washing with DMF and dichloromethane, in a peptide solid phase synthesis method with Fmoc strategy employing a PreludeTM peptide synthesizer, a protected peptide was synthesized on a resin. The condensation reaction was performed in DMF using HCTU as the condensation agent.
The Fmoc protecting group was removed by treating with 20% piperidine in DMF. After washing with DMF and dichloromethane, TFA:water:triisopropylsilane:ethanedithiol (=90:2.5:5:2.5) was added, and this was shaken for 3 hours at room temperature. The resin was filtered off, cold diethyl ether was added to the filtrate, and crude peptide was obtained as precipitate. A part of the crude peptide was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=72:28→68.5:31.5, 20 minutes, linear gradient elution] to obtain peptide 44 (SEQ ID NO. 62) represented by the following formula (44) (30.7 mg).




ESI-MS: (m/z) calcd for C146H224N44O41S5: [M+3H]3+ 1138.3, [M+4H]4+ 854.0, [M+5H]5+ 683.4. found 1138.2, 853.9, 683.1.
20-2 Glycosylation Reaction of Thiol
Peptide 44 obtained in the method described in the above 20-1 (45.8 mg, 13.4 μmol) and compound a represented by the above formula (a) (125.8 mg, 53.7 μmol, 4.0 equivalents to peptide 44) were dissolved in 33 mM phosphate buffer (pH 7.4, 4.0 mL), and reacted at room temperature for 30 minutes. The reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=83:17→72:28, 15 minutes, linear gradient elution] to obtain glycopeptide 45 represented by the following formula 45 (SEQ ID NO. 63) (44.5 mg, 5.61 μmol, yield 42%).




20-3 Deprotection of Acm Group

To glycopeptide 45 obtained in the method described in the above 20-2 (44.5 mg, 5.61 μmol) was added an aqueous solution (2.2 mL) of silver(I) acetate (14.8 mg, 88.7 μmol), and reacted at room temperature for 30 minutes. DTT (33.2 mg, 215 μmol) dissolved in 200 mM phosphate buffer (pH 7.4, 2.2 mL) and 100 mM ascorbic acid aqueous solution (561 μL) were added, and this was promptly filtered with a filter. The filtrate was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=83:17→72:28, 15 minutes, linear gradient elution] to obtain glycopeptide 46 (SEQ ID NO. 64) represented by the following formula (46) (34.4 mg, 4.41 μmol, yield 79%).




ESI-MS: (m/z) calcd for C312H492N56O163S5: [M+4H]4+ 1950.0, [M+5H]5+ 1560.2, [M+6H]6+ 1300.3. found 1949.8, 1560.1, 1300.2.
20-4 Formation of Disulfide Bond
Glycopeptide 46 obtained in the method described in the above 20-3 (34.4 mg, 4.41 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO (1/1, v/v, 8.8 mL), and reacted at room temperature for 2 days. The reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=77:23→65:35, 16 minutes, linear gradient elution] to obtain a fraction containing compound (S1-2C(disialo)-SRIF28) represented by the following formula (47) (SEQ ID NO. 21).




This fraction was further purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=83:17→72:28, 15 minutes, linear gradient elution] to obtain S1-2C(disialo)-SRIF28 (20.1 mg, 2.58 μmol, yield 58%).
ESI-MS: (m/z) calcd for C312H490N56O163S5: [M+4H]4+ 1949.5, [M+5H]5+ 1559.8, [M+6H]6+ 1300.0. found 1949.4, 1559.7, 1299.9.
Example 21
Synthesis of S1C(disialo).N5C(disialo)-SRIF28
A compound represented by the following formula (49) (S1C(disialo).N5C(disialo)-SRIF28) (SEQ ID NO. 22) was synthesized similarly to Example 20, except that a compound represented by the following formula (48) (peptide 48) (SEQ ID NO. 65) was employed instead of peptide 44.




Example 22
Synthesis of S1C(disialo).R13C(disialo)-SRIF28
A compound represented by the following formula (51) (S1C(disialo) R13C(disialo)-SRIF28) (SEQ ID NO. 23) was synthesized similarly to Example 20, except that a compound represented by the following formula (50) (peptide 50) (SEQ ID NO. 66) was employed instead of peptide 44.




Example 23
Synthesis of N5C(disialo).A9C(disialo)-SRIF28
A compound represented by the following formula (53) (N5C(disialo).A9C(disialo)-SRIF28) (SEQ ID NO. 24) was synthesized similarly to Example 20, except that a compound represented by the following formula (52) (peptide 52) (SEQ ID NQ. 67) was employed instead of peptide 44.




Example 24
Synthesis of S1-3C(disialo)-SRIF28
24-1 Synthesis of Peptide
2-chlorotrityl chloride resin (100 μmol) was taken in a column for solid phase synthesis, and after washing with DMF and dichloromethane, a solution of Fmoc-Cys(Acm)-OH (49.7 mg, 120 μmol) and DIPEA (104.5 μL, 600 μmol) in dichloromethane (3.0 mL) was added, and this was shaken for 1 hour. After washing with dichloromethane and DMF, the Fmoc protecting group was removed by treating with 20% piperidine in DMF. After washing with DMF, in a peptide solid phase synthesis method with Fmoc strategy employing a PreludeTM peptide synthesizer, a protected peptide was synthesized on the resin. The condensation reaction was performed in DMF using HCTU as the condensation agent.
The Fmoc protecting group was removed by treating with 20% piperidine in DMF. After washing with DMF and dichloromethane, TFA:water:triisopropylsilane:ethanedithiol (=90:2.5:5:2.5) was added, and this was shaken at room temperature for 3 hours. The resin was filtered off, cold diethyl ether was added to the filtrate, and crude peptide was obtained as precipitate. A part of the crude peptide was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ50×250 mm, flow rate: 43.7 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=73:27→65:35, 14 minutes, linear gradient elution] to obtain peptide 54 (SEQ ID NO. 68) represented by the following formula (54) (41.7 mg).




ESI-MS: (m/z) calcd for C149H229N45O42S6: [M+3H]3+ 1172.7, [M+4H]4+ 879.8, [M+5H]5+ 704.0. found 1172.5, 879.4, 703.9.
24-2 Glycosylation Reaction of Thiol
Peptide 54 obtained in the method described in the above 24-1 (10.7 mg, 3.04 μmol) and compound a represented by the above formula (a) (36.6 mg, 15.6 μmol, 5.2 equivalents to peptide 54) were dissolved in 33 mM phosphate buffer (pH 7.4, 0.91 mL) containing 10 μM TCEP, and reacted at room temperature for 100 minutes. The reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=80:20→70:30, 5 minutes, then A:B=70:30→65:35, 12 minutes, linear gradient elution] to obtain glycopeptide 55 (SEQ ID NO. 69) represented by the following formula (55) (11.7 mg, 1.14 μmol, yield 38%).




ESI-MS: (m/z) calcd for C407H646N66O228S6: [M+5H]5+ 2061.8, [M+6H]6+ 1718.4, [M+7H]7+ 1473.0, [M+8H]8+ 1289.0, [M+9H]9+ 1145.9, [M+10H]10+ 1031.4. found 2061.8, 1718.2, 1472.9, 1289.0, 1145.8, 1031.3.
24-3 Deprotection of Acm Group
To glycopeptide 55 obtained in the method described in the above 24-2 (11.7 mg, 1.14 μmol) was added an aqueous solution (0.46 mL) of silver(I) acetate (4.7 mg, 28 μmol), and reacted at room temperature for 2 hours. DTT (11.3 mg, 73 μmol) dissolved in 200 mM Tris-HCl buffer (pH 7.4, 0.46 mL) and 100 mM ascorbic acid aqueous solution (0.11 mL) were added, and this was promptly filtered with a filter. The filtrate was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=80:20→70:30, 5 minutes, then 70:30→55:45, 15 minutes, linear gradient elution] to obtain glycopeptide 56 (SEQ ID NO. 70) represented by the following formula (56) (7.4 mg, 0.73 μmol, yield 64%).




ESI-MS: (m/z) calcd for C401H636N64O226S6: [M+6H]6+ 1694.7, [M+7H]7+ 1452.7, [M+8H]8+ 1271.3, [M+9H]9+ 1130.1, [M+10H]10+ 1017.2. found 1694.6, 1452.5, 1271.4, 1130.0, 1017.2.
24-4 Formation of Disulfide Bond
Glycopeptide 56 obtained in the method described in the above 24-3 (7.4 mg, 0.73 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO (1/1, v/v, 1.8 mL), and reacted at room temperature for 25 hours. The reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=80:20→70:30, 5 minutes, then 70:30→69.3:30.7, 5 minutes, linear gradient elution] to obtain a fraction containing compound (S1-3C(disialo)-SRIF28) represented by the following formula (57) (SEQ ID NO. 25).




This fraction was further purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=80:20→40:60, 20 minutes, linear gradient elution] to obtain S1-3C(disialo)-SRIF28 (4.7 mg, 0.46 μmol, yield 63%).
ESI-MS: (m/z) calcd for C401H634N64O226S6: [M+3H]3+ 3387.7, [M+4H]4+ 2541.0, [M+5H]5+ 2033.0, [M+6H]6+ 1694.3, [M+7H]7+ 1452.4. found 3387.6, 2540.9, 2032.7, 1694.2, 1452.3.
Example 25
Synthesis of S1C(disialo) N5C(disialo).A9C(disialo)-SRIF28
A compound represented by the following formula (59) (S1C(disialo).N5C(disialo).A9C(disialo)-SRIF28) (SEQ ID NO. 26) was synthesized similarly to Example 24, except that peptide 58 (SEQ ID NO. 71) represented by the following formula (58) was employed instead of peptide 54.




Example 26
Synthesis of S1C(monosialo)-SRIF28
A compound represented by the following formula (60) (S1C(monosialo)-SRIF28) (SEQ ID NO. 27) was synthesized similarly to Example 1, except that compound b represented by the following formula (b) (bromoacetamidated oligosaccharide: from Otsuka Chemical Co., Ltd.) was employed instead of compound a.




In the final product, the ratio between the glycosylated polypeptide having the sugar chain of the following formula b1 and the glycosylated polypeptide having the sugar chain of the following formula b2 was 45:55. Note that it is possible to manufacture a glycosylated polypeptide having substantially uniform sugar chain structure by using monosialo sugar chain derivatives having identical structure.




Example 27
Synthesis of S1C(asialo)-SRIF28
A compound represented by the following formula (61) (S1C(asialo)-SRIF28) (SEQ ID NO. 28) was synthesized similarly to Example 1, except that compound c represented by the following formula (c) (bromoacetamidated oligosaccharide: from Otsuka Chemical Co., Ltd.) was employed instead of compound a.




Example 28
Synthesis of S1-2C(asialo)-SRIF28
28-1 Glycosylation reaction of thiol Peptide 44 (21.2 mg, 6.21 μmol) and compound c (44.5 mg, 25.3 μmol, 4.1 equivalents to peptide 44) were dissolved in 33 mM phosphate buffer (pH 7.4, 1.9 mL), and reacted at room temperature for 1 hour. The reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=77:23→62:38, 15 minutes, linear gradient elution] to obtain glycopeptide 62 (SEQ ID NO. 72) represented by the following formula (62) (24.0 mg, 3.54 μmol, yield 57%).




ESI-MS: (m/z) calcd for C274H434N54O133S5: [M+4H]4+ 1694.3, [M+5H]5+ 1355.6, [M+6H]6+ 1129.8. found 1694.3, 1355.6, 1130.0.
28-2 Deprotection of Acm Group
To glycopeptide 62 obtained in the method described in the above 28-1 (24.0 mg, 3.54 μmol) was added an aqueous solution (1.4 mL) of silver(I) acetate (6.0 mg, 36 μmol), and reacted at room temperature for 3 hours. DTT (14.0 mg, 90.8 μmol) dissolved in 500 mM phosphate buffer (pH 7.4, 0.57 mL) and 100 mM ascorbic acid aqueous solution (0.35 mL) were added, and this was promptly filtered with a filter. The filtrate was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=75:25→65:35, 15 minutes, linear gradient elution] to obtain glycopeptide 63 (SEQ ID NO. 73) represented by the following formula (63) (20.1 mg, 3.03 μmol, yield 86%).




ESI-MS: (m/z) calcd for C268H424N52O131S5: [M+4H]4+ 1658.7, [M+5H]5+ 1327.2. found 1658.8, 1327.0.
28-3 Formation of Disulfide Bond
Glycopeptide 63 obtained in the method described in the above 28-2 (20.1 mg, 3.03 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO (1/1, v/v, 6.1 mL), and reacted at room temperature for 2 days. The reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=77:23→65:35, 16 minutes, linear gradient elution] to obtain a fraction containing compound (S1-2C(asialo)-SRIF28) represented by the following formula (64) (SEQ ID NO. 29).




This fraction was further purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=92:8→80:20, 16 minutes, linear gradient elution] to obtain S1-2C(asialo)-SRIF28 (11.0 mg, 1.66 μmol, yield 55%).
ESI-MS: (m/z) calcd for C268H422N52O131S5: [M+4H]4+ 1658.2, [M+5H]5+ 1326.8, [M+6H]6+ 1105.8, [M+7H]7+ 948.0, [M+8H]8+ 829.6. found 1658.1, 1326.7, 1105.6, 947.8, 829.4.
Example 29
Synthesis of S1-3C(asialo)-SRIF28
29-1 Glycosylation Reaction of Thiol
Peptide 54 (21.3 mg, 6.06 μmol) and compound c (53.4 mg, 30.3 μmol, 5.0 equivalents to peptide 54) were dissolved in 33 mM phosphate buffer (pH 7.4, 1.8 mL), and reacted at room temperature for 1 hour. The reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=75:25→70:30, 20 minutes, linear gradient elution] to obtain glycopeptide 65 (SEQ ID NO. 74) represented by the following formula (65) (39.3 mg, 4.59 μmol, yield 76%).




ESI-MS: (m/z) calcd for C341H544N60O180S6: [M+4H]4+ 2140.2, [M+5H]5+ 1712.3, [M+6H]6+ 1427.1. found 2140.2, 1712.4, 1427.2.
29-2 Deprotection of Acm Group
To glycopeptide 65 obtained in the method described in the above 29-1 (39.3 mg, 4.59 μmol) was added an aqueous solution (1.8 mL) of silver(I) acetate (18.7 mg, 112 μmol), and reacted at room temperature for 90 minutes. DTT (43.4 mg, 28.1 μmol) dissolved in 200 mM phosphate buffer (pH 7.4, 1.8 mL) and 100 mM ascorbic acid aqueous solution (0.46 mL) were added, and this was promptly filtered with a filter. The filtrate was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=75:25→68:32, 18 minutes, linear gradient elution] to obtain glycopeptide 66 (SEQ ID NO. 75) represented by the following formula (66) (27.6 mg, 3.28 μmol, yield 71%). [Chemical Formula 86]




ESI-MS: (m/z) calcd for C355H534N58O178S6: [+4H]4+ 2104.6, [M+5H]5+ 1683.9, [M+6H]6+ 1403.4. found 2104.6, 1684.0, 1403.3.
29-3 Formation of Disulfide Bond
Glycopeptide 66 obtained in the method described in the above 29-2 (27.6 mg, 3.28 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO (1/1, v/v, 8.2 mL), and reacted at room temperature for 2 days. The reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=72:28→70.5:29.5, 15 minutes, linear gradient elution] to obtain a fraction containing compound (S1-3C(asialo)-SRIF28) represented by the following formula (67) (SEQ ID NO. 30).




This fraction was further purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=96:4→82:18, 20 minutes, linear gradient elution] to obtain S1-3C(asialo)-SRIF28 (14.5 mg, 1.72 μmol, yield 52%).
ESI-MS: (m/z) calcd for C335H532N58O178S6: [M+4H]4+ 2104.1, [M+5H]5+ 1683.5, [M+6H]6+ 1403.1. found 2103.7, 1683.3, 1403.0.
Example 30
Synthesis of N5N(disialo)-SRIF28
30-1 Solid Phase Synthesis of Glycopeptide
2-chlorotrityl chloride resin (100 μmol) was taken in a column for solid phase synthesis, and after washing with DMF and dichloromethane, a solution of Fmoc-Cys(Trt)-OH (72.5 mg, 120 μmol) and DIPEA (104.6 μL, 600 μmol) in dichloromethane (3.0 mL) was added, and this was shaken for 1 hour. After washing with dichloromethane and DMF, the Fmoc protecting group was removed by treating with 20% piperidine in DMF. After washing with DMF, in a peptide solid phase synthesis method with Fmoc strategy employing a PreludeTM peptide synthesizer, a protected peptide 68 (SEQ ID NO. 76) represented by the following formula (68) was synthesized in a state bound to the resin. The condensation reaction was performed in DMF using HCTU as the condensation agent.




Next, the Fmoc protecting group was removed by treating with 20% piperidine in DMF. After washing with DMF, compound d represented by the following formula (d) (from Otsuka Chemical Co., Ltd.) (411.9 mg, 150.4 μmol), DMSO-DMF (1/1, v/v, 871 μL) solution, TBTU (64.2 mg, 200 μmol), and DIPEA (52.3 μL, 300 μmol) were sequentially added to the resin, and this was shaken at room temperature for 3 hours to allow condensation.




After washing with DMF, this condensation operation was repeated once. After washing the resin with DMF and dichloromethane, this was shaken with 20% piperidine in DMF for 20 minutes to deprotect the Fmoc group, and the resin was washed with DMF to synthesize a protected compound represented by the following formula (69) (peptide 69) (SEQ ID NO. 77) on the resin.




To this resin, employing HOBt/DIC as the condensation agent, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ala, and Fmoc-Ser(tBu)-OH were condensed in sequence. After condensation, the Fmoc protecting group was removed by treating with 20% piperidine in DMF. After washing with DMF and dichloromethane, TFA:water:triisopropylsilane:ethanedithiol (=90:2.5:5:2.5) was added, and this was shaken at room temperature for 3 hours. Cold diethyl ether was added to the filtrate, and crude peptide was obtained as precipitate. This crude peptide was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, A:B=70:30] to obtain glycopeptide 70 (SEQ ID NO. 78) represented by the following formula (70) (29.1 mg, 5.26 μmol).




ESI-MS: (m/z) calcd for C235H357N47O100S3: [M+3H]3+ 1846.6, [M+4H]4+ 1385.2, [M+5H]5+ 1108.4. found 1846.5, 1385.1, 1108.3.
30-2 Formation of Disulfide Bond
Glycopeptide 70 obtained in the method described in the above 30-1 (12.2 mg, 2.20 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO (1/1, v/v, 5.5 mL), and reacted at room temperature for 2 days. The reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=80:20→66:35, 30 minutes, linear gradient elution] to obtain glycopeptide 71 (SEQ ID NO. 79) represented by the following formula (71) (8.3 mg, 1.5 μmol, yield 68%).




ESI-MS: (m/z) calcd for C235H355N47O100S3: [M+3H]3+ 1846.5, [M+4H]4+ 1384.7, [M+5H]5+ 1108.0. found 1846.5, 1384.7, 1108.1.
30-3 Deprotection of Benzyl Group
Glycopeptide 71 obtained in the method described in the above 30-2 (7.5 mg, 1.4 μmol) was dissolved in 50 mM sodium hydroxide aqueous solution (20.6 mL), and reacted at 0° C. for 80 minutes. 200 mM acetic acid aqueous solution (5.1 mL) was added, and the mixed solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=73:27→66.3:33.7, 20 minutes, linear gradient elution] to obtain a fraction containing compound (N5N(disialo)-SRIF28) represented by the following formula (72) (SEQ ID NO. 31)




This fraction was further purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=80:20→60:40, 30 minutes, linear gradient elution] to obtain N5N(disialo)-SRIF28 (1.4 mg, yield 19%).
ESI-MS: (m/z) calcd for C221H343N47O100S3: [M+3H]3+ 1785.9, [M+4H]4+ 1339.6, [M+5H]5+ 1071.9. found 1785.7, 1339.5, 1071.8.
Example 31
Synthesis of S1N(disialo)-SRIF28
31-1 Solid Phase Synthesis of Glycopeptide
2-chlorotrityl chloride resin (100 μmol) was taken in a column for solid phase synthesis, and after washing with DMF and dichloromethane, a solution of Fmoc-Cys(Trt)-OH (72.5 mg, 120 μmol) and DIPEA (104.6 μL, 600 μmol) in dichloromethane (3.0 mL) was added, and this was shaken for 1 hour. After washing with dichloromethane and DMF, the Fmoc protecting group was removed by treating with 20% piperidine in DMF. After washing with DMF, in a peptide solid phase synthesis method with Fmoc strategy employing a PreludeTM peptide synthesizer, a protected peptide 73 (SEQ ID NO. 80) represented by the following formula (73) was synthesized in a state bound to the resin. The condensation reaction was performed in DMF using HCTU as the condensation agent.




Next, the Fmoc protecting group was removed by treating with 20% piperidine in DMF. After washing with DMF, compound d (420.2 mg, 153.3 μmol), DMSO-DMF (1/1, v/v, 871 μL) solution, TBTU (64.2 mg, 200 μmol), and DIPEA (52.3 μL, 300 μmol) were sequentially added to the resin, and this was shaken at room temperature for 2 hours to allow condensation. After washing with DMF, this condensation operation was repeated once. After washing the resin with DMF and dichloromethane, this was shaken with 20% piperidine in DMF for 20 minutes to deprotect the Fmoc group, and the resin was washed with DMF to synthesize a protected peptide 74 (SEQ ID NO. 81) represented by the following formula (74) on the resin.




After washing with DMF and dichloromethane, TFA:water:triisopropylsilane:ethanedithiol (=90:2.5:5:2.5) was added, and this was shaken at room temperature for 3 hours. Cold diethyl ether was added to the filtrate, and crude peptide was obtained as precipitate. This crude peptide was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, A:B=71:29] to obtain glycopeptide 75 (SEQ ID NO. 82) represented by the following formula (75) (65.7 mg, 11.8 μmol).




ESI-MS: (m/z) calcd for C236H358N48O100S3: [M+4H]4+ 1392.0, [M+5H]5+ 1113.8. found 1391.9, 1113.8.
31-2 Formation of Disulfide Bond
Glycopeptide 75 obtained in the method described in the above 31-1 (20.3 mg, 3.65 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO (1/1, v/v, 9.0 mL), and reacted at room temperature for 2 days. The reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=72:28→67:33, 30 minutes, linear gradient elution] to obtain glycopeptide 76 (SEQ ID NO. 83) represented by the following formula (76) (17.0 mg, 3.06 μmol, yield 84%).




ESI-MS: (m/z) calcd for C236H356N48O100S3: [M+4H]4+ 1391.5, [M+5H]5+ 1113.4. found 1391.3, 1113.2.
31-3 Deprotection of Benzyl Group
Glycopeptide 76 obtained in the method described in the above 31-2 (7.0 mg, 1.3 μmol) was dissolved in 50 mM sodium hydroxide aqueous solution (19.1 mL), and reacted at 0° C. for 1 hour. 200 mM acetic acid aqueous solution (9.6 mL) was added, and the mixed solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=85:15→77.5:22.5, 20 minutes, linear gradient elution] to obtain a compound represented by the following formula (77) (S1N(disialo)-SRIF28) (SEQ ID NO. 32) (2.7 mg, 0.50 μmol, yield 40%).




ESI-MS: (m/z) calcd for C222H344N48O100S3: [M+3H]3+ 1794.9, [M+4H]4+ 1346.4, [M+5H]5+ 1077.3, [M+6H]6+ 897.9. found 1794.7, 1346.2, 1077.2, 897.7.
Example 32
Synthesis of S1C(disialo).N19C(GlcNAc)-SRIF28
32-1 Synthesis of Peptide
2-chlorotrityl chloride resin (100 μmol) was taken in a column for solid phase synthesis, and after washing with DMF and dichloromethane, a solution of Fmoc-Cys(Acm)-OH (49.7 mg, 120 μmol) and DIPEA (104.5 μL, 600 μmol) in dichloromethane (3.0 mL) was added, and this was shaken for 1 hour. After washing with dichloromethane and DMF, the Fmoc protecting group was removed by treating with 20% piperidine in DMF. After washing with DMF and dichloromethane, in a peptide solid phase synthesis method with Fmoc strategy employing a PreludeTM peptide synthesizer, a protected peptide was synthesized on a resin. The condensation reaction was performed in DMF using HCTU as the condensation agent.
The Fmoc protecting group was removed by treating with 20% piperidine in DMF. After washing with DMF and dichloromethane, TFA:water:triisopropylsilane:ethanedithiol (=90:2.5:5:2.5) was added, and this was shaken at room temperature for 3 hours. The resin was filtered off, cold diethyl ether was added to the filtrate, and crude peptide was obtained as precipitate. A part of the crude peptide was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=72:28→64:36, 20 minutes, linear gradient elution] to obtain peptide 78 (SEQ ID NO. 84) represented by the following formula (78) (28.9 m).




ESI-MS: (m/z) calcd for C146H226N42O39S6: [M+3H]3+ 1129.7, [M+4H]4+ 847.5. found 1129.5, 847.4.
32-2 Glycosylation of Thiol and Deprotection of StBu Group
Peptide 78 obtained in the method described in the above 32-1 (10.0 mg, 2.95 μmol) and compound e represented by the following formula (e) (bromoacetamidated monosaccharide: from Otsuka Chemical Co., Ltd.) (2.0 mg, 5.90 μmol, 2.0 equivalents to peptide 78) were dissolved in 33 mM phosphate buffer (pH 7.4, 0.89 mL) containing 20 μM TCEP, and reacted at room temperature for 2 hours.




After the reaction, DTT (45.5 mg, 295 μmol) dissolved in 0.1 M phosphate buffer (pH 7.4, 3.0 mL) was added, and reacted at room temperature for 3 hours. The reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=75:25→65:35, 20 minutes, linear gradient elution] to obtain glycopeptide 79 (SEQ ID NO. 85) represented by the following formula (79) (6.8 mg, 1.9 μmol, yield 64%).




ESI-MS: (m/z) calcd for C152H234N44O45S5[M+3H]3+ 1187.0, [M+4H]4+ 890.5. found 1187.0, 890.5.
32-3 Glycosylation Reaction of Thiol
Peptide 79 obtained in the method described in the above 32-2 (6.8 mg, 1.9 μmol) and compound a represented by the above formula (a) (22.4 mg, 9.56 μmol, 5.0 equivalents to peptide 79) were dissolved in 0.1 M phosphate buffer (pH 7.4, 2.0 mL) containing 7.6 mM DTT, and reacted at room temperature for 2 hours. The reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=85:15→65:35, 20 minutes, linear gradient elution] to obtain glycopeptide 80 (SEQ ID NO. 86) represented by the following formula (80) (3.4 mg, 0.58 μmol, yield 31%).




ESI-MS: (m/z) calcd for C238H373N51O107S5: [M+5H]5+ 1165.2, [M+6H]6+ 971.2, [M+7H]7+ 832.6, [M+8H]8+ 728.6. found 1165.2, 971.1, 832.6, 728.6.
32-4 Deprotection of Acm Group
To glycopeptide 80 obtained in the method described in the above 32-3 (3.8 mg, 0.65 μmol) was added an aqueous solution (262 μL) of silver(I) acetate (2.7 mg, 16 μmol), and reacted at room temperature for 1 hour. DTT (10.0 mg, 64.8 μmol) dissolved in 100 mM phosphate buffer (pH 7.4, 426 μL) and 100 mM ascorbic acid aqueous solution (66 μL) were added, and this was promptly filtered with a filter. The filtrate was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=90:10→60:40, 30 minutes, linear gradient elution] to obtain glycopeptide 81 (SEQ ID NO. 87) represented by the following formula (81) (2.5 mg, 0.44 μmol, yield 67%).




ESI-MS: (m/z) calcd for C232H363N49O105S5: [M+3H]3+ 1894.0, [M+4H]4+ 1420.7, [M+5H]5+ 1136.8. found 1893.8, 1420.6, 1136.7.
32-4 Formation of Disulfide Bond
Glycopeptide 81 obtained in the method described in the above 32-3 (2.5 mg, 0.44 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO (1/1, v/v, 1.1 mL), and reacted overnight at room temperature. The reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=90:10→70:30, 30 minutes, linear gradient elution] to obtain a compound represented by the following formula (82) (S1C(disialo).N19C(GlcNAc)-SRIF28) (SEQ ID NO. 33) (1.5 mg, 0.26 μmol, yield 59%).




ESI-MS: (m/z) calcd for C232H361N49O105S5: [M+3H]3+ 1893.3, [M+4H]4+ 1420.2, [M+5H]5+ 1136.4. found 1893.5, 1420.1, 1136.3.
Example 33
Synthesis of S1C(disialo).N19C(diMan)-SRIF28
A compound represented by the following formula (83) (S1C(disialo).N19C(diMan)-SRIF28) (SEQ ID NO. 34) was synthesized similarly to Example 32, except that compound f represented by the following formula (f) (bromoacetamidated oligosaccharide: from Otsuka Chemical Co., Ltd.) was employed instead of compound e.




Example 34
Synthesis of S1-5C(disialo)-SRIF28
34-1 Synthesis of Peptide
2-chlorotrityl chloride resin (100 μmol) was taken in a column for solid phase synthesis, and after washing with DMF and dichloromethane, a solution of Fmoc-Cys(Acm)-OH (49.7 mg, 120 μmol) and DIPEA (104.5 μL, 600 μmol) in dichloromethane (3.0 mL) was added, and this was shaken for 1 hour. After washing with dichloromethane and DMF, the Fmoc protecting group was removed by treating with 20% piperidine in DMF. After washing with DMF, in a peptide solid phase synthesis method with Fmoc strategy employing a PreludeTM peptide synthesizer, a protected peptide was synthesized on the resin. The condensation reaction was performed in DMF using HCTU as the condensation agent.
The Fmoc protecting group was removed by treating with 20% piperidine in DMF. After washing with DMF and dichloromethane, TFA:water: triisopropylsilane: ethanedithiol (=90:2.5:5:2.5) was added, and this was shaken at room temperature for 3 hours. The resin was filtered off, cold diethyl ether was added to the filtrate, and crude peptide was obtained as precipitate. The crude peptide was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ50×250 mm, flow rate: 43.7 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=73:27→65:35, 35 minutes, linear gradient elution] to obtain peptide 84 (SEQ ID NO. 90) represented by the following formula (84).




ESI-MS: (m/z) calcd for C155H239N47O44S8: [M+3H]3+ 1241.5, [M+4H]4+ 931.3, [M+5H]5+ 745.3. found 1241.2, 931.2, 745.1.
34-2 Glycosylation Reaction of Thiol
Peptide 84 obtained in the method described in the above 34-1 (33.7 mg, 9.06 μmol) and compound a represented by the above formula (a) (bromoacetamidated oligosaccharide: from Otsuka Chemical Co., Ltd.) (160.9 mg, 68.6 μmol, 7.5 equivalents to peptide 84) were dissolved in 33 mM phosphate buffer (pH 7.4, 2.7 mL) containing 10 μM TCEP, and reacted overnight at room temperature. The reaction solution was purified with HPLC [column: SHISEIDO Proteonavi (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=80:20→78.4:21.6, 20 minutes, linear gradient elution] to obtain glycopeptide 85 (SEQ ID NO. 91) represented by the following formula (85) (35.1 mg, 2.33 μmol, yield 26%).
ESI-MS: (m/z) calcd for C585H934N82O354S8: [M+6H]6+ 2507.1, [M+7H]7+ 2149.1, [M+8H]8+ 1880.6. found 2506.9, 2149.0, 1880.6.




34-3 Deprotection of Acm Group

To glycopeptide 85 obtained in the method described in the above 34-2 (20.6 mg, 1.37 μmol) was added an aqueous solution (0.55 mL) of silver(I) acetate (5.6 mg, 34 μmol), and reacted at room temperature for 50 minutes. Then, DTT (13.8 mg, 89 μmol) dissolved in 200 mM phosphate buffer (pH 7.4, 0.55 mL) and 100 mM ascorbic acid aqueous solution (137 μL) were added, and this was promptly filtered with a filter. The filtrate was purified with HPLC [column: SHISEIDO Proteonavi (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=80:20→73.6:26.4, 16 minutes, linear gradient elution] to obtain glycopeptide 86 (SEQ ID NO. 92) represented by the following formula (86) (13.6 mg, 0.913 μmol, yield 67%).




ESI-MS: (m/z) calcd for C579H924N80O352S8: [M+6H]6+ 2483.4, [M+7H]7+ 2128.8, [M+8H]8+ 1862.8. found 2483.2, 2128.8, 1862.9.
34-4 Formation of Disulfide Bond
Glycopeptide 86 obtained in the method described in the above 34-3 (13.6 mg, 0.913 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO (1/1, v/v, 2.2 mL), and reacted at room temperature for 2 days. Then, the reaction solution was purified with HPLC [column: Proteonavi (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 10 mM ammonium acetate aqueous solution, B: 10 mM ammonium acetate-acetonitrile (1/9, v/v), gradient A:B=78:22→70.8:29.2, 15 minutes, linear gradient elution] to obtain S1-5C(disialo)-SRIF28 (SEQ ID NO. 88) represented by the following formula (87) (10.4 mg, 0.698 μmol, yield 76%).




ESI-MS: (m/z) calcd for C579H922N80O352S8: [M+4H]5+ 3724.1, [M+5H]5+ 2979.5, [M+6H]6+ 2483.1, [M+7H]7+ 2128.5, [M+8H]8+ 1862.6. found 3723.7, 2979.1, 2482.9, 2128.2, 1862.4.
Example 35
Synthesis of S1-10C(disialo)-SRIF28
35-1 Synthesis of Peptide
2-chlorotrityl chloride resin (100 μmol) was taken in a column for solid phase synthesis, and after washing with DMF and dichloromethane, a solution of Fmoc-Cys(Acm)-OH (49.7 mg, 120 μmol) and DIPEA (104.5 μL, 600 μmol) in dichloromethane (3.0 mL) was added, and this was shaken for 1 hour. After washing with dichloromethane and DMF, the Fmoc protecting group was removed by treating with 20% piperidine in DMF. After washing with DMF, in a peptide solid phase synthesis method with Fmoc strategy employing a PreludeTM peptide synthesizer, a protected peptide was synthesized on the resin. The condensation reaction was performed in DMF using HCTU as the condensation agent.
The Fmoc protecting group was removed by treating with 20% piperidine in DMF. After washing with DMF and dichloromethane, TFA:water:triisopropylsilane:ethanedithiol (=90:2.5:5:2.5) was added, and this was shaken at room temperature for 3 hours. The resin was filtered off, cold diethyl ether was added to the filtrate, and crude peptide was obtained as precipitate. The crude peptide was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=69:31→66.6:33.4, 15 minutes, linear gradient elution] to obtain peptide 88 (SEQ ID NO. 93) represented by the following formula (88) (16.8 mg).




ESI-MS: (m/z) calcd for C170H264N52O49S13: [M+3H]3+ 1413.4, [M+4H]4+ 1060.3, [M+5H]5+ 848.4. found 1412.9, 1060.2, 848.1.
35-2 Synthesis of Disialo Sugar Chain Having Bromoacetamidated Benzyl Protecting Group
To compound a (28.9 mg, 12.3 μmol) were sequentially added DMF (0.58 mL), lithium bromide (21.5 mg, 248 μmol), and benzyl bromide (14.6 μL, 122 μmol), and reacted at 30° C. for 20 hours. Benzyl bromide (14.6 μL, 122 μmol) was further added and reacted for 20 hours. To the reaction solution was added toluene (30 mL), and after centrifugal separation (10,000×g, 10 minutes), the precipitate was dissolved in water (100 μL) and purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 8.0 mL/min, eluent: water:acetonitrile=95:5→70:30, 20 minutes, linear gradient elution] to obtain compound g represented by the following formula (g) (bromoacetamidated disialo sugar chain: 7.6 mg, 3.0 μmol, yield 24%).




MALDI-MS: (m/z) calcd for C100H152BrN7O62: [M+Na]+ 2544.8. found 2544.4.
35-3 Glycosylation Reaction of Thiol
Peptide 88 obtained in the method described in the above 35-1 (8.1 mg, 1.9 μmol) and compound g obtained in the method described in the above 35-2 (58.0 mg) were dissolved in 66 mM phosphate buffer (pH 6.8, 0.57 mL) containing 2.3 M guanidine hydrochloride, and reacted overnight at room temperature. The reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=80:20→74.7:25.3, 30 minutes, linear gradient elution] to obtain glycopeptide 89 (SEQ ID NO. 94) represented by the following formula (89) (12.3 mg, 0.429 μmol, yield 23%).




ESI-MS: (m/z) calcd for C1170H1774N122O669S13: [M+8H]8+ 3584.7, [M+9H]9+ 3186.5, [M+10H]10+ 2868.0, [M+11H]11+ 2607.4, [M+12H]12+ 2390.2, [M+13H]13+ 2206.4, [M+14H]14+ 2048.9. found 3585.0, 3186.6, 2867.9, 2607.6, 2390.1, 2206.4, 2048.9.
35-4 Deprotection of Acm Group
To glycopeptide 89 obtained in the method described in the above 35-3 (49.8 mg, 1.74 μmol) was added an aqueous solution (0.70 mL) of silver(I) acetate (10.2 mg), and reacted at room temperature for 1 hour. A solution of DTT (16.6 mg) in 200 mM phosphate buffer (pH 7.4, 0.70 mL) and 100 mM ascorbic acid aqueous solution (0.17 mL) were added, and this was promptly filtered with a filter. The filtrate was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=78:22→73:27, 20 minutes, linear gradient elution] to obtain glycopeptide 90 (SEQ ID NO. 95) represented by the following formula (90) (33.0 mg, 1.16 μmol, yield 67%).




ESI-MS: (m/z) calcd for C1164H1764N120O867S13: [M+9H]9+ 3170.8, [M+10H]10+ 2853.8, [M+11H]11+ 2594.4, [M+12H]12+ 2378.3, [M+13H]13+ 2195.4, [M+14H]14+ 2038.7, [M+15H]15+ 1902.9. found 3170.9, 2853.7, 2594.4, 2378.3, 2195.4, 2038.7, 1902.9.
35-5 Formation of Disulfide Bond
Glycopeptide 90 obtained in the method described in the above 35-4 (2.1 mg, 0.074 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO (1/1, v/v, 185 μL), and reacted at room temperature for 2 days. Then, the reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ4.6×250 mm, flow rate: 0.7 mL/min, eluent A: 10 mM ammonium acetate aqueous solution, B: 10 mM ammonium acetate-acetonitrile (1/9, v/v), gradient A:B=69:31→65:35, 20 minutes, linear gradient elution] to obtain glycopeptide 91 (SEQ ID NO. 96) represented by the following formula (91) (1.0 mg, 0.035 μmol, yield 47%).




ESI-MS: (m/z) calcd for C1164H1762N120O667S13: [M+8H]8+ 3566.7, [M+9H]9+ 3170.5, [M+10H]10+ 2853.6. found 3566.6, 3170.4, 2853.6.
35-6 Deprotection of Benzyl Group
Glycopeptide 91 obtained in the method described in the above 35-5 (17.1 mg, 0.599 μmol) was dissolved in 50 mM sodium hydroxide aqueous solution (12 mL), and reacted at 0° C. for 90 minutes. 100 mM acetic acid aqueous solution 12 mL was added, and the mixed solution was purified with HPLC [column: SHISEIDO Proteonavi, φ4.6×250 mm, flow rate: 0.7 mL/min, eluent A: 10 mM ammonium acetate aqueous solution, B: 10 mM ammonium acetate-acetonitrile (1/9, v/v), gradient A:B=72:18→80:20, 20 minutes, linear gradient elution] to obtain glycopeptide 92 (SEQ ID NO. 89) represented by the following formula (92) (5.8 mg, 0.22 μmol, yield 37%).




ESI-MS: (m/z) calcd for C1024H1642N120O667S13: [M+7H]7+ 3818.6, [M+8H]8+ 3341.4, [M+9H]9+ 2970.3, [M+10H]10+ 2673.3. found 3818.3, 3341.0, 2970.1, 2673.1.
Example 36
Synthesis of C(disialo)-SRIF28
A compound represented by the following formula (94) (C(disialo)-SRIF28) (SEQ ID NO. 98) was synthesized similarly to Example 1, except that a compound represented by the following formula (93) (peptide 93) (SEQ ID NO. 97) was employed instead of peptide 1.




Example 37
Synthesis of R11C(disialo)-SRIF28
A compound represented by the following formula (96) (R11C(disialo)-SRIF28) (SEQ ID NO. 100) was synthesized similarly to Example 1, except that a compound represented by the following formula (95) (peptide 95) (SEQ ID NO. 99) was employed instead of peptide 1.




Example 38
Synthesis of F20C(disialo)-SRIF28
A compound represented by the following formula (98) (F20C(disialo)-SRIF28) (SEQ ID NO. 102) was synthesized similarly to Example 1, except that a compound represented by the following formula (97) (peptide 97) (SEQ ID NO. 101) was employed instead of peptide 1.




Example 39
Synthesis of T24C(disialo)-SRIF28
A compound represented by the following formula (100) (T24C(disialo)-SRIF28) (SEQ ID NO. 104) was synthesized similarly to Example 1, except that a compound represented by the following formula (99) (peptide 99) (SEQ ID NO. 103) was employed instead of peptide 1.




Example 40
Synthesis of F25C(disialo)-SRIF28
A compound represented by the following formula (102) (F25C(disialo)-SRIF28) (SEQ ID NO. 106) was synthesized similarly to Example 1, except that a compound represented by the following formula (101) (peptide 101) (SEQ ID NO. 105) was employed instead of peptide 1.




Example 41
Synthesis of S27C(disialo)-SRIF28
A compound represented by the following formula (104) (S27C(disialo)-SRIF28) (SEQ ID NO. 108) was synthesized similarly to Example 1, except that a compound represented by the following formula (103) (peptide 103) (SEQ ID NO. 107) was employed instead of peptide 1.




Example 42
Synthesis of C(disialo)-K-SRIF14
A compound represented by the following formula (106) (C(disialo)-K-SRIF14) (SEQ ID NO. 110) was synthesized similarly to Example 1, except that a compound represented by the following formula (105) (peptide 105) (SEQ ID NO. 109) was employed instead of peptide 1.




Example 43
Synthesis of S1C(disialo)-F25Y-SRIF28
A compound represented by the following formula (108) (S1C(disialo)-F25Y-SRIF28) (SEQ ID NO. 112) was synthesized similarly to Example 1, except that a compound represented by the following formula (107) (peptide 107) (SEQ ID NO. 111) was employed instead of peptide 1.




Example 44
Synthesis of S1C(disialo)-SRIF28-amide
A compound represented by the following formula (110) (S1C(disialo)-SRIF28-amide) (SEQ ID NO. 114) was synthesized similarly to Example 1, except that a compound represented by the following formula (109) (peptide 109) (SEQ ID NO. 113) was employed instead of peptide 1.




Example 45
Synthesis of C(disialo)-PEG linker-SRIF14
45-1 Synthesis of Peptide
The Fmoc protecting group of the protected peptide 39 (SEQ ID NO. 58) (50 μmol) bound to the resin, obtained in the method described in the above 19-1, was removed by treating with 20% piperidine in DMF. After washing with DMF, employing HCTU as the condensation agent, Fmoc-NH-(PEG)2-COOH (from Merck) and Fmoc-Cys(Trt)-OH were condensed in sequence. The Fmoc protecting group was removed by treating with 20% piperidine in DMF. After washing with DMF and dichloromethane, TFA:water:triisopropylsilane:ethanedithiol (=90:2.5:5:2.5) was added, and this was shaken for 3 hours at room temperature. The resin was filtered off, cold diethyl ether was added to the filtrate, and crude peptide was obtained as precipitate. A part of the crude peptide was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=74:26→69:31, 1 minute, then 69:31→62:38, 30 minutes, linear gradient elution] to obtain a compound represented by the following formula (111) (peptide 111) (SEQ ID NO. 115) (43.1 mg).




ESI-MS: (m/z) calcd for C94H138N22O26S3: [M+2H]2+ 1045.2, [M+3H]3+ 697.1. found 1045.0, 697.0.
45-2 Synthesis of C(disialo)-PEG linker-SRIF14
A compound represented by the following formula (112) (C(disialo)-PEG linker-SRIF14) (SEQ ID NO. 116) was synthesized similarly to Example 1, except that peptide 111 obtained in the method described in the above 45-1 was employed instead of peptide 1.




Example 46
Synthesis of Biotin-S1C(disialo)-SRIF28
46-1 Synthesis of Peptide
2-chlorotrityl chloride resin (100 μmol) was taken in a column for solid phase synthesis, and after washing with DMF and dichloromethane, a dichloromethane (3.0 mL) solution containing Fmoc-Cys(Acm)-OH (49.7 mg, 120 μmol) and DIPEA (104.5 μL, 600 μmol) was added, and this was shaken for 1 hour. After washing with dichloromethane and DMF, the Fmoc protecting group was removed by treating with 20% piperidine in DMF. After washing with DMF, in a peptide solid phase synthesis method with Fmoc strategy employing a PreludeTM peptide synthesizer, a protected peptide 113 (SEQ ID NO. 117) represented by the following formula (113) was synthesized in a state bound to the resin. The condensation reaction was performed in DMF using HCTU as the condensation agent.




The Fmoc protecting group was removed by treating with 20% piperidine in DMF. After washing with DMF, employing HCTU as the condensation agent, biotin was condensed. After washing with DMF and dichloromethane, TFA:water:triisopropylsilane:ethanedithiol (=90:2.5:5:2.5) was added, and this was shaken at room temperature for 3 hours. This leads to the detachment of the protecting group of the amino acid side chain (other than the Acm group), as well as cleaving between the peptide and the resin. The resin was filtered off, cold diethyl ether was added to the filtrate, and crude peptide was obtained as precipitate. The crude peptide was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=75:25→61:39, 18 minutes, linear gradient elution] to obtain peptide 114 (SEQ ID NO. 118) represented by the following formula (114).




ESI-MS: (m/z) calcd for C153H233N45O42S5: [M+3H]3+ 1179.4, [M+4H]4+ 884.8, [M+5H]5+ 708.0. found 1179.2, 884.4, 707.9.
46-2 Synthesis of Biotin-S1C(disialo)-SRIF28
A compound represented by the following formula (115) (Biotin-S1C(disialo)-SRIF28) (SEQ ID NO. 119) was synthesized similarly to Example 1, except that peptide 114 obtained in the method described in the above 46-1 was employed instead of peptide 1.




Example 47
Synthesis of Biotin-PEG linker-S1C(disialo)-SRIF28
47-1 Synthesis of Peptide
The Fmoc protecting group of the protected peptide 113 (SEQ ID NO. 117) bound to the resin, obtained in the above 46-1, was removed by treating with 20% piperidine in DMF. After washing with DMF, employing HCTU as the condensation agent, Fmoc-NH-(PEG)2-COOH (from Merck) and biotin were condensed in sequence. After washing with DMF and dichloromethane, TFA:water:triisopropylsilane:ethanedithiol (=90:2.5:5:2.5) was added, and this was shaken at room temperature for 3 hours. The resin was filtered off, cold diethyl ether was added to the filtrate, and crude peptide was obtained as precipitate. The crude peptide was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=75:25→61:39, 22 minutes, linear gradient elution] to obtain peptide 116 (SEQ ID NO. 120) represented by the following formula (116).




ESI-MS: (m/z) calcd for C162H250N46O46S5: [M+3H]3+ 1247.1, [M+4H]4+ 935.6, [M+5H]5+ 748.7. found 1246.9, 935.4, 748.6.
47-2 Synthesis of Biotin-PEG linker-S1C(disialo)-SRIF28
A compound represented by the following formula (117) (Biotin-PEG linker-S1C(disialo)-SRIF28) (SEQ ID NO. 121) was synthesized similarly to Example 1, except that peptide 116 obtained in the method described in the above 47-1 was employed instead of peptide 1.




Example 48
Synthesis of Azido-S1C(disialo)-SRIF28
48-1 Synthesis of Peptide
The Fmoc protecting group of the protected peptide 113 (SEQ ID NO. 117) bound to the resin, obtained in the above 46-1, was removed by treating with 20% piperidine in DMF. After washing with DMF, employing HCTU as the condensation agent, 5-Azido-pentanoic acid was condensed. After washing with DMF and dichloromethane, TFA:water:triisopropylsilane:ethanedithiol (=90:2.5:5:2.5) was added, and this was shaken at room temperature for 3 hours. The resin was filtered off, cold diethyl ether was added to the filtrate, and crude peptide was obtained as precipitate. The crude peptide was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=70:30→60:40, 20 minutes, linear gradient elution] to obtain peptide 118 SEQ ID NO. 122) represented by the following formula (118).




ESI-MS: (m/z) calcd for C148H226N46O41S4: [M+3H]3+ 1145.6, [M+4H]4+ 859.5, [M+5H]5+ 687.8. found 1145.5, 859.2, 687.5.
48-2 Synthesis of Azido-S1C(disialo)-SRIF28
A compound represented by the following formula (119) (Azido-S1C(disialo)-SRIF28) (SEQ ID NO. 123) was synthesized similarly to Example 1, except that peptide 118 obtained in the method described in the above 48-1 was employed instead of peptide 1.




Example 49
Synthesis of S1C(disialo) E12C(disialo)-SRIF28
A compound represented by the following formula (121) (S1C(disialo).E12C(disialo)-SRIF28) (SEQ ID NO. 125) was synthesized similarly to Example 20, except that a compound represented by the following formula (120) (peptide 120) (SEQ ID NO. 124) was synthesized and employed instead of peptide 44.




Example 50
Synthesis of 2C(disialo)-R—K-SRIF14
A compound represented by the following formula (123) (2C(disialo)-R—K-SRIF14) (SEQ ID NO. 127) was synthesized similarly to Example 20, except that a compound represented by the following formula (122) (peptide 122) (SEQ ID NO. 126) was synthesized and employed instead of peptide 44.




Example 51
Synthesis of 3C(disialo)-R—K-SRIF14
A compound represented by the following formula (125) (3C(disialo)-R—K-SRIF14) (SEQ ID NO. 129) was synthesized similarly to Example 24, except that a compound represented by the following formula (124) (peptide 124) (SEQ ID NO. 128) was synthesized and employed instead of peptide 54.




Example 52
Synthesis of S1C(diGlcNAc)-SRIF28
52-1 Glycosylation Reaction of Thiol
Peptide 1 (SEQ ID NO. 38) (from APC, Inc.) (25.0 mg, 7.56 μmol) and compound h represented by the following formula (h) (bromoacetamidated oligosaccharide: from Otsuka Chemical Co., Ltd.) (15.6 mg, 15.1 μmol, 2.0 equivalents to peptide 1) were dissolved in 33 mM phosphate buffer (pH 7.4, 2.3 mL), and reacted at room temperature for 30 minutes.




The reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, A:B=75:25→62:38, 13 minutes, linear gradient elution] to obtain glycopeptide 126 (SEQ ID NO. 130) represented by the following formula (126) (25.6 mg, 6.01 μmol, yield 79%).




ESI-MS: (m/z) calcd for C195H304N48O76S4: [M+3H]3+ 1556.0, [M+4H]4+ 1167.3. found 1555.7, 1167.0.
52-2 Deprotection of Acm Group
To glycopeptide 126 obtained in the method described in the above 52-1 (28.3 mg, 6.07 μmol) was added an aqueous solution (2.4 mL) of silver(I) acetate (12.5 mg, 74.5 μmol), and reacted at room temperature for 30 minutes. DTT (28.8 mg, 187 μmol) dissolved in 200 mM Tris-HCl buffer (pH 7.4, 2.4 mL) and 100 mM ascorbic acid aqueous solution (0.6 mL) were added, and this was promptly filtered with a filter. The filtrate was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=73:27→60:40, 13 minutes, linear gradient elution] to obtain glycopeptide 127 (SEQ ID NO. 131) represented by the following formula (127) (19.8 mg, 4.38 μmol, yield 72%).




ESI-MS: (m/z) calcd for C189H294N46O74S4: [M+3H]3+ 1508.6, [M+4H]4+ 1131.7, [M+5H]5+ 905.6. found 1508.3, 1131.5, 905.4.
52-3 Formation of Disulfide Bond
Glycopeptide 127 obtained in the method described in the above 52-2 (19.8 mg, 4.38 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO (1/1, v/v, 8.8 mL), and reacted at room temperature for 2 days. The reaction solution was crudely purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=73:27→60:40, 13 minutes, linear gradient elution] to obtain a fraction containing compound (S1C(diGlcNAc)-SRIF28) represented by the following formula (128) (SEQ ID NO. 132).




This fraction was further purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=90:10→78:22, 12 minutes, linear gradient elution] to obtain S1C(diGlcNAc)-SRIF28 (11.9 mg, 2.63 μmol, yield 60%).
ESI-MS: (m/z) calcd for C189H292N46O74S4: [M+3H]3+ 1508.0, [M+4H]4+ 1131.2, [M+5H]5+ 905.2. found 1507.7, 1131.0, 905.0.
Example 53
Synthesis of S1C(diMan)-SRIF28
A compound represented by the following formula (129) (S1C(diMan)-SRIF28) (SEQ ID NO. 133) was synthesized similarly to Example 52, except that compound f was employed instead of compound h.




Example 54
Synthesis of N19C(diMan)-SRIF28
A compound represented by the following formula (130) (N19C(diMan)-SRIF28) (SEQ ID NO. 134) was synthesized similarly to Example 52, except that peptide 21 was employed instead of peptide 1 and compound f was employed instead of compound h.




Example 55
Synthesis of S1C(GlcNAc)-SRIF28
A compound represented by the following formula (131) (S1C(GlcNAc)-SRIF28) (SEQ ID NO. 135) was synthesized similarly to Example 52, except that compound e was employed instead of compound h.




Example 56
Synthesis of N19C(GlcNAc)-SRIF28
A compound represented by the following formula (132) (N19C(GlcNAc)-SRIF28) (SEQ ID NO. 136) was synthesized similarly to Example 52, except that peptide 21 was employed instead of peptide 1 and compound e was employed instead of compound h.




Example 57
Synthesis of S1C(trisialo)-SRIF28
A compound represented by the following formula (133) (S1C(trisialo)-SRIF28) (SEQ ID NO. 137) was synthesized similarly to Example 1, except that compound i represented by the following formula (i) (bromoacetamidated oligosaccharide: from Otsuka Chemical Co., Ltd.) was employed instead of compound a.




Example 58
Synthesis of S1C(tetrasialo)-SRIF28
A compound represented by the following formula (134) (S1C(tetrasialo)-SRIF28) (SEQ ID NO. 138) was synthesized similarly to Example 1, except that compound j represented by the following formula (j) (bromoacetamidated oligosaccharide: from Otsuka Chemical Co., Ltd.) was employed instead of compound a.




Example 59
Synthesis of S1C(disialo(aminoethylamide))-SRIF28
59-1 Synthesis of Bromoacetamidated Disialo Sugar Chain Derivative
To compound k represented by the following formula (k) (from Otsuka Chemical Co., Ltd.) (204.1 mg, 92.1 μmol) were added water (2 mL) and tert-butyl N-(2-aminoethyl)carbamate (0.29 mL, 0.18 mmol), and this was stirred at room temperature for 1 hour. After lyophilization, to the lyophilizate obtained were added DMF (5 mL), HATU (349 mg, 921 μmol), and DIPEA (161 μL, 921 μmol), and reacted at 37° C. for 18 hours. Toluene (50 mL) was added to the solution, and the deposited precipitate was collected by filtration. The precipitate was dissolved in DMF (5 mL), purified with gel filtration purification [column: Sephadex G-25, φ20×200 mm, flow rate: 30 mL/h], and the fraction of interest was collected and lyophilized to obtain compound 1 represented by the following formula (1) (152.4 mg, 60.8 μmol, yield 66%).




MALDI-MS: (m/z calcd for C98H166N10O64: [M+Na]+ 2530.0. found 2529.4.
Compound 1 (100 mg, 39.8 μmol) and ammonium hydrogen carbonate (31.4 mg, 398 μmol) were dissolved in water (1 mL), and reacted at room temperature for 7 days. After lyophilization, to the lyophilizate obtained were sequentially added water (1 mL), DCC (41.0 mg, 199 μmol), and bromoacetic acid (27.7 mg, 199 μmol) dissolved in DMF (1 mL). After 1 hour of reaction under ice cooling, the solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 8.0 mL/min, eluent: water:acetonitrile=84:16] to obtain compound m represented by the following formula (m) (bromoacetamidated disialo sugar chain derivative: 100 mg, 38.2 μmol, yield 96%).




MALDI-MS: (m/z) (m/z) calcd for C100H168BrN11O64: [M+Na]+ 2648.9. found 2648.5.
59-2 Glycosylation Reaction of Thiol
Compound m obtained in the method described in the above 59-1 (14.2 mg, 5.40 μmol) and peptide 1 (15.0 mg, 4.53 μmol were dissolved in 33 mM phosphate buffer (pH 7.4, 1.3 mL), and reacted at room temperature for 30 minutes. The reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% acetic acid (AcOH) water, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=86:14→82:18, 20 minutes, linear gradient elution] to obtain glycopeptide 135 (SEQ ID NO. 139) represented by the following formula (135) (13.4 mg, 2.29 μmol, yield 51%).
ESI-MS: (m/z) calcd for C243H386N54O104S4: [M+4H]4+ 1465.1, [M+5H]5+ 1172.2, [M+6H]6+ 977.0. found 1464.9, 1172.1, 977.1.




59-3 Deprotection of Boc Group

Glycopeptide 135 obtained in the method described in the above 59-2 (13.4 mg, 2.29 μmol) was dissolved in 95% TFA aqueous solution (458 μL), and this was shaken at room temperature for 5 minutes. After adding 50 mM ammonium acetate aqueous solution (pH 6.8, 33 mL), the reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=73:27→65:35, 10 minutes, linear gradient elution] to obtain glycopeptide 136 (SEQ ID NO. 140) represented by the following formula (136) (12.7 mg, 98 μmol, yield 98%).




ESI-MS: (m/z) calcd for C233H370N54O100S4: [M+4H]4+ 1415.1, [M+5H]5+ 1132.2, [M+6H]6+ 943.7, [M+7H]7+ 809.0. found 1414.9, 1132.1, 943.6, 808.9.
59-4 Deprotection of Acm Group
To glycopeptide 136 obtained in the method described in the above 59-3 (12.7 mg, 2.25 μmol) was added an aqueous solution (0.9 mL) of silver(I) acetate (9.2 mg, 55 μmol), and reacted at room temperature for 30 minutes. Then, DTT (21.2 mg, 137 μmol) dissolved in 200 mM phosphate buffer (pH 7.4, 0.9 mL) and 100 mM ascorbic acid aqueous solution (225 μL) were added, and this was promptly filtered with a filter. The filtrate was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=73:27→60:40, 13 minutes, linear gradient elution] to obtain glycopeptide 137 (SEQ ID NO. 141) represented by the following formula (136) (5.2 mg, 0.94 μmol, yield 42%).




ESI-MS: (m/z) calcd for C227H360N52O98S4: [M+4H]4+ 1379.5, [M+5H]5+ 1103.8, [M+6H]6+ 920.0, [M+7H]7+ 788.7. found 1379.4, 1103.7, 919.9, 788.6.
59-5 Formation of Disulfide Bond
Glycopeptide 137 obtained in the method described in the above 59-4 (5.2 mg, 0.94 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO (1/1, v/v, 1.9 mL), and reacted at room temperature for 2 days. The reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=70:30→65:35, 13 minutes, linear gradient elution] to obtain a fraction containing compound (S1C(disialo(aminoethylamide))-SRIF28) represented by the following formula (138) (SEQ ID NO. 142).




This fraction was further purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=92:8→85:15, 14 minutes, linear gradient elution] to obtain S1C(disialo(aminoethylamide))-SRIF28 (3.8 mg, 0.69 μmol, yield 73%).
ESI-MS: (m/z) calcd for C227H358N52O98S4: [M+3H]3+ 1838.3, [M+4H]4+ 1379.0, [M+5H]5+ 1103.4, [M+6H]6+ 919.6, [M+7H]7+ 788.4. found 1838.0, 1378.5, 1103.2, 919.5, 788.2.
Example 60
Synthesis of S1C(disialo(amide))-SRIF28
60-1 Synthesis of Bromoacetamidated Disialo Sugar Chain Derivative
To compound k (from Otsuka Chemical Co., Ltd.) (152 mg, 68.6 μmol) were sequentially added DMF (1.9 mL), lithium bromide (357 mg, 4.12 mmol), and phenacyl chloride (273 mg, 1.37 mmol), and reacted at 37° C. After 10 hours, water (19 mL) was added and the precipitate was removed by filtration. To the filtrate was added 25% ammonium water (5 mL), and reacted at room temperature for 18 hours, and then 100 mM phosphate buffer (pH 7.4, 80 mL) was added to allow neutralization. The solution was purified with HPLC [column: YMC Hydrosphere C18 (5 μm), φ20×250 mm, flow rate: 8.0 mL/min, eluent: 0.1% aqueous TFA:acetonitrile=98:2→92:8, 30 minutes, linear gradient elution] and lyophilized to obtain compound n represented by the following formula (n) (60.9 mg, 27.4 μmol, yield 40%).
MALDI-MS: (m/z) calcd for C84H140N8O60: [M+Na]+ 2243.8. found 2243.6.




The obtained intermediate n (37.3 mg, 16.8 μmol) and ammonium hydrogen carbonate (13.3 mg, 168 μmol) were dissolved in water (0.37 mL), and reacted at room temperature for 7 days. After lyophilization, to the lyophilizate obtained were sequentially added water (0.37 mL), DCC (17.3 mg, 84 μmol), and bromoacetic acid (11.7 mg, 84 μmol) dissolved in DMF (0.37 mL). The solution after 1 hour of reaction under ice cooling was purified with HPLC [column: YMC Hydrosphere C18 (5 μm), φ20×250 mm, flow rate: 8.0 mL/min, eluent: 0.1% aqueous TFA:acetonitrile=98:2→92:8, 30 minutes, linear gradient elution] to obtain compound o represented by the following formula (o) (bromoacetamidated disialo sugar chain derivative: 29.0 mg, 12.4 μmol, yield 74%).




MALDI-MS: (m/z) calcd for C86H142BrN9O60: [M+Na]+ 2362.7. found 2362.5.
60-2 Synthesis of S1C(disialo(amide))-SRIF28
A compound represented by the following formula (139) (S1C(disialo(amide))-SRIF28) (SEQ ID NO. 143) was synthesized similarly to Example 1, except that compound o obtained in the method described in the above 60-1 was employed instead of compound a.




Example 61
Synthesis of S1C(disialo(Bn))-SRIF28
A compound represented by the following formula (140) (S1C(disialo(Bn))-SRIF28) (SEQ ID NO. 144) was synthesized similarly to Example 1, except that compound g was employed instead of compound a.




Example 62
Synthesis of S1C(disialo (hexadecylamide))-SRIF28
62-1 Synthesis of Bromoacetamidated Disialo Sugar Chain Derivative
To compound k (from Otsuka Chemical Co., Ltd.) (140 mg, 63.0 μmol) were added water (1.5 mL), methanol (1.5 mL), and hexadecylamine (300 mg, 126 μmol), and this was stirred at room temperature for 1 hour. After lyophilization, to the lyophilizate obtained were added DMF (5 mL), HATU (239 mg, 630 μmol), and DIPEA (110 μL, 630 μmol), and reacted at 37° C. After 18 hours, diethyl ether (100 mL) was added to the solution, and the deposited precipitate was collected by filtration. This precipitate was dissolved in DMF (5 mL) and purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 8.0 mL/min, eluent: water:acetonitrile=40:60→10:90, 30 minutes, linear gradient elution] to obtain compound p represented by the following formula (p) (71.1 mg, 26.6 μmol, yield 42%).




MALDI-MS: (m/z) calcd for C116H204N8O60: [M+Na]+ 2692.3. found 2691.9.

The obtained compound p (71.7 mg, 26.6 μmol) and ammonium hydrogen carbonate (21.8 mg, 266 μmol) were dissolved in water (0.7 mL) and methanol (0.7 mL), and reacted at room temperature. After 7 days, to the lyophilizate obtained by lyophilization were sequentially added water (0.7 mL), methanol (0.7 mL), DCC (27.4 mg, 133 μmol), and bromoacetic acid (18.5 mg, 133 μmol) dissolved in DMF (0.7 mL). After 1 hour of reaction under ice cooling, the solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 8.0 mL/min, eluent: water:acetonitrile=40:60→10:90, 30 minutes, linear gradient elution] to obtain compound q represented by the following formula (q) (bromoacetamidated disialo sugar chain: 24.9 mg, 8.9 μmol, yield 33%).




MALDI-MS: (m/z) calcd for C118H206BrN9O60: [M+Na]+ 2811.2. found 2811.0.
62-2 Glycosylation Reaction of Thiol
Peptide 1 (10.4 mg, 3.14 μmol) was dissolved in 0.5 M phosphate buffer (pH 7.4, 62 μL) containing 30 μM TCEP. To this solution was added a solution of compound q obtained in the method described in the above 62-1 (79.4 mg, 28.5 μmol) in DMSO (3.7 mL), and reacted at room temperature for 20 minutes. The reaction solution was purified with HPLC [column: SHISEIDO Proteonavi (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=50:50→22:78, 14 minutes, linear gradient elution] to obtain glycopeptide 141 (SEQ ID NO. 145) represented by the following formula (141) (6.4 mg, 1.1 μmol, yield 35%).




ESI-MS: (m/z) calcd for C261H424N52O100S4: [M+3H]3+ 2007.2, [M+4H]4+ 1505.7, [M+5H]5+ 1204.7, [M+6H]6+ 1004.1. found 2007.4, 1505.5, 1204.8, 1004.0.
62-3 Deprotection of Acm Group
To glycopeptide 141 obtained in the method described in the above 62-2 (6.4 mg, 1.1 μmol) was added an aqueous solution (0.8 mL) of silver(I) acetate (3.8 mg, 23 μmol), and reacted at room temperature for 40 minutes. Then, DTT (8.8 mg, 57 μmol) dissolved in 200 mM phosphate buffer (pH 7.4, 377 μL) and 100 mM ascorbic acid aqueous solution (106 μL) were added, and this was promptly filtered with a filter. The filtrate was purified with HPLC [column: SHISEIDO Proteonavi (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=48:52→38:62, 3 minutes, then 38:62→30:70, 8 minutes, linear gradient elution] to obtain glycopeptide 142 (SEQ ID NO. 146) represented by the following formula (142) (3.2 mg, 0.54 μmol, yield 49%).




ESI-MS: (m/z) calcd for C255H414N50O98S4: [M+3H]3+ 1959.9, [M+4H]4+ 1470.1, [M+5H]5+ 1176.3, [M+6H]6+ 980.4. found 1959.6, 1469.9, 1176.1, 980.5.
62-3 Formation of Disulfide Bond
Glycopeptide 142 obtained in the method described in the above 62-2 (3.2 mg, 0.54 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO (1/1, v/v, 1.1 mL), and reacted at room temperature for 2 days. The reaction solution was purified with HPLC [column: SHISEIDO Proteonavi (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=48:52→38:62, 3 minutes, then 38:62→30:70, 8 minutes, linear gradient elution] to obtain a compound represented by the following formula (143) (S1C(disialo (hexadecylamide))-SRIF28) (SEQ ID NO. 147) (2.8 mg, 0.48 μmol, yield 89%).




ESI-MS: (m/z) calcd for C255H412N50O98S4: [M+3H]3+ 1959.2, [M+4H]4+ 1469.6, [M+5H]5+ 1175.9, [M+6H]6+ 980.1. found 1958.9, 1469.4, 1175.7, 979.9.
Example 63
Synthesis of S1-2C(disialo(amide))-SRIF28
A compound represented by the following formula (144) (S1-2C(disialo(amide))-SRIF28) (SEQ ID NO. 148) was synthesized similarly to Example 28, except that compound o was employed instead of compound c.




Example 64
Synthesis of S1-2C(disialo(Bn))-SRIF28
A compound represented by the following formula (145) (S1-2C(disialo(Bn))-SRIF28) (SEQ ID NO. 149) was synthesized similarly to Example 28, except that compound g was employed instead of compound c.




Example 65
Synthesis of S1C(Asn(disialo))-SRIF28
A compound represented by the following formula (146) (S1C(Asn(disialo))-SRIF28) (SEQ ID NO. 150) was synthesized similarly to Example 1, except that compound r represented by the following formula (r) (bromoacetylated glycosylated Asn: from Otsuka Chemical Co., Ltd.) was employed instead of compound a.




Example 66
Synthesis of S1N(disialo).N19C(diMan)-SRIF28
66-1 Solid Phase Synthesis of Glycopeptide
2-chlorotrityl chloride resin (100 μmol) was taken in a column for solid phase synthesis, and after washing with DMF and dichloromethane, a solution of Fmoc-Cys(Trt)-OH (72.5 mg, 120 μmol) and DIPEA (104.6 μL, 600 μmol) in dichloromethane (3.0 mL) was added, and this was shaken for 1 hour. After washing with dichloromethane and DMF, the Fmoc protecting group was removed by treating with 20% piperidine in DMF. After washing with DMF, in a peptide solid phase synthesis method with Fmoc strategy employing a PreludeTM peptide synthesizer, a protected peptide 147 (SEQ ID NO. 151) represented by the following formula (147) was synthesized in a state bound to the resin. The condensation reaction was performed in DMF using HCTU as the condensation agent.




Next, the Fmoc protecting group was removed by treating with 20% piperidine in DMF. After washing with DMF, compound d (141.0 mg, 51.5 μmol), DMSO-DMF (1/1, v/v, 1.1 mL) solution, TBTU (21.2 mg, 66.0 μmol), and DIPEA (17.2 μL, 98.7 μmol) were sequentially added to the resin, and this was shaken at room temperature for 4 hours to allow condensation. After washing with DMF, this condensation operation was repeated once. After washing the resin with DMF and dichloromethane, this was shaken with 20% piperidine in DMF for 20 minutes to deprotect the Fmoc group, and the resin was washed with DMF to synthesize a protected peptide 148 (SEQ ID NO. 152) represented by the following formula (148) in a state bound to the resin.




After washing with DMF and dichloromethane, TFA:water:triisopropylsilane:ethanedithiol (=90:2.5:5:2.5) was added, and this was shaken at room temperature for 3 hours. The resin was filtered off, cold diethyl ether was added to the filtrate, and crude peptide was obtained as precipitate. This crude peptide was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, A:B=70:30] to obtain glycopeptide 149 (SEQ ID NO. 153) represented by the following formula (149) (5.8 mg, 1.1 μmol).




ESI-MS: (m/z) calcd for C241H367N49O101S4: [M+4H]4+ 1424.8, [M+5H]5+ 1140.0. found 1424.6, 1139.9.
66-2 Glycosylation Reaction of Thiol
Glycopeptide 149 obtained in the method described in the above 66-1 (10.8 mg, 1.90 μmol) and compound f (5.3 mg, 5.1 μmol) were dissolved in 100 mM phosphate buffer (pH 7.4, 0.8 mL) containing 141 μM TCEP, and reacted at room temperature for 24 hours. The reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, A:B=75:25→60:40, 30 minutes, linear gradient elution] to obtain glycopeptide 150 (SEQ ID NO. 154) represented by the following formula (150) (4.9 mg, 0.74 μmol, yield 39%).




ESI-MS: (m/z) calcd for C277H426N52O127S4: [M+3H]3+ 2216.0, [M+4H]4+ 1662.2, [M+5H]5+ 1330.0. found 2215.6, 1661.9, 1330.1.
66-3 Deprotection of Acm Group
To glycopeptide 150 obtained in the method described in the above 66-2 (4.9 mg, 0.74 μmol) was added an aqueous solution (148 μL) of silver(I) acetate (1.5 mg, 9.0 μmol), and reacted at room temperature for 1.5 hours reaction. DTT (3.5 mg, 23 μmol) dissolved in 200 mM phosphate buffer (pH 7.4, 145 μL) and 100 mM ascorbic acid aqueous solution (37 μL) were added, and this was promptly filtered with a filter. The filtrate was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=74:26→64:36, 30 minutes, linear gradient elution] to obtain glycopeptide 151 (SEQ ID NO. 155) represented by the following formula (151) (3.7 mg, 0.57 μmol, yield 77%).




ESI-MS: (m/z) calcd for C271H416N50O125S4: [M+3H]3+ 2168.6, [M+4H]4+ 1626.7, [M+5H]5+ 1301.5. found 2168.6, 1626.4, 1301.3.
66-4 Formation of Disulfide Bond
Glycopeptide 151 obtained in the method described in the above 66-3 (3.7 mg, 0.54 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO (1/1, v/v, 1.4 mL), and reacted at room temperature for 31 hours. The reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=75:25→60:40, 30 minutes, linear gradient elution] to obtain glycopeptide 152 (SEQ ID NO. 156) represented by the following formula (152) (2.9 mg, 0.45 μmol, yield 78%).




ESI-MS: (m/z) calcd for C271H414N50O125S4: [M+3H]3+ 2167.9, [M+4H]4+ 1626.2, [M+5H]5+ 1301.1. found 2167.9, 1626.0, 1301.2.
66-5 Deprotection of Benzyl Group
Glycopeptide 152 obtained in the method described in the above 66-4 (2.9 mg, 0.45 μmol) was dissolved in 50 mM sodium hydroxide aqueous solution (13.6 mL), and reacted at 0° C. for 1 hour. 200 mM acetic acid aqueous solution (3.4 mL) was added, and the mixed solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=75:25→60:40, 20 minutes, linear gradient elution] to obtain a fraction containing compound (S1N(disialo).N19C(diMan)-SRIF28) represented by the following formula 153 (SEQ ID NO. 157).




This fraction was further purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ4.6×250 mm, flow rate: 0.7 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=95:5→85:15, 2 minutes, then 85:15→65:35, 20 minutes, linear gradient elution] to obtain S1N(disialo).N19C(diMan)-SRIF28 (1.6 mg, 0.25 μmol, yield 57%).
ESI-MS: calcd for C257H402N50O125S4: [M+3H]3+ 2107.8, [M+4H]4+ 1581.1, [M+5H]5+ 1265.1. found 2107.9, 1580.9, 1265.1.
Example 67
Synthesis of C(disialo(aminoethylamide)) S1C(disialo)-SRIF28
67-1 Synthesis of Peptide
2-chlorotrityl chloride resin (100 μmol) was taken in a column for solid phase synthesis, and after washing with DMF and dichloromethane, a solution of Fmoc-Cys(Acm)-OH (49.7 mg, 120 μmol) and DIPEA (104.5 μL, 600 μmol) in dichloromethane (3.0 mL) was added, and this was shaken for 1 hour. After washing with dichloromethane and DMF, the Fmoc protecting group was removed by treating with 20% piperidine in DMF. After washing with DMF, in a peptide solid phase synthesis method with Fmoc strategy employing a PreludeTM peptide synthesizer, a protected peptide 154 (SEQ ID NO. 158) represented by the following formula (154) was synthesized in a state bound to the resin. The condensation reaction was performed in DMF using HCTU as the condensation agent.




A part of the resin (50 μmol) was taken, and the Fmoc protecting group was removed by treating with 20% piperidine in DMF. After washing with DMF and dichloromethane, TFA:water:triisopropylsilane:ethanedithiol (=90:2.5:5:2.5) was added, and this was shaken at room temperature for 3 hours. The resin was filtered off, cold diethyl ether was added to the filtrate, and crude peptide was obtained as precipitate. The crude peptide was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=73:27→63:37, 30 minutes, linear gradient elution] to obtain peptide 155 (SEQ ID NO. 159) represented by the following formula (155) (30.4 mg).




ESI-MS: (m/z) (m/z) calcd for C150H232N44O41S6: [M+3H]3+ 1167.7, [M+4H]4+ 876.0. found 1167.5, 875.9.
67-2 Deprotection of Boc Group in Sugar Chain Derivative
Compound m (50.0 mg, 19.0 μmol) was dissolved in a TFA-H2O (95/5, v/v, 2.5 mL) solution, and this was shaken at room temperature. After 10 minutes, diethyl ether (15 mL) was added, and the deposited precipitate was centrifuged (10,000×g10 minutes). The precipitate was dissolved in water and lyophilized to obtain compound s represented by the following formula (s) (bromoacetamidated disialo sugar chain: 46.0 mg, 18.9 μmol, yield 99%).




ESI-MS: (m/z) calcd for C90H152BrN11O60: [M+2H]2+ 1215.1, [M+3H]3+ 810.4. found 1214.9, 810.3.
67-3 Glycosylation of Thiol
Peptide 155 obtained in the method described in the above 67-1 (20.6 mg, 5.89 μmol) and compound s obtained in the method described in the above 67-2 (28.6 mg, 11.8 μmol) were dissolved in 33 mM phosphate buffer (pH 7.4, 1.8 mL), and reacted at room temperature for 30 minutes. The reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=74:26→64:36, 20 minutes, linear gradient elution] to obtain glycopeptide 156 (SEQ ID NO. 160) represented by the following formula (156) (17.1 mg, 2.93 μmol, yield 50%).




ESI-MS: (m/z) calcd for C240H383N55O101S6: [M+3H]3+ 1950.1, [M+4H]4+ 1462.8, [M+5H]5+ 1170.5, [M+6H]6+ 975.6. found 1949.9, 1462.6, 1170.3, 975.4.
67-4 Deprotection of StBu Group
To glycopeptide 156 obtained in the method described in the above 67-3 (17.1 mg, 2.93 μmol) was added DTT (52.9 mg, 343 μmol) dissolved in 0.1 M phosphate buffer (pH 7.4, 3.4 mL), and reacted at room temperature for 3 hours. The reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=95:5→75:25, 20 minutes, linear gradient elution] to obtain glycopeptide 157 (SEQ ID NO. 161) represented by the following formula (157) (8.6 mg, 1.5 μmol, yield 51%).




ESI-MS: (m/z) calcd for C236H375N55O101S5: [M+3H]3+ 1920.7, [M+4H]4+1440.8, [M+5H]5+ 1152.8, [M+6H]6+ 960.9, [M+7H]7+ 823.7. found 1920.5, 1440.6, 1152.7, 960.6, 823.6.
67-5 Glycosylation Reaction of Thiol
Peptide 157 obtained in the method described in the above 67-4 (6.2 mg, 1.1 μmol) and compound a (3.8 mg, 1.6 μmol) were dissolved in 0.36 M phosphate buffer (pH 7.4, 339 mL) containing 1.6 mM DTT, and reacted at room temperature for 2.5 hours The reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=90:10→75:25, 30 minutes, linear gradient elution] to obtain glycopeptide 158 (SEQ ID NO. 162) represented by the following formula (158) (6.4 mg, 0.80 μmol, yield 73%).




ESI-MS: (m/z) calcd for C322H514N62O163S5: [M+4H]4+ 2006.5, [M+5H]5+ 1605.4, [M+6H]6+ 1338.0. found 2006.6, 1605.3, 1338.0.
67-6 Deprotection of Acm Group
To glycopeptide 158 obtained in the method described in the above 67-5 (8.2 mg, 1.0 μmol) was added an aqueous solution (225 μL) of silver(I) acetate (2.1 mg, 13 μmol), and reacted at room temperature for 1 hour. DTT (4.8 mg, 31 μmol) dissolved in 100 mM phosphate buffer (pH 7.4, 204 μL) and 100 mM ascorbic acid aqueous solution (51 μL) were added, and this was promptly filtered with a filter. The filtrate was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=90:10→75:25, 30 minutes, linear gradient elution] to obtain glycopeptide 159 (SEQ ID NO. 163) represented by the following formula (159) (5.5 mg, 0.70 μmol, yield 70%).




ESI-MS: (m/z) calcd for C316H504N60O161S5: [M+4H]4+ 1971.0, [M+5H]5+ 1577.0, [M+6H]6+ 1314.3. found 1970.6, 1576.8, 1314.2.
67-7 Formation of Disulfide Bond
Glycopeptide 159 obtained in the method described in the above 67-6 (5.4 mg, 0.69 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO (1/1, v/v, 1.7 mL), and reacted overnight at room temperature. The reaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=75:25→65:35, 30 minutes, linear gradient elution] to obtain a fraction containing compound (C(disialo(aminoethylamide))/S1C(disialo)-SRIF28) represented by the following formula 160 (SEQ ID NO. 164).




This fraction was further purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=90:10→75:25, 20 minutes, linear gradient elution] to obtain C(disialo(aminoethylamide))/S1C(disialo)-SRIF28 (3.1 mg, 0.40 μmol, yield 58%).
ESI-MS: calcd for C316H502N60O161S5: [M+4H]4+ 1970.5, [M+5H]5+1576.6, [M+6H]6+ 1314.0, [M+7H]7+ 1126.4, [M+8H]8+985.8, [M+9H]9+ 876.3. found 1970.3, 1576.4, 1313.9, 1126.4, 985.5, 876.1.
Example 68
Synthesis of S1-4C(disialo)-SRIF28
68-1 Synthesis of Peptide
2-chlorotrityl chloride resin (100 μmol) was taken in a column for solid phase synthesis, and after washing with DMF and dichloromethane, a solution of Fmoc-Cys(Acm)-OH (49.7 mg, 120 μmol) and DIPEA (104.5 μL, 600 μmol) in dichloromethane (3.0 mL) was added, and this was shaken for 1 hour. After washing with dichloromethane and DMF, the Fmoc protecting group was removed by treating with 20% piperidine in DMF. After washing with DMF, in a peptide solid phase synthesis method with Fmoc strategy employing a PreludeTM peptide synthesizer, a protected peptide 161 (SEQ ID NO. 165) represented by the following formula (161) was synthesized in a state bound to the resin. The condensation reaction was performed in DMF using HCTU as the condensation agent.




The Fmoc protecting group was removed by treating with 20% piperidine in DMF. After washing with DMF and dichloromethane, TFA:water: triisopropylsilane:ethanedithiol (=90:2.5:5:2.5) was added, and this was shaken at room temperature for 3 hours. The resin was filtered off, cold diethyl ether was added to the filtrate, and crude peptide was obtained as precipitate. The crude peptide was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ50×250 mm, flow rate: 43.7 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=75:25→65:35, 20 minutes, linear gradient elution] to obtain peptide 162 (SEQ ID NO. 166) represented by the following formula (162) (127.2 mg).




ESI-MS: (m/z) calcd for C152H234N46O43S7: [M+3H]3+ 1207.1, [M+4H]4+ 905.6, [M+5H]5+ 724.6. found 1206.9, 905.1, 724.5.
68-2 Glycosylation Reaction of Thiol
Peptide 162 obtained in the method described in the above 68-1 (30.4 mg, 8.40 μmol) and compound a (128 mg, 54.7 μmol) were dissolved in 33 mM phosphate buffer (pH 7.4, 2.5 mL), and reacted overnight at room temperature. The reaction solution was purified with HPLC [column: SHISEIDO Proteonavi (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=82:18→71:29, 20 minutes, linear gradient elution] to obtain glycopeptide 163 (SEQ ID NO. 167) represented by the following formula (163) (30.9 mg, 2.44 μmol, yield 29%).
ESI-MS: (m/z) calcd for C496H790N74O291S7: [M+6H]6+ 2112.7, [M+7H]7+ 1811.1. found 2112.8, 1811.0.




68-3 Deprotection of Acm Group

To glycopeptide 163 obtained in the method described in the above 68-2 (30.9 mg, 2.44 μmol) was added an aqueous solution (0.98 mL) of silver(I) acetate (5.0 mg, 30 μmol), and reacted at room temperature for 20 minutes. Then, DTT (11.8 mg, 76.5 μmol) dissolved in 200 mM Tris-HCl buffer (pH 7.4, 0.98 mL) and 100 mM ascorbic acid aqueous solution (244 μL) were added, and this was promptly filtered with a filter. The filtrate was purified with HPLC [column: SHISEIDO Proteonavi (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradient A:B=82:18→70:30, 20 minutes, linear gradient elution] to obtain glycopeptide 164 (SEQ ID NO. 168) represented by the following formula (164) (20.6 mg, 1.64 μmol, yield 67%).




ESI-MS: (m/z) calcd for C490H780N72O289S7: [M+5H]5+ 2506.6, [M+6H]6+ 2089.0, [M+7H]7+ 1790.7. found 2506.5, 2088.8, 1790.4.
68-4 Formation of Disulfide Bond
Glycopeptide 164 obtained in the method described in the above 68-3 (20.6 mg, 1.64 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO (1/1, v/v, 2.1 mL), and reacted at room temperature for 2 days. Then, the reaction solution was purified with HPLC [column: SHISEIDO Proteonavi (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 10 mM ammonium acetate aqueous solution, B: 10 mM ammonium acetate-acetonitrile (1/9, v/v), gradient A:B=75:25→72:28, 15 minutes, linear gradient elution] to obtain S1-4C(disialo)-SRIF28 (SEQ ID NO. 169) represented by the following formula (165) (11.6 mg, 0.93 μmol, yield 57%).




ESI-MS: (m/z) calcd for C490H778N72O289S7: [M+5H]5+ 2506.2, [M+6H]6+2088.7, [M+7H]7+ 1790.5. found 2506.1, 2088.6, 1790.4.
Tables 1-1 to 1-7 show the MS spectrum data (ESI-MS) of the glycosylated SRIF peptides obtained in the methods described in Examples 1-68. The molecular mass was obtained by performing deconvolution of polyvalent protein mass spectrometry with MassLynx version 4.1 (from Waters).











TABLE 1-1







Calculated

Observed


Exam-

molecular

molecular


ple
Compound
weight
Observed ions (m/z)
weight




















1
S1C(disialo)-
5427.7
[M + 3H]3+
1810.0
5427.0



SRIF28

[M + 4H]4+
1357.5






[M + 5H]5+
1086.4



2
N5C(disialo)-
5400.6
[M + 3H]3+
1801.0
5400.1



SRIF28

[M + 4H]4+
1350.8






[M + 5H]5+
1081.2



3
A9C(disialo)-
5443.7
[M + 3H]3+
1815.4
5443.1



SRIF28

[M + 4H]4+
1361.8






[M + 5H]5+
1089.6



4
E12C(disialo)-
5385.6
[M + 3H]3+
1796.0
5384.0



SRIF28

[M + 4H]4+
1347.0






[M + 5H]5+
1078.0



5
R13C(disialo)-
5358.6
[M + 3H]3+
1787.0
5358.0



SRIF28

[M + 4H]4+
1340.5






[M + 5H]5+
1072.6



6
K14C(disialo)-
5386.6
[M + 3H]3+
1796.4
5386.0



SRIF28

[M + 4H]4+
1347.5






[M + 5H]5+
1078.2



7
A15C(disialo)-
5443.7
[M + 3H]3+
1815.4
5443.1



SRIF28

[M + 4H]4+
1361.8






[M + 5H]5+
1089.6



8
G16C(disialo)-
5457.7
[M + 3H]3+
1820.0
5457.1



SRIF28

[M + 4H]4+
1365.3






[M + 5H]5+
1092.2



9
K18C(disialo)-
5386.6
[M + 3H]3+
1796.4
5386.1



SRIF28

[M + 4H]4+
1347.5






[M + 5H]5+
1078.2






[M + 6H]6+
898.7






[M + 7H]7+
770.5




















TABLE 1-2







Calculated

Observed




molecular

molecular


Example
Compound
weight
Observed ions (m/z)
weight




















10
N19C(disialo)-SRIF28
5400.6
[M + 3H]3+
1801.0
5400.1





[M + 4H]4+
1351.0






[M + 5H]5+
1080.8



11
F21C(disialo)-SRIF28
5367.6
[M + 3H]3+
1790.0
5366.9





[M + 4H]4+
1342.7






[M + 5H]5+
1074.4



12
T26C(disialo)-SRIF28
5413.6
[M + 3H]3+
1354.3
5412.1





[M + 4H]4+
1083.4






[M + 5H]5+
903.0






[M + 6H]6+
774.3



13
29C(disialo)-SRIF28
5514.7
[M + 3H]3+
1839.1
5514.1





[M + 4H]4+
1379.5






[M + 5H]5+
1103.8



14
30C(disialo)-SRIF28
5615.8
[M + 3H]3+
1872.7
5615.1





[M + 4H]4+
1404.8






[M + 5H]5+
1124.0



15
S1C(disialo)-D-Trp22-
5427.7
[M + 3H]3+
1810.1
5427.2



SRIF28

[M + 4H]4+
1357.8






[M + 5H]5+
1086.4






[M + 6H]6+
905.5



16
A9C(disialo)-D-Trp22-
5443.7
[M + 3H]3+
1815.4
5442.9



SRIF28

[M + 4H]4+
1361.8






[M + 5H]5+
1089.6



17
C(disialo)-SRIF14
4004.1
[M + 2H]2+
2002.9
4003.3





[M + 3H]3+
1335.4






[M + 4H]4+
1001.8



18
C(disialo)-R-K-SRIF14
4288.4
[M + 3H]3+
1430.2
4287.5





[M + 4H]3+
1072.9



19
C(disialo)-C12linker-SRIF14
4201.4
[M + 3H]3+
1401.2
4200.5





[M + 4H]4+
1051.2






[M + 5H]5+
841.1




















TABLE 1-3







Calculated

Observed




molecular

molecular


Example
Compound
weight
Observed ions (m/z)
weight




















20
S1-2C(disialo)-SRIF28
7793.8
[M + 4H]4+
1949.4
7793.4





[M + 5H]5+
1559.7






[M + 6H]6+
1299.9



21
S1C(disialo)•N5C(disialo)-
7679.7
[M + 3H]3+
1920.4
7678.7



SRIF28

[M + 4H]4+
1536.8






[M + 5H]5+
1280.9



22
S1C(disialo)•R13C(disialo)-
7637.7
[M + 3H]3+
1910.2
7636.6



SRIF28

[M + 4H]4+
1528.3






[M + 5H]5+
1273.8



23
N5C(disialo)•A9C(disialo)-
7695.7
[M + 5H]5+
1540.1
7695.2



SRIF28

[M + 6H]6+
1283.6






[M + 7H]7+
1100.3






[M + 8H]8+
962.9



24
S1-3C(disialo)-SRIF28
10160.0
[M + 3H]3+
3387.6
10158.2





[M + 4H]4+
2540.9






[M + 5H]5+
2032.7






[M + 6H]6+
1694.2






[M + 7H]7+
1452.3



25
S1C(disialo)•N5C(disialo)•A9C(disialo)-
9974.8
[M + 5H]5+
1995.9
9974.3



SRIF28

[M + 6H]6+
1663.2



26
S1C(monosialo)-SRIF28
5136.4
[M + 3H]3+
1713.0
5134.9





[M + 4H]4+
1284.7






[M + 5H]5+
1028.2






[M + 6H]6+
856.8




















TABLE 1-4







Calculated






molecular

Observed


Example
Compound
weight
Observed ions (m/z)
molecular weight




















27
S1C(asialo)-SRIF28
4845.1
[M + 3H]3+
2615.7
4843.9





[M + 4H]4+
1212.0






[M + 5H]5+
969.8



28
S1-2C(asialo)-SRIF28
6628.8
[M + 4H]4+
1658.1
6627.2





[M + 5H]5+
1326.7






[M + 6H]6+
1105.6






[M + 7H]7+
947.8






[M + 8H]8+
829.4



29
S1-3C(asialo)-SRIF28
8412.5
[M + 4H]4+
2103.7
8411.7





[M + 5H]5+
1683.3






[M + 6H]6+
1403.0



30
N5N(disialo)-SRIF28
5354.5
[M + 3H]3+
1785.7
5354.0





[M + 4H]4+
1339.5






[M + 5H]5+
1071.8



31
S1C(disialo)-SRIF28
5381.6
[M + 3H]3+
1794.7
5380.9





[M + 4H]4+
1346.2






[M + 5H]5+
1077.2



32
S1C(disialo)•N19C(GlcNAc)-
5676.9
[M + 3H]3+
1893.5
5676.4



SRIF28

[M + 4H]4+
1420.1






[M + 5H]5+
1136.3



33
S1C(disialo)•N19C(diMan)-
6366.6
[M + 3H]3+
2122.9
6366.7



SRIF28

[M + 4H]4+
1592.5






[M + 5H]5+
1274.4



34
S1-5C(disialo)-SRIF28
14892.4
[M + 4H]4+
3723.7
14891.3





[M + 5H]5+
2979.1






[M + 6H]6+
2482.9






[M + 7H]7+
2128.2






[M + 8H]8+
1862.4



35
S1-10C(disialo)-SRIF28
26723.2
[M + 7H]7+
3818.3
26721.8





[M + 8H]8+
3341.0






[M + 9H]9+
2970.1






[M + 10H]10+
2673.1




















TABLE 1-5







Calculated






molecular

Observed


Example
Compound
weight
Observed ions (m/z)
molecular weight




















36
C(disialo)-SRIF28
5514.7
[M + 4H]4+
1379.7
5514.6





[M + 5H]5+
1103.9






[M + 6H]6+
920.1






[M + 7H]7+
788.8



37
R11C(disialo)-SRIF28
5358.5
[M + 3H]3+
1787.4
5358.6





[M + 4H]4+
1340.7






[M + 5H]5+
1072.7



38
F20C(disialo)-SRIF28
5367.6
[M + 3H]3+
1790.1
5367.1





[M + 4H]4+
1342.8






[M + 5H]5+
1074.4



39
T24C(disialo)-SRIF28
5413.6
[M + 3H]3+
1805.7
5412.6





[M + 4H]4+
1354.2






[M + 5H]5+
1083.7



40
F25C(disialo)-SRIF28
5367.6
[M + 3H]3+
1790.1
5367.1





[M + 4H]4+
1342.8






[M + 5H]5+
1074.4






[M + 6H]6+
895.4



41
S27C(disialo)-SRIF28
5427.7
[M + 3H]3+
1810.1
5427.4





[M + 4H]4+
1357.8



42
C(disialo)-K-SRIF14
4131.2
[M + 2H]2+
2066.9
4131.7





[M + 3H]3+
1378.2






[M + 4H]4+
1033.9



43
S1C(disialo)-F25Y-SRIF28
5443.7
[M + 3H]3+
1815.4
5443.1





[M + 4H]4+
1361.6






[M + 5H]5+
1089.6



44
S1C(disialo)-SRIF28-amide
5426.7
[M + 3H]3+
1809.8
5426.2





[M + 4H]4+
1357.6






[M + 5H]5+
1086.2






[M + 6H]6+
905.2



45
C(disialo)-PEGlinker-SRIF14
4207.3
[M + 3H]3+
1403.2
4206.6





[M + 4H]4+
1052.7






[M + 5H]5+
842.3



46
Biotin-S1C(disialo)-SRIF28
5654.0
[M + 3H]3+
1885.4
5653.2





[M + 4H]4+
1414.3






[M + 5H]5+
1131.7






[M + 6H]6+
943.2



47
Biotin-PEGlinker-
5857.2
[M + 3H]3+
1953.2
5856.4



S1C(disialo)-SRIF28

[M + 4H]4+
1465.1






[M + 5H]5+
1172.1






[M + 6H]6+
977.1






[M + 7H]7+
837.6




















TABLE 1-6







Calculated






molecular

Observed


Example
Compound
weight
Observed ions (m/z)
molecular weight




















48
Azido-S1C(disialo)-SRIF28
5552.8
[M + 3H]3+
1851.8
5552.3





[M + 4H]4+
1389.1






[M + 5H]5+
1111.7



49
S1C(disialo)•E12C(disialo)-
7664.7
[M + 4H]4+
1917.1
7664.1



SRIF28

[M + 5H]5+
1533.8






[M + 6H]6+
1278.4



50
2C(disialo)-R-K-SRIF14
6654.6
[M + 3H]3+
2219.2
6653.4





[M + 4H]4+
1664.4






[M + 5H]5+
1331.9






[M + 6H]6+
1109.9



51
3C(disialo)-R-K-SRIF14
9020.8
[M + 4H]4+
2256.1
9020.2





[M + 5H]5+
1805.1






[M + 6H]6+
1504.4



52
S1C(diGlcNAc)-SRIF28
4520.9
[M + 3H]3+
1507.7
4519.9





[M + 4H]4+
1131.0






[M + 5H]5+
905.0



53
S1C(diMan)-SRIF28
4114.5
[M + 3H]3+
1372.3
4113.8





[M + 4H]4+
1029.5






[M + 5H]5+
823.8



54
N19C(diMan)-SRIF28
4087.5
[M + 3H]3+
1363.3
4086.8





[M + 4H]4+
1023.0



55
S1C(GlcNAc)-SRIF28
3424.9
[M + 2H]2+
1713.3
3424.5





[M + 3H]3+
1142.5






[M + 4H]4+
856.9






[M + 5H]5+
686.1



56
N19C(GlcNAc)-SRIF28
3397.8
[M + 2H]2+
1699.8
3397.5





[M + 3H]3+
1133.5






[M + 4H]4+
850.4



57
S1C(trisialo)-SRIF28
6084.2
[M + 3H]3+
2028.8
6083.4





[M + 4H]4+
1521.9






[M + 5H]5+
1217.7






[M + 6H]6+
1014.9



58
S1C(tetrasialo)-SRIF28
6740.8
[M + 3H]3+
2247.7
6739.6





[M + 4H]4+
1685.9






[M + 5H]5+
1349.1






[M + 6H]6+
1124.3






[M + 7H]7+
963.8



59
S1C(disialo(aminoethylamide))-
5511.8
[M + 3H]3+
1838.0
5511.1



SRIF28

[M + 4H]4+
1378.5






[M + 5H]5+
1103.2






[M + 6H]6+
919.5






[M + 7H]7+
788.2




















TABLE 1-7







Calculated






molecular

Observed


Example
Compound
weight
Observed ions (m/z)
molecular weight




















60
S1C(disialo(amide))-SRIF28
5425.7
[M + 3H]3+
1809.3
5423.9





[M + 4H]4+
1357.0






[M + 5H]5+
1085.8






[M + 6H]6+
905.2



61
S1C(disialo(Bn))-SRIF28
5607.9
[M + 3H]3+
1870.0
5605.9





[M + 4H]4+
1402.5






[M + 5H]5+
1122.2






[M + 6H]6+
935.5



62
S1C(disialo(hexadecylamide))-
5874.5
[M + 3H]3+
1958.9
5873.6



SRIF28

[M + 4H]4+
1469.4






[M + 5H]5+
1175.7






[M + 6H]6+
979.9



63
S1-2C(disialo(amide))-SRIF28
7789.9
[M + 4H]4+
1948.2
7789.9





[M + 5H]5+
1559.0






[M + 6H]6+
1299.2



64
S1-2C(disialo(Bn))-SRIF28
8154.3
[M + 4H]4+
2039.3
8153.0





[M + 5H]5+
1631.6






[M + 6H]6+
1360.2



65
S1C(Asn(disialo))-SRIF28
5542.7
[M + 3H]3+
1848.4
5542.2





[M + 4H]4+
1386.6






[M + 5H]5+
1109.4






[M + 6H]6+
924.9



66
S1N(disialo)•N19C(diMan)-SRIF28
6320.5
[M + 3H]3+
2107.9
6319.5





[M + 4H]4+
1580.9






[M + 5H]5+
1265.1



67
C(disialo(aminoethylamide))•S1C(disialo)-
7878.0
[M + 4H]4+
1970.3
7878.1



SRIF28

[M + 5H]5+
1576.4






[M + 6H]6+
1313.9






[M + 7H]7+
1126.4






[M + 8H]8+
985.5






[M + 9H]9+
876.1



68
S1-4C(disialo)-SRIF28
12526.2
[M + 5H]5+
2506.1
12525.5





[M + 6H]6+
2088.6






[M + 7H]7+
1790.4









Example 69-1
Calculation of Receptor Binding Affinity
Competitive binding assay was performed with the method below to calculate receptor binding affinity.
Reagents employed in the competitive binding assay and their proper chemical names are as follows: HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and BSA (bovine serum albumin).
Competitive binding assay was consigned to Ricerca Biosciences, and experiment and data analysis was performed. The receptor subtypes and receptor membrane samples employed are shown in Table 2. Common in each binding assay, [125I]Tyr11-Somatostatin 14 (Tyr11-SRIF14) was used as the labeled ligand, Somatostatin-14 (SRIF14) was used as the unlabeled ligand, and 25 mM HEPES containing 5 mM MgCl2, 1 mM CaCl2, and 0.1% BSA, pH 7.4 was used as the buffer. The test substance was used at a concentration of 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, or 1000 nM, and mixed with the membrane sample into a reaction solution. Moreover, the concentrations of the labeled and unlabeled ligands added to the reaction solution are shown in Table 2. The incubation condition of the reaction solution was at 25° C. for 4 hours for SSTR2 and at 25° C. for 2 hours for SSTR1, SSTR3, SSTR4, and SSTR5. For each experiment run, SRIF14 was used as the positive control. For data analysis, 50% inhibitory concentration (IC50 value) was determined using MathIQTM (ID Business Solutions, UK) in a nonlinear least squares method based on the numerical data of the binding inhibition rate. The binding inhibition constant (Ki value) was calculated by the method of Cheng, Y et al. (Biochem Pharmacol, 22, 3099-3108, 1973).












TABLE 2






SSTR1
SSTR2
SSTR3
SSTR4
SSTR5







Lobelod ligand
0.1 nM
0.03 nM
0.1 nM
0.1 nM
0.1 nM


Unlabeled ligand
  1 μM
  1 μM
  1 μM
  1 μM
  1 μM


Source
Human
Human
Human
Human
Human



recombinant
recombinant
recombinant
recombinant
recombinant



CHO-K1 cells
CHO-K1 cells
CHO-K1 cells
Chem-1 cells
Chem-1 cells









Compounds subjected to binding assay and the results of the binding experiment are shown in Table 3A. Moreover, octreotide, SRIF14 and SRIF28 were similarly evaluated as control compounds. Note that for octreotide, IC50 values could not be calculated for SSTR1 and SSTR4 since the maximum concentration was 100 nM, and is thus shown as >100 nM.
FIGS. 1A and 1B show examples of the structures of glycosylated peptides (glycosylated forms) corresponding to the compound names in Table 3A.













TABLE 3A









SSTR1
SSTR2
SSTR3
SSTR4
SSTR5


















Ex-

IC50
Ki
IC50
Ki
IC50
Ki
IC50
Ki
IC50
Ki


ample

(nM)
(nM)
(nM)
(nM)
(nM)
(nM)
(nM)
(nM)
(nM)
(nM)





















1
S1C(disialo)-
2.19
1.81
1.109
0.0576
0.480
0.164
2.30
2.07
0.442
0.250



SRIF28












2
N5C(disialo)-
1.10
0.912
0.0945
0.0502
0.278
0.0990
1.30
1.17
0.458
0.259



SRIF28












3
A9C(disialo)-
2.08
1.72
0.0741
0.0394
0.593
0.203
1.72
1.54
0.471
0.266



SRIF28












4
E12C(disialo)-
1.70
1.40
0.112
0.0596
0.362
0.124
2.04
1.84
0.376
0.212



SRIF28












5
R13C(disialo)-
15.4
13.6
0.310
0.165
2.87
0.981
0.43
5.79
3.28
1.85



SRIF28












6
K14C(disialo)-
13.8
11.4
0.481
0.256
2.59
0.887
8.02
7.22
4.01
2.27



SRIF28












10
N19C(disialo)-
10.1
13.3
1.21
0.645
1.17
0.503
12.2
11.0
3.69
2.09



SRIF28












13
29C(disialo)-
4.43
3.67
0.738
0.392
7.00
2.39
6.11
5.50
3.73
2.11



SRIF28












14
30C(disialo)-
4.21
3.48
0.517
0.275
6.91
2.36
2.53
2.28
1.86
1.05



SRIF28












15
S1C(disialo)-D-
2.23
1.85
0.0324
0.0172
0.834
0.285
5.85
5.26
0.565
0.319



Trp22-SRIF28












16
A9C(disialo)-D-
6.92
5.73
0.0396
0.0210
0.256
0.0876
4.87
4.38
0.194
0.110



Trp22-SRIF28












17
C(disialo)-SRIF14
32.3
26.7
0.336
0.179
4.62
1.56
8.24
7.41
5.70
3.22


18
C(disialo)-R-K-
3.09
2.56
0.104
0.0551
0.231
0.0791
1.06
0.951
0.187
0.106



SRIF14












19
C(disialo)-
15.5
12.8
0.502
0.267
3.12
1.07
2.92
2.62
2.29
1.29



C12linker-













SRIF28












20
S1-2C(disialo)-
18.8
15.5
0.338
0.179
1.28
0.439
5.61
5.05
0.607
0.343



SRIF28












21
S1C(disialo)•
6.89
5.70
0.224
0.119
1.58
0.539
4.00
3.60
0.728
0.411



N5C(disialo)-













SRIF28












22
S1C(disialo)•R13C
177
147
6.28
3.33
11.0
3.77
45.0
40.5
14.7
8.33



(disialo)- SRIF28












23
N5C(disialo)•A9C
30.0
24.9
0.793
0.421
4.09
1.40
14.2
12.8
1.81
1.02



(disialo)-SRIF28












24
S1-3C(disialo)-
20.4
16.9
0.421
0.224
3.30
1.13
7.69
6.92
0.927
0.524



SRIF28












25
S1C(disialo)• 
69.2
57.3
0.985
0.523
7.93
2.71
25.2
22.7
4.14
2.34



N5C(disialo)•













A9C(disialo)-













SRIF28












26
S1C(monosialo)-
1.43
1.19
0.0465
0.0247
0.322
0.110
1.14
1.03
0.372
0.210



SRIF28












27
S1C(asialo)-
0.851
0.704
0.0224
0.0119
0.155
0.0531
0.627
0.564
0.460
0.260



SRIF28












28
S1-2C(asialo)-
0.860
0.712
0.0632
0.0336
0.177
0.0604
0.780
0.702
0.190
0.107



SRIF28












29
S1-3C(asialo)-
4.21
3.49
0.0978
0.0519
0.581
0.199
1.44
1.29
0.369
0.209



SRIF28












30
N5N(disialo)-
3.02
2.50
0.0685
0.0364
0.579
0.198
2.86
2.57
0.465
0.263



SRIF28












31
SIN(disialo)-
2.68
2.21
0.0754
0.0401
0.314
0.107
1.07
0.960
0.272
0.154



SRIF28












—
SRIF14
0.438
0.362
0.0142
0.00755
0.131
0.0450
0.303
0.273
0.578
0.326


—
Octreotide
>100
—
0.0628
0.0334
6.41
2.19
>100
—
9.39
5.31


—
SRIF28
0.353
0.292
0.0271
0.144
0.0368
0.0297
0.294
0.423
0.423
0.239









The control octreotide bound to each of receptors SSTR2, SSTR3, and SSTR5, and SRIF14 and SRI28 bound to all SSTRs. As shown in Table 3A, the compounds according to the present invention potently bound to all SSTRs. The binding affinity of the positive control SRIF14 against SSTR1 in terms of Ki value was 0.362 nM, and where compounds having one sugar chain was 0.704-26.7 nM, the compounds of the present invention having two sugar chains (the compounds of Examples 20-23 and 28) were 0.712-147 nM, showing similarly sufficient receptor binding affinity. Moreover, the compounds of the present invention having three sugar chains (the compounds of Examples 24, 25, and 29) also had sufficient receptor binding affinity, showing 3.49 nM, 16.9 nM, and 57.3 nM. Since bioavailability (BA) will be considerably increased due to the extension of its half-life in blood, they can work effectively on receptors in vivo even if the Ki value of the binding affinity is somewhat high. Similarly, where the binding affinity of SRIF14 against SSTR2, SSTR3, and SSTR4 in terms of Ki value were each 0.00755 nM, 0.0450 nM, and 0.273 nM, the compounds of the present invention having two or more sugar chains were each 0.0336-3.33 nM, 0.0604-3.77 nM, and 0.702-40.5 nM, all showing sufficient receptor binding affinity. Moreover, where the binding affinity of SRIF14 against SSTR5 was 0.326 nM, the compounds of the present invention having two or more sugar chains showed a sufficient receptor binding affinity of up to 8.33 nM.
In this way, it was found that Examples 20-23 and 28 are diglycosylated modified forms having affinity towards all receptors SSTR1-SSTR5, and Examples 24, 25, and 29 are triglycosylated modified forms having affinity towards all receptors SSTR1-SSTR5.
Example 69-2
Calculation of Receptor Binding Affinity-2
Competitive binding assay was performed with each compound shown in Table 3B with the method described in Example 69-1, and receptor binding affinity was calculated. Moreover, SRIF14 and SRIF28 were similarly evaluated as control compounds. The results of the binding assay are shown in Table 3B.
FIGS. 1C and 1D show examples of the structures of glycosylated peptides corresponding to the compound names in Table 3B.













TABLE 3B









SSTR1
SSTR2
SSTR3
SSTR4
SSTR5


















Ex-

IC50/
Ki 
IC50/
Ki
IC50
Ki 
IC50
Ki 
IC50/
Ki


ample

(nM)
(nM)
(nM)
(nM)
(nM)
(nM)
(nM)
(nM)
(nM)
(nM)





















1
S1C(disialo)-
7.71
6.45
0.543
0.289
1.98
0.676
7.10
6.39
1.61
0.911



SRIF28












7
A15C(disialo)-
11.4
9.43
0.938
0.408
3.49
1.19
8.18
7.36
3.84
2.17



SRIF28












8
G16C(disialo)-
17.4
14.4
1.22
0.646
3.12
1.07
12.2
11.0
5.67
3.21



SRIF28












9
K18C(disialo)-
45.4
37.6
12.2
6.51
14.7
5.03
62.7
56.5
160
95.0



SRIF28












12
T26C(disialo)-
21.6
17.9
310
160
21.7
7.44
26.8
24.1
400
230



SRIF28












26
S1C(monosialo)-
5.38
4.46
0.213
0.112
1.18
0.405
2.35
2.12
0.816
0.409



SRIF28












27
S1C(asialo)-
3.97
3.29
0.200
0.105
1.02
0.349
2.98
2.68
1.04
0.588



SRIF28












32
S1C(disialo)•N19C
<10
<8.27
2.20
1.17
3.05
1.04
8.55
7.69
2.37
1.34



(GlcNAc)













SRIF28












33
S1C(disialo)-N19C
44.0
37.0
3.73
1.98
3.63
1.24
25.0
23.0
5.69
3.22



(diMan)•SRIF28












34
S1-5C(disialo)-
110
91.9
12.7
6.58
21.3
7.29
62.3
56.4
7.23
4.09



SRIF28












35
S1-10C(disialo)-
117
56.5
10.1
5.35
40.4
13.8
101
172
34.8
19.7



SRIF28












36
C(disialo)-SRIF28
5.57
4.61
0.478
0.255
2.05
0.870
8.65
7.79
2.62
1.48


37
R11C(disialo)-
22.0
18.2
1.38
0.735
6.34
2.17
20.2
18.1
3.65
2.07



SRIF28












40
F25C(disialo)-
>1000
>828
>1000
>531
>1000
>342
130
110
58.0
33.0



SRIF28












41
S27C(disialo)-
28.4
23.5
0.326
0.170
6.77
2.31
33.4
30.1
4.94
2.80



SRIF28












42
C(disialo)-K-
20.7
17.2
1.97
1.05
3.65
1.21
11.0
9.77
3.52
1.99



SRIF14












43
S1C(disialo)-
1.77
1.46
0.310
0.160
0.480
0.160
1.55
1.39
1.09
0.620



F25Y-SRIF28












44
S1C(disialo)-
4.87
4.03
1.32
0.700
5.91
2.02
6.88
6.19
3.75
2.12



SRIF28-amide












45
C(disialo)-
36.0
30.0
4.67
2.48
8.71
2.98
21.0
19.0
>10
>5.65



PEGlinker-SRIF14












46
Biotin-S1C 
11.0
9.05
1.68
0.890
2.92
1.00
9.28
8.35
2.42
1.37



(disialo)-SRIF28












47
Biotin-PEGlinker-
9.41
7.79
1.96
1.04
3.41
1.17
9.31
8.38
2.00
1.13



S1C(disialo)-













SRIF28












48
amino-S1C(disialo)-
8.82
7.30
1.29
0.690
3.17
1.09
5.34
4.81
0.840
0.470



SRIF28












49
S1C(disialo)-E12C
22.0
19.0
2.70
1.44
4.62
1.58
20.0
18.0
2.11
1.19



(disialo)-SRIF28












50
2C(disialo)-RK-
48.0
40.0
5.35
2.84
9.74
3.33
49.0
44.0
6.16
3.48



SRIF14












51
3C(disialo)-RK-
270
230
32.0
17.0
40.0
14.0
>100
>90.0
41.0
23.0



SRIF14












52
S1C(diGlcNAc)-
5.04
4.17
0.190
0.100
2.02
0.690
3.91
3.52
1.45
0.820



SRIF28












53
S1C(diMan)-
5.06
4.18
0.140
0.0750
1.32
0.450
3.01
2.70
0.550
0.310



SRIF28












54
N19C(diMan)-
6.58
5.44
1.17
0.620
1.55
0.530
8.02
7.22
2.94
1.66



SRIF38












55
S1C(GlcNAc)-
5.91
4.89
0.225
0.120
1.65
0.565
2.89
2.60
1.15
0.650



SRIF28












56
N19C(GlcNAc)-
2.60
2.15
0.840
0.500
<1
<0.342
2.38
2.15
1.89
1.07



SRIF28












57
S1C(trisialo)-
17.0
14.0
0.780
0.410
5.59
1.91
15.0
14.0
1.95
1.10



SRIF28












58
S1C(tetrasialo)-
17.3
14.2
1.12
0.597
5.63
1.93
14.3
12.6
2.12
1.20



SRIF28












59
S1C(disialo)(amino-
2.35
1.94
0.101
0.0536
0.766
0.264
2.24
2.01
2.68
1.18



ethylamide))-SRIF28












60
S1C(disialo 
5.09
4.21
0.207
0.110
1.53
0.522
6.12
2.60
2.60
1.47



(amide))-SRIF28












61
S1C(disialo(Bn))-
6.20
5.12
0.247
0.130
2.15
0.733
6.01
5.41
3.25
1.83



SRIF28












62
S1C(disialo
5.53
4.57
1.04
0.550
11.0
3.75
26.0
24.0
37.0
21.0



(hexadexylamide))-













SRIF28












63
S1-2C(disialo
6.92
5.73
0.830
0.440
2.06
0.710
4.76
2.67
2.67
1.51



(amide))-SRIF28












64
S1-2C(disialo
6.01
4.97
0.830
0.440
2.59
0.880
8.20
7.38
2.60
1.47



(Bn))-SRIF28












65
S1C(Asn(disialo))-
15.0
13.0
0.610
0.330
5.57
1.91
15.0
14.0
1.61
0.910



SRIF28












66
S1N(disialo)-N19C
35.0
29.0
5.65
3.00
3.90
1.33
25.0
22.0
8.53
4.82



(diMan)-SRIF28












67
C(disialo(aminoethyl-
6.20
5.13
0.900
0.510
1.80
0.650
6.12
5.51
1.81
1.02



amide)); S1C













(disialo)-SRIF28












68
S1-4C(disialo)-
130
100
3.76
2.00
10.0
3.43
66.0
59.0
10.0
5.32



SRIF28












—
SRIF28
0.533
1.80
0.0680
0.0565
0.106
0.116
0.776
1.23
0.189
0.666


—
SRIF14
1.25
2.69
0.0329
0.0993
0.0697
0.403
0.823
1.95
0.416
0.813









As shown in Table 3B, the controlss SRIF14 and SRIF27 bound to all receptors SSTR1-SSTR5. Since the number of test runs etc. was different from Example 69-1, the Ki value of SRIF14 against each receptor was different at a high value of 2.5 to 13.2. The Ki values of the compounds of Examples 1, 7, 8, 9, 26, 27, 32, 33, 34, 35, 36, 37, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, and 68 against SSTR1 were 1.46-100 nM, and similarly 0.0536-6.58 nM for SSTR2, 0.160-13.8 nM for SSTR3, 1.39-172 nM for SSTR4, and 0.310-95.0 nM for SSTR5, showing high binding affinity towards all the receptors. The Ki values of the compound of Example 12 against SSTR2 and SSTR5 were 280-1600-folds that of SRIF14 but the Ki values against SSTR1, SSTR3, and SSTR4 were 17.9, 7.44, and 24.1 nM, and it was thought that binding affinity was retained. The affinity of the compound of Example 40 against SSTR1, SSTR2, and SSTR3 was significantly reduced with Ki values of 310-5300-folds that of SRIF14 but the Ki values against SSTR4 and SSTR5 were 110 and 33.0 nM, and it was thought that binding affinity was retained. The Ki values of the compound of Example 51 against SSTR2 and SSTR4 were 170-folds and 46-folds or more that of SRIF14 but the Ki values against SSTR1, SSTR3, and SSTR5 were each 270 nM, 14.0 nM, and 23.0 nM, and it was thought that binding affinity was retained.
Example 69-3
Agonist Activity Evaluation Employing Receptor Expression Cells-1
Somatostatin receptor is a G-protein-coupled receptor (GPCR). SSTR1-SSTR5 all suppress adenyl cyclase activity via G-protein subfamily Gi/Go and reduces intracellular cAMP concentration. In this experimental line, each somatostatin receptor expression cells was employed to calculate the IC50 value of the cAMP production suppressing action of the test substance to evaluate agonist activity. Moreover, SRIF14, SRIF28 were similarly evaluated as control compounds.
Reagents employed in the present experiment and their proper chemical names are as follows: DMEM (Dulbecco's Modified Eagle's Medium), IBMX (3-isobutyl-1-methylxanthine), and HBSS (Hank's Buffered Salt Solution).
For evaluations of SSTR2-SSTR5, experiments were performed under the following conditions. DMEM containing 0.3% BSA and 0.5 mM IBMX was employed as the buffer, and receptor expression cells shown in Table 3C were seeded at 104 cells/well. The test compounds was treated by mixing at a concentration of 0.00001 nM, 0.0001 nM, 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, or 1000 nM with 10 μM forskolin, and allowed to react at room temperature for 30 minutes. After the reaction, the cells were dissolved with 0.2 N HCl, and the amount of cAMP accumulated in the cell was measured with Cayman cyclic AMP EIA kit (Cayman, 582002).
Evaluation of SSTR1 was consigned to Cerep, and the experiment was performed. HBSS containing 20 mM HEPES (pH 7.4) and 500 μM IBMX was used as the buffer, and SSTR1 receptor expression cells were seeded at 104 cells/well. The test substance was treated by mixing at a concentration of 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, or 100 nM with 1 μM NKH477, and allowed to react at 37° C. for 20 minutes. After the reaction, the amount of cAMP accumulated in the cell was measured with Cisbio cAMP dynamic2 kit (Cisbio, 62AM4PE).











TABLE 3C






SSTR2
SSTR3
SSTR4
SSTR5
















Distributer
Perkin Elmer











Serial Number
ES-521-CF
ES-523-CF
ES-524-CF
ES-522-CF








Derived cell
CHO-K1 cells









The experimental results (IC50 (nM)) of the agonist activity evaluation test when employing the compounds of Examples 1, 18, 27, 28, 59, and 60 as the glycosylated form are shown in Table 3D and FIG. 1E. IC50 of the control compounds SRIF14 and SRIF28 was 0.83-0.89 nM against SSTR1, and 0.0076-0.073 nM, 0.029-0.21 nM, 0.015-0.074 nM, and 0.066-0.12 nM against SSTR2, SSTR3, SSTR4, and SSTR5, respectively. IC50 of the agonist activity of the glycosylated compounds against SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5 were 1.0-2.4 nM, 0.041-0.10 nM, 0.12-0.30 nM, 0.039-0.085 nM, and 0.043-0.081 nM, respectively, and potent agonist activity was shown against all receptors SSTR1-SSTR5. From the results shown in Examples 69-1 and 69-2, it is apparent that these compounds have receptor binding affinity, and it became clear that these compounds exert proper agonistic action by binding to a receptor.













TABLE 3D





Example

SSTR1
SSTR2
SSTR3
SSTR4
SSTR5







—
SRIF14
0.83 ± 0.52
0.0076 ± 0.0012
0.029 ± 0.017
0.015 ± 0.006
0.12 ± 0.03


—
SRIF28
0.89 ± 0.41
0.073 ± 0.014
0.21 ± 0.08
0.074 ± 0.058
0.066 ± 0.033


1
S1C(disialo)-SRIF28
2.2 ± 1.1
0.075 ± 0.030
0.13 ± 0.06
0.065 ± 0.042
0.056 ± 0.028


18
Cys(disialo)-R-K-SRIF14
1.2 ± 0.6
0.055 ± 0.015
0.18 ± 0.10
0.050 ± 0.025
0.052 ± 0.032


27
S1C(asialo)-SRIF28
1.6 ± 0.6
0.041 ± 0.006
0.12 ± 0.05
0.039 ± 0.021
0.055 ± 0.031


28
S1-2C(asialo)-SRIF28
1.5 ± 0.6
0.079 ± 0.033
0.15 ± 0.08
0.051 ± 0.046
0.064 ± 0.029


59
S1C(disialo(aminoethylamide))-SRIF28
1.0 ± 0.9
0.10 ± 0.02
0.30 ± 0.15
0.085 ± 0.011
0.081 ± 0.019


60
S1C(disialo(amide))-SRIF28
2.4 ± 1.7
0.055 ± 0.013
0.14 ± 0.06
0.043 ± 0.023
0.043 ± 0.015









Example 69-4
Agonist Activity Evaluation Employing Receptor Expression Cells-2
Agonist activity evaluation test was carried out similarly to Example 69-3 employing each of the compounds described in Table 3E as the glycosylated form. The experimental results are shown in Table 3E. In Table 3E, fields with “- (hyphen)” shown indicates that the test was not performed.













TABLE 3E





Experiment

SSTR1
SSTR2
SSTR3
SSTR4
SSTR5





















—
SRIF14
0.83
0.0076
0.029
0.015
0.12


—
SRIF28
0.89
0.073
0.21
0.074
0.066


4
E12C(disialo)-SRIF28
2.3
—
—
—
—


10
N19C(disialo)-SRIF28
22
—
—
—
—


15
S1C(disialo)-D-Trp22-SRIF28
3.5
—
—
—
—


17
C(disialo)-SRIF14
—
0.029
0.18
0.10
0.23


19
C(disialo)-C12linker-SRIF14
—
—
—
0.11
—


20
S1-2C(disialo)-SRIF28
—
—
0.55
0.14
0.22


21
S1C(disialo)•N5C(disialo)-SRIF28
—
0.072
0.37
0.041
0.090


23
N5C(disialo)•A9C(disialo)-SRIF28
—
0.096
0.29
0.091
0.13


24
S1-3C(disialo)-SRIF28
7.5
0.19
0.79
0.36
0.97


25
S1C(disialo)•N5C(disialo)•A9C(disialo)-SRIF28
—
1.4
2.4
0.16
1.1


26
S1C(monosialo)-SRIF28
1.5
0.059
0.15
0.046
0.062


29
S1-3C(asialo)-SRIF28
—
0.19
—
0.10
—


31
S1N(disialo)-SRIF28
2.3
—
—
—
—


32
S1C(disialo)•N19C(GlcNAc)-SRIF28
—
0.080
0.039
0.033
0.088


34
S1-5C(disialo)-SRIF28
18
0.81
2.0
0.27
—


42
C(disialo)-K-SRIF14
—
0.019
0.10
0.059
—


44
S1C(disialo)-SRIF28-amide
1.7
—
—
—
—


47
C(disialo)-PEG2linker-SRIF14 
—
0.034
0.15
0.066
0.18


50
2C(disialo)-R-K-SRIF14
—
0.15
0.55
1.1
—


51
3C(disialo)-R-K-SRIF14
—
1.4
3.8
1.6
—


52
S1C(diGlcNAc)-SRIF28
1.2
0.045
0.14
0.050
0.033


53
S1C(diMan)-SRIF28
1.2
0.035
0.11
0.043
0.037


54
N19C(diMan)-SRIF28
—
—
—
0.11
—


55
S1C(GlcNAc)-SRIF28
1.4
0.040
0.14
0.049
0.031


56
N19C(GlcNAc)-SRIF28
—
—
—
0.092
—


57 
S1C(trisialo)-SRIF28
4.9
0.11
0.38
0.092
0.096


58
S1C(tetrasialo)-SRF28
2.8
0.13
0.25
0.081
0.11


61
S1C(disialo(Bn))-SRIF28
1.4
—
—
—
—


62
S1C(disialo(hexadecylamide))-SRIF28
1.3 
—
—
—
—


65
S1C(Asn(disialo))-SRIF28
2.6
—
—
0.13
—


67
C(disialo(aminoethylamide))•S1C(disialo)-SRIF28
—
0.039
0.17
0.047
0.034





(—: Not performed)






In Table 3E, IC50 of the agonist activity of the glycosylated compounds against SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5 were 1.2-22 nM, 0.019-1.4 nM, 0.039-3.8 nM, 0.033-1.6 nM, and 0.031-1.1 nM, respectively, and agonist activity was shown against receptors SSTR1-SSTR5. From the results shown in Examples 69-1 and 69-2, it became clear that these compounds have receptor binding affinity, and these compounds exert agonist activity by binding to a receptor.
Example 70
Pharmacokinetics Test with Rats 1
In order to confirm that the glycosylated polypeptide of the present invention (glycosylated form) had improved pharmacokinetics profiles such as drug plasma concentration—area under the time curve (AUC), half-life in blood (t1/2), mean retention time (MRT), and bioavailability compared to a non-glycosylated SRIF28, pharmacokinetics analysis by intravenous and subcutaneous administrations was performed with rats.
70-1 Preparation of Administration Solution and Reagent
The glycosylated form (S1C(disialo)-SRIF28) was dissolved in Japanese Pharmacopeia saline (from Otsuka Pharmaceutical factory, Inc.) to prepare a 40 μM solution to make an administration solution. PBS solution was prepared by dissolving 1 tablet of Phosphate buffered saline (P4417 from Sigma) in 200 mL of ultrapure water. EDTA-PBS was prepared by dissolving EDTA-2Na (from Wako Pure Chemical Industries, Ltd.) in PBS to 2 mg/mL. Aprotinin-containing EDTA-PBS solution was prepared by dissolving aprotinin (010-11834 from Wako Pure Chemical Industries, Ltd.) in EDTA-PBS to 0.142 mg/mL, and was employed as an anticoagulant for collected blood.
70-2 Preparation of Plasma Sample
To the tail vain or dorsal subcutaneous of male SD rats (Crl: CD (SD), Charles River Japan, 6 weeks-old, n=3, body weight 161.3-239.3 g), the administration solution prepared in the above 70-1 was administered under nonfasting condition at a dosage of 1 mL/kg with a glass syringe and a 26 G injection needle (all from Terumo Corporation) (40 nmol/kg as S1C(disialo)-SRIF28). Blood was collected from the rat cervical vein before administration, as well as at 2 minutes, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 8 hours after administration. 0.2 mL of the collected blood was promptly mixed with 0.2 mL of the aprotinin-containing EDTA-PBS solution prepared in the above 70-1, and left on ice for 30 minutes or more. After centrifugal separation (1,870×g, 4° C., 10 minutes), 250 μL of the supernatant was taken as the plasma sample. As blank plasma, plasma obtained by similarly treating the blood collected from untreated rat cervical vein was employed. Plasma samples were frozen in storage until employed for measurement. Tips and tubes used were low absorbent products from BM Equipment Co., Ltd.
70-3 Measurement of Concentration in Plasma
Measurement of plasma concentration of S1C(disialo)-SRIF28 in the plasma sample obtained in the above 70-2 was performed with Phoenix Pharmaceuticals somatostatin EIA kit (Phoenix Pharmaceuticals Inc, EK-060-03). The plasma sample was diluted with the assay buffer supplied in the EIA kit to 5, 20, 100, 400, and 1600-folds as measurement samples. The standard solution for creating a standard curve was prepared as follows. First, the blank plasma obtained in the above 70-2 was diluted with the assay buffer supplied in the EIA kit similarly to the plasma sample, and this was used as the assay buffer for standard solution preparation (for example, when diluting the plasma sample to 100-fold, 1/100 amount of blank plasma was added to the assay buffer supplied in the EIA kit, and this was used as the assay buffer for standard solution preparation). S1C(disialo)-SRIF28 was diluted with PBS solution to prepare a 100 μM solution, and a 2 μM solution was prepared from the 100 μM solution. The 2 μM S1C(disialo)-SRIF28 solution obtained was serially diluted with the assay buffer for standard solution preparation to prepare standard solutions of 20 nM, 10 nM, 2 nM, 0.4 nM, 0.08 nM, and 0.04 nM. By multiplying the results obtained and the dilution ratio, and further multiplying the dilution ratio 2 in the aprotinin-containing EDTA-PBS solution used as anticoagulant treatment, the plasma concentration was calculated. As a control, a similar operation was carried out employing non-glycosylated SRIF28 instead of the glycosylated form. The transition of plasma S1C(disialo)-SRIF28 concentration obtained is shown in FIG. 2.
70-4 Estimation of Pharmacokinetics Parameter
From the transition of S1C(disialo)-SRIF28 concentration obtained, AUC was calculated by the moment analysis method and the trapezoidal rule. Moreover, the predicted initial concentration (C0) was determined by the extrapolation method for intravenous administration, t1/2 and MRT were calculated, and the maximum plasma concentration (Cmax) was determined from the actual value for subcutaneous administration. The pharmacokinetics parameters obtained are shown in Table 4.









TABLE 4








Intravenous administration
Subcutaneous administration
















t1/2
AUC
MRT
C0
t1/2
AUC
MRT 
Cmax


















S1C
18.1
5086
20.4
780
26.0
1273
43.8
19.9


(disialo)-










SRIF28










SRIF28
0.8
358
2.3
678
1.5
15
3.2
4.4





(t1/2: min, AUC: min · nM, MRT: min, C0: nM, Cmax: nM)






As is clear from the results shown in FIG. 2 and Table 4, S1C(disialo)-SRIF28 has significantly extended t1/2 and MRT compared to the non-glycosylated SRIF28, and an increase in AUC and Cmax was recognized. These are thought to be due to the increase in resistance to degradation activity in blood by glycosylation. It is clear that the glycosylated form has improved stability in vivo compared to the non-glycosylated form. Moreover, as factors for the increase in AUC and Cmax, the improvement of bioavailability according to the present invention, as well as improvement of stability thereof in vivo are thought to be factors.
Example 71
Pharmacokinetics Test with Rats 2
A pharmacokinetics test was carried out similarly to Example 70, except that S1C(disialo)-SRIF28, N5C(disialo)-SRIF28, and S1C(disialo).N5C(disialo)-SRIF28 were employed as the glycosylated forms. As a control, non-glycosylated SRIF28 was employed instead of the glycosylated form. The compound plasma concentration transition obtained is shown in FIG. 3.
Example 72
Pharmacokinetics Test with Rats 3
A pharmacokinetics test was carried out similarly to Example 70, except that S1C(disialo)-SRIF28, S1C(disialo).R13C(disialo)-SRIF28, and S1C(disialo).N5C(disialo) A9C(disialo)-SRIF28 were employed as the glycosylated forms. As a control, non-glycosylated SRIF28 was employed instead of the glycosylated form. The compound plasma concentration transition obtained is shown in FIG. 4.
Example 73
Pharmacokinetics Test with Rats 4
A pharmacokinetics test was carried out similarly to Example 70, except that S1C(disialo)-SRIF28, S1C(disialo).N5C(disialo).A9C(disialo)-SRIF28, S1-3C(disialo)-SRIF28, S1-5C(disialo)-SRIF28, and S1-10C(disialo)-SRIF28 were employed as the glycosylated forms. The compound plasma concentration transition obtained is shown in FIG. 5.
Example 74
Pharmacokinetics Test with Rats 5
A pharmacokinetics test was carried out similarly to Example 70, except that S1C(disialo)-SRIF28, N5C(disialo)-SRIF28, A9C(disialo)-SRIF28, S1C(disialo) N5C(disialo)-SRIF28, N5C(disialo).A9C(disialo)-SRIF28, and S1C(disialo).N5C(disialo).A9C(disialo)-SRIF28 were employed as the glycosylated forms. The compound plasma concentration transition obtained is shown in FIG. 6.
Example 75
Pharmacokinetics Test with Rats 6
A pharmacokinetics test was carried out similarly to Example 70, except that S1C(disialo)-SRIF28, S1-2C(disialo)-SRIF28, and S1-3C(disialo)-SRIF28 were employed as the glycosylated forms. The compound plasma concentration transition obtained is shown in FIG. 7.
From the compound plasma concentration transition obtained in the pharmacokinetics tests of Examples 71-75, the pharmacokinetics parameters of each compound were calculated similarly to Example 70. Moreover, the parameters obtained were employed to calculate bioavailability from the following mathematical formula. The results are shown in Table 5A.

BA (%)=(AUC(sc)/Dose(sc))/(AUC(iv)/Dose(iv))

AUC(sc): AUC for subcutaneous administration (min·nM)
Dose(sc): Administration dose for subcutaneous administration (nmol/kg)
AUC(iv): AUC for intravenous administration (min·nM)
Dose(iv): Administration dose for intravenous administration (nmol/kg)













TABLE 5









The number of
Value
Intravenous administration
Subcutaneous administration



















Compound
sugar chains
of n
t1/2
AUC
MRT
C0
t1/2
AUC
MRT
Cmax
BA






















—
SRIF28
None
6
1.00 ± 
472 ± 
2.20 ±
554 ± 
2.94 ± 
25.5 ±
4.76 ±
4.47 ± 
5






0.20
166
0.11
221
1.59
13.8
1.80
1.21



1
S1C(disialo)-SRIF28
 1
15
16.9 ± 
4855 ±
18.6 ±
686 ± 
29.8 ±
1773 ±
47.9 ±
28.9 ±
37






1.02
1030
1.72
363
2.68
281
2.68
3.23



2
N5C(disialo)-SRIF28
 1
6
20.1 ± 
4421 ±
22.9 ±
496 ±
28.1 ±
2392 ±
51.9 ± 
32.4 ±
54






1.49
1366
2.16
312
3.72
443
5.92
4.87



3
A9C(disialo)-SRIF28
 1
3
19.9 ±
4083 ±
25.2 ±
547 ± 
30.3 ±
2462 ±
55.6 ±
30.2 ±
60






0.33
515
0.38
211
2.45
64.0
5.81
5.05

















Average value
19.0
4453
22.2
576.3
29.4
2209
51.8
30.5
50



















20
S1-2C(disialo)-SRIF28
 2
3
28.8 ± 
7306 ±
23.3 ±
1382 ± 
40.9 ±
4046 ±
67.9 ±
46.3 ± 
55




(dense)

3.11
625
2.63
321
12.2
248
15.0
9.75



21
S1C(disialo)•N5C(disialo)-
 2
6
25.8 ±
4099 ±
24.6 ±
558 ±
38.4 ±
3344 ±
67.9 ±
35.5 ±
82



SRIF28
(with intervals)

3.41
634
5.97
248
4.93
663
8.39
6.38



22
S1C(disialo)•R13C(disialo)-
 2
3
23.4 ±
4029 ±
20.6 ±
822 ±
39.6 ±
3407 ±
67.7 ±
38.7 ±
85



SRIF28
(with intervals)

0.66
184
1.32
106
2.50
436
5.39
8.73



23
N5C(disialo)•A9C(disialo)-
 2
3
32.4 ±
4465 ±
26.6 ± 
664 ±
42.7 ±
3450 ±
75.0 ±
32.5 ±
77



SRIF28
(with intervals)

1.66
833
0.27
104
9.76
784
12.4
7.06


















27.2
4198
23.9
681.3
40.2
3400
70.2
35.6
81



















24
S1-3C(disialo)-SRIF28
 3
6
38.5 ± 
5660 ±
24.0 ±
1061 ±
87.7 ±
3884 ±
126 ±
27.5 ±
69




(dense)

2.83
1354
3.92
209
21.4
543
31.7
10.8



25
S1C(disialo)•N5C(disialo)• 
 3
9
39.8 ±
4402 ±
25.8 ±
938 ± 
87.4 ±
4015 ±
132 ± 
24.8 ±
91



A9C(disialo)-SRIF28
(with intervals)

1.87
999
2.87
145
17.1
944
19.8
9.03



34
S1-5C(disialo)-SRIF28
 5
3
99.5 ±
6016 ±
49.0 ± 
752 ±
271 ±
5524 ±
389 ±
13.5 ±
92




(dense)

14.0
873
6.06
81.4
38.7
633
50.0
2.85



35
S1-10C(disialo)-SRIF28
10
3
219 ± 
100259 ± 
272 ± 
821 ±
>480
—
—
45.9 ±
—




(dense)

6.27
10463
14.3
264



2.32






(t1/2: min, AUC: min · nM, MRT: min, C0: nM, Cmax: nM, BA: %)






As is clear from the results shown in Table 5A, the bioavailability of the glycosylated form of the present invention increased as the number of modifying sugar chains increased. In other words, what was 5% in the non-glycosylated form was 37-60% in the glycosylated polypeptide having one sugar chain added, an increase of 50% on average. On the other hand, it was found that this was increased to 77-85%, 81% on average in those having two sugar chains added with intervals, and to 91% in those having three sugar chains added with intervals. Moreover, comparing those with dense glycosylation intervals, it was found that there was a 37% increase in those having one added, 55% in those having two added, 69% in those having three added, and 92% in those having five added (the compounds of Examples 1, 20, 24, and 34). For those having ten sugar chains added, as shown in FIG. 5, AUC could not be measured because the rise in plasma concentration was maintained throughout the measurement time, and as a result bioavailability could not be calculated. From these results, it was proven that bioavailability is significantly improved by glycosylation according to the present invention compared to somatostatin without any glycosylation, and a further improved bioavailability could be obtained by adding two or more sugar chains compared to those having one sugar chain added.
As factors for the increase in bioavailability for subcutaneous administration, various pharmacokinetic factors exist. Among these factors, some are thought to be the stability of the compound in blood, or transitivity into the blood (from the administration site). As shown in Table 5A, it is recognized that the AUC for subcutaneous administration which will be an indicator for speculating the transit to blood for subcutaneous administration will increase with the increase in the number of modifying sugar chains (1: 2209 min·nM, 2 (with intervals): 3400 min·nM, and 3 (with intervals): 4015 min·nM), and it was speculated to contribute to improvement bioavailability.
Moreover, it was recognized that as the number of modifying sugar chains increased, t1/2 and MRT extension effect for intravenous and subcutaneous administrations (for example, t1/2 for intravenous administration was 1: 19.0 min, 2 (with intervals): 27.2 min, and 3 (with intervals): 39.8 min), and it was speculated that stability in blood was improved. On the other hand, AUC for intravenous administration was not an increase in proportion to the number of modifying sugar chains (1: 4453 min·nM, 2 (with intervals): 4198 min·nM, and 3 (with intervals): 4402 min-nM), and it is thought that the increase in the stability in blood of the compound by the increase in the number of modifying sugar chains is not the only factor contributing to the increase in bioavailability.
Cmax for subcutaneous administration increased compared to the non-glycosylated form up until the number of modifying sugar chains was two (0: 4.47 nM, 1: 30.5 nM, and 2 (with intervals): 35.6 nM), and three (with intervals) (24.8 nM) resulted in an adverse decrease. When considering blood transit from the administration site (subcutaneous administration in the present Example), speculated factors for the increase in AUC are two formats of rapid transit to blood, or mild but continuous transit to blood. The former has the advantage of avoiding degradation at the administration site, but if there is no problem in stability, it is speculated that the latter format has a higher transit in blood. The fact that Cmax had decreased in those having three modifying sugar chains with high bioavailability is thought to be the result showing not a sudden but a mild and continuous migration in blood. From this, it is thought that the increase in the number of modifying sugar chains related to the present invention shows a continuous migration in blood without a rapid rise in plasma concentration (may generally be referred to as absorption delaying effect).
As is clear from the results shown in Table 5A, there was no significant difference in t1/2 and MRT when the modifying positions had intervals and when they were dense (for example, tl/2 for intravenous administration was 2 (dense): 28.8 min, 2 (with intervals): 27.2 min, 3 (dense): 38.5 min, and 3 (with intervals): 39.8 min).
Moreover, with respect to AUC, for intravenous administration, AUC was increased when glycosylation was dense. In other words, for two sugar chains, this was 4029-4465 min·nM for compounds 21, 22, and 23 with intervals as opposed to 7306 min·nM for a dense compound 20, and for three sugar chains, compound 25 with intervals was 4402 min·nM whereas a dense compound 24 was 5660 min·nM.
On the other hand, bioavailability increased when there were intervals in the glycosylation positions compared to when they were dense. In other words, for two sugar chains, a dense compound 20 was 55% whereas compounds 21, 22, and 23 with intervals were 77-85% (81% on average), and for three sugar chains, a dense compound 24 was 69% whereas compound 25 with intervals was 91%. From this, it is thought that for glycosylation positions related to the present invention, multiple modifications with intervals contribute more to the improvement of bioavailability than dense multiple modifications.
Example 76
Pharmacokinetics Test with Rats 7
A pharmacokinetics test was carried out similarly to Example 70, except that SiC(disialo)-SRIF28, S1C(disialo(amide))-SRIF28, and S1C(disialo(aminoethylamide))-SRIF28 were employed as the glycosylated forms. The compound plasma concentration transition obtained is shown in FIG. 8.
Example 77
Pharmacokinetics Test with Rats 8
A pharmacokinetics test was carried out similarly to Example 70, except that S1C(disialo)-SRIF28, S1C(disialo(Bn))-SRIF28, and S1C(disialo)-D-Trp22-SRIF28 were employed as the glycosylated forms. The compound plasma concentration transition obtained is shown in FIG. 9.
Example 78
Pharmacokinetics Test with Rats 9
A pharmacokinetics test was carried out similarly to Example 70, except that S1C(disialo)-SRIF28, R13C(disialo)-SRIF28, and K14C(disialo)-SRIF28 were employed as the glycosylated forms. The compound plasma concentration transition obtained is shown in FIG. 10.
Example 79
Pharmacokinetics Test with Rats 10
A pharmacokinetics test was carried out similarly to Example 70, except that S1C(disialo)-SRIF28, E12C(disialo)-SRIF28, N19C(disialo)-SRIF28, 29C(disialo)-SRIF28, S1C(monosialo)-SRIF28, and S1C(asialo)-SRIF28 were employed as the glycosylated forms. The compound plasma concentration transition obtained is shown in FIG. 11.
Example 80
Pharmacokinetics Test with Rats 11
A pharmacokinetics test was carried out similarly to Example 70, except that S1C(disialo)-SRIF28, K14C(disialo)-SRIF28, and C(disialo)-SRIF14 were employed as the glycosylated forms. The compound plasma concentration transition obtained is shown in FIG. 12.
Example 81
Pharmacokinetics Test with Rats 12
A pharmacokinetics test was carried out similarly to Example 70, except that C(disialo)-SRIF14, C(disialo)-C12 linker-SRIF14, and C(disialo)-PEG linker-SRIF14 were employed as the glycosylated forms. The compound plasma concentration transition obtained is shown in FIG. 13.
Example 82
Pharmacokinetics Test with Rats 13
A pharmacokinetics test was carried out similarly to Example 70, except that SRIF28, S1C(disialo)-SRIF28, S1C(asialo)-SRIF28, S1C(diGlcNAc)-SRIF28, S1C(diMan)-SRIF28, and S1C(GlcNAc)-SRIF28 were employed as the glycosylated forms. The compound plasma concentration transition obtained is shown in FIG. 14.
Example 83
Pharmacokinetics Test with Rats 14
A pharmacokinetics test was carried out similarly to Example 70, except that S1C(disialo)-SRIF28, S1C(tetrasialo)-SRIF28, S1C(trisialo)-SRIF28, S1C(Asn(disialo))-SRIF28, and S1-2C(disialo)-SRIF28 were employed as the glycosylated forms. The compound plasma concentration transition obtained is shown in FIG. 15.
Example 84
Pharmacokinetics Test with Rats 15
A pharmacokinetics test was carried out similarly to Example 70, except that SRIF28, S1C(disialo)-SRIF28, S1-2C(disialo)-SRIF28, S1-3C(disialo)-SRIF28, and S1-4C(disialo)-SRIF28 were employed as the glycosylated forms. The compound plasma concentration transition obtained is shown in FIG. 16.
Example 85
Pharmacokinetics Test with Rats 16
A pharmacokinetics test was carried out similarly to Example 70, except that SRIF14, C(disialo)-SRIF14, C(disialo)-K-SRIF14, C(disialo)-R—K-SRIF14, 2C(disialo)-R—K-SRIF14, and 3C(disialo)-R—K-SRIF14 were employed as the glycosylated forms. The compound plasma concentration transition obtained is shown in FIG. 17.
Example 86
Pharmacokinetics Test with Rats 17
A pharmacokinetics test was carried out similarly to Example 70, except that S1C(asialo)-SRIF28, S1-2C(asialo)-SRIF28, and S1-3C(asialo)-SRIF28 were employed as the glycosylated forms. The compound plasma concentration transition obtained is shown in FIG. 18.
Example 87
Pharmacokinetics Test with Rats 18
A pharmacokinetics test was carried out similarly to Example 70, except that S1C(disialo)-SRIF28, S1-2C(disialo)-SRIF28, S1-2C(asialo)-SRIF28, C(disialo(aminoethylamide)/S1C(disialo)-SRIF28, S1-2C(disialo(Bn))-SRIF28, and S1-2C(disialo(amide))-SRIF28 were employed as the glycosylated forms. The compound plasma concentration transition obtained is shown in FIG. 19.
From the compound plasma concentration transition obtained in pharmacokinetics tests 7-18 of Examples 76-87, the pharmacokinetics parameters of each compound were calculated similarly to Example 70. The pharmacokinetics parameters obtained are shown in Tables 5B-5F.











TABLE 5B









Intravenous administration
Subcutaneous administration


















Example

T1/2
AUC
MRT
C0
T1/2
AUC
MRT
Cmax
BA




















—
SRIF14
0.500
119
2.00
819
2.30
8
4.30
1.80
7


—
SRIF28
1.32
467
2.46
518
2.38
22
4.18
4.65
5


1
S1C(disialo)-SRIF28
17.7
5190
19.1
633
31.2
1946
49.2
30.1
38


4
E12C(disialo)-SRIF28
19.5
10521
27.5
1086
38.2
2891
57.5
34.7
27


5
R13C(disialo)-SRIF28
20.3
4926
22.8
481
35.4
5528
55.9
65.9
78


6
K14C(disialo)-SRIF28
20.4
6278
20.0
884
35.1
3945
55.1
57.5
62


10
N19C(disialo)-SRIF28
18.8
3854
21.5
407
37.9
2624
55.3
41.7
56


13
29C(disialo)-SRIF28
23.7
3736
25.3
359
39.3
2176
57.6
29.4
51


15
S1C(disialo)-D-Trp-SRIF28
18.3
4924
20.6
588
31.0
2220
51.9
34.0
45


18
C(disialo)-R-K-SRIF14
18.9
6571
22.5
429
28.4
1996
45.2
32.5
30


42
C(disialo)-K-SRIF14
18.8
5277
19.4
646
26.4
2446
43.7
40.5
46


17
C(disialo)-SRIF14
16.5
5614
18.3
725
27.2
3641
40.0
73.6
66


19
C(disialo)-C12linker-SRIF14
11.0
1686
7.60
500
14.4
345
22.0
13.4
21


45
C(disialo)-PEGlinker-SRIF14
16.4
3538
18.8
412
20.7
2725
39.2
47.8
77









As is clear from the results shown in Table 5B, S1C(disialo)-SRIF28, E12C(disialo)-SRIF28, R14C(disialo)-SRIF28, K14C(disialo)-SRIF28, N19C(disialo)-SRIF28, and 29C(disialo)-SRIF28, S1C(disialo)-D-Trp22-SRIF28 extended t1/2 by 13-18-folds and increased AUC by 8-23-folds compared to the non-glycosylated form SRIF28 when administered intravenously. Moreover, C(disialo)-SRIF14, C(disialo)-K-SRIF14, and C(disialo)-R—K-SRIF14 extended t1/2 by 33-38-folds and increased AUC by 44-55-folds compared to the non-glycosylated form SRIF14 when administered intravenously. Moreover, C(disialo)-C12 linker-SRIF14 and C(disialo)-PEG linker-SRIF14 extended t1/2 by 22-33-folds and increased AUC by 14-30-folds compared to the non-glycosylated form SRIF14. In other words, similarly to Example 70, this was thought to be the result of improved stability in blood by glycosylation.











TABLE 5C









Intravenous administration
Subcutaneous administration


















Example

T1/2
AUC
MRT
C0
T1/2
AUC
MRT
Cmax
BA




















—
SRIF28
1.32
467
2.46
518
2.38
22
4.18
4.65
5


55
S1C(GlcNAc)-SRIF28
1.80
690
3.00
666
5.90
63
7.90
7.10
9


53
S1C(diMan)-SRIF28
5.50
1445
5.20
423
9.10
124
13.9
7.30
9


52
S1C(diGlcNAc)-SRIF28
10.5
3588
12.3
495
16.5
445
25.7
13.2
12


27
S1C(asialo)-SRIF28
13.4
4602
12.5
749
19.7
710
29.0
17.2
16


26
S1C(monosialo)-SRIF28
15.9
6618
15.3
1061
24.5
995
37.8
19.4
17


1
S1C(disialo)-SRIF28
17.7
5190
19.1
633
31.2
1946
49.2
30.1
38


57
S1C(trisialo)-SRIF28
21.1
4428
22.0
423
36.6
1979
56.9
24.9
45


58
S1C(tetrasialo)-SRIF28
20.9
4851
23.0
471
40.5
2624
59.4
32.2
54


65
S1C(Asn(disialo))-SRIF28
27.9
6488
35.9
365
34.0
2935
55.3
33.9
45


59
S1C(disialo(aminoethylamide))-SRIF28
18.6
3473
12.1
746
27.1
284
33.2
9.60
8


60
S1C(disialo(amide))-SRIF28
18.0
5814
12.6
1048
30.0
961
42.5
19.1
17


61
S1C(disialo(Bn))-SRIF28
14.4
3708
14.5
526
45.5
1047
52.6
18.7
28









As is clear from the results shown in Table 5C, when the size of the modifying sugar chain is altered to GlcNAc (monosaccharide), diMan (5 sugars), diGlcNAc (7 sugars), asialo (9 sugars), monosialo (10 sugars), disialo (11 sugars), trisialo (14 sugars), and tetrasialo (17 sugars), the t1/2, AUC, and bioavailability when administered subcutaneously increased to 2-17-folds, 3-120-folds, and 2-11-folds, respectively, according to the size of the modifying sugar chain. From this, it became clear that altering the size of the modifying sugar chain not only improved the stability in blood, but allowed change in its increase rate. Moreover, since it is known that dimannose or asialo sugar chains etc. among these sugar chains interact with particular proteins, they are thought to be utilizable for targeting a particular protein or organs and cells having the particular protein. Moreover, as modification of the sialic acid at the non-reducing terminal, S1C(disialo(aminoethylamide))-SRIF28, SlC(disialo(amide))-SRIF28, and S1C(disialo(Bn))-SRIF28 having added a sugar chain with altered charge by e.g. the introduction of an aminoethylamide, amide, and Bn to the carboxy group extended t1/2 by 11-14-folds and increased AUC by 7-12-folds, and improved stability in blood compared to the non-glycosylated form when administered intravenously. This shows that the carboxy group of the sialic acid has a large influence on retentivity in blood, and shows the possibility for (usage in) blood clearance or body distribution control by abstraction of the negative charge of the carboxy group (disialo(Bn) and disialo(amide)) or conversion into a positive charge (disialo(aminoethylamide)).











TABLE 5D









Intravenous administration
Subcutaneous administration


















Example

T1/2
AUC
MRT
C0
T1/2
AUC
MRT
Cmax
BA




















—
SRIF14
0.500
119
2.00
819
2.30
8
4.30
1.80
7


—
SRIF28
1.32
467
2.46
518
2.38
22
4.18
4.65
5


18
C(disialo)-R-K-SRIF14
18.9
6571
22.5
429
28.4
1996
45.2
32.5
30


50
2C(disialo)R-K-SRIF14
31.7
5041
24.1
704
47.7
3310
73.4
36.7
66


51
3C(disialo)R-K-SRIF14
35.7
4288
22.2
1013
70.6
3515
112
23.5
82


1
S1C(disialo)-SRIF28
17.7
5190
19.1
633
31.2
1946
49.2
30.1
38


20
S1-2C(disialo)-SRIF28
28.2
5804
23.0
950
40.8
3091
65.3
34.6
53


24
S1-3C(disialo)-SRIF28
39.5
5834 
23.8
1083
79.3
4174
115
30.7
72


68
S1-4C(disialo)-SRIF28
63.5
7070
25.2
1007
119
4554
172
22.0
64









As is clear from the results shown in Table 5D, C(disialo)-R—K-SRIF14 modified with one disialosugar chain, 2C(disialo)-R—K-SRIF14 modified with two disialosugar chains, and 3C(disialo)-R—K-SRIF14 modified with three disialosugar chains on SRIF14 each extended t1/2 by 38, 63, and 71-folds and increased AUC by 55, 42, and 36-folds compared to the non-glycosylated form when administered intravenously. Similarly, S1C(disialo)-SRIF28 modified with one disialosugar chain, S1-2C(disialo)-SRIF28 modified with two disialosugar chains, S1-3C(disialo)-SRIF28 modified with three disialosugar chains, and S1-4C(disialo)-SRIF28 modified with four disialosugar chains on SRIF28 each extended t1/2 by 13, 21, 30, and 48-folds and increased AUC by 11, 12, 12, and 15-folds compared to the non-glycosylated form when administered intravenously. In either of SRIF14 and SRIF28, it became clear that stability in blood is improved according to the number of modifying disialosugar chain.











TABLE 5E









Intravenous administration
Subcutaneous administration


















Example

T1/2
AUC
MRT
C0
T1/2
AUC
MRT
Cmax
BA




















—
SRIF28
1.32
467
2.46
518
2.38
22
4.18
4.65
5


27
S1C(asialo)-SRIF28
13.4
4602
12.5
749
19.7
710
29.0
17.2
16


28
S1-2C(asialo)-SRIF28
8.30
2664
9.60
741
19.2
1049
36.4
19.8
39


29
S1-3C(asialo)-SRIF28
6.60
2733
6.50
1147
26.4
885
42.1
15.0
32









As is clear from the results shown in Table 5E, S1C(asialo)-SRIF28 modified with one asialosugar chain, S1-2C(asialo)-SRIF28 modified with two asialosugar chains, and S1-3C(asialo)-SRIF28 modified with three asialosugar chains on SRIF28 each extended t1/2 by 10, 6, and 5-folds and increased AUC by 10, 6, and 6-folds compared to the non-glycosylated form when administered intravenously. It became clear that stability in blood is also improved when the modifying sugar chain is an asialosugar chain.











TABLE 5F









Intravenous administration
Subcutaneous administration


















Example

T1/2
AUC
MRT
C0
T1/2
AUC
MRT
Cmax
BA




















—
SRIF28
1.32
467
2.46
518
2.38
22
4.18
4.65
5


1
S1C(disialo)-SRIF28
17.7
5190
19.1
633
31.2
1946
49.2
30.1
38


20
S1-2C(disialo)-SRIF28
28.2
5804
23.0
950
40.8
3091
65.3
34.6
53


28
S1-2C(asialo)-SRIF28
8.30
2664
9.60
741
19.2
1049
36.4
19.8
39


63
S1-2C(disialo(amide))-SRIF28
23.8
5268
20.5
737
33.1
1297
55.8
16.3
25


64
S1-2C(disialo(Bn))-SRIF28
25.6
5525
20.7
807
33.9
1843
59.4
21.5
33


67
C(disialo(aminoethylamide))•
22.5
3742
20.5
518
42.3
1289
64.7
16.2
34



S1C(disialo)-SRIF28


















As is clear from the results shown in Table 5F, S1-2C(disialo)-SRIF28, S1-2C(asialo)-SRIF28, C(disialo(aminoethylamide))/S1C(disialo)-SRIF28, S1-2C(disialo(Bn))-SRIF28, and S1-2C(disialo(amide))-SRIF28 each extended t1/2 by 6-21-folds and increased AUC by 6-12-folds compared to the non-glycosylated form when administered intravenously. In other words, stability in blood improved by modifying a sugar chain onto SRIF28. Moreover, t1/2, AUC, Cmax, and BA increased in both intravenous and subcutaneous administrations according to the increase in the number of sialic acid sugar chains. On the other hand, when the negative charge of the carboxy group of the sialic acid was removed (2C(disialo(Bn))/2C(disialo(amide))), or when a positive charge was adjacently added (C(disialo(aminoethylamide))/S1C(disialo)), although t1/2 was extended when administered subcutaneously, AUC or Cmax did not increase. Since the carboxy group of the sialic acid has a large influence on retentivity in blood or blood migration, this shows the possibility for (usage in) blood clearance or body distribution control by conversion of charge at the sugar chain terminal.
Example 88
Plasma Stability Test with Rat Plasma
88-1 Preparation of Compound Solution, Reagent, and Rat Plasma
The glycosylated form and the non-glycosylated SRIF28 were dissolved in PBS solution to prepare 2 μM solutions as treatment solutions. 10% TFA was prepared by dissolving trifluoroacetic acid (208-02746 from Wako Pure Chemical Industries, Ltd.) in water to 10 v/v %. Rat plasma was prepared from Wistar rats (Crlj: Wistar, male, Charles River Japan, 7 weeks-old) as heparin-added plasma (heparin: Japanese Pharmacopeia heparin sodium injectable solution (Mochida Pharmaceutical Co., Ltd.)).
88-2 Preparation of Plasma-Added Sample
To 0.27 mL of rat plasma (n=3), 0.03 mL of the glycosylated compound solution prepared in the above 88-1 was promptly mixed as plasma-added sample, and kept warm in a 37° C. thermostat bath. After mixing, 0.04 mL of plasma-added sample was taken at 0 minute and over time at 1-24 hours, and then promptly mixed with 0.01 mL of 10% TFA. After centrifugal separation (20,000×g, 4° C., 10 minutes), 0.04 mL of the supernatant was taken as plasma stability measurement samples. Sampling time was 0, 1, 2, and 4 hours, or 0, 4, 8, and 24 hours. As blank plasma, plasma obtained from a similar treatment except that PBS solution was employed as the treatment solution was employed. Plasma samples were frozen in storage until employed for measurement. For each experiment run, non-glycosylated SRIF28 was employed as the positive control.
88-3 Measurement of Concentration in Sample and Calculation of Plasma Stability Index
In a method similar to plasma concentration measurement of Example 70-3, the concentration of the glycosylated form remaining in the plasma stability measurement samples obtained in the above 88-2 was measured. The concentration of the glycosylated form at 0 minute after mixing was set as 100%, the residual rate over time was represented in percentage (%), and the half-life was calculated from the elimination rate constant of the following exponential formula (1) employing the calculation formula (2). Then, the half-life of the non-glycosylated SRIF28 for each experiment run was set as 1, and the plasma stability index (PS index) of the glycosylated form was calculated. The results are shown in Table 6 and FIG. 20. Moreover, those having a plasma stability index of 20 or higher are shown as >20. In FIG. 20, those having a plasma stability index of higher than 20 are also shown with 20 as the upper limit.

Residual rate (%)=100·e(k·t)  (1)

e: Base of natural logarithm
k: elimination rate constant
t: Time (hours)

Half-life(hours)=0.693/k  (2)










TABLE 6





Example

PS index

















 1
S1C(disialo)-SRIF28
4.5


 2
N5C(disialo)-SRIF28
4.7


 3
A9C(disialo)-SRIF28
6.7


 4
E12C(disialo)-SRIF28
6.2


 5
R13C(disialo)-SRIF28
15.9


 6
K14C(disialo)-SRIF28
19.1


10
N19C(disialo)-SRIF28
>20


13
29C(disialo)-SRIF28
>20


14
30C(disialo)-SRIF28
16.8


15
S1C(disialo)-D-Trp-SRIF28
12.3


16
A9C(disialo)-D-Trp-SRIF28
17.2


21
S1C(disialo)•N5C(disialo)-SRIF28
17.8


22
S1C(disialo)•R13C(disialo)-SRIF28
>20


24
S1-3C(disialo)-SRIF28
>20


25
S1C(disialo)•N5C(disialo)•A9C(disialo)-SRIF28
>20


26
S1C(monosialo)-SRIF28
3.5


27
S1C(asialo)-SRIF28
2.6


—
Octreotide
>20


—
SRIF28
1









As is clear from the results shown in FIG. 20, the glycosylated polypeptide of the present invention was increased in plasma stability compared to SRIF28. In other words, this was 4.5-6.7-folds in those having one sugar chain added at positions 1, 5, 9, and 12 (the compounds of Examples 1, 2, 3, and 4), 15.9-folds for those having a sugar chain added at position 13 (the compound of Example 5), 19.1-folds for those having a sugar chain added at position 14 (the compound of Example 6), and 16.8-folds for those having a sugar chain added at position 30 (the compound of Example 14), compared to that of SRIF28. Moreover, this was 20-folds or more for those having one glycosylated amino acid further added at position 19 which is the C-terminal side (the compounds of Examples 10 and 13). It was shown that in regards to the position for glycosylation, plasma stability increases from position 1 towards the C-terminal.
Example 89
GH Production Suppression Test with Rats
As shown in Example 69, the glycosylated polypeptide of the present invention had affinity towards each receptor of SSTR. On the other hand, some were seen to have attenuated affinity towards each SSTR, but in order to prove that even in such cases pharmacologically effective action is shown towards receptors in vivo by e.g. extension of half-life in blood or increase in bioavailability as shown in Examples 70-88, a test for evaluating the administration effect of the glycosylated polypeptide of the present invention on growth hormone (GH) production was carried out as an in vivo test employing rats. Increase of GH production amount into the blood is thought to cause proliferation or differentiation of cells and activation or suppression of the biosynthetic system in various organs via the paracrine effect of GH, and thereby influence biological reactions. Somatostatin released from the hypothalamus suppresses GH secretion from the anterior pituitary gland into the blood. This experimental line was carried out as a test line that can evaluate the pharmacological action of the glycosylated form on SSTR and its residence in blood after administration, with blood GH production amount as an indicator.
89-1 Preparation of Administration Solution and Reagent
Employing S1C(disialo)-SRIF28, N19C(disialo)-SRIF28, 29C(disialo)-SRIF28, SiC(disialo).N5C(disialo)-SRIF28, and SiC(disialo) N5C(disialo).A9C(disialo)-SRIF28 as the glycosylated forms, these were dissolved in Japanese Pharmacopeia saline (from Otsuka Pharmaceutical factory, Inc.) to prepare 1-100 μM solutions as the administration solution. Moreover, GRF (GH releasing hormone, Growth hormone releasing factor) was used as the GH release enhancer. GRF was prepared by dissolving GRF injectable solution (GRF Sumitomo 50 for injection, Lot. 2006C, from Dainippon Sumitomo Pharma Co., Ltd.) in 1 mL of water for injection (Lot. 09H18C, from Fuso Pharmaceutical Industries, Ltd.), and then diluting to 25-folds with saline to obtain 2 μg/mL. As heparin employed as the anticoagulant when collecting blood, Japanese Pharmacopeia heparin sodium injectable solution (Lot. B084, from Mochida Pharmaceutical Co., Ltd.) was used directly as stock solution.
89-2 Preparation of Plasma Sample
To the dorsal subcutaneous of male SD rats (Crl: CD (SD), Charles River Japan, 6 weeks-old, n=3, body weight 145.2-163.9 g), the administration solution prepared in the above 89-1 was administered under nonfasting condition at a dosage of 1 mL/kg with a glass syringe and a 26 G injection needle (all from Terumo Corporation). As the control group, saline without glycosylated polypeptide was similarly administered (vehicle group). Then, i.e. between 5-6 minutes after the administration of the glycosylated form, pentobarbital sodium (somnopentyl, Lot. 0608101, from Kyoritsuseiyaku Corporation) was intraperitoneally administered as a general anesthetic with a glass syringe and an injection needle to give 50 mg/kg. One hour after administration of the glycosylated form, i.e. after 50 minutes or more had passed under anesthesia, GRF was administered as the GH release enhancer to the tail vain at a dosage of 1 mL/kg with a glass syringe and an injection needle. Five minutes after administration of GRF, blood was collected from the rat cervical vein with a glass syringe containing heparin and an injection needle. 0.4 mL of the collected blood was left on ice for 20 minutes or more, then centrifuged (1,870×g, 4° C., 10 minutes), and 100 μL of the supernatant was taken as the plasma sample. As blank plasma, plasma obtained by similarly treating the blood collected from untreated rat cervical vein was employed. Plasma samples were frozen in storage until employed for measurement.
89-3 Measurement of GH Concentration in Plasma
Measurement of GH concentration in the plasma sample obtained in the above 89-2 was performed with rat Growth Hormone EIA kit from SPI-Bio (SPI-Bio, A05104). The plasma sample was diluted with the assay buffer supplied in the EIA kit to 20, 100, and 500-folds as measurement samples. The standard solution for creating a standard curve followed the attached instructions by preparing a 40 ng/mL solution with distilled water, and then subjecting to serial dilution with the assay buffer to prepare 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.63 ng/mL, and 0.31 ng/mL. By multiplying the results obtained and the dilution ratio, the GH concentration was calculated. The GH concentration in the plasma sample obtained is shown in Table 7.










TABLE 7








GH


Exam-


(ng/


ple

Dose
mL)


















 1
S1C(disialo)-SRIF28
 1 nmol/kg
490


 1
S1C(disialo)-SRIF28
 10 nmol/kg
17.1


10
N19C(disialo)-SRIF28
 10 nmol/kg
563


10
N19C(disialo)-SRIF28
100 nmol/kg
1.5


13
29C(disialo)-SRIF28
 10 nmol/kg
497


13
29C(disialo)-SRIF28
100 nmol/kg
88.5


21
S1C(disialo)•N5C(disialo)-
 1 nmol/kg
776



SRIF28




21
S1C(disialo)•N5C(disialo)-
 10 nmol/kg
181



SRIF28




25
S1C(disialo)•N5C(disialo)•A9C(disialo)-
 10 nmol/kg
833



SRIF28




25
S1C(disialo)•N5C(disialo)•A9C(disialo)-
100 nmol/kg
151



SRIF28




—
vehicle
—
861









As is clear from the results shown in Table 7, the glycosylated form of the present invention suppressed GH production in rats. The ratio between the Ki value of glycosylated forms of Examples 21 and 25 against each receptor and the Ki value of unglycosylated SRIF14 is shown to be in the range of 15.7:1-1.2:1 (the compound of Example 21) and 158:1-7.1:1 (the compound of Example 25), respectively, as measured in the method of Example 69-1. Moreover, the glycosylated forms of Examples 21 and 25 are shown to have 25-folds or more (the compound of Example 21) or 30-folds or more (the compound of Example 25) increase in half-life in blood, respectively, as measured in the method of Example 70. On the other hand, from the present Example having two sugar chains, it was shown that the glycosylated form effectively exerts pharmacological action even in vivo.
Moreover, the glycosylated form of the present invention had GH production suppressing action even when administered 1 hour before GRF administration.
It was shown that the effective administration dose for rat GH production ability between the glycosylated forms of Examples 1 and 21 and Example 25 was different by approximately 10-folds. This is similar to the receptor affinities thereof having a difference of approximately 10-folds in terms of Ki value, as measured in the method of Example 69-1. It was shown that they have pharmacological activity even when the affinity of the glycosylated form was somewhat attenuated.
Example 90
GH Production Suppression Test with Rats 2
Similarly to Examples 89-1-89-3, rat GH production suppression tests of the glycosylated compounds shown below were carried out. S1C(disialo)-SRIF28, N5C(disialo)-SRIF28, A9C(disialo)-SRIF28, E12C(disialo)-SRIF28, R13C(disialo)-SRIF28, K14C(disialo)-SRIF28, S1C(disialo)-D-Trp22-SRIF28, S1C(disialo(Bn))-SRIF28, S1C(disialo(amide))-SRIF28, S1C(disialo(aminoethylamide))-SRIF28, S1C(monosialo)-SRIF28, S1C(asialo)-SRIF28, C(disialo)-R—K-SRIF14, S1-2C(asialo)-SRIF28, S1C(disialo) N5C(disialo)-SRIF28, S1C(disialo) N5C(disialo) A9C(disialo)-SRIF28, and S1-5C(disialo)-SRIF28 were employed as the glycosylated forms. The GH concentration in the plasma sample obtained is shown in Table 8.











TABLE 8









The number


Example

Dose
GH (ng/mL)
of runs



















 1
S1C(disialo)-SRIF28
 1 nmol/kg
787
4




 3 nmol/kg
182
3




10 nmol/kg
15
5




30 nmol/kg
8
1




100 nmol/kg 
7
2


 2
N5C(disialo)-SRIF28
 1 nmol/kg
969
1




10 nmol/kg
12
1


 3
A9C(disialo)-SRIF28
 1 nmol/kg
577
1




10 nmol/kg
20
1


 4
E12C(disialo)-SRIF28
10 nmol/kg
10
1


 5
R13C(disialo)-SRIF28
10 nmol/kg
136
1


 6
K14C(disialo)-SRIF28
10 nmol/kg
55
1


15
S1C(disialo)-D-Trp22-SRIF28
0.3 nmol/kg 
986
3




 1 nmol/kg
335
3




 3 nmol/kg
50
1




10 nmol/kg
6
2




30 nmol/kg
2
1


18
C(disialo)-R-K-SRIF14
 1 nmol/kg
912
4




 3 nmol/kg
291
4




10 nmol/kg
30
4




30 nmol/kg
12
1


21
S1C(disialo)•N5C(disiolo)-SRIF28
 1 nmol/kg
776
1




10 nmol/kg
181
1


25
S1C(disialo)•N5C(disialo)•A9C(disialo)-SRIF28
100 nmol/kg 
151
1


26
S1C(monosialo)-SRIF28
10 nmol/kg
47
1


27
S1C(asialo)-SRIF28
 1 nmol/kg
879
4




 3 nmol/kg
515
4




10 nmol/kg
146
4


28
S1-2C(asialo)-SRIF28
 3 nmol/kg
997
3




10 nmol/kg
175
3


34
S1-5C(disialo)-SRIF28
10 nmol/kg
842
1


59
S1C(disialo(aminoethylamide))-SRIF28
10 nmol/kg
314
1


60
S1C(disialo(amide))-SRIF28
10 nmol/kg
14
1


61
S1C(disialo(Bn))-SRIF28
10 nmol/kg
49
1


—
vehicle
—
1012
19









As is clear from the results shown in Table 8, the glycosylated polypeptide of the present invention suppressed GH production in rats. In the present test line, S1C(disialo)-SRIF28 suppressed GH production from 1 nmol/kg, and showed 82-99% of the GH production suppression effect at 3-10 nmol/kg. This is thought to be due to the gain in affinity towards SSTR1-SSTR5 and improvement in the retentivity in blood as shown in Examples 69-1, 69-2, and 70-87.
In Examples 69-1 and 69-2, N5C(disialo)-SRIF28, A9C(disialo)-SRIF28, E12C(disialo)-SRIF28, S1C(disialo)-D-Trp-SRIF28, S1C(disialo(Bn))-SRIF28, and C(disialo)-R—K-SRIF14 showing affinity equivalent to S1C(disialo)-SRIF28 showed 95-99% of the GH production suppression effect at 10 nmol/kg, showing an effect equivalent to S1C(disialo)-SRIF28.
From the results shown in the methods of Examples 70-87, S1C(monosialo)-SRIF28, S1C(asialo)-SRIF28, S1-2C(asialo)-SRIF28, and S1C(disialo(amide))-SRIF28 had an AUC for subcutaneous administration which was 1⁄3-1⁄2 of S1C(disialo)-SRIF28, and S1C(disialo(aminoethylamide))-SRIF28 was 1/7. Meanwhile, all of these showed higher affinity than S1C(disialo)-SRIF28 by the methods shown in Examples 69-1 and 69-2. Accordingly, in this experimental line, S1C(monosialo)-SRIF28, S1C(asialo)-SRIF28, S1-2C(asialo)-SRIF28, S1C(disialo(aminoethylamide))-SRIF28, and S1C(disialo(amide))-SRIF28 suppressed 70-99% of the GH production at 10 nmol/kg, and is thought to have shown an effect equivalent to S1C(disialo)-SRIF28.
R13C(disialo)-SRIF28, K14C(disialo)-SRIF28, S1C(disialo) N5C(disialo)-SRIF28, S1C(disialo) N5C(disialo) A9C(disialo)-SRIF28, and S1-5C(disialo)-SRIF28 showed a lower affinity than S1C(disialo)-SRIF28 by the methods shown in Examples 69-1 and 69-2. Meanwhile, from the results shown in the methods of Examples 70-87, these compounds have AUC for subcutaneous administration that was improved to 1.8-3.1-folds of S1C(disialo)-SRIF28. In this experimental line, R13C(disialo)-SRIF28, K14C(disialo)-SRIF28, S1C(disialo) N5C(disialo)-SRIF28, S1C(disialo) N5C(disialo) A9C(disialo)-SRIF28, and S1-5C(disialo)-SRIF28 showed GH production suppression activity by administration of 10 or 100 nmol/kg. These results show that somatostatin activity can be compensated or increased by the increase in stability in blood even when receptor affinity is reduced.
Example 91
Drug Effect Test in Gastrointestinal Obstruction Model
As shown in Examples 89 and 90, the glycosylated polypeptides of the present invention proved to have effective action as agonists even in rats in vivo. Next, in order to prove that they also show efficacy in disease models, an evaluation in rat gastrointestinal obstruction model was carried out. In gastrointestinal obstructions such as ileus, gastrointestinal symptoms such as sense of abdominal fullness, vomiting, and abdominal pain are shown by gastrointestinal tract tissue disorder and reduction in absorption ability of e.g. water or electrolyte. Their pathologies are known to be caused by obstruction of gastrointestinal content or release of digestive juice or biologically active material into the gastrointestinal tract. Somatostatin show the effect of decreasing the gastrointestinal content by secretory suppression of various digestive juices or promotion of water and electrolytes absorption via SSTR expressed in the gastrointestinal system, and is thought to be effective for improvement of the symptoms. This experimental line was carried out as a test line to evaluate the promotion of intestinal fluid absorption or the secretory suppressing action, using the change in the intestinal fluid weight in the jejunum after bowel obstruction as an indicator. Moreover, blood parameters of deviation enzymes amylase (pancreas), lactate dehydrogenase (LDH, liver), and creatine phosphokinase (CPK, skeletal muscle, cardiac muscle etc.) were measured as indicators of tissue disorders.
Example 91-1
Production of Gastrointestinal Obstruction Model
The present test was consigned to Mitsubishi Chemical Medience Corporation and carried out. Male SD rats (Crl: CD (SD), Charles River Japan, 8 weeks-old, n=5 or more, body weight 251.1-278.1 g) were fasted for 12 hours or more. Anesthesia was introduced with inhalation of 2% isoflurane and laughing gas:oxygen=7:3, and this was maintained throughout the surgery. A median incision was made in the abdomen, and the jejunum at about 10 cm from the musculus suspensorius duodeni was ligated with a surgical suture. Then, the incision site was promptly sutured, and the rats were fasted until compound administration. In the sham treatment group, ligation of the jejunum was not performed, but the treatment of suturing the incision site after a median incision was made in the abdomen was performed.
Example 91-2
Preparation of Compound and Administration
Employing S1C(disialo)-SRIF28, C(disialo)-R—K-SRIF14, and S1-2C(asialo)-SRIF28 as the glycosylated forms, these were dissolved in Japanese Pharmacopeia saline (from Otsuka Pharmaceutical factory, Inc.) to prepare a 40 μM solution as the administration solution. Eighteen hours after the gastrointestinal obstruction surgery, this was subcutaneously administered into the dorsal cervix at a dosage of 1 mL/kg with a glass syringe and a 25 G injection needle (all from Terumo Corporation). As the vehicle group, saline without glycosylated polypeptide was similarly administered. Moreover, octreotide was administered as the control group.
Example 91-3
Measurement of Intestinal Fluid Weight
One hour after compound administration, a median incision was made in the abdomen under inhalation anesthesia, and 1.5 mL of blood was collected from the abdominal vena cava. Then, the ligated jejunum on the musculus suspensorius duodeni side was resected. The fluid and blood on the jejunum surface were removed with a paper towel, nerves, blood vessels, and fat attached to the jejunum was removed, this was cut into 6 cm lengths, and wet weight was measured. This was then dried at 36 degrees for 24 hours, and dry weight was measured. The intestinal fluid weight (mg) was calculated by wet weight-dry weight. The intestinal fluid weight of the jejunum obtained is shown in Table 9.








TABLE 9






Intestinal fluid weight(mg)


















Sham treatment
285 ± 27



vehicle
481 ± 31



octreotide
693 ± 48



S1C(disialo)-SRIF28
630 ± 83



C(disialo)-R-K-SRIF14
584 ± 95



S1-2C(asialo)-SRIF28
566 ± 34









As apparent from Table 9, the vehicle had significantly increased intestinal fluid weight compared to the sham treatment, and it was recognized that enhancement of intestinal fluid secretion accompanying gastrointestinal obstruction due to ligation was caused. Somatostatin or octreotide are shown to have the secretory suppression effect of the intestinal fluid into the intestinal tract and the promotional effect of intestinal fluid absorption into the bowel tissue side in such gastrointestinal obstruction models (Scand. J. Gastroenterol. 1995 May; 30 (5): 464-9), and octreotide was confirmed to have this effect also in this experimental line. S1C(disialo)-SRIF28, C(disialo)-R—K-SRIF14, and S1-2C(asialo)-SRIF28 were all recognized to have increase in the intestinal fluid weight compared to the vehicle, and it became clear that the glycosylated forms of the present invention also show efficacy such as secretory suppression of the intestinal fluid and promotion of water and electrolyte absorption. Moreover, it has become clear that in Examples 86 and 87, the AUC for subcutaneous administration of S1-2C(asialo)-SRIF28 was 1⁄2 compared to S1C(disialo)-SRIF28, but in Example 69-1, the affinity towards SSTR1-SSTR5 is approximately 1.7-2.9-folds higher than S1C(disialo)-SRIF28. This is thought to be the reason why improvement of receptor affinity causes the drug effects in the present model to be similar even when plasma concentration is low. Similarly, this is thought to be the reason why drug effects are similar because receptor affinity is approximately 0.7-2.4-folds higher even though C(disialo)-R—K-SRIF14 has a slightly low AUC for subcutaneous administration compared to that of S1C(disialo)-SRIF28.
Example 91-4
Measurement of Blood Parameter
Employing the blood collected in Example 91-3, amylase (IU/L), LDH (IU/L) and CPK concentrations (IU/L) were measured with an autoanalyzer 7170 (Hitachi, Ltd.). The measuring methods employed were BG5B, UV-rate, and JSCC methods, respectively. The results obtained are shown in Table 10.










TABLE 10






Amylase (IU/L)
LDH (IU/L)
CPK (IU/L)







Sham treatment
 669 ± 164
168 ± 82
353 ± 68 


vehicle
1672 ± 143
341 ± 68
377 ± 51 


octreotide
1732 ± 774
 269 ± 141
472 ± 215


S1C(disialo)-SRIF28
1164 ± 224
338 ± 97
455 ± 202


C(disialo)-R-K-SRIF14
 985 ± 238
 228 ± 155
415 ± 137


S1-2C(asialo)-SR1F28
1594 ± 573
318 ± 44
728 ± 665









As apparent from Table 10, the vehicle had increased amylase activity and LDH activity compared to the sham treatment, and it was speculated that tissue disorder of the gastrointestinal system had developed accompanying gastrointestinal obstruction. In S1C(disialo)-SRIF28 and C(disialo)-R—K-SRIF14, the amylase activity had a low value compared to the vehicle. On the other hand, the amylase activity of octreotide was equivalent to the vehicle. As apparent from Examples 69-1 and 69-2, S1C(disialo)-SRIF28, C(disialo)-R—K-SRIF14, and S1-2C(asialo)-SRIF28 had binding affinity toward all the receptors from SSTR1-SSTR5, whereas octreotide is a compound having specific affinity towards SSTR2, SSTR3, and SSTR5. In rat pancreas, since there is a report that all the receptors SSTR1-SSTR5 are expressed (J. Histochem. Cytochem. 2004 March; 52 (3): 391-400), the possibility that the glycosylated form alleviated the tissue disorder by acting on a receptor different from that of octreotide was conceived. Moreover, in octreotide and C(disialo)-R—K-SRIF14, the LDH activity had a low value compared to the vehicle. There were no others that reduced each parameter by administration of the glycosylated form.

## Claims

1. A glycosylated polypeptide selected from the group consisting of:
(A) a SRIF14 consisting of the amino acid sequence of SEQ ID NO:1;
(B) a polypeptide having 1 to 3 amino acids deleted from, substituted with, or added to SRIF14, SRIF14 consisting of the amino acid sequence of SEQ ID NO:1; and
(C) a polypeptide having 80% or more homology with SRIF14 consisting of the amino acid sequence of SEQ ID NO: 1, wherein said glycosylated polypeptide further comprises
(i) N amino acids at the N-terminal side, wherein N is an integer from 1 or more to 20 or less and/or
(ii) M amino acids at the C-terminal side, wherein M is an integer from 1 or more to 6 or less,

wherein said glycosylated polypeptide further comprises at least two amino acids substituted with a glycosylated amino acids, at least one of said at least two amino acids substituted with glycosylated amino acids is present in said N amino acids and/or at least one of said at least two amino acids substituted with glycosylated amino acids is present in said M amino acids, and the glycosylated polypeptide has affinity towards somatostatin receptors,
wherein each of said glycosylated amino acids comprises a sugar chain represented by the following formula:





wherein R1 and R2 are identical or different and are:




and Ac is an acetyl group.

2. A pharmaceutical composition comprising a glycosylated polypeptide of claim 1 and/or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

3. A method for treating or preventing a somatostatin-related disease, comprising administering to a patient in need thereof an effective amount of a glycosylated polypeptide of claim 1.

4. The method of claim 3, wherein said somatostatin-related disease is at least one disease selected from the group consisting of acromegaly, gigantism, Alzheimer's disease and other forms of dementia, cancer, hormone-producing tumor, endocrine tumor, carcinoid, VIPoma, insulinoma, glucagonoma, Cushing's disease, hormone secretion defect, diabetes and complications thereof, pains, arthritis, diarrhea, gastric ulcer, inflammatory bowel disease, irritable bowel syndrome, gastrointestinal obstruction, ileus, postoperative restenosis, radiation damage, eye disease, dry eye, glaucoma, interstitial keratitis, iritis, cataract, and conjunctivitis.

5. A glycosylated polypeptide selected from the group consisting of:
(A) a SRIF28 consisting of the amino acid sequence of SEQ ID NO:2;
(B) a polypeptide having one or more amino acids deleted from, substituted with, or added to SRIF28, SRIF28 consisting of the amino acid sequence of SEQ ID NO:2;
(C) a polypeptide having 80% or more homology with SRIF28 consisting of the amino acid sequence of SEQ ID NO:2;
(D) a polypeptide of one of (A), (B), or (C) and comprising J amino acids at the N-terminal side of (A)-(C), wherein J is an integer from 1 or more to 6 or less; and
(E) a polypeptide of one of (A), (B), or (C) comprising K amino acids at the C-terminal side of (A)-(C), wherein K is an integer from 1 or more to 6 or less;
wherein at least two amino acids are substituted with glycosylated amino acids, and the polypeptide has affinity towards somatostatin receptors, and each of said glycosylated amino acids comprise a sugar chain represented by the following formula:





wherein R1 and R2 are identical or different and are:




Ac is an acetyl group.

6. A pharmaceutical composition comprising a glycosylated polypeptide according to claim 5 and/or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

7. A method for treating or preventing a somatostatin-related disease, comprising administering to a patient in need thereof an effective amount of a glycosylated polypeptide according to claim 5.

8. The method of claim 7, wherein said somatostatin-related disease is at least one disease selected from the group consisting of acromegaly, gigantism, Alzheimer's disease and other forms of dementia, cancer, hormone-producing tumor, endocrine tumor, carcinoid, VIPoma, insulinoma, glucagonoma, Cushing's disease, hormone secretion defect, diabetes and complications thereof, pains, arthritis, diarrhea, gastric ulcer, inflammatory bowel disease, irritable bowel syndrome, gastrointestinal obstruction, ileus, postoperative restenosis, radiation damage, eye disease, dry eye, glaucoma, interstitial keratitis, iritis, cataract, and conjunctivitis.

9. A glycosylated polypeptide selected from the group consisting of SEQ ID NOs: 127 and 129.

10. A glycosylated polypeptide selected from the group consisting of:
(A) a SRIF14 consisting of the amino acid sequence of SEQ ID NO: 1;
(B) a polypeptide having one or a few amino acids deleted from, substituted with, or added to SRIF14, SRIF14 consisting of the amino acid sequence of SEQ ID NO:1;
(C) a polypeptide having 80% or more homology with SRIF14 consisting of the amino acid sequence of SEQ ID NO:1;
(D) a polypeptide of one of (A), (B), or (C) comprising N amino acids at the N-terminal side of (A)-(C), wherein N is an integer from 1 or more to 20 or less at the N-terminal side of (A)-(C); and
(E) a polypeptide of one of (A), (B), or (C) comprising M amino acids at the C-terminal side of (A)-(C), wherein M is an integer from 1 or more to 6 or less;

wherein at least two amino acids are substituted with glycosylated amino acids, and the polypeptide has affinity towards somatostatin receptors, and
wherein each of said glycosylated amino acids comprises a sugar chain represented by the following formula:





wherein R1 and R2 are identical or different and are:




and Ac is an acetyl group.

11. A glycosylated polypeptide selected from the group consisting of:
(A) a SRIF14 consisting of the amino acid sequence of SEQ ID NO:1;
(B) a polypeptide having one or a few amino acids deleted from, substituted with, or added to SRIF14, SRIF14 consisting of the amino acid sequence of SEQ ID NO:1;
(C) a polypeptide having 80% or more homology with SRIF14 consisting of the amino acid sequence of SEQ ID NO: 1;
(D) a polypeptide further comprising N amino acids at the N-terminal side of (A)-(C), wherein N is an integer from 1 or more to 20 or less) at the N-terminal side of (A)-(D); and
(E) a polypeptide further comprising M amino acids at the C-terminal side of (A)-(C),

wherein M is an integer from 1 or more to 6 or less;
wherein at least two amino acids are substituted with glycosylated amino acids, and the polypeptide has affinity towards somatostatin receptors, and
wherein said sugar chain has at least one sialic acid at the non-reducing terminal, and the carboxy group of said sialic acid is modified by an alkylamino group, a benzyl group, an amino group, or an aminoethylamino group, having 1-30 carbons.

12. A polypeptide selected from the group consisting of:
(A) a SRIF28 consisting of the amino acid sequence of SEQ ID NO:2;
(B) a polypeptide having one or a few amino acids deleted from, substituted with, or added to SRIF28, SRIF28 consisting of the amino acid sequence of SEQ ID NO:2;
(C) a polypeptide having 80% or more homology with SRIF28 consisting of the amino acid sequence of SEQ ID NO:2;
(D) a polypeptide further comprising J amino acids at the N-terminal side of (A)-(C), wherein J is an integer from 1 or more to 6 or less; and
(E) a polypeptide further comprising K amino acids at the C-terminal side of (A)-(C), wherein K is an integer from 1 or more to 6 or less;
wherein at least two amino acids are substituted with glycosylated amino acids, and the polypeptide has affinity towards somatostatin receptors,
wherein each of said glycosylated amino acids comprises a sugar chain represented by the following formula:





wherein R1 and R2 are identical or different and are:




and Ac is an acetyl group.

13. A polypeptide selected from the group consisting of:
(A) a SRIF28 consisting of the amino acid sequence of SEQ ID NO:2;
(B) a polypeptide having one or a few amino acids deleted from, substituted with, or added to SRIF28, SRIF28 consisting of the amino acid sequence of SEQ ID NO:2;
(C) a polypeptide having 80% or more homology with SRIF28 consisting of the amino acid sequence of SEQ ID NO:2;
(D) a polypeptide further comprising J amino acids at the N-terminal side of (A)-(C), wherein J is an integer from 1 or more to 6 or less; and
(E) a polypeptide further comprising K amino acids at the C-terminal side of (A)-(C),

wherein K is an integer from 1 or more to 6 or less;
wherein at least two amino acids are substituted with glycosylated amino acids, and the polypeptide has affinity towards somatostatin receptors, and
wherein said sugar chain has at least one sialic acid at the non-reducing terminal, and the carboxy group of said sialic acid is modified by an alkylamino group, a benzyl group, an amino group, or an aminoethylamino group, having 1-30 carbons.

14. A glycosylated polypeptide, which is selected from the group consisting of SEQ ID NOs: 21-26, 29, 30, 33, 34, 88, 89, 125, 148, 149, 157, 164 and 169.

15. A pharmaceutical composition comprising a glycosylated polypeptide according to claim 9 and/or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

16. A method for treating or preventing a somatostatin-related disease, comprising administering to a patient in need thereof an effective amount of a glycosylated polypeptide according to claim 9.

17. The method of claim 16, wherein said somatostatin-related disease is at least one disease selected from the group consisting of acromegaly, gigantism, Alzheimer's disease and other forms of dementia, cancer, hormone-producing tumor, endocrine tumor, carcinoid, VIPoma, insulinoma, glucagonoma, Cushing's disease, hormone secretion defect, diabetes and complications thereof, pains, arthritis, diarrhea, gastric ulcer, inflammatory bowel disease, irritable bowel syndrome, gastrointestinal obstruction, ileus, postoperative restenosis, eye disease, radiation damage, dry eye, glaucoma, interstitial keratitis, iritis, cataract, and conjunctivitis.

18. A pharmaceutical composition comprising a glycosylated polypeptide according to claim 10 and/or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

19. A method for treating or preventing a somatostatin-related disease, comprising administering to a patient in need thereof an effective amount of a glycosylated polypeptide according to claim 10.

20. The method of claim 19, wherein said somatostatin-related disease is at least one disease selected from the group consisting of acromegaly, gigantism, Alzheimer's disease and other forms of dementia, cancer, hormone-producing tumor, endocrine tumor, carcinoid, VIPoma, insulinoma, glucagonoma, Cushing's disease, hormone secretion defect, diabetes and complications thereof, pains, arthritis, diarrhea, gastric ulcer, inflammatory bowel disease, irritable bowel syndrome, gastrointestinal obstruction, ileus, postoperative restenosis, eye disease, radiation damage, dry eye, glaucoma, interstitial keratitis, iritis, cataract, and conjunctivitis.

21. A pharmaceutical composition comprising a glycosylated polypeptide according to claim 11 and/or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

22. A method for treating or preventing a somatostatin-related disease, comprising administering to a patient in need thereof an effective amount of a glycosylated polypeptide according to claim 11.

23. The method of claim 22, wherein said somatostatin-related disease is at least one disease selected from the group consisting of acromegaly, gigantism, Alzheimer's disease and other forms of dementia, cancer, hormone-producing tumor, endocrine tumor, carcinoid, VIPoma, insulinoma, glucagonoma, Cushing's disease, hormone secretion defect, diabetes and complications thereof, pains, arthritis, diarrhea, gastric ulcer, inflammatory bowel disease, irritable bowel syndrome, gastrointestinal obstruction, ileus, postoperative restenosis, eye disease, radiation damage, dry eye, glaucoma, interstitial keratitis, iritis, cataract, and conjunctivitis.

24. A pharmaceutical composition comprising a glycosylated polypeptide according to claim 12 and/or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

25. A method for treating or preventing a somatostatin-related disease, comprising administering to a patient in need thereof an effective amount of a glycosylated polypeptide according to claim 12.

26. The method of claim 25, wherein said somatostatin-related disease is at least one disease selected from the group consisting of acromegaly, gigantism, Alzheimer's disease and other forms of dementia, cancer, hormone-producing tumor, endocrine tumor, carcinoid, VIPoma, insulinoma, glucagonoma, Cushing's disease, hormone secretion defect, diabetes and complications thereof, pains, arthritis, diarrhea, gastric ulcer, inflammatory bowel disease, irritable bowel syndrome, gastrointestinal obstruction, ileus, postoperative restenosis, eye disease, radiation damage, dry eye, glaucoma, interstitial keratitis, iritis, cataract, and conjunctivitis.

27. A pharmaceutical composition comprising a glycosylated polypeptide according to claim 13 and/or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

28. A method for treating or preventing a somatostatin-related disease, comprising administering to a patient in need thereof an effective amount of a glycosylated polypeptide according to claim 13.

29. The method of claim 28, wherein said somatostatin-related disease is at least one disease selected from the group consisting of acromegaly, gigantism, Alzheimer's disease and other forms of dementia, cancer, hormone-producing tumor, endocrine tumor, carcinoid, VIPoma, insulinoma, glucagonoma, Cushing's disease, hormone secretion defect, diabetes and complications thereof, pains, arthritis, diarrhea, gastric ulcer, inflammatory bowel disease, irritable bowel syndrome, gastrointestinal obstruction, ileus, postoperative restenosis, eye disease, radiation damage, dry eye, glaucoma, interstitial keratitis, iritis, cataract, and conjunctivitis.