JP2002226377A - Glycoside-containing peripheral blood stem cell increasing agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a peripheral blood stem cell increasing agent in a peripheral blood stem cell transplantation therapy by G-CSF. SOLUTION: This peripheral blood stem cell increasing agent in the peripheral blood stem cell transplantation therapy by the G-CSF comprises at least one kind of a glycoside compound represented by the formula (A) or its salt [preferable example: (2S,3S,4R)-1-(α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4- octadecanediol] as an active ingredient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an agent for increasing peripheral blood stem cells and a medicament 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. Composition.

[0002]

2. Description of the Related Art Conventionally, bone marrow transplantation (allogeneic and autologous) and peripheral blood stem cell transplantation therapy are known for the treatment of this kind of disease. In bone marrow transplantation, cancer cells and bone marrow cells are killed by intense chemo / radiotherapy, and then the prepared bone marrow cells are transplanted. 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 using cells previously collected from the patient's own bone marrow does not have this problem.However, when cancer cells have infiltrated the bone marrow, it is difficult to collect the required amount of bone marrow without cancer cells. Accompany. Furthermore, there is considerable danger since bone marrow collection 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., H.
ematology / Oncology Clinic
sof North America, 9, 1-22
(1995)] is more convenient and safer. This method is based on the finding that hematopoietic stem cells are present in peripheral blood in a very small amount compared to bone marrow, and that they increase transiently and significantly after myeloablative treatment. There is no. Nevertheless, the hematopoietic stem cell abundance is small and generally requires a large number of cell harvests.

To address this problem by increasing the number of hematopoietic stem cells in peripheral blood, for example, granulocyte colony stimulating factor (G-
CSF) and stem cell factor (SCF) [McNie
See, et al., Stem Cell, 11 (suppl.
2), 36 (1993); Yan et al., Blood, 8
No. 4,795 (1994)] and a combination of IL-1 and G-CSF [Japanese Patent Laid-Open No. 8-127538].

[0005] In vivo, β-galactosylceramide and β-glucosylceramide in which various sugars are β-linked to ceramide exist [Svennerholm et al., B.
iochim. Biophys. Acta, 280, 6
26-636 (1972); Karlsson et al., Bi.
ochim. Biophys. Acta, 316, 31
7-335 (1973)]. On the other hand, α-galactosylceramide and α-glucosylceramide have remarkable immunostimulatory and antitumor effects [Morita et al., J. Am. Me
d. Chem. , 38, 2176-2187 (199).
5), WO 93/05055, WO 94/09020 and WO 94/24142], which are much more potent than in the case of β-galactosylceramide or β-glucosylceramide [Motoki et al., Biol. Ph
arm. Bull. , 18, 1487-1491 (19)
95)] is also known. Furthermore, when a compound having an α-glycosylceramide structure is administered to a living body, it has a radioprotective effect [Motoki et al., Bioorg. Med. C
hem. Lett. , 5, 2413 (1995)], and the effect of increasing platelet count, leukocyte count and bone marrow cells [M
Otoki et al., Biol. Pharm. Bull. , 1
9, 952-955 (1996) and WO 94/02
168]. However, in vivo administration of a compound having an α-glycosylceramide structure,
The effect on hematopoietic progenitor cells in peripheral blood is not known. Of course, the combined use of such a compound with G-CSF,
There is no report that it is effective as a peripheral blood stem cell increasing agent.

[0006]

SUMMARY OF THE INVENTION The present inventors have shown that the above combination can significantly increase the number of hematopoietic progenitor cells in peripheral blood, and that the present invention has When peripheral blood from a donor mouse to which the enhancer of the present invention was administered was transferred, the survival time was significantly prolonged, and that the transferred hematopoietic progenitor cells engrafted and proliferated in the recipient mouse. Find it,
The present invention has been reached. The enhancer of the present invention is also excellent in safety as described below.

[0007]

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

[0008]

Embedded image Wherein R ₁ is H or OH; X is an integer of 7 to 25; R ₂ is a substituent defined by any of the following (a) to (e), wherein Y is 5 It is -17 _{integer; (a) -CH 2 (CH} 2) Y CH 3 (b) -CH (OH) (CH 2) Y CH 3 (c) -CH (OH) (CH 2) Y CH ( _{CH 3) 2 (d) -CH} = CH (CH 2) Y CH 3 (e) -CH (OH) (CH 2) Y CH (CH 3) CH
_{One of 2} CH ₃ R ₃ and R ₄ is H, the other is H,
OH, NH ₂ , NHCOCH ₃ or

[0009]

Embedded image One of R ₅ and R ₆ is H and the other is OH,

[0010]

Embedded image One of R ₇ and R ₈ is H and the other is OH,

[0011]

Embedded image R ₉ is H, CH ₃ , CH ₂ OH or

[0012]

Embedded image And an agent for increasing peripheral blood stem cells in peripheral blood stem cell transplantation therapy with G-CSF, which comprises at least one glycoside compound represented by the formula: or a salt thereof as an active ingredient. In a preferred embodiment of the present invention, the following formula (B):

[0013]

Embedded image [Wherein R ₁ , X and R ₂ have the same meanings as in formula (A); R _{3 to} R ₉ are substituents selected so as to correspond to any of the following i) to v) I) [galactose type] R ₃ , R ₆ and R ₈ are all H, and R ₄ is H, OH, NH ₂ , NHCOCH ₃ or

[0014]

Embedded image R ₅ is OH,

[0015]

Embedded image R ₇ is OH and R ₉ is H, CH ₃ , CH ₂
OH or

[0016]

Embedded image Ii) [Glucose-based] R ₃ , R ₆ and R ₇ are all H, R ₄ , R ₅ and R ₉ are each the same as defined in i), and R ₈ is OH,

[0017]

Embedded image Iii) [mannose type] R ₄ , R ₆ and R ₇ are all H, R ₅ and R ₉ are each the same as defined in i), and R _{3 is}
Is H, OH, NH ₂ , NHCOCH ₃ or

[0018]

Embedded image And R ₈ is OH or

[0019]

Embedded image Iv) [Althrose type] R ₄ , R ₅ and R ₇ are all H, and R ₃ , R
₆ and R ₈ are both OH and R ₉ is H, CH
₃₎ or CH ₂ OH, or v) [allose type] R ₃ , R ₅ and R ₇ are all H, and R ₄ , R
₆ and R ₈ are both OH and R ₉ is H, CH
₃ or CH ₂ OH], or a glycoside compound represented by the formula:
An agent for increasing peripheral blood stem cells in peripheral blood stem cell transplantation therapy by SF is provided. The present invention also provides a pharmaceutical composition for increasing peripheral blood stem cells, comprising G-CSF, at least one glycoside compound defined by formula A or B or a salt thereof, and a pharmaceutically acceptable carrier. Also provide.

The present invention also provides a pharmaceutical kit for increasing peripheral blood stem cells, comprising G-CSF and at least one glycoside compound defined by the formula A or B or a salt thereof.

The glycoside compounds defined by formula A or B consist of a sugar moiety and an aglycone moiety, some of which are α-
Also referred to as cerebroside, α-glycosylceramide, α-glucosylceramide, α-galactocerebreside or α-galactosylceramide. These compounds are characterized in that the anomeric configuration is α.

In the sugar moiety of the glycoside compound,
R ₃ , R ₆ and R ₈ are all H, and R ₄ , R
₅ and R ₇ are both OH and R ₉ is CH
Those which are ₂ OH, that is, those whose sugar moiety is α-galactopyranosyl, are preferred.

In the aglycone portion of the glycoside compound, R ₂ is preferably a substituent (b), (c) or (e). R ₁ is H (meaning kerasin type) and R ₂ is a substituent (b),
More preferred. X is 21 to 25 and Y is 11
~ 15 are preferred.

Preferred examples of the glycoside compounds are as listed below. Here 1) ~
9) is an example in which R ₂ is a substituent (a).
0) to 24) are (b), 25) to 31) are (c), 3
2) and 33) correspond to (d) and 34) to (e), respectively. The alphabets described after each compound name indicate the literature in which the synthesis method is described, A is WO93 / 05055, B is WO94 / 02168, C
Means that WO 94/09020 and D means that WO 94/24142, respectively. Among the glycoside compounds, compound 14), that is, (2S, 3S, 4R) -1
Most preferred is-(α-D-galactopyranosyloxy) -2-hexacosanoylamino-3,4-octadecanediol (hereinafter referred to as KRN7000). An example of a method for synthesizing this compound is shown in the following Production Examples and [Chemical Formulas 25 and 26].

1) (2S, 3R) -1- (α-D-galactopyranosyloxy) -2-[(R) -2-hydroxytetracosanoylamino] -3-octadecanol A 2) (2S, 3R) -1- (α-D-galactopyranosyloxy) -2-tetracosanoylamino-3-octadecanol A 3) (2S, 3R) -1- (α-D-galactopyranosyloxy) ) -2-Tetradecanoylamino-3-octadecanol A 4) (2S, 3R) -1- (α-D-glucopyranosyloxy) -2-tetradecanoylamino-3-octadecanol C 5) (2S, 3R) -1- (6'-deoxy-α-D-galactopyranosyloxy) -2-tetradecanoylamino-3-octadecanol C 6) (2S, 3R) -1- (β -L-arabinopyranosyloxy) -2-tetradecanoylamino-3-octadecanol C 7) (2S, 3S) -1- (α-D-galactopyranosyloxy) -2-tetradeca Noylamino-3-hexadecanol A 8) (2R, 3R) -1- (α-D-galactopyranosyloxy) ) -2-Tetradecanoylamino-3-hexadecanol A 9) (2R, 3S) -1- (α-D-galactopyranosyloxy) -2-tetradecanoylamino-3-hexadecanol A 10) (2S, 3S, 4R) -1- (α-D-galactopyranosyloxy) -2-[(R) -2-hydroxytetracosanoylamino] -3,4-octadecanediol A11) (2S, 3S, 4R) -1- (α-D-galactopyranosyloxy) -2-[(R) -2-hydroxytetracosanoylamino] -3,4-undecanediol A 12) (2S , 3S, 4R) -1- (α-D-galactopyranosyloxy) -2-[(R) -2-hydroxyhexacosanoylamino] -3,4-icosanediol A 13) (2S, 3S, 4R) -1- (α-D-galactopyranosyloxy) -2-[(S) -2-hydroxytetracosanoylamino] -3,4-heptadecanediol A 14) (2S, 3S , 4R) -1- (α-D-Galactopyranosyloxy) -2-hexacosanoylamino-3,4-octadecanediol Production Example 15) (2S, 3S, 4R) -1- (α-D-galactopyranosyloxy) -2-octacosanoylamino-3,4-heptadecanediol B 16) (2S, 3S, 4R) -1- (α-D-galactopyranosyloxy) ) -2-Tetracosanoylamino-3,4-octadecanediol A 17) (2S, 3S, 4R) -1- (α-D-galactopyranosyloxy) -2-tetracosanoylamino-3 , 4-Undecanediol A 18) (2S, 3S, 4R) -1- (α-D-glucopyranosyloxy) -2-hexacosanoylamino-3,4-octadecanediol C 19) O-β- D-galactofuranosyl- (1 → 3) -O-α-D-galactopyranosyl- (1 → 1)-(2S, 3S, 4R) -2-amino-N-[(R) -2- Hydroxytetracosanoyl] -1,3,4-octadecanetriol D 20) O-α-D-galactopyranosyl- (1 → 6) -O-α-D-glucopyranosyl- (1 → 1)-( 2S, 3S, 4R) -2-Amino-N-hexacosanoyl-1,3,4-octadecanetriol D 21) O-α-D-Galactopyranosyl- (1 → 6) -O-α-D-gal Ctopyranosyl- (1 → 1)-(2S, 3S, 4R) -2-amino-N-hexacosanoyl-1,3,4-octadecanetriol D 22) O-α-D-glucopyranosyl- (1 → 4) -O -α-D-glucopyranosyl- (1 → 1)-(2S, 3S, 4R) -2-amino-N-hexacosanoyl-1,3,4-octadecanetriol D23) O- (N-acetyl-2-amino -2-deoxy-α-D-galactopyranosyl)-(1 → 3) -O- [α-D-glucopyranosyl- (1 → 2)]-O-α-D-galactopyranosyl- (1 → 1)-(2S, 3S, 4R) -2-Amino-N-[(R) -2-hydroxyhexacosanoyl-1,3,4-octadecanetriol D 24) O- (N-acetyl-2 -Amino-2-deoxy-α-D-galactopyranosyl)-(1 → 3) -O- [α-D-glucopyranosyl- (1 → 2)]-O-α-D-galactopyranosyl- (1 → 1)-(2S, 3S, 4R) -2-amino-N-[(R) -2-hydroxytetracosanoyl-1,3,4-hexadecanediol D 25) (2S, 3S, 4R ) -1- (α-D-Galactopyranosyloxy) -2-[(R) -2-hydr Xito ricosanoylamino] -16-methyl-3,4-heptadecanediol A 26) (2S, 3S, 4R) -1- (α-D-galactopyranosyloxy) -2-[(S)- 2-hydroxytetracosanoylamino] -16-methyl-3,4-heptadecanediol A 27) (2S, 3S, 4R) -1- (α-D-galactopyranosyloxy) -16-methyl- 2-tetracosanoylamino] -3,4-heptadecanediol A 28) O-β-D-galactofuranosyl- (1 → 3) -O-α-D-galactopyranosyl- (1 → 1)-( 2S, 3S, 4R) -2-Amino-N-[(R) -2-hydroxytetracosanoyl] -17-methyl-1,3,4-octadecanetriol D 29) O-β-D-galactofuranosyl -(1 → 3) -O-α-D-galactopyranosyl- (1 → 1)-(2S, 3S, 4R) -2-amino-N-[(R) -2-hydroxytetracosanoyl] -15-Methyl-1,3,4-hexadecanediol D 30) O- (N-acetyl-2-amino-2-deoxy-α-D-galactopyranosyl)-(1 → 3) -O- [ α-D-glucopi Nosyl- (1 → 2)]-O-α-D-galactopyranosyl- (1 → 1)-(2S, 3S, 4R) -2-amino-N-[(R) -2-hydroxyhexacosa Noyl-16-methyl-1,3,4-octadecanetriol D 31) O- (N-acetyl-2-amino-2-deoxy-α-D-galactopyranosyl)-(1 → 3) -O- [α-D-glucopyranosyl- (1 → 2)]-O-α-D-galactopyranosyl- (1 → 1)-(2S, 3S, 4R) -2-amino-N-[(R)- 2-hydroxytetracosanoyl-16-methyl-1,3,4-heptadecanetriol D 32) (2S, 3S, 4E) -1- (α-D-galactopyranosyloxy) -2-octadecanoyl Amino-4-octadecene-3-ol A 33) (2S, 3S, 4E) -1- (α-D-galactopyranosyloxy) -2-tetradecanoylamino-4-octadecene-3-ol A 34) (2S, 3S, 4R) -1- (α-D-galactopyranosyloxy) -2-[(R) -2-hydroxypentacosanoylamino] -16-methyl-3,4- Octadecanediol A as defined by formula A or B The glycoside compound may be capable of forming an acid addition salt with a pharmaceutically acceptable acid. Acids to form acid addition salts include inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, or acetic acid, propionic acid, maleic acid,
Organic acids such as oleic acid, palmitic acid, citric acid, succinic acid, tartaric acid, fumaric acid, glutamic acid, pantothenic acid, laurylsulfonic acid, methanesulfonic acid and phthalic acid can be mentioned.

The G-CSF according to the present invention has the following amino acid sequence or an amino acid sequence in which one or more amino acid residues in the sequence are deleted, substituted, added and / or inserted. And G-CS
Polypeptides having F activity can be mentioned. Such a convenient method of obtaining G-CSF is described in, for example, JP-A-63-500636 and Biochem. Biophy
s. Res. Commun. , 159, 103 (198
9). Furthermore, not only sugar-free G-CSF produced in Escherichia coli but also G-CSF having a sugar chain provided by a eukaryotic host such as CHO cells can be used. Human G-CSF is particularly suitable.

[0027] Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu 1 5 10 15 Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu 20 25 30 Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu 35 40 45 Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser 50 55 60 Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His 65 70 75 Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile 80 85 90 95 Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala 100 105 110 Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala 115 120 125 Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala 130 135 140 Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser 145 150 155 Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro 160 165 170 174 Here, deletion, substitution, addition and / or insertion of an amino acid residue can be performed by, for example, DFMark et al., Proc. Natl. Acad. Sci. U
SA, Vol. 81, p. 5662-5666, 1984, S. Inouye et al., Proc. Nat.
l.Acad.Sci.USA, Vol.79, p.3438-3441,1982, PCT W
O85 / 00817, published February 28, 1985, RPWharton et al., Na
, Vol. 316, pp. 601-605, Aug. 15, 1985, a site-directed mutagenesis method, which is a well-known technique before the present application, and the like.

The agent for increasing peripheral blood stem cells according to the present invention may take any route of administration that meets its purpose. Specifically, in the case of non-human animals, 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,
It can be administered transdermally or rectally.

The drug of the present invention can be administered in an appropriate dosage form determined by the administration method and administration purpose, specifically, in the form of injection, suspension, emulsifier, ointment, cream and the like. In these preparations, additives such as a pharmaceutically acceptable carrier or a diluent, specifically, a solvent, a solubilizer, an isotonic agent, a preservative, an antioxidant, an excipient, a binder, Stabilizers and the like can be added. Such additives include:
Serum albumin and mannitol, cytokines such as human IL-1, human IL-3, human IL
-11, human SCF, human LIF, human EPO, human G
Includes M-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. The specific dose varies depending on the administration method and various conditions of the patient, for example, age, body weight, sex, sensitivity, administration time, concomitant drug and the like. In general, the dose required for expressing the activity of the glycoside compound is, for example, in the case of intravenous administration, about 0.01 to 10 mg per day for a human adult, but preferably 0.1 to 1 mg. The dose required for the expression of G-CSF activity is
Allograft is different from autologous transplantation, but 1 per day for human
-20 μg / kg, and many cases of subcutaneous administration of 2-16 μg / kg for 4-6 days have been reported. These may be separately administered (sequentially, simultaneously, separately) in any order, but the peripheral blood stem cell expansion containing the glycoside compound, the G-CSF, and a pharmaceutically acceptable carrier is included. It is preferable to use a pharmaceutical composition for medical use. Among them, the glycoside compound is G-
A pharmaceutical composition for increasing peripheral blood stem cells, which is contained in a weight ratio of 1:10 to 10: 1 with CSF, is preferable, and furthermore, a glycoside compound is contained in an almost equal amount by weight with G-CSF. Are most preferred. In addition, existing G-CSF
When using a formulation, in order to avoid accidents and errors at the medical site, a vial of the glycoside compound and an existing G-
It is most preferable to make a contrivance such as preparing a kit in which the CSF preparation is set.

[0031]

The peripheral blood stem cell-increasing agent of the present invention is used for the purpose of increasing the number of stem cells when collecting peripheral blood stem cells.
When peripheral blood stem cells are collected from a healthy person, the time of administration can be arbitrarily selected before that. When collecting from a patient such as a cancer patient, the recovery period from the cytopenia period is preferably set. However, it may be used before or immediately after radiation / chemotherapy treatment.

[0032]

[Example] Manufacturing example

[0033]

Embedded image

[0034]

Embedded image Synthesis of Compound G1 To a solution of D-lyxose (200 g, 1.33 mol) in calcium chloride-dried acetone (3.0 L), add sulfuric acid (0.5 mL),
Stirred at room temperature for 8 hours. Molecular sieves 4A powder
(100 g) was added, the reaction solution was neutralized, filtered through celite, and the residue was washed with acetone. The filtrate and the washing were combined and concentrated under reduced pressure to obtain a crude G1 product. Yield 240 g (95
%). Used for the next step without further purification. The sample for analysis was purified by silica gel chromatography using hexane: acetone (9: 1) as an elution solvent.

Mp 76-78 ° C .; FDMS m / z 191 (M + 1) ⁺ ; ¹ H-
NMR (500MHz, CDCl ₃ ) δ5.45 (1H, 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), 1.4
8 (3H, s), 1.32 (3H, s). Synthesis of Compound G2 A solution of G1 (239 g, about 1.26 mol) in methylene chloride (168 m
l), pyridine (10 ml), trityl chloride (39.0 g)
Was added and 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 bicarbonate and brine, and then concentrated under reduced pressure. The residue was dissolved in ethyl acetate and crystallized by cooling to 0 ° C. Yield 501 g (87 from D-Lixose)
%).

Mp 174-176 ° C .; FDMS m / z 432 M ⁺ ; ¹ H-NM
R (500MHz, CDCl ₃ ) δ7.21-7.49 (15H, m), 5.38 (1
H, 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 tri de country tetraphenylphosphonium bromide compound G3 (962 g,
1.16 mol; prepared by heating 1-bromotridecane and triphenylphosphine to 140 ° C for 4.5 hours) in a THF solution (1500 ml) under an argon atmosphere under a 2.5 M hexane solution of n-butyllithium (462 ml). ; 1.16 mol) was added dropwise at 0 ° C. After completion of the dropwise addition, the mixture was stirred for 15 minutes, and G2 (250 g, 579
mmol) in a THF solution (450 ml) 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 hexane: methanol: water (10: 7: 3, 1000 m
l) was added, and the mixture was washed with a saturated aqueous ammonium chloride solution. The aqueous layer was extracted with hexane (500 ml), and all the organic layers were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain a crude G3 product. Used for the next step without further purification. Yield 339 g (98%). The sample for analysis was purified by silica gel chromatography using hexane: ethyl acetate (9: 1) as an elution solvent.

FDMS m / z 598 M ⁺ ; ¹ H-NMR (500 MHz, CDC
l ₃ ) δ7.21-7.45 (15H, m), 5.48-5.59 (2H, m), 4.9
1 (0.7H, t, J = 7.3 Hz), 4.44 (0.3H, t, J = 7.3 H
z), 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), H1bE overlapped], 1.75-2.03 (2H,
m), 1.49 (3H, s), 1.39 and 1.38 (3H, each s), 1.21-
1.34 (20H, m), 0.88 (3H, t, J = 6.7 Hz) Synthesis of compound G4 A solution of G3 (338 g, about 565 mmol) in methylene chloride (1500 m
l), add pyridine (500 ml) to methanesulfonyl chloride
(49 ml, 633 mmol) was added dropwise, and the mixture was stirred at 31 ° C for 24 hours.
Ethanol (40 ml) was added dropwise, and the mixture was stirred at room temperature for 1 hour.
After concentration under reduced pressure, hexane: methanol: water (10: 7: 3,
(1000 ml) was added, and the mixture was separated. The aqueous layer is hexane (2
(00 ml) three times, and all the organic layers were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain a crude G4 product. Used for the next step without further purification. Yield 363 g (95%). The sample for analysis is hexane: ethyl acetate
Purification was performed by silica gel chromatography using (9: 1) as an elution solvent.

FDMS m / z 676 M ⁺ ; ¹ H-NMR (500 MHz, CDC
l ₃ ) δ7.21-7.47 (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, d
t, 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), 1.47and 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 A solution of G4 (362 g, about 536 mmol) in methylene chloride (1500 m
l), add methanol (350 ml), and add concentrated hydrochloric acid (200 ml
l) was added dropwise and stirred at room temperature for 5 hours. The reaction solution was neutralized by adding sodium hydrogen carbonate, and then filtered. The filtrate was concentrated under reduced pressure, ethyl acetate was added to the residue, and the mixture was washed with brine. 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 hexane. Yield 161 g (70% over G2).

Mp 66-67 ° C .; FDMS m / z 377 (M-H ₂ O) ⁺ ;
¹ H-NMR (500 MHz, CDCl ₃ + D ₂ O) δ 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, 1
4.7 Hz), 5.49 (0.7H, bt, J = 9.8 Hz), 4.91-4.97 (1
H, m), 4.51 (0.7H, bt, J = 9.8 Hz), 4.11 (0.3H, b
t, 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 (2
H, m), 1.22-1.43 (20H, m), 0.88 (3H, t, J = 6.7 H
z) Synthesis of compound G6 5% palladium-barium sulfate (16 g) was added to a THF solution (780 ml) of G5 (160 g, 405 mmol), and the reaction vessel was replaced 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 chloroform: methanol (1: 1). The filtrate and the washing were combined and concentrated under reduced pressure. The residue was crystallized from ethyl acetate. Yield 146 g (91%).

[Α] ₂₃ ^D + 12 ° (c 1, CHCl ₃ / MeOH = 1:
1); mp 124-126 ° C .; FDMS m / z 397 (M + 1) ⁺ ; ¹ H-NMR (5
00MHz, CDCl ₃ / CD ₃ OD = 1: 1) δ4.93-4.96 (1H, m, H
2), 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, d
d, J = 1.8, 8.5 Hz), 3.19 (3H, s), 1.75-1.83 (1H,
m), 1.53-1.62 (1H, m), 1.21-1.45 (24H, m), 0.89 (3
H, t, J = 6.7 Hz. Synthesis of compound G7 To a solution of G6 (145 g, 365 mmol) in DMF (1000 ml) was added sodium azide (47 g, 730 mmol) and 95. The mixture was stirred at C for 4 hours. The reaction solution 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 a saline solution, 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. The sample for analysis was purified by silica gel chromatography using hexane: ethyl acetate (9: 1) as an elution solvent.

[Α] ₂₃ ^D + 16.5 ° (c 0.5, CHCl ₃ / MeOH =
1: 1); mp 92-93 ° C .; FDMS m / z 344 (M + 1) ⁺ ; ¹ H-NMR
(500MHz, CD ₃ OD) δ3.91 (1H, dd, J = 3.7, 11.6 H
z), 3.75 (1H, dd, J = 7.9, 11.6 Hz), 3.49-3.61 (3
H, m), 1.50-1.71 (2H, m), 1.22-1.46 (24H, m), 0.90
(3H, t, J = 6.7 Hz) Synthesis of compound G8 A solution of G7 (121 g, about 352 mmol) in methylene chloride (750 ml)
Pyridine (250 ml), trityl chloride (124 g, 445 mmol)
Was added and 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 hydrogencarbonate, a saturated aqueous solution of ammonium chloride and brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by silica gel chromatography using hexane: ethyl acetate (10: 1) as an eluent. Yield 34.4
g (52% over G6).

[Α] ₂₄ ^D + 11.9 ° (c 0.9, CHCl ₃ ), FDMS
m / z 585 M ⁺ ; ¹ H-NMR (500 MHz, CDCl ₃ + D ₂ O) δ 7.
24-7.61 (15H, m), 3.62-3.66 (2H, m), 3.51-3.57 (2
H, 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 G8 (33.5 g, 57.3 mmol) in DMF (300 ml) was added with 60% sodium hydride (5.5 g, about NaH). 138 mmol) and stirred at room temperature for 40 minutes. The reaction was cooled to 0 ° C. and benzyl chloride (15 ml, 120 mmol) was added dropwise. The mixture was stirred for 18 hours while gradually raising the temperature to room temperature. Add ice water (1
(00 ml) to terminate the reaction, followed by extraction with ethyl acetate. The extract was washed three times with a saline solution, and all the organic layers were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain a crude product of G9. Used for the next step without further purification. Yield 42.2 g (96%). The sample for analysis was purified by silica gel chromatography using hexane: ethyl acetate (100: 1) as an elution solvent.

[Α] ₂₄ ^D + 9.8 ° (c 1.0, CHCl ₃ ), FDMS
m / z 738 (MN ₂ ) ⁺ ; ¹ H-NMR (500 MHz, CDCl ₃ ) δ
7.07-7.48 (25H, m), 4.57 (1H, d, J = 11.6 Hz), 4.4
4 (1H, d, J = 11.6 Hz), 4.41 (2H, s), 3.73-3.79 (1
H, m), 3.46-3.56 (2H, m), 3.37 (1H, dd, J = 8.6, 1
0.4 Hz), 1.20-1.64 (26H, m), 0.88 (3H, t, J = 6.7H
z) Synthesis of compounds G10 and G11 A solution of G9 (41.2 g, about 54 mmol) in 1-propanol (250 ml)
Methanol (30 ml), and further 5% palladium on carbon
(4.1 g) and ammonium formate (27.1 g, 4.3 mol) were 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 and dissolved in ethyl acetate.
Wash three times with saturated aqueous sodium bicarbonate solution and brine,
All the organic layers were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain a crude product of G10. Yield 38.9 g
(98%). The obtained G10 was used in the next step without further purification.

In a methylene chloride solution (300 ml) of G10, hexacosanoic acid (22.4 g, 56.5 mmol) and WSC hydrochloride (12.6 g,
64.6 mmol), and the mixture was heated under reflux for 2 hours. 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 0.5 M aqueous hydrochloric acid, brine, saturated aqueous sodium hydrogen carbonate, and brine. Combine all organic layers, dry over anhydrous magnesium sulfate, concentrate under reduced pressure,
A crude product of 1 was obtained. Yield 53.2 g (88%). G11 obtained
Was used for the next step without further purification. The sample for analysis was purified by silica gel chromatography using hexane: ethyl acetate (100: 1) as an elution solvent.

[Α] ₂₄ ^D + 5.3 ° (c 0.4, CHCl ₃ ); F
DMS m / z 1118 M ⁺ ; ¹ H-NMR (500 MHz, CDCl ₃ ) δ7.2
0-7.38 (25H, m), 5.57 (1H, d, J = 9.1 Hz), 4.80 (1
H, d, J = 11.6 Hz), 4.48-4.50 (3H, m), 4.24-4.32 (1
H, m), 3.83 (1H, dd, J = 3.0, 6.7 Hz), 3.43-3.51
(2H, m, H1a), 3.29 (1H, dd, J = 4.3, 9.8 Hz), 1.92
(2H, t, J = 7.3 Hz), 1.28-1.60 (72H, m), 0.88 (6
(H, t, J = 6.7 Hz) Synthesis of compound G12 G11 (52.2 g, about 47 mmol) in methylene chloride (180 ml)
To the mixture was added methanol (36 ml), and then a 10% methanolic hydrochloric acid solution (3.0 ml) was added dropwise, followed by stirring at room temperature 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 the washing 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 3
8.6 g (77% over G9).

[Α] ₂₄ ^D -29.7 ° (c 0.7, CHCl ₃ ); mp
75-76.5 ° C; FDMS m / z 876 M ⁺ ; ¹ H-NMR (500 MHz, CDCl
₃ ) δ7.30-7.47 (10H, m), 6.03 (1H, d, J = 7.9H
z), 4.72 (1H, d, J = 11.6 Hz), 4.66 (1H, d, J = 1
1.6 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, dd
d, J = 4.3, 8.6, 11.6Hz), 1.94-2.05 (2H, m), 1.15-
1.69 (72H, m), 0.88 (6H, t, J = 6.1 Hz). Synthesis of Compound G13 1) 2,3,4,6-Tetra-O-benzyl-D-galactopyranosyl acetate (79.8 g) Was dissolved in a mixture of toluene (160 ml) and isopropyl ether (520 ml), and -10
Cooled to ~ 0 ° C. To this was added an isopropyl ether solution containing 2.0 equivalents of HBr (2.8 mmol / ml, about 100 m2).
l). 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. After transferring the whole amount to a separating funnel, the aqueous layer was discarded and washed twice with a 10% aqueous sodium chloride solution. After concentration under reduced pressure, a syrup of 2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl bromide (Gal-Br) was obtained.

2) A toluene solution (420 ml) of G12 (60.0 g, 68.6 mmol), tetrahexylammonium bromide (89.4 g, 206 mmol), and molecular sieves 4A (60 g)
To this was added DMF (140 ml) and then a solution of GalBr (about 137 mmol) in toluene (250 ml), and the mixture was stirred at room temperature for 72 hours. Methanol (12 ml) was added to the reaction solution, and the mixture was stirred for 2 hours.
After filtration through celite, the extract was washed with a saturated aqueous solution of sodium hydrogen carbonate and brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. Acetonitrile was added to the residue and stirred for 2 hours to obtain a precipitate. The obtained precipitate was dried under reduced pressure to obtain a dry powder. This was purified by silica gel chromatography using hexane: ethyl acetate (8: 1) as an eluting solvent. Yield 70.9 g (74%).

[Α] ₂₄ ^D + 18.8 ° (c 0.9, CHCl ₃ ); mp
74-75 ° C; FDMS m / z 1399 (M + 1) ⁺ ; ¹ H-NMR (500 MHz,
CDCl ₃ ) δ7.21-7.37 (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 (4
H, m), 3.73 (1H, dd, J = 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). (2S, 3S, 4R) -1-O- (α-D-galactopyranosyl) -N-hexa
Cosanoyl-2-amino-1,3,4-octadecanetriol
(KRN7000) G13 (60.0 g, 42.9 mmol) was added and suspended in ethanol (960 ml), and 20% palladium hydroxide (6.0 g) was added as an ethanol suspension. Further, 4-methylcyclohexene (120 ml, 93.5 mmol) serving as a hydrogen source was added, and 4
After heating under reflux for an hour, the mixture was filtered to remove the catalyst. The residue was washed with warm ethanol. A white precipitate obtained by allowing the filtrate to stand at room temperature was filtered and dried under reduced pressure. The obtained powder was suspended in ethanol: water (92: 8, 3.5 L), dissolved by heating with stirring, and left to stand at room temperature to precipitate again. The precipitate was filtered, and the collected cake was dried under reduced pressure to obtain a white powder. Yield 35.0 g (95%).

[Α] ₂₃ ^D + 43.6 ° (c 1.0, pyridine); m
p 189.5-190.5 ° C; negative FABMS m / z 857 (MH) ^- ; I
R (cm ^-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). 13 C-NMR
(125 MHz, C ₅ D ₅ N) δ 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), 30.0
0 (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 or KRN7000
Hematopoietic progenitors in peripheral blood by in vivo administration of both G-CSF
Experiments were carried out using the C57BL / 6 mice were purchased from increased Japan SLC Co., Ltd. of cells. KRN7000 was used in the following experiments as a representative of glycoside compounds. G-CSF includes sugar-free G-CSF
(Generic name: Gran, manufactured by Kirin Brewery) was used.

C57BL / 6 mice (6 weeks old, female) were
The experiment was divided into four groups. That is, (A) control (untreated) group. (B) KRN7000 (100μg / kg) for day -4
G-CSF (100 μg / kg) on days -3, -2,
-1 and 0 (4 hours before blood collection) were administered subcutaneously (KR
(N7000 + G-CSF group), (C) KRN7000 (100μg / kg) on day-4
Group (KRN7000 group), (D) G-CSF (100
μg / 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 heart using heparin, and the blood of each group was pooled. After diluting the blood twice using RPMI 1640 medium, the blood was overlaid on Lympholite M (Cedalane) and centrifuged to obtain a mononuclear cell layer from which red blood cells had been removed. 8 x 10 mononuclear cells obtained in this way
Diluted to ⁵ cells / ml, cultured in 1 ml of 0.8% methylcellulose semi-solid medium containing 20 ng / ml of mouse IL-3 and 2 U / ml of erythropoietin for 6 days, and formed leukocyte cell mass Hematopoietic progenitor cells (CFU-
GM). The number of CFU-GM per 1 ml of the original peripheral blood was calculated from the dilution. Table 1 shows the results.

[0051]

[Table 1] CFU-GM (cells / ml peripheral blood) (A) Control 58 ± 22 (B) KRN7000 + G-CSF 1421 ± 246 ^{a, b, c} (C) KRN7000 210 ± 28 ^a (D) G- CSF 658 ± 7 ^{a, b} a: Indicates that there is a significant difference at 5% risk relative to control.

B: Significantly different from KRN7000 at a risk rate of 5%.

C: Significantly different from G-CSF at a risk rate of 5%.

As shown in Table 1, the administration of KRN7000 and the administration of G-CSF significantly increased CFU-GM in peripheral blood. Furthermore, by co-administering KRN7000 and G-CSF, CFU-GM in peripheral blood can be reduced to KRN7000 or G-CSF.
-Significantly increased compared to CSF alone.

(Pharmacological test 2): Inject KRN7000 and G-CSF
Donor mouse peripheral blood was irradiated with whole body radiation.
Effect on survival of the recipient mice C57BL / 6 mice (6 weeks old, female) were the donor mice,
The experiment was conducted in the following seven groups. That is, (A) control (untreated) group. (B) KRN7000 (100μg / kg)
Intravenous administration on day -4 and G-CSF (100μg / kg) on days-
Groups administered subcutaneously at 3, 2, -1, and 0 (4 hours before blood collection) (KRN7000 + G-CSF group), (C) KRN7000 (100 μg / kg)
Iv on day -4 (KRN7000 group), (D) G-
CSF (100 μg / kg) was administered subcutaneously on days -3, -2, -1, and 0 (4 hours before blood collection) (G-CSF sc group), and (E) G-CSF as a positive control (200 μg / kg) and SCF (25 μg / kg) administered intravenously 7 times from days -7 to -1 (G-CSF + SCF group), (F) G-CSF (200 μg / kg)
Administered intravenously 7 times from days -7 to -1 (G-CSFiv group)
, And (G) SCF (25 μg / kg) in days -7 to -1
Drugs were administered in seven groups of intravenous administration (SCF group), blood was collected from donor mice of each experimental group on day 0, and the blood was pooled. Since the above group (E) is known as a drug that effectively increases peripheral blood stem cells, an experiment was performed as a positive control group to compare the effect with the group (B).

Recipient mice (6 weeks old, female C57
9 Gy (900 rad) against 10 BL / 6 mice per group)
X-ray whole body irradiation was performed, and peripheral blood (100 μl / mouse) collected from each group as described above within 2 hours after the irradiation was transferred to recipient mice via vein. Then, the survival of each recipient mouse was observed.

FIG. 1 shows the results.

As shown in FIG. 1, (A) control group,
All recipient mice to which peripheral blood of (C) KRN7000 group, (D) G-CSF sc group, and (G) SCF group had been transferred 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) G-CSF + SCF
One and two survivors were observed, respectively, in the recipient mice to which the group of peripheral blood had been transferred. And (B) KR
Five (50%) of the recipient mice to which the peripheral blood of the N7000 + G-CSF group had been transferred survived. This result suggests that among the above seven groups, the most hematopoietic progenitor cells are present in the peripheral blood of the donor mice to which KRN7000 + G-CSF was administered.

(Pharmacological experiment 3): Inject KRN7000 and G-CSF
Donor mouse peripheral blood was irradiated with whole body radiation.
Action then for survival of the recipient mice were, using BALB / c female mice in which the mouse and the system is different from (6 weeks old) was subjected to the same experiment as pharmacological experiments 1.

FIG. 2 shows the results.

As shown in FIG. 2, (A) a control group,
All recipient mice to which peripheral blood of (C) KRN7000 group and (G) SCF group had been transferred died within 15 days after whole body irradiation with X-rays. Also, 40 days after whole body irradiation with X-rays,
In each of the recipient mice to which the peripheral blood of the (D) G-CSF sc group and the (F) G-CSF iv group had been transferred, survival of one mouse was observed. (E) In the recipient mice to which the peripheral blood of the G-CSF + SCF group was transferred, four survivors were observed, respectively, while (B) the peripheral blood of the KRN7000 + G-CSF group was transferred. Six (60%) of the recipient mice survived. This result indicates that the hematopoietic progenitor cells in the peripheral blood of the donor mice to which KRN7000 + G-CSF was administered were the most significant among the above seven groups even when mice of a different strain from those used in pharmacological experiment 1 were used. It strongly suggests that there are many.

(Pharmacological experiment 4): Inject KRN7000 and G-CSF
Of hematopoietic progenitor cells in peripheral blood of a given donor mouse
Investigation on Engraftment and Proliferation in the Entrance Mice Next, it was examined whether or not hematopoietic progenitor cells in peripheral blood transplanted from donor mice would engraft and proliferate in recipient mice. In other words, using male BALB / c mice as donor mice, the above seven groups were prepared, and peripheral blood collected from each group was transferred to female BALB / c mice that had been subjected to whole body irradiation with 9 Gy of radiation. Populated.

First, the survival time of the recipient mice to which the peripheral blood of each group was transferred is shown in FIG.

As shown in FIG. 3, (A) control group,
(C) KRN7000 group, (D) G-CSF sc group, (E) G-CSF + SCF group,
All the recipient mice to which the peripheral blood of the (F) G-CSF iv group and the (G) SCF group were transferred died within 40 days after whole body irradiation with X-rays. However, surprisingly, even after 50 days of whole-body irradiation, 9 (90%) of the (B) KRN7000 + G-CSF group recipient mice that had peripheral blood transferred survived. Was. Furthermore, these mice in group (B)
All animals survived 150 days later. The result is
In experiments using LB / c mice, pharmacological experiments 2 and 3 described above showed that among the above 7 groups, hematopoietic progenitor cells were most abundant in peripheral blood of donor mice to which KRN7000 + G-CSF had been administered. I support the results.

Next, it was examined whether hematopoietic progenitor cells in the peripheral blood of the donor mouse survived in the recipient mouse and further proliferated. Male and female BALB / c
From mice (positive and negative controls) and individual surviving mice (females) in group (B), irradiated 60, 90, 120,
And 150 days later, orbital blood was collected, and male (
(Donor) The presence or absence of mouse-derived blood cells was determined by PC
The study was performed using the R method (Yan et al. (1994) Blood, 84, 795-799). That is, the genomic DN is obtained from the blood of each mouse described above.
A was prepared and amplified by the PCR method, followed by electrophoresis, and Srylo present on the male Y chromosome in each genomic DNA.
The presence or absence of cus was examined. FIG. 4 shows positive and negative control male and female mice, and 150 days after irradiation.
(B) shows the results of PCR performed using the blood of the surviving mice (9 mice) in the group.

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 male mice.
The presence of Sry locus derived from the male Y chromosome was clearly observed in the blood of all the surviving mice in the group (B). Similar results were obtained in experiments using blood of recipient mice 60, 90, and 120 days after irradiation. These results indicate that donor mouse-derived blood cells have engrafted in the recipient mouse, and that the donor mouse-derived blood cells have proliferated in the recipient mouse 150 days after irradiation.

[0067]

[Sequence List] SEQ ID NO: 1 Sequence length: 174 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu 1 5 10 15 Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu 20 25 30 Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu 35 40 45 Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser 50 55 60 Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His 65 70 75 Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile 80 85 90 95 Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala 100 105 110 Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala 115 120 125 Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala 130 135 140 Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser 145 150 155 Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro 160 165 170 174

[Brief description of the drawings]

FIG. 1 shows the survival time (No. 1) of recipient mice.

FIG. 2 shows the survival time (part 2) of recipient mice.

FIG. 3 shows the survival time (part 3) of recipient mice.

FIG. 4 is an electrophoretogram showing engraftment and proliferation of hematopoietic progenitor cells.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4C057 BB02 BB03 BB04 CC03 CC04 DD02 JJ09 JJ11 4C086 AA01 AA02 EA05 FA03 GA16 MA01 MA02 MA04 MA06 MA17 MA66 NA14 ZB02 ZB21 ZB22 ZB26 ZB27 ZC75

Claims (20)

[Claims] 1. The following formula (A): Wherein R ₁ is H or OH; X is an integer of 7 to 25; R ₂ is a substituent defined by any of the following (a) to (e), wherein Y is 5 It is -17 _{integer; (a) -CH 2 (CH} 2) Y CH 3 (b) -CH (OH) (CH 2) Y CH 3 (c) -CH (OH) (CH 2) Y CH ( _{CH 3) 2 (d) -CH} = CH (CH 2) Y CH 3 (e) -CH (OH) (CH 2) Y CH (CH 3) CH
_{One of 2} CH ₃ R ₃ and R ₄ is H, the other is H,
OH, NH ₂ , NHCOCH ₃ or One of R ₅ and R ₆ is H, the other is OH, One of R ₇ and R ₈ is H, the other is OH, R ₉ is H, CH ₃ , CH ₂ OH or A peripheral blood stem cell increasing agent in peripheral blood stem cell transplantation therapy with G-CSF, comprising at least one glycoside compound represented by the formula: or a salt thereof as an active ingredient. 2. The glycoside compound represented by the following formula (B): Wherein R ₁ , X and R ₂ have the same meanings as in claim 1; R _{3 to} R ₉ are substituents selected so as to correspond to any of the following i) to v): I) R ₃ , R ₆ and R ₈ are all H, and R ₄ is H, OH, NH ₂ , NHCOCH ₃ or R ₅ is OH, R ₇ is OH; R ₉ is H, CH ₃ , CH ₂ OH or Ii) R ₃ , R ₆ and R ₇ are all H, R ₄ , R ₅ and R ₉ are each as defined in i), and R ₈ is OH; Iii) R ₄ , R ₆ and R ₇ are all H, R ₅ and R ₉ are each as defined in i), and R ₃ is H, OH, NH ₂ , NHCOCH ₃ or Embedded image R ₈ is OH or Iv) R ₄ , R ₅ and R ₇ are all H, R ₃ , R ₆ and R ₈ are all OH and R ₉ is H, CH ₃ or CH ₂ OH Or v)
R ₃ , R ₅ and R ₇ are all H, R ₄ , R ₆ and R ₈ are all OH and R ₉ is H, CH ₃ or CH ₂ OH]; The agent for increasing peripheral blood stem cells according to claim 1. 3. The glycoside compound according to claim 1, wherein R ₃ , R ₆
And R ₈ are all H; R ₄ , R ₅ and R ₇ are all OH;
_{3. The} agent for increasing peripheral blood stem cells according to claim _{2, wherein 9} is CH ₂ OH. 4. The peripheral blood stem cell increasing agent according to claim ₂ , wherein R ₂ in the glycoside compound is a substituent (b), (c) or (e). 5. The peripheral blood stem cell increasing agent according to claim 4, wherein in the glycoside compound, R ₁ is H and R ₂ is a substituent (b). 6. The peripheral blood stem cell increasing agent according to claim 5, wherein in the glycoside compound, the configuration of the hydroxyl group in the substituent (b) is R. 7. In the glycoside compound, X is 21 to
The agent for increasing peripheral blood stem cells according to claim 5, wherein the number is 25 and Y is 11 to 15. 8. The method according to claim 1, wherein the glycoside compound is (2S, 3S, 4)
R) -1- (α-D-galactopyranosyloxy) -2
The agent for increasing peripheral blood stem cells according to claim 6 or 7, which is -hexacosanoylamino-3,4-octadecanediol. 9. The agent for increasing peripheral blood stem cells according to claim 1, 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 according to claim 1 or a salt thereof, and a pharmaceutically acceptable carrier. 11. The method according to claim 11, wherein the glycoside compound is G-CSF.
The pharmaceutical composition for increasing peripheral blood stem cells according to claim 10, which is contained at a weight ratio of 1:10 to 10: 1. 12. The method according to claim 12, wherein the glycoside compound is G-CSF.
The pharmaceutical composition for increasing peripheral blood stem cells according to claim 11, which is contained in a substantially equal amount by weight with respect to the total amount of the peripheral blood stem cells. 13. The pharmaceutical composition for increasing peripheral blood stem cells according to claim 10, wherein the G-CSF is human G-CSF. 14. The G-CSF, wherein one or more amino acid residues are deleted, substituted, added and / or inserted in the amino acid sequence of SEQ ID NO: 1, and the G-CSF is G-CSF.
The pharmaceutical composition for increasing peripheral blood stem cells according to any one of claims 10 to 12, which is a protein having F activity. 15. A pharmaceutical kit for increasing peripheral blood stem cells, comprising G-CSF and at least one glycoside compound according to claim 1 or a salt thereof. 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, wherein the G-CSF is human G-CSF. 18. The G-CSF, wherein one or more amino acid residues are deleted, substituted, added and / or inserted in the amino acid sequence of SEQ ID NO: 1, and the G-CSF is G-CSF.
16. A protein having F activity.
7. The pharmaceutical kit for increasing peripheral blood stem cells according to 6. 19. The method according to claim 19, wherein the glycoside compound is the G-CSF.
The pharmaceutical kit for increasing peripheral blood stem cells according to any one of claims 15 to 18, which is contained at a weight ratio of 1:10 to 10: 1. 20. The glycoside compound, wherein the G-CSF
20. The pharmaceutical kit for increasing peripheral blood stem cells according to claim 19, wherein the pharmaceutical kit is contained in an approximately equal amount by weight with respect to the total amount of peripheral blood stem cells.