WO1998016235A1

WO1998016235A1 - Peripheral blood stem cell-increasing agents containing glycosides

Info

Publication number: WO1998016235A1
Application number: PCT/JP1997/003698
Authority: WO
Inventors: Yasuhiko Koezuka; Kazuhiro Motoki
Original assignee: Kirin Brewery Company, Limited
Priority date: 1996-10-14
Filing date: 1997-10-14
Publication date: 1998-04-23
Also published as: AU4473297A; JP2002226377A

Abstract

Peripheral blood stem cell-increasing agents to be used in the peripheral blood stem cell transplantation therapy with the use of G-CSF, which contain as the active ingredient at least one glycoside compound represented by general formula (A) or its salt preferably exemplified by (2S,3S,4R)-1-(α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octadecanediol; and medicinal compositions or medicinal kits for increasing peripheral blood stem cells which contain these agents together with G-CSF.

Description

Specification

Glycoside-Containing Peripheral Blood Stem Cell Enhancer Background of the Invention

TECHNICAL FIELD OF THE INVENTION

The present invention relates to an agent for increasing peripheral blood stem cells and a pharmaceutical composition for increasing peripheral blood stem cells, which is useful for treating cancer such as solid cancer and leukemia, or for treating hematologic diseases such as aplastic anemia.

Disclosure of related technology

Conventionally, bone marrow transplantation (allogeneic and autologous) and peripheral blood stem cell transplantation therapy are known as treatments for this kind of disease. Bone marrow transplantation involves the transplantation of prepared bone marrow cells after killing cancer cells and bone marrow cells by intense chemo / radiation therapy. For allogeneic bone marrow transplantation, normal blood stem cells collected from the bone marrow of the donor are used as the bone marrow cells, but there is a high possibility that a graft-versus-host disease reaction will occur. Autologous bone marrow transplantation, which uses cells previously collected from the patient's own bone marrow, does not have this problem, but if the bone marrow is infiltrated with cancer cells, collect the required amount of bone marrow without cancer cells Comes with difficulties. In addition, there is considerable danger since bone marrow sampling requires general anesthesia. On the other hand, peripheral blood stem cell transplantation therapy in which peripheral blood stem cells are transplanted into a patient after myeloablative treatment [Lee et al., Hematology / Oncology Clinics of North America, 9, 1, 1-22 (1 995 )] Is said to be simpler and more secure. This method is based on the finding that hematopoietic stem cells are also present in peripheral blood in a small amount compared to bone marrow, and that they are transiently increased significantly after myeloablative treatment. No need for general anesthesia. Nevertheless, hematopoietic stem cells are rarely present and generally require multiple cell harvests.

To address this issue by increasing the number of hematopoietic stem cells in peripheral blood, for example, a combination of granulocyte colony stimulating factor (G-CSF) and stem cell factor (SCF) [Mc Niese et al. Stem Cell, 11 (supp 1.2), 36 (1993); Yan et al., B1ood, 84, 795 (1994)], and IL-l G—CS

Attempts such as the use with F [Japanese Patent Application Laid-Open No. 8-1275339] have shown that in vivo, various forms of /? The presence of silceramide or /?

[S vennerholm et al., Biochim. Biophys. Acta, 280, 626-6336 (1972), Kar lsson et al., Biochim. Biophys. Aca, 316, 317-335 (1973)]]. On the other hand, hi-galactosylceramide and hi-glucosylceramide have significant immunostimulatory and antitumor effects [Morita et al., J. Med. Chem., 38 , 2176-2187 (1995), W093 / 05505, WO94 / 09002 and W094 / 241442] And that they are much more powerful than those of 1-galactosylceramide or /?-Glucosylceramide [Motoki et al.

B i o l. P h a r m. B u l l 18, 1 4 8 7-1 4 9

1 (1995)] is also known. In addition, when a compound having a glycosylcerad structure is administered to a living body, a radiation protective effect [Motoki et al., Biorg.Med.Chem.

Lett., 5, 2413 (1995)], and the effect of increasing platelet count, leukocyte count, and bone marrow cells [Motoki et al., Bio

1. P h a r m. B ul l 9, 95 2-95 5 (1 9 9

6) and W 094/0 2 1 6 8] are known. However, the effect of in vivo administration of a compound having a glycosylceramide structure on hematopoietic progenitor cells in peripheral blood is not known. Of course, the use of such compounds in combination with G-CSF is There is no report that it is effective as a peripheral blood stem cell increasing agent. The present inventors have found that the combined use of a glycoside compound having a specific structure and G—CSF significantly increased hematopoietic progenitor cells in peripheral blood. And the ability of donor mice to administer the additive to a recipient mouse whose bone marrow has been treated by whole body irradiation. When peripheral blood is transferred, the survival time is significantly prolonged, and the transferred hematopoietic progenitor cells can survive and multiply in the recipient mouse. And have reached the present invention. The increasing agent of the present invention is also excellent in safety as described later.

Summary of the Invention

The present invention provides the following formula (A):

Where R is H or 0H;

X is? An integer of from 25 to 25;

R ₂ is a substituent defined by any of the following (a) to (e) Where Y is an integer from 5 to 17

(a) — CH ₂ (CH _? ) CH

(b) — CH (OH) (CH ₉ ) CH

(C) one _{CH (OH) (CH 2)} Y CH (CH,)

(d)-CH = CH (CH ₂ ) _Y CH

(e) One CH (OH) (CH ₂ ) _Y CH (CH,) CH ₉ CH

One of R ₃ and R ₄ is H, the other is H, 0 H

NH, NHC 0 CH or

Is;

One of R ₅ and R ₆ Ri H Der, the other is 0 H

And

One or R ₇ your good beauty R ₈ Noi deviation Ri Oh in H, the other is 0 H

And

R _q is H, CHCH ₂ 〇 H or

Is

A peripheral blood stem cell augmentation agent in a peripheral blood stem cell transplantation therapy using G—CSF, comprising at least one glycoside compound or a salt thereof represented by the following formula: .

The present invention also relates to G-CSF and a glycoconjugate compound as defined in formula A. It is also intended to provide a pharmaceutical composition for increasing peripheral blood stem cells, comprising at least one kind of a substance or a salt thereof, and a pharmaceutically acceptable carrier.

The present invention further relates to an increase in peripheral blood stem cells comprising G-CSF and at least one glycoside compound or a salt thereof as defined in formula A. It also provides a pharmaceutical kit for medical use.

The invention also requires the use of G-CSF in combination with at least one glycoside compound or a salt thereof as defined in Formula A to increase peripheral blood stem cells. Methods for treating humans or other animals are provided.

The present invention further relates to the use of at least one glycoside compound defined by formula A or a salt thereof for the manufacture of a medicament having a peripheral blood stem cell-enhancing action. provide .

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows donors receiving KRN 7000 and G-CSF. C57BL / 6 Mouse (mes) Recipient C57B L / 6 with whole body radiation of mouse (mes) peripheral blood. It shows the effect of extending the survival time of the recipient mouse when transferred to the mouse (mes).

Figure 2 shows a donor BALB / c mouse treated with KRN 7000 and G-CSF (mess). A recipient BALB / c mouse irradiated with whole body radiation of peripheral blood. ), The recipe at the time Demonstrates the effect of prolonging the survival of the event mouse

FIG. 3 shows a donor BA receiving KRN 7000 and G—CSF.

LB / c mouse (os) Recipient mouse when peripheral blood is transferred to a whole-body irradiated recipient BALB / c mouse (mes) This shows the effect of prolonging the survival time.

Figure 4 is an electrophoretogram showing the engraftment and proliferation of hematopoietic progenitor cells when donor mouse peripheral blood was transferred to a recipient mouse.

Detailed description of the invention

According to the present invention, the following formula (A)

Where R i is H or 〇 H

X? An integer of ~ 25;

R ₂ is a substituent defined by any of the following (a) to (e), wherein Y is an integer of 5 to 17; (a) — CH ₂ (CH ₂ ) CH

(B) - CH (OH) (CH 2) CH

(C) one _{CH (OH) (CH 2)} Y CH (CH 3)

(d) CH 2 CH (CH 2) _Y CH

(e) One CH (OH) (CH ₂ ) _Y CH (CH ₃ ) CH ₂ CH

One of R ₃ and R ₄ is H, and the other is H or OH

N H N H C O C H or

Is;

One of R _¾ and R _e Ri H Der, the other is 0 H

Is; One of R ₇ and R ₈ is H and the other is 0 H

And

R _q is H, CHCH ₂₀ H or

Is]

A peripheral blood stem cell enriching agent for peripheral blood stem cell transplantation therapy by G—CSF, comprising at least one glycoside compound or a salt thereof represented by the following formula:

In a preferred embodiment of the present invention, the following formula (B):

[Where RX and R ₂ have the same meaning as in equation (A).

RR _g is a substituent selected to be applicable in any of the following i) to v);

) [Galaxy]

RR _s you and R ₈ cups displaced even Tsu H Der

R ₄ is H, 〇H, NH ₂ , NHCOCH ₃ or

And

R ₅ is 0 H,

And

R ₇ is 0 H

R q is H, CHCH ₂ 〇 H or

,

i i) [Glucose type]

RR ₆ and R ₇ are both H

RR ₅ and R ₉ are the same as defined in i), respectively.

The _R Β 0 H,

,

111) [Mannose type]

RR ₆ and R ₇ are both H

R ₅ and R 9 are the same as defined in i)

R ₃ is H, 〇 HNHNHCOCH ₃ or

And

R ₈ is 〇H or T

14

,

i v) [Althrose type]

RR ₅ and R ₇ are both H

RR ₆ and R ₈ are both 0 H

R ₉ is H, CH ₃ or CH ₂₀ H, or

V) [Aloose type]

RR ₅ and R ₇ are both H

RR ₆ and R ₈ are both 〇H

R _g is H, CH ₃ or CH ₂ 〇H]

A peripheral blood stem cell enriching agent in peripheral blood stem cell transplantation therapy by G-CSF, comprising at least one glycoside compound or a salt thereof represented by the following formula:

Glycoside compounds as defined by Formula A or B consist of a sugar moiety and an aglycone moiety, some of which are in the form of a hyselev mouth side or a single glycan. Also referred to as co-silcer lamid, hi-glu co-silcer amid, single-glu co-ser-b-side, single-glu-co-sel-bro-side or single-glu-co-to-sil-se-lamid It is called. These compounds are characterized by the fact that they have an anomeric configuration. In the sugar moiety of the glycoside compound, R ₃ , R ₆ and R ₈ are all H, R ₄ , R ₅ and R ₇ are all 0 H, and R ₉ is preferably CH ₂ OH, that is, the sugar moiety is preferably 1-galactovilanosyl. In the aglycone portion of the glycoside compound, R ₂ is preferably a substituent (b), (c) or (e). It is even more preferred that R i is H (meaning that it is a kerasin type) and R ₂ is a substituent (b). X is preferably 21 to 25, and Y is preferably 11 to 15. Preferred examples of the glycoside compound are listed below.

1) (2S, 3R) -l- (a -D-galactopyrano siloxy) -2- [(R)-2 -Hydroxyte tracosanoylamino]- 3-Ok Evening A

2) (2S, 3R) -l- (a-D-galactopyranosiloxy) -2-tetracosanoylamino-3 -octadecanol A ^

3) (2S, 3R) -l- (a-D-galactopyranosiloxy) -2-Tetradecanoylamino-3--3-octanedecanol A

4) (2S, 3R)-(H-D-Glucobylanosiloxy) -2-Tetradecanoylamino-3 -octadecanol C

5) (2S, 3R) _1- (6'-deoxy-hi-D-galacto-ylano-siloxy) -2-tetradecanoylaminino-3-ok Le C

6) (2S, 3R) _1- (?-L-arapinovirano silo kishi)-2-tetradecanoylamino-3-oku evening decanol C

7) (2S, 3S)-1- (H-D-Galactoviranosiloxy)-2-Tetradecanoylamino-3 -Hexadecanol A

8) (2 R, 3R) _1- (H-D-Galactoylano siloxy) -2-tetradecanoylamino-3 -Hexadecanol A

9) (2R, 3S)-1- (H-D-galactopyranosiloxy) -2-tetradecanoylamino-3-3-hexadecanol A

10) (2S, 3S, 4R)-1- (H-D-galactoylanosiloxy) -2-[(R)-2-hydroxyltra Mino] -3,4-octadecanediol A

11) (2S, 3S, 4R) -l- (a-D-galactosilane) 2-[(R)-2-hydroxyltracosano Illumino]-3, 4-ゥ decane Diol A

12) (2S, 3S, 4R) -l- (a-D-galactolanoxy) -2-[(R) -2-Hydroxyhexanosano Rua mino] _3,4-Icosandiol A

13) (2S, 3S, 4R) -l- (a-D-galactopyranosiloxy) -2-[(S) -2-hydroxy citrate Ilamino] -3,4-hepcane decandiol A

14) Manufacture of (2S, 3S, 4R) -l-(-D-galactopyrano siloxy) -2-hexacosanoyluamino-3,4-octadecanediol An example

15) (2S, 3S, 4R)-1- (H-D-G-V-A-L-A-L-L) -2-D Diol B

16) (2S, 3S, 4R) _1- (HI-D-G-ACT) Diol A

17) (2S, 3S, 4R)-1- (H-D-Galacto-Virano-siloxy) -2-Tetracosanoylamino-3,4- ゥ decane Diol A

18) (2S, 3S, 4R)-1- (H-D-Gluco-Vilanosiloxy) -2-hexa-sanoylamino-3,4-Ok Diol C

19) 0-D-Galactofuranosyl-(1 → 3)-0-H-D-Garactofuranosyl-(1 → 1)-(2S, 3S, 4R)- 2-amino-N-[(R)-2- Tracosanol] -1,3,4-octadecanol triol D

20) 0_H-D-galacto lanosyl-(1 → 6) -0—H-D -glucon lanosyl-(1 → 1)-(2S, 3S, 4R) -2-Amino-N-Hexagonal-

1,3,4-octadecant real D

21) 0-a-D-galactosylanosyl-(1 → 6)-0-hi-D-galactopyranosyl- (1 → 1)-(2S, 3S, 4R) -2-Amino -N-Hexaco-sanoyl-

1, 3, 4-octadecantory D

22) 0- a-D-Glucolanosyl-(1 → 4) -0-H-D-Glucolanosyl-(1 → 1)-(2S, 3S, 4R)- 2-amino-N-hexacosanol-1,3,4-octadecantriol D

23) 0- (N-Acetyl-2-Amino-2-Doxo-α-D-Galactolananosyl)-(1 → 3) -0- [H- D-glucoviranosyl-(1 → 2)]-0-H -D-galactosylanosil-(1 → 1)-(2S, 3S, 4R)-2-amino -N- [(R)-2-hydroxyhexanol-1,3,4-octane Decantor

D

24) 0- (N-Acetyl-2-Amino-2-Doxy-H-D-galactopyranosyl)-(1 ~> 3)-0- [H-D-gluco Vilanosyl- (1 → 2)]-0-H-D-Galactovilanosyl- (1 → 1)-(2S, 3S, 4R)-2-Amino-N- [ (R) -2 -Hydroxycitrate tracosanoyl-1,3,4-hexadecanediol D 25) (23,33,410_1_ (h-0-galactopyranosiloxy)) -2-[(R) -2-Hydroxycitrinosylamino] -16 -Methyl-3,4-heptanol decandiol A

26) (2 S, 3 S, 4 R)-1 (--D-ピ-ラ-)-2 _ [(S)-2-ヒNOLAMINO] -16-methyl

-3,4-heptadecane diol A

27) (2S, 3S, 4R)-1- (H-D-galactoylano) 16-methyl-2-tetracosanoylamino] -3, 4-heptanol decane diol A

28) 0- 5 -D-Galactofuranosyl-(1 → 3) _0- a-D-Garactofuranosyl _ (1 → 1)-(2S, 3S, 4R)- 2-Amino-N-[(R) -2-Hex mouth tracosanoyl] -17-Methyl-1,3,4-octane Decantor Le

D

29) 0-?-D-Galactofuranosyl-(1 → 3) -0-H-D-Garactofuranosyl-(1 → 1)-(2S, 3S, 4R) 2-Amino-N-[(R)-2-Hexole tracosanoyl] -15-Methyl-1, 3,4-hexadenediol D

30) 0- (N-Acetyl-2-amino-2-D-hydroxy-H-D-galacto-Vylanosyl)-(1 → 3)-0- [H-D-glucobi Lanosyl- (1 → 2)]-0-H-D-Galactoyllanosyl- (1 → 1)-(2S, 3S, 4R)-2-Amino-N- [ (R) -2_ hydroxyhexanol-16-methyl-1,3,4-octane decan Triol D

31) 0- (N-Acetyl-2-amino-2-Doxy-H-D-galacto-Vyranosyl)-(1 → 3) —0— [H-D-Gluco-Villa Nosyl— (1 → 2)] — 0—Hi—D—Galactoyllanosyl- (1 → 1)-(2S, 3S, 4R) -2-Amino-N-[(R ) -2 -Hydroxy titracosanoyl-16 -Methyl-1,3,4-Heavy decanter D

32) (2S, 3S, 4E) -l- (α-D-galactopyranosiloxy)-2-octane Decanoylamino-4-octadecene-3 -All A

33) (2S, 3S, 4E) -1- (H-D-Galac-to-Vila-no-Siloki-S) -2 -Tetradecanoylamino- 4-Octo-Dec -3 -All A

34) (2S, 3S, 4R) -1- (H-D-galactoylanosiloxy) -2-[(R) -2-Hydroxysipene ] -16-Methyl-3,4-octadecanediol A where 1) to 9) are examples in which R ₂ is a substituent (a); hereinafter, 10) 24) correspond to (h), 25) to 31) correspond to (〇), 32) and 33) correspond to (d), and 34) correspond to (e). The alphabet described after each compound name indicates the literature in which the synthesis method is described, A is WO93 / 055, B is WO9 4/0 2 1 6 8, C is W〇 9 4/0 9 0 2 0, D is ,

W 094/2 4 1 4 2 means to refer to each. Of the glycoside compounds described above, the conjugated compound 14), immediately, (2S, 3S, 4R)-1- (HIIC-D—galacto-vilano-siloxy)- 2-Hexacosanoylamino 3, 4-The most preferred is decandiol (hereinafter referred to as KRN700). An example of the synthesis of this compound is shown in the following Production Examples and Reaction Schemes.

Glycoside compounds as defined by Formula A or B may be capable of forming acid addition salts with pharmaceutically acceptable acids. Acids that form acid addition salts include inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, and phosphoric acid, or acetic acid, propionic acid, maleic acid, oleic acid, and pallic acid. Mitic acid, citric acid, conodic acid, tartaric acid, fumaric acid, glutamate, pantothenic acid, raurylsulfonate, methansulfonate, Organic acids such as phthalic acid can be mentioned.

The compound of the present invention can be obtained by subjecting the above compound 14) to known chemical techniques, if necessary, using known raw materials in the same manner as shown in the following Production Examples and Reaction Schemes. They can be combined and combined.

The present invention relates to a G-CSF and a glycoside defined by formula A or B Also provided is a pharmaceutical composition for increasing peripheral blood stem cells, which comprises at least one compound or a salt thereof and a pharmaceutically acceptable carrier.

The present invention also provides a peripheral blood stem cell expansion comprising a G-CSF and at least one glycoside compound or a salt thereof as defined by Formula A or B. We also provide pharmaceutical kits for medical use.

The G-CSF according to the present invention has the following amino acid sequence, or lacks or substitutes one or more amino acid residues in the sequence. In addition, a polypeptide having an added and / or inserted amino acid sequence and having G-CSF activity can be obtained. A convenient method for obtaining such a G-CSF is disclosed in, for example,

6 3 5 0 0 6 3 6 and B i o c h e m. B i o p h y s

Res. Commun 159, 103 (19989). In addition to the sugar-free G-CSF produced by Escherichia coli, G-CSF with sugar chains provided by eukaryotic host cells such as CHO cells can be used. Wear . Human G-CSF having the amino acid sequence shown below (SEQ ID NO: 1) is particularly suitable.

UI Oil 59ΐ 091 oJd "10 E IV" si sin 3v ηθΐ ΙΒΛ3iV 丄] BA nio nsq sqj

SSI 091 5H

3S "ID no sin J9S EIV Ι ^ Λ nsq IB ^ Xio iO e [v 3JV 3JV uiO aqd

0Π 58ΐ 08Ϊ IV J3S v 9 d ojj BIV 3 uio J¾l OJJ UJQ n9q BI OJJ

¾ZI OZT SIT

I 'f 10 ngq ni nio UIQ uio (丄 9i i 丄丄 eiv sqd dsv

0Π SOI 00Ϊ

IV Ι ^ Λ dsy naq ngq 丄 dsv naq 丄 cud λιο naq nio ^0J d J9S

56 06 38 08

911 10 nio na BIV "10" 91 na Xio UJOΐ 丄 sqd noq λΐο iss

Si Oi S9

s! H uio J3S naq S ^ Q ΛΙΟ BI naq UJO nsq BIV ^ IO J ^g S OJJ s 3

09 59 OS J9S -I9S Π9Ί OJJ BIV d_i 丄 cud 9ii 10 "91 sin 10 ^η3 Ί ^η9 Ί Ι ^Β Λ

Si '0 S £

n = nig "ID OJJ S IH SXQ ngq s q J jqi B IV Π9 s nio ujg

OS 5Ζ ΟΖ

Π9Ί BIV BIV 19 ds (ΙΟ "10 ¾Ι I s ^ gjy (ΒΛ UIQ ηθΐ s ^ o sX

SI Οΐ 5 I

nsi naq aqj jss u \ r, OJJ ns jas J9S ^ IV OJJ Λΐΰ Π9 OJJ

^Z画 OAV „,

24 Here, deletion, substitution, addition and / or insertion of an amino acid residue is carried out, for example, by DFMark et al., Proc. Natl. Acad. Sci. USA, Vol. .5662-5666, 1984, S. Inouy et al., Proc. Natl. Acad. Sci. USA, Vol. 79: p. 3438-3441, 1982, PCT W085 / 00817, published February 28, 1985, RP Wharton. Et al., Nature, Vol. 316, p. 601-605, Aug. 15, 1985, which is a well-known technique prior to the filing of the present application, and which is implemented by a site-specific mutagenesis method or the like. And can be done.

The agent for increasing peripheral blood stem cells according to the present invention may be administered by any administration route that meets its purpose. Specifically, in the case of animals other than humans, methods such as intraperitoneal administration, subcutaneous administration, intravenous administration to a vein or artery, and local administration by injection are possible. In the case of humans, intravenous administration, intraarterial administration, local administration by injection, intraperitoneal or pleural administration, subcutaneous administration, intramuscular administration, sublingual administration, transdermal administration Alternatively, it can be administered by rectal administration.

The drug of the present invention is administered in an appropriate dosage form determined by the administration method and administration purpose, specifically, in the form of injections, suspensions, emulsifiers, ointments, creams, etc. can do . These preparations contain additives such as carriers or diluents which are pharmaceutically acceptable, specifically, solvents, solubilizers, isotonic agents, preservatives, antioxidants, excipients and the like. Excipient, binding Additives, stabilizers, etc. can be added. Such additives include serum albumin and mannitol, as well as site potions, such as human IL-11, human IL-13, and human IL-13. Includes IL-11, human SCF, human LIF, human EPO, human GM-CSF and human M-CSF.

Each active ingredient in the medicament of the present invention can be administered continuously or intermittently according to individual circumstances. Specific dosages will vary depending on the mode of administration, patient conditions, eg, age, weight, sex, sensitivity, time of administration, concomitant medications, etc. In general, the dose required for the expression of the glycoside compound activity is, for example, about 0.01 to 1 Omg per day for a human adult by intravenous administration; ~ 1 mg is preferred. The dose required for the expression of G-CSF activity is different for allogeneic and autologous transplantation, but is about 1 to 20 mg / kg / day for humans. There have been many reports of subcutaneous administration of 2 to 16 mg / kg for 4 to 6 days. These may be separately administered in any order (sequentially, simultaneously, separately), but include the glycoside compound, G-CSF, and a pharmaceutically acceptable carrier. It is preferable to use a pharmaceutical composition for increasing peripheral blood stem cells. Among them, the glycoside compound is contained in a weight ratio of 1:10 to 10: 1 with G-CSF. --

Pharmaceutical compositions for increasing peripheral blood stem cells which are described above are suitable, and moreover, glycoside compounds are almost always contained in the same amount by weight as G—CSF. I like it. In addition, when using an existing G-CSF formulation, set aside the glycoside compound vial and the existing G-CSF formulation to avoid accidents and errors at the medical site. It is most preferable to create a customized kit.

The present invention therefore relates to the use of G-CSF in combination with at least one glycoside compound defined by formula A or B or a salt thereof to increase peripheral blood stem cells. It also includes methods for treating humans or other animals in need thereof.

The present invention also relates to at least one glycoside compound or a salt thereof defined by formula A or B for the manufacture of a medicament having a peripheral blood stem cell-enhancing action. Includes the use of

The agent for increasing peripheral blood stem cells of the present invention is used for increasing stem cells when collecting peripheral blood stem cells. When peripheral blood stem cells are collected from a healthy subject, the timing of administration can be arbitrarily selected before that. When it is collected from a patient such as a cancer patient, it is preferable to set the recovery period from the cytopenia period. However, radiation / chemotherapy It may be used before or immediately after legal action.

Example

Manufacturing example

(2 S, 3 S, 4 R)-1-(Hiichi D-Garakto Villano Silo Kishi) 1, 2-Hexacosanoylamino 1, 3-4-year old Kutadecanediol An example of manufacturing (referred to as “KRN 7000”) will be described based on the following scheme.

H ₃

G1 (R = H)

G2 (R = Tr) aq.HCI I CH ₂ CI ₂

G3 G4

GS (70% yield from G2) G6

G7 (R = H) G9

G8 (R = Tr)

Replacement form (Rule 26) , (CH ₂ ) ₂₃ CH ₃

10% HCI-MeOH

H ₂ N OBn, Xacosanoic acid HN OBn / CH ₂ CI ₂ f?

TrO WSC-HCl / CH ₂ C! ₂ ? !

"(CH ₂ ) ₁₃ CH ₃ ^ΤΓθ -eight ^ (CH ₂₎ ^l3 CH ₃

OBn OBn

G10 Gil

G1 2 (Yield from G9 77%) G13

KRN7000

Replacement form (Rule 26) Synthesis of compound G 1

Sulfuric acid (0.5 mL) was added to a solution of D-lyxose (200 g, 1.33 mol) in a calcium chloride-dried acetate (3.0 L) solution, and the mixture was stirred at room temperature for 18 hours. After adding Molecular Sieves 4A powder (100 g) to neutralize the reaction mixture, the mixture was filtered through celite and the residue was washed with acetone. The filtrate and the washings were combined and concentrated under reduced pressure to obtain a crude G1 product. Yield 240g (95%). Used for the next step without further purification. Analytical samples were purified by silica gel chromatography using hexane: aceton (9: 1) as the elution solvent.

mp 76-78 ° C; FDMS m / z 191 (M + l) ⁺ ; ¹ H-NMR (500 MHz, CDCL) δ 5.45 (1Η, d, J = 1.8 Hz), 4.83 (1H, dd, J = 3.7) , 5.5 Hz), 4.64 (1H, d, J = 6.1 Hz), 4.27-4.30 (1H, m), 3.90-3.99 (2H, m), 1.48 (3H, s), 1.32 (3H, s).

Synthesis of compound G2

Pyridine (10 ml) and trityl chloride (39.0 g) were added to a methylene chloride solution (168 ml) of Gl (239 g, about 1.26 mol), and the mixture was stirred at 32 ° C for 4 hours. Ethanol (8 ml) was added dropwise, and the mixture was stirred at room temperature for 2 hours. The extract was washed with a saturated aqueous solution of ammonium chloride, a saturated aqueous solution of sodium hydrogen carbonate, and a saline solution, and then concentrated under reduced pressure. The residue is ethyl acetate And crystallized upon cooling to 0 ° C. Yield 501 g (87% over D-lyxose).

m 174 - 176 ° C; FDMS m / z 432 M +; 1 H-NMR (500MHz, CDC1 3) ά 7.21-7.49 (15H, m), 5.38 (1H, d, J = 2.4 Hz), 4.75 (1H , dd, J = 3.7, 6.1 Hz), 4.59 (1H, d, J = 6.1 Hz), 4.31-4.35 (1H, m), 3.43 (1H, dd, J = 4.9, 9.8 Hz), 3.39 (1H, dd, J: 6.7, 9.8 Hz), 1.29 (3H, s), 1.28 (3H, s).

Synthesis of compound G3

Tridecane triphenylphosphine bromide (962 g, 1.16 mol; 1-bromotridecane, triphenylphosphine was heated to 140 ° C for 4.5 hours. A 2.5 M hexane solution of n-butyllithium (462 mL; 1.16 mol) was added dropwise at 0 ° C to a THF solution (prepared) of THF (1500 ml) under argon atmosphere. After completion of the dropwise addition, the mixture was stirred for 15 minutes, and a THF solution (450 ml) of G2 (250 g, 579 mmol) was added dropwise. The mixture was stirred for 18 hours while gradually raising the temperature to room temperature. The reaction solution was concentrated under reduced pressure, and a residue mixture of hexane: methanol: water (10: 7: 3, 1000 ml) was added to the residue, and the mixture was washed with a saturated aqueous solution of ammonium chloride. The aqueous layer was extracted with hexane (500 ml), and all the organic layers were combined, dried over magnesium sulfate anhydride, and concentrated under reduced pressure to obtain a crude G3 product. _nr

32 Used for the next step without further purification. Yield 339 g (98%) The analytical sample was purified by silica gel chromatography using hexane: ethyl acetate (9: 1) as the elution solvent.

^{FDMS m / z 598 M +;} 1 H-NMR (500MHz, CDC1 3) (57.21-7.45 (15H, m), 5.48-5.59 (2H, m), 4.91 (0.7H, t, J = 7.3 Hz), 4.44 (0.3H, t, J = 7.3 Hz), 4.26 (0.3H, dd, J = 4.3, 7.3 Hz), 4.21 (0.7H, dd, J = 4.3, 6.7 Hz), 3.75 (0.7H, m) , 3.69 (0.3H, m), 3.24 (0.3H, dd, J = 4.9, 9.8 Hz), 3.17 (0.7H, dd, J = 4.9, 9.8 Hz), 3.09-3.14 [1H, (3.11, dd, J = 4.9, 9.2 Hz), HlbE overlapped], 1.75-2.03 (2H, m), 1.49 (3H, s), 1.39 and 1.38 (3H, each s), 1.2t 1.34 (20H, m), 0.88 (3H , t, J = 6.7 Hz). Synthesis of compound G4

To a solution of G3 (338 g, about 565 minol) in methylene chloride (1500 ml) was added pyridin (500 ml), and methansulfonyl chloride (49 ml, 633 mmol) was added dropwise at 31 ° C. Stirred for hours. Ethanol (40 ml) was added dropwise, and the mixture was stirred at room temperature for 1 hour. After concentration under reduced pressure, a mixture of hexane: methanol: water (10: 7: 3, 1000 ml) was added to the residue, and the mixture was separated. The aqueous layer was extracted three times with hexane (200 ml), and all organic layers were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to give crude G4. I got something. It was used for the next step without further purification. Yield 363 g (95%). An analytical sample, to hexa emissions: acetate Echiru (9: 1) Li mosquitoes gel click as the eluent The filtrate Conclusions graph _c FDMS was by Ri purified I over ^{m / z 676 M +; 1} H- _{NMR (500MHz, CDC1 3) 57.21-7.47} (15H, m), 5.41 (0.7H, ddd, J = 5.5, 9.2, 11.0 Hz), 5.32 (0.7H, bt, J = 11.0 Hz), 5.22 (0.3H , bdd, J = 9.2, 15.0 Hz), 5.02 (0.3H, dt, J _t = 7.3 Hz, J _d = 15.0 Hz), 4.8 (0.7H, ddd, J = 3.1, 5.5, 7.9 Hz), 4.73 ( 0.7H, dd, J = 5.5, 9.8 Hz), 4.64-4.67 (0.3H, m), 4.61 (0.3H, dd, J = 5.5, 9.2 Hz), 4.48 (0.7H, dd, J = 5.5, 7.9) Hz), 4.22 (0.3H, dd, J = 5.5, 9.2 Hz), 3.55 (0.3H, dd, J = 2.4, 11.6 Hz), 3.45 (0.7H, dd, J = 3.2, 11.0 Hz), 3.06- 3.12 [4H, (3.12, s), (3.11, s), (3.09, dd, J = 3.1, 11.0 Hz)], 1.66-1.82 (2H, m), 1.47 and 1.46 (3H, each s), 1.39 (3H, s), 1.13- 1.35 (20H, m), 0.88 (3H, t, J = 6.8 Hz). Synthesis of compound G5

To a methylene chloride solution (1500 ml) of G4 (362 g, about 536 mmol) was added methanol (350 ml), to which concentrated hydrochloric acid (200 ml) was added dropwise, and the mixture was stirred at room temperature for 5 hours. The reaction mixture was neutralized with sodium hydrogen carbonate and filtered. The filtrate was concentrated under reduced pressure, and ethyl acetate was added to the residue. Washed with water. The aqueous layer was extracted with ethyl acetate, and all the organic layers were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. Crystallized from hexan. Yield 16 lg (70% over G2).

mp 66 - 67 ° C; FDMS m / z 377 (MH 2 0) +; 1 H-NMR (500MHz, CDC 1 3 + D 2 0) 6 5.86 (0.3H, dt, J t = 7.3 Hz, J d = 14.7 Hz), 5.77 (0.7H, dt, J _t = 7.3, J _d = 10.4 Hz), 5.55 (0.3H, br.dd, J = 7.3, 14.7 Hz), 5.49 (0.7H, bt, J = 9.8 Hz), 4.91-4.97 (1H, m), 4.51 (0.7H, bt, J = 9.8Hz), 4.11 (0.3H, bt, J = 7.3Hz), 3.94-4.03 (2H, m), 3.67- 3.73 [1H, (3.70, dd, J = 3.1, 6.7 Hz), (3.69, dd, J = 3.1, 7.3 Hz)], 3.20 and 3.19 (3H, each s), 2.05-2.22 (2H, m), 1.22- 1.43 (20H, m), 0.88 (3H, t, J = 6.7 Hz).

Synthesis of compound G6

To a solution of G5 (160 g, 405 mmol) in THF (780 ml) was added 5% nordium-barium sulfate (16 g), and the reaction vessel was purged with hydrogen gas, followed by 20 hours at room temperature. Stirred. After the reaction solution was filtered through celite, it was washed with a mixed solution of formaldehyde and methanol (1: 1). The filtrate and the washing were combined, and concentrated under reduced pressure. The residue was crystallized from ethyl acetate. Yield 146g (91%) ₀ _{^{[A] 2 3 D + 12}} ° (c 1, CHC1 3 / MeOH = 1: 1); m 124-126 ° C; FDMS m / z 397 (M + l) +; 1 H-NMR (500MHz, CDC1 ₃ / CD ₃ OD = l: 1) δ 4.93-4.96 (1H, m, H2), 3.91 (1H, dd, J = 6.7, 12.2 Hz), 3.85 (1H, dd, J = 4.9, 12.2 Hz), 3.54-3.60 (1H, m), 3.50 (1H, dd, J = 1.8, 8.5 Hz), 3.19 (3H, s), 1.75-1.83 (1H, m), 1.53- 1.62 (1H, i), 1.21- 1.45 (24H, m), 0.89 (3H, t, J = 6.7 Hz). Synthesis of compound G7

To a DMF solution (1000 ml) of G6 (145 g, 365 mmol) was added sodium azide (47 g, 730 mmol), and the mixture was stirred at 95 ° C for 4 hours. The reaction mixture was concentrated, ethyl acetate (450 ml) was added to the residue, and the mixture was washed with water. The aqueous layer was re-extracted with ethyl acetate. All the organic layers were combined, washed with brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain a crude G7 product. Yield 122 g (97%). Used for the next step without further purification. Analytical samples were purified by silica gel chromatography using hexane: ethyl acetate (9: 1) as the elution solvent.

_{^{[Shed] 2 3 D + 16.5 ° (}} c 0.5, CHC1 3 / MeOH = 1: 1); mp 92-93 ° C; FDMS m / z 344 (M + l) +; 1 H-NMR (500MHz, CD ₃ 0D) 53.91 (1H, dd, J = 3.7, 11.6 Hz), 3.75 (1H, dd, J = 7.9, 11.6 Hz), 3.49-3.61 (3H, m), 1.50-1.71 (2H, m), 1.22-1.46 (24H, i), 0.90 (3H, t, J = 6.7 Hz).

Synthesis of compound G8

To a methylene chloride solution (750 ml) of G7 (121 g, about 352 mmol) were added pyridin (250 ml) and trityl chloride (124 g, 445 mmol), and the mixture was stirred at room temperature for 16 hours. Ethanol (30 ml) was added dropwise, and the mixture was stirred at room temperature for 30 minutes, washed with a saturated aqueous solution of sodium bicarbonate, a saturated aqueous solution of ammonium chloride, and brine, and then dried over anhydrous magnesium sulfate. It was dried with lime and concentrated under reduced pressure. The residue was purified by silica gel chromatography using hexane: ethyl acetate (10: 1) as a solvent. Yield 34.4g (52% over G6).

_{[Shed] 2 4 D + 11.9 ° (} c 0.9, CHC13), FDMS m / z 585M +; 1 H-NMR (500MHz, CDC13 + D 2 0) 6 7.24-7.61 (15H, m), 3.62-3.66 (2H , M), 3.51-3.57 (2H, m), 3.42 (1H, dd, J = 6.0, 10.4 Hz), 1.23-1.56 (26H, m), 0.88 (3H, t, J = 6.7 Hz).

Synthesis of compound G9

To a DMF solution (300 ml) of G8 (33.5 g, 57.3 mmol) was added 60% sodium hydride (5.5 g, about 138 mmol as NaH), and the mixture was stirred at room temperature for 40 minutes. The reaction solution was cooled to 0 ° C, and benzyl chloride (15 ml, 120 ml mmol) was added dropwise. The mixture was stirred for 18 hours while gradually increasing the temperature to room temperature. The reaction solution was quenched with ice water (100 ml) and extracted with ethyl acetate. The extract was washed three times with saline, all organic layers were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain a crude G9 product. It was used for the next step without further purification. Yield 42.2 g (96%). Analytical samples were purified by silica gel chromatography using hexane: ethyl acetate (100: 1) as the elution solvent.

_{^{[A] 2 4 D +9.8 °}} (c 1.0, CHC13), FDMS m / z 738 (MN 2) +; 1 H-NMR (500MHz, CDC ") d 7.07-7.48 (25H, m), 4.57 (1H , D, J = 11.6 Hz), 4.44 (1H, d, J = 11.6 Hz), 4.41 (2H, s), 3.73-3.79 (1H, m), 3.46 -3.56 (2H, m), 3.37 (1H, dd, J = 8.6, 10.4 Hz), 1.20-1.64 (26H, m), 0.88 (3H, t, J = 6.7 Hz). Synthesis of compounds G 10 and G 11

To a solution of G9 (41.2 g, about 54 mmol) in 1-pronol / knol (250 ml) was added methanol (30 ml), and further 5% paradium carbon (4.1 g) and formic acid Water (27. lg, 4.3 mol) was added. After stirring at room temperature for 16 hours, the mixture was diluted with ethyl acetate and filtered through celite. The filtrate was concentrated under reduced pressure, dissolved in ethyl acetate, and then saturated aqueous sodium bicarbonate was added. After washing with brine three times, all the organic layers were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain a crude product of G10. Yield 38.9g (98%). The obtained G10 was used for the next step without further purification.

Hexacosanoic acid (22.4 g, 56.5 mmol) and WSC hydrochloride (12.6 g, 64.6 mmol) were added to a methylene chloride solution (300 ml) of G10, and the mixture was heated under reflux for 1 hour. After cooling to room temperature, the mixture was concentrated under reduced pressure. Ethyl acetate (500 ml) was added to the residue, and the mixture was washed with a 0.5 M aqueous hydrochloric acid solution, a saline solution, a saturated aqueous sodium hydrogen carbonate solution, and further with a saline solution. All the organic layers were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain a crude G11 product. Yield 53.2g (88%). The obtained Gil was used for the next step without further purification. The analytical sample was purified by silica gel chromatography using hexane: ethyl acetate (100: 1) as the elution solvent.

[H 1. _{^{4 D + 5.3 ° (c 0.4}} , CHC13); FDMS m / z 1118 M +; 1 H-NMR (500MHz, CDC1 3) (57.20-7.38 (25H, m), 5.57 (1H, d, J = 9.1 Hz ), 4.80 (1H, d, J = 11.6 Hz), 4.48-4.50 (3H, m), 4.24- 4.32 (1H, m), 3.83 (1H, dd, J = 3.0, 6.7 Hz), 3.43-3.51 ( 2H, m, Hla), 3.29 (1H, dd, J = 4.3, 9.8 Hz), 1.92 (2H, t, J = W

39

(7.3 Hz), 1.28-1.60 (72H, m), 0.88 (6H, t, J = 6.7 Hz). Synthesis of compound G12

To a solution of Gil (52.2 g, about 47 mmol) in methylene chloride (180 ml) was added methanol (36 ml), and then a 10% solution of methanol in hydrochloric acid (3.0 ml) was added dropwise. The mixture was stirred for 2 hours. The reaction solution was neutralized with powdery sodium hydrogen carbonate (18 g) and filtered through celite. The residue was washed with methylene chloride. The filtrate and washing solution were combined, washed with brine, and the organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was dissolved in hot acetone, cooled to 0 ° C, and purified by precipitation. Yield 38.6g (77% over G9).

_{^{[A] 7 4 D - 29.7}} ° (c 0.7, CHC1 3); mp 75-76.5 ° C; FDMS m / z 876 M +; 1 H-NMR (500MHz, CDC1 3) 57.30-7.47 (10H, m), 6.03 (1H, d, J = 7.9 Hz), 4.72 (1H, d, J = 11.6 Hz), 4.66 (1H, d, J = 11.6 Hz), 4.61 (1H, d, J = 11.6 Hz), 4.45 ( 1H, d, J = 11.6 Hz ), 4.12-4.17 (1H, m), 4.00 (1H, dt, J t = 4.3, J d = 7.3 Hz), 3.67-3.72 (2H, m), 3.61 (1H, ddd, J = 4.3, 8.6, 11.6 Hz), 1.94-2.05 (2H, m), 1.15-1.69 (72H, m), 0.88 (6H, t, J = 6.1 Hz). . „

40 Synthesis of Compound G13

1) 2, 3, 4, 6-Tetra-0_ benzyl-D-galactopyranosyl

Acetate (79.8 g) was dissolved in a mixture of toluene (160 ml) and isopropyl acetate (520 ml) and cooled to -10 to 0 ° C. To this was added an isopropyl ether solution containing 2.0 equivalents of HBr (2.8 mmol / ml, about 100 ml). After stirring at _10 to 0 ° C for about 90 minutes, a 5% aqueous sodium hydrogen carbonate solution was poured into the reaction solution, and the mixture was stirred to neutralize excess HBr. The whole amount was transferred to a separating funnel, and after separation, the aqueous layer was discarded and washed twice with a 10% aqueous sodium chloride solution. Concentrate under reduced pressure and concentrate to 2,3,4,6-Tetra -0-benzyl-hi-D-galactosilane silo mouth mid (Ga 1 -Br) I got

2) G12 (60.0 g, 68.6 mmol), tetrahexyl ammonium bromide (89.4 g, 206 mmol), toluene of molecular sieves 4A (60 g) To the solution (420 ml) were added DMF (UOml) and then a solution of GalBr (about 137 mmol) in toluene (250 ml), and the mixture was stirred at room temperature for 72 hours. To the reaction solution was added methanol (12 ml), and the mixture was stirred for 2 hours. After filtration through celite, the mixture was washed with a saturated aqueous solution of sodium hydrogencarbonate and brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. Acetonitrile was added to the residue, and the mixture was stirred for 2 hours to obtain a precipitate. Reduce the resulting precipitate „ _Λ

It was dried under pressure to obtain a dry powder. This was purified by silica gel chromatography using hexane: ethyl acetate (8: 1) as the eluting solvent. Yield 70.9g (74%).

_{^{[Shed] 2 4 D + 18.8 ° (}} c 0.9, CHC1 3); mp 74-75 ° C; FDMS m / z 1399 (M + l) +; 1 H-NMR (500MHz, CDC1 3) 57.21 -7.37 ( 30H, m), 6.12 (1H, d, J = 9.0 Hz), 4.91 (1H, d, J = 11.6 Hz), 4.84 (1H, d, J = 3.7 Hz), 4.72-4.80 (4H, m), 4.35-4.65 (7H, m), 4.12-4.18 (1H, m), 3.99-4.05 (2H, m), 3.84-3.93 (4H, m), 3.73 (1H, dd, J2 3.7, 11.0 Hz), 3.47-3.51 (2H, m), 3.42 (1H, dd, J = 6.1, 9.1 Hz), 1.87-1.99 (2H, m), 1.18-1.70 (72H, m), 0.88 (6H, t, J = 7.4 Hz).

(23,33,41 -1-0- (hi_0_galactosylanosil)) -N-hexacosanyl-2-amino-1,3,4- Evening decanter (KRN7000)

G13 (60.0 g, 42.9 mmol) was added to ethanol (960 ml) and suspended, and then 20% palladium hydroxide (6.0 g) was added to the ethanol suspension. And I got it. Further, 4-methylcyclohexene (120 ml, 93.5 mmol) as a hydrogen source was added, and the mixture was heated under reflux for 4 hours, filtered, and the catalyst was removed. The residue was washed with warm ethanol. The white precipitate obtained by allowing the filtrate to stand at room temperature was filtered and dried under reduced pressure. It was dry. The obtained powder is suspended in ethanol: water (92: 8, 3.5 L), heated and dissolved with stirring, and left again at room temperature to precipitate again. did. The precipitate was filtered, and the cake collected by filtration was dried under reduced pressure to obtain a white powder. Yield 35.0 g (95%).

_{^{[Shed] 2 3 D +43.6 ° (c}} 1.0, pyridine); mp 189.5- 190.5. C; negative FABMS m / z 857 (M- H广; IR (cm one ^{1, KBr) 3300, 2930,} 2850, 1640, 1540, 1470, 1070; 1 H-NMR (500 MHz, C 5 D 5 N) δ 8.47 (1H, d, J = 8.5 Hz), 5.58 (1H, d, J = 3.7 Hz), 5.27 (1H, m), 4.63-4.70 (2H, m), 4.56 (1H, m), 4.52 ( 1H, t, J = 6.1 Hz), 4.37- 4.47 (4H, m), 4.33 (2H, m), 2.45 (2H, t, J = 7.3 Hz), 2.25-2.34 (1H, m), 1.87-1.97 (2H, m), 1.78-1.85 ( 2H, m), 1.62-1.72 (1H, m), 1.26-1.45 (66H, m), 0.88 (6H, t, J = 6.7 Hz). 1 3 C-NMR (125 MHz, C ₅ D ₅ N) c5 "173.2 (s), 101.5 (d), 76.7 (d), 73.0 (d), 72.5 (d), 71.6 (d), 71.0 (d), 70.3 (d ), 68.7 (t), 62.7 (t), 51.4 (d), 36.8 (t), 34.4 (t), 32.1 (t), 30.4 (t), 30.2 (t), 30.03 (t), 30.00 (t ), 29.93 (t), 29.87 (t), 29.81 (t), 29.76 (t), 29.6 (t), 26.5 (t), 26.4 (t), 22.9 (t), 14.3 (q). Pharmacological test example

(Pharmacological test 1): KRN7000, G—CSF is present. Increase in hematopoietic progenitor cells in peripheral blood by in vivo administration of both KRN7000 and G—CSF.

The experiment was performed using a C57BL / 6 mouse purchased from SLC Japan. KRN7000 was used as a representative glycoside compound in the following experiments. As G-CSF, sugar-free G-CSF (generic name: Gran, manufactured by Kirin Brewery) was used.

C57BL / 6 mice (6 weeks old, female) were divided into the following four groups to conduct experiments. That is, (A) a control (unprocessed) group. (B) KRN7000 (100 g / kg) was administered intravenously on day-and G-CSF (100 juLg / H) was administered on days -3, -2, -1, and 0 (4 hours before blood collection). Subcutaneously administered group (KRN7000 + G-CSF group), (C) KRN7000 (100 / g / kg) intravenously administered on day-(KRN7000 group), (D) G-CSF (100 g / kg) kg) was administered subcutaneously on days -3, -2, -1, and 0 (4 hours before blood collection) (G-CSF sc group). Peripheral blood was aseptically collected from the mouse's heart using parylene, and the blood in each group was removed. After diluting this blood two-fold using RPMI 1640 medium, the blood is overlaid on Linholite M (Sedaren) and centrifuged to remove mononuclear cells from erythrocytes. A spherical layer was obtained. like this The resulting mononuclear cells were diluted to 8 x 10 ⁵ cells / ml and contained mouse I 3 20 ng / ml and erythropoietin 2 U / l. After culturing in 0.8% methylcellulose semi-solid medium lm1 for 6 days, the formed leukocyte cell mass (hematopoietic progenitor cells

(CFU-GM) was measured, and the number of CFU-GM per 1 ml of the original peripheral blood was calculated by conversion from the dilution. The results are shown in Table 1 "9.

Table 1 KRN 7000, G-CSF Available for both KRN 7000 and G-CSF

On the increase of hematopoietic progenitor cells in peripheral blood by in vivo administration of mouse

Processing CFU-GM (pcs / ml peripheral blood)

(A) Control 58 Sat 22

(B) KRN7000 + G-CSF a, b

1421 ± 246

(C) KRN7000 210 Sat 28 ^a

(D) G-CSF 658 Sat 7 ^a ' ^b

a: There is a significant difference at 5% risk relative to control. b: Indicates that there is a significant difference at a risk rate of 5% with respect to KRN7000. c: Indicates that there is a significant difference at a risk rate of 5% with respect to G-CSF. As shown in Table 1, administration of KRN7000 and administration of G-CSF significantly increased CFU-GM in peripheral blood. Furthermore, the co-administration of KRN7000 and G-CSF significantly increased the CFU-GM in peripheral blood compared to KRN7000 or G-CSF alone. (Pharmacological test 2): Effect of donor blood from peripheral blood administered KRN7000 and G-CSF on the survival time of whole-body irradiated recipient mice

C57BL / 6 mice (6 weeks old, female) were used as donor mice, and the experiments were performed in the following seven groups. That is, the (A) content port (unprocessed) group. (B) KRN7000 (100〃g / kg) administered intravenously on day-and G-CSF (100 / g / kg) on days -3, -1, -1, -1 and 0 (4 hours before blood collection) ) Subcutaneously (KRN7000 + G-CSF group), (C) KRN7000 (lOO / zg / kg) intravenously on day-(KRN7000 group), (D) G-CSF (lOO g / kg) was administered subcutaneously on days -3, -2, -1 and 0 (4 hours before blood collection) (G-CSF sc group), as well as positive control. (E) G-CSF (200 juLg / g) and SCF (25 〃g / kg) administered intravenously 7 times from days-6 to 0 (G-CSF + SCF group), (F) G-CSF (200 ig / kg) (G-CSF iv group), and (G) SCF (25 / g / kg) administered intravenously 7 times from days-6 to 0 Drugs were administered in 7 groups (SCF group), and on day 0, blood was collected from donna mice in each experimental group, and the blood was pooled. The group (E) described above is known as a drug that effectively increases peripheral blood stem cells, and thus the positive control was used to compare the effect with the group (B). The experiment was carried out as a group of mouths.

Recipient mice (6 weeks old, C57BL / 6 mouse, Mess, 10 mice per group) were subjected to 9 Gy (900 rad) X-ray whole-body irradiation, and 2 days after irradiation. Peripheral blood (100 1 / mouse) collected from each group as described above was transferred by vein into the recipient mouse within the above time. Then, the survival of each recipient mouse was observed. The results are shown in Figure 1.

As shown in Fig. 1, peripheral blood from (A) control group, (C) KRN7000 group, (D) G-CSF sc group, and (G) SCF group was transferred to peripheral blood. All point mice died within 20 days after whole body irradiation with X-rays. Also, 40 days after whole body irradiation with X-rays, (F) G-CSF iv group And (E) In the mice receiving peripheral blood from the G-CSF + SCF group, one and two mice were observed to survive. (B) Five (50%) mice survived in the recipient mice to which peripheral blood was transferred from the KRN7000 + G-CSF group. This result indicates that among the seven groups described above, there is more hematopoietic progenitor cells in the peripheral blood of donor mice treated with KRN7000 + G-CSF I suggest this.

(Pharmacological experiment 3): Effects of peripheral blood of Donau mouse treated with KRN70Q0 and G-CSF on the survival time of whole-body irradiated recipient mice

Next, an experiment similar to the pharmacological experiment 2 was performed using a BALB / c mouse (6 weeks old) having a strain different from that of the mouse described above. The result is shown in figure 2.

As shown in Fig. 2, the recipient mice to which the peripheral blood of (A) the control group, (C) the KRN7000 group, and (G) the SCF group were transferred were: All died within 15 days after whole body irradiation with X-rays. Also, 40 days after whole-body irradiation with X-rays, in the recipient mice to which peripheral blood of (D) G-CSF sc group and (F) G-CSF iv group had been transferred, Survival of one of them was observed. Then, (E) peripheral blood of G-CSF + SCF group was _Λη

48 In each of the recipient mice that had been transfected, four survivors were observed, but (B) the recipient mice that received peripheral blood from the KRN7000 + G-CSF group. In mouse control, six (60%) mice survived. This result indicates that, even when a mouse strain different from that in pharmacological experiment 2 was used, in the 7 groups above, donor mice receiving KRN7000 + G-CSF were not used. This strongly suggests that hematopoietic progenitor cells are most abundant in the peripheral blood of humans.

(Pharmacological experiment 4): Engraftment and proliferation of hematopoietic progenitor cells in donor mouse peripheral blood administered to KRN7000 and G-CSF in recipient mice Consideration

Next, we examined whether hematopoietic progenitor cells in peripheral blood transplanted from Donner mouse can survive and proliferate in the recipient mouse. . That is, 7 groups were prepared as in pharmacological experiment 2 using Drosophila BALB / c mouse as the mouse, and peripheral blood collected from each group was collected. The whole body of Gy radiation was transferred to the BALB / c mouse.

First, Fig. 3 shows the survival time of the recipient mice to which the peripheral blood of each group was transferred.

As shown in Fig. 3, (A) control group, (C) KRN7000 group, (D) G-CSF sc group, (E) G-CSF + SCF group, (F) G-CSF iv group and (G) SCF group All horses died within 40 days after whole body irradiation with X-rays. However, surprisingly, 50 days after whole-body irradiation with X-rays, (B) in the recipient mouse to which peripheral blood of the KRN7000 + G-CSF group had been transferred, Nine (90%) mice survived. In addition, all mice in group (B) survived even after 150 days. These results indicate that in the experiment using BALB / c mice, in the 7 groups described above, the peripheral blood of donor mice receiving KRN7000 + G-CSF was not used. We support the results of pharmacological experiments 2 and 3 above, in which hematopoietic progenitor cells are the most abundant.

Next, it was examined whether hematopoietic progenitor cells in Donor mouse peripheral blood survive in the recipient mouse and further proliferate. And ALB / c mice (positive and negative controls) and from individual surviving mice (mes) in group (B), irradiated 60, 90, Orbital blood was collected 120 and 150 days later, and the presence of os (dona) mouse-derived blood cells in each peripheral blood was determined by the PCR method (Yan et al. (1994) Blood, 84, 795-799). That is, genomic DNA is prepared from the blood of each mouse described above. After performing _CA and amplifying by PCR, electrophoresis was performed to examine the presence or absence of Sry locus present on the os Y chromosome in each genomic DNA. Figure 4 shows the results of PCR performed on the positive and negative control mice and mice, and the blood of the surviving mouse (nine mice) in group (B) 150 days after irradiation. The results are shown.

As shown in Fig. 4, the presence of Sry locus was not observed in the blood of female mice, but a band indicating the presence of Sry locus was observed in the blood of female mice. It was done. The presence of Sry locus derived from os chromosome Y was clearly found in the blood of all the surviving mice in group (B). Similar results were obtained in experiments using blood from recipe mice 60, 90, and 120 days after irradiation. These results indicate that blood cells derived from donna mouse engraft in the body of the recipient mouse and that the recipient mouse remains 150 days after irradiation. This indicates that donor mouse-derived blood cells are growing in the body. Sequence Listing SEQ ID NO: 1

Sequence length: 1 7 4 Amino acid Sequence type: Amino acid

Topology: linear

Sequence type: Protein

Origin:

Organism name: Homo sapiens

Array

OLl 59T

o d u D Gxv ΠΘΤ: STH 5-i na; 丁 6JV JA JSS Τ ^ Λ ηχο nai

09ΐ ςχ οςχ S

eiV 6u V 6av uxo οι ςετ οει a <I STV u as BI aqd STV O d ¾sw εχ χο U "3 ji, OJJ UTD naq BTV

031 ςττ

OJd Bi ¾aw ^ TD ΠΘΤ: ηχο ηχο ¾sw u o u "D djj, 3 cho eiV s d οπ ςοι ooi

dsv ¾TV Τ ^ Λ dsv nsi U ^ D naq; jqj, dsv ΠΘΊ jrqj, OJJ A f) ΠΘΊ ηχο OJ <J

S6 06 58

As as en AID ηχο U3T exv UTD na ^ nai Λχο UTD J ^ i nsq; sy <i ΠΘΊ AID

08 S 0 .59

• iss STH ηθΊ uio ass naq sAo Λχο e-cho v ΠΘΊ U O nsq; Βχν UTD J3S OJd

09 ςς 05

SAD JSS JSS ΠΘΙ OJ <J BXV O OJ <J STi Α 3 ΠΘΊ JSS STH AID nsi nai si 'o ςε Π9 Ί nTD ri te 3 OJ <J STH SAQ ns S ^ T jjj, εχν sAo ΠΘΤs people χ ηχο

Οε S3 02

UTO ηθτ: STV Βχ d3 dsv Αχο u ^ Ι sAq; £>: rv Τ ^ Λ UTD ηχο ΠΘΊ ΞΑΟ ο οτ ς sAq; nsi; nai aqd J3S uxo oj <i ΠΘΊ BS SS EJ OJ <J χο ΠΘΤ; OJ <I ji,

2S

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Claims

B 冃 l l The following equation (A)

Where R is H or 〇H

X? An integer of from 25 to 25;

Is a substituent defined by any of the following (a) to (e), and Y is an integer of 5 to 17;

(a) — CH ₂ (CH ₂ ) CH

(b) CH (OH) (CH,) _Y CH

(C) - CH (OH) (CH 2) Y CH (CH 3)

(d)-CH = CH (CH 2) _Y CH

(e) One CH (OH) (CH ₂ ) _Y CH (CH ₃ ) CH ₂ CH

_R Ί your good beauty of R ₄ Les, one or deviation Ri Oh in H, the other is H, 0 H

NH. , NHCOCH ₃ or

Is;

One of R ₅ and R ₆ Ri H Der, the other is 0 H

Is;

One of R ₇ and R ₈ is Η, and the other is 0 H

And

R. Is H, CHCH ₂ 〇 H or

Is

A peripheral blood stem cell augmentation agent in a peripheral blood stem cell transplantation therapy using G-CSF, comprising at least one glycoside compound or a salt thereof represented by the following formula:

2. The glycoside compound is represented by the following formula (B):

[Wherein, R i, X and R ₂ are as defined in claim 1;

RR ₉ may fall under any of i) to v) below A substituent selected for;

i) RR ₆ and R ₈ are both H

R ₄ is H, 0 H, NHNHCOCH ₃ or

And

R 5 is 0 H

And

R ₇ is 0 H,

R ₉ is H, CH ₃ , CH. 0 H or Or

OCH ₂ -OCH ₂

Is

ii) RR ₆ and R ₇ are both H

RR ₅ and R ₉ are each the same as defined in i),

R _s is 0 H,

Is

iii) RR ₆ and R ₇ are both H

And R ₉ is Ri same der defined as in each i) H, 〇 H, _NH?, NHC 〇 CH ₃ or

And

R ₈ is 0 H or

Is

iv) RR ₅ and R ₇ are both H

RR ₆ and R ₈ are both 0 H

R ₈ is H, CH ₃ or CH ₂₀ H, or

V) RR ₅ and R ₇ are both H

R ₄ , R ₆ and R ₈ are all 〇H

R ₉ is H, CH ₃ or CH ₂ OH] The peripheral blood stem cell increasing agent according to claim 1, which is represented by:

3. In the glycoside compound, R ₃ , R ₆ and R ₈ are all H,

R 4 s R ₅ and R ₇ are both 0 H, and

3. The agent for increasing peripheral blood stem cells according to claim _{2, wherein} R ₉ is CH ₂ OH.

4. In the above glycoside compound, R ₂ is substituent (b), (c) or (e) der Ru, claim 2 or peripheral blood stem cell increase pressure agent according 3.

5. The peripheral blood stem cell enhancer according to claim 4, wherein in the glycoside compound, R i is H and R ₂ is a substituent (b).

6. The agent for increasing peripheral blood stem cells according to claim 5, wherein in the glycoside compound, the configuration of the hydroxyl group in the substituent (b) is R.

7. The agent of claim 5, wherein X is 21 to 25 and Y is 11 to 15 in the glycoside compound.

8. The glycoside compound is (2S, 3S, 4R) -1-1 (H-D-galacto-vilanosyloxy) -1-2-hexacosanoylua The agent for increasing a peripheral blood stem cell according to claim 6 or 7, which is bu mino 13-, 4-year-old katadecanediol.

9. The peripheral blood stem cell increasing agent according to any one of claims 1 to 8, wherein the glycoside compound is dissolved in an aqueous medium.

10. A pharmaceutical composition for increasing peripheral blood stem cells, comprising: G—CSF, at least one glycoside compound or a salt thereof according to claim 1, and a pharmaceutically acceptable carrier. object.

11. The pharmaceutical composition for increasing peripheral blood stem cells according to claim 10, wherein the glycoside compound is contained in a weight ratio of 1:10 to 10: 1 with the G-CSF.

12. The pharmaceutical composition for increasing peripheral blood stem cells according to claim 11, wherein the glycoside compound is contained in a substantially equal amount to the G-CSF in a weight ratio.

13. The pharmaceutical composition _c for increasing peripheral blood stem cells according to any one of claims 10 to 12, wherein the G—CSF is human G—CSF.

14. The G—CSF has a deletion, substitution, addition and / or insertion of one or more amino acid residues in the amino acid sequence of SEQ ID NO: 1; — An increase in peripheral blood stem cells according to any one of claims 10 to 12, which is a protein having CSF activity. Pharmaceutical composition for bl.

15. A pharmaceutical kit for increasing peripheral blood stem cells, comprising a G—CSF and at least one glycoside compound or a salt thereof according to claim 1.

16. The pharmaceutical kit for increasing peripheral blood stem cells according to claim 15, wherein the G-CSF is contained as a solution suitable for injection administration.

17. The pharmaceutical kit for increasing peripheral blood stem cells according to claim 15 or 16, wherein the G-CSF is human G-CSF.

18. The G—CSF has one or more amino acid residues deleted, substituted, added and / or inserted in the amino acid sequence of SEQ ID NO: 1. The pharmaceutical kit for increasing peripheral blood stem cells according to claim 15 or 16, which is a protein having G-CSF activity.

19. The peripheral blood stem cell according to any one of claims 15 to 18, wherein the glycoside compound is contained in a weight ratio of 1:10 to 10: 1 with the G-CSF. Pharmaceutical kit for increase.

20. The drug kit for increasing peripheral blood stem cells according to claim 19, wherein the glycoside compound is contained in a substantially equal amount to the G-CSF in a weight ratio. b

21. At least one kind of the glycoside compound or the salt thereof according to claim 1 and G-CSF are used in combination with a human or other animal in need of peripheral blood stem cell expansion. How to treat.

22. Use of at least one glycoside compound according to claim 1 or a salt thereof for the manufacture of a medicament having a peripheral blood stem cell increasing effect.