JP2003192692A - Vegf antisense compound - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new VEGF antisense compound effective for suppressing the action of VEGF and useful for the treatment of diseases in which the vascularization or vascular permeation promotion by VEGF is participated. <P>SOLUTION: The compound is expressed by general formula H0-B1-B2-B3-B4- B5-B6-B7-B8-B9-B10-B11-B12-B13-B14-B15-B16-B17-B18-B19-B20-B21-B22-H (I) (B1 to B3, B8, B11 and B20 are each independently C<SP>p</SP>or the like; B4, B5, B7, B9, B12, B14 to B16 and B21 are each independently A<SP>p</SP>or the like; B6, B10, B13 and B17 are each independently G<SP>p</SP>or the like; B18 and B19 are each independently T<SP>p</SP>or the like; and B22 is T<SP>t</SP>or the like) and its pharmacologically permissible salt. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel VEGF antisense compound which suppresses the action of VEGF and is effective in treating a disease associated with angiogenesis or vascular permeability enhancement by VEGF.

[0002]

2. Description of the Related Art VEGF (vascular e growth factor)
ndothelial growth factor) is a growth factor that acts specifically on vascular endothelial cells and promotes angiogenesis. Angiogenesis is performed by vascular endothelial cells being stimulated by growth factors, physiologically active substances or mechanical damage to proliferate, and plays an important role in development, wound healing, inflammation and the like. For solid guns, the diameter of the cancer tissue is 1-2 mm
In order to grow beyond this, it is necessary for new blood vessels to extend from the existing blood vessels to reach the cancer tissue in order to supply oxygen and nutrients necessary for the tumor (J. Folkman,
J. Natl. Cancer Inst., 82: 4 (1990)), and it is known that the growth of cancer tissue is explosively accelerated when the blood vessel reaches the cancer tissue. Done. VEGF is believed to play a central role in tumor angiogenesis. Regarding the involvement of VEGF in cancer, 1) Many cancer cells secrete VEGF (S.
Kondoet al., Bichem. Biophys. Res. Commun., 194: 1
234 (1993)), 2) When a cancer tissue section is stained with an anti-VEGF antibody, the cancer tissue and the new blood vessels around it are strongly stained (HF Dvorak et al., J. Exp. Med. 174: 1275 (199).
1)., LF Brown at al., Cancer Res., 53: 4727 (199
3)), 3) In mice in which one of the VEGF receptors is genetically inactivated, the growth of transplanted cancer is suppressed (B. M
illauer et al., Nature, 367: 576 (1994)), 4) anti-VEG
F-neutralizing antibody shows antitumor activity against cancer-bearing mice (KJ Kim et al., Nature, 362: 841 (1993), S. Kon.
do et al., Bichem. Biophys. Res. Commun., 194: 1234
(1993)) etc. have been reported. Based on the above facts, it is considered that VEGF secreted by cancer cells plays a major role in tumor angiogenesis, and it is expected that substances that suppress VEGF angiogenic activity may suppress cancer growth and metastasis. Has been done. In addition, diabetic retinopathy, which is an eye disease, is recognized as a complication in nearly half of diabetic patients. In diabetic retinopathy, shortage of oxygen promotes the formation of microcapillaries,
It is believed that this eventually ruptures and bleeds to form scar tissue, which eventually leads to retinal detachment and blindness. Therefore,
It is expected that the severity of retinopathy can be suppressed by suppressing angiogenesis. Miller et al. (Miller et al .:
Am. J. Pathol. 145, pp. 574-584 (1994)) have reported that VEGF is closely related to the development of proliferative retinopathy from the results of experiments using monkeys. Therefore, substances that suppress the angiogenic activity of VEGF are considered to be useful for the prevention or treatment of diabetic retinopathy. Rheumatoid arthritis, psoriasis, hemangiomas, scleroderma,
Angiogenic diseases such as glaucoma are accompanied by pathological neovascularization, which is one of the main symptoms (J. Folkma.
n, N. Engle. J. Med., 320: 1211 (1989)). Therefore VEG
A substance that suppresses the action of F may be used for the treatment of various diseases related to the above-mentioned angiogenesis. Also,
It is known that VEGF is involved in not only angiogenesis but also vascular hyperpermeability. VEGF-induced vascular hyperpermeability is associated with cerebral edema in patients with cancerous ascites and ischemia-reperfusion injury (Ni
cholas van Bruggen et al: J. Clin. Invest. 104: 1613-1
620. (1999)) and other pathological conditions have been implicated. Therefore, a substance that suppresses the action of VEGF is considered to be useful for the treatment of the above-mentioned diseases in which angiogenesis or vascular permeability enhancement by VEGF is involved.

As such a compound, the following sequence 5'-cccaagacagcagaaagttcat-3 '(SEQ ID NO: in the sequence listing)
1), and an antisense molecule (hereinafter referred to as “compound A”) in which each base is bound by a phosphorothioate is known (J. Biol. Chem. (1995) 270, 28316-2832).
Four).

However, the compound A does not have a sufficient VEGF inhibitory activity, and a compound having a stronger VEGF inhibitory activity has been expected.

[0005]

[Non-Patent Document 1] J. Biol. Chem. (1995) 270, 28316-2.
8324

[0006]

DISCLOSURE OF THE INVENTION The inventors of the present invention have studied the substance that suppresses the action of VEGF and found that it has the general formula (I)
It was found that the compound having the formula (1) has a strong VEGF inhibitory activity, and is useful for the treatment of diseases associated with VEGF-induced angiogenesis or vascular permeability enhancement, and completed the present invention.

[0007]

[Means for Solving the Problem] The following general formula (I) HO-B1-B2-B3-B4-B5-B6-B7-B8-B9-B10-B11-B12-B13-B14-B15-B16-B17 -B18-B19-B2 0-B21-B22-H (I) (wherein B1 to B3, B8, B11 and B20 are the same or different,

[0008]

[Chemical 16]

A group represented by (hereinafter referred to as "C ^p "), a formula

[0010]

[Chemical 17]

A group represented by (hereinafter referred to as "C ^s ") or a general formula C ^e

[0012]

[Chemical 18]

(Wherein l represents an integer of 1 to 5), B4, B5, B7, B9, B12, B14 to B16 and B21 are the same or different and

[0014]

[Chemical 19]

A group represented by (hereinafter referred to as "A ^p "), a formula

[0016]

[Chemical 20]

A group represented by (hereinafter referred to as "A ^s ") or a general formula A ^e

[0018]

[Chemical 21]

(Wherein, m represents an integer of 1 to 5), B6, B10, B13 and B17 are the same or different and

[0020]

[Chemical formula 22]

A group represented by (hereinafter referred to as "G ^p "), a formula

[0022]

[Chemical formula 23]

A group represented by (hereinafter referred to as "G ^s "), a formula

[0024]

[Chemical formula 24]

A group represented by (hereinafter referred to as "G ^m ") or a general formula

[0026]

[Chemical 25]

(Wherein n represents an integer of 1 to 5), B18 and B19 are the same or different and

[0028]

[Chemical formula 26]

A group represented by (hereinafter referred to as "T ^p "), a formula

[0030]

[Chemical 27]

A group represented by (hereinafter referred to as "T ^s ") or a general formula

[0032]

[Chemical 28]

(In the formula, o represents an integer of 1 to 5), B22 represents a group

[0034]

[Chemical 29]

A group represented by (hereinafter referred to as "T ^t ") or a general formula T ^et

[0036]

[Chemical 30]

(In the formula, p represents an integer of 1 to 5) and represents a group. However, B1 to B3, B8, B11 and B20 are the same or different and are C ^p or C ^s , and B4, B5, B7, B9, B1.
2, B14 to B16 and B21 are the same or different and A ^p or A ^s, B6, B10, B13 and B17 are the same or different and G ^p, G ^s also G
^s is G ^m , and B18 and B19 are the same or different and T ^p or T ^s
And B22 is T ^t , it is excluded. And a pharmacologically acceptable salt thereof.

In the compound represented by the above general formula (I) or a pharmacologically acceptable salt thereof, preferably,
(1) B1 to B3, B8, B11 and B20 are the same or different and C ^s
Or C ^e , B4, B5, B7, B9, B12, B14 to B16 and B
21 is the same or different and is A ^s or A ^e , B6, B10, B13 and B17 are the same or different and is G ^s , G ^m or G ^e , and B
A compound in which 18 and B19 are the same or different and are T ^s or T ^e ,
And a pharmacologically acceptable salt thereof (2) B1 to
A compound in which B3 is C ^e , a pharmacologically acceptable salt thereof, (3) a compound in which B4 is A ^e , a pharmacologically acceptable salt thereof, and (4) B5 is A ^e A compound, a pharmacologically acceptable salt thereof, (5) a compound wherein B6 is G ^m or G ^e , a pharmacologically acceptable salt thereof, (6) a compound wherein B7 is A ^e , It is a pharmacologically acceptable salt, (7)
A compound wherein B8 is C ^e , a pharmacologically acceptable salt thereof, (8) a compound wherein B22 is T ^et , B21 is A ^e , and B20 is C ^e , a drug thereof A pharmaceutically acceptable salt, (9) a compound in which B19 is T ^e , a pharmacologically acceptable salt thereof, and (10) a compound in which B18 is T ^e , a pharmacologically acceptable salt thereof. And (11) B17 is G ^m or G
a compound which is ^e , a pharmacologically acceptable salt thereof,
(12) A compound in which B16 is A ^e , a pharmacologically acceptable salt thereof, (13) a compound in which B15 is A ^e , a pharmacologically acceptable salt thereof, and (15) B14 is A 14. a compound of ^e , a pharmacologically acceptable salt thereof, (16) l, m,
a compound in which n, o and p are the same or different and are 1 or 2,
It is a pharmacologically acceptable salt of (17) l, m, n, o
And p are the same and are 1 or 2, a pharmacologically acceptable salt thereof, wherein (18) l, m, n, o and p
Is a compound or a pharmacologically acceptable salt thereof.

Further, the above (2) to (7) and (8) to
The numbers in (14) and (15) to (17) are larger.
According to the formula, more preferable compounds are shown in the general formula (I)
, B1 to B22 are arbitrarily selected from (1), and B1 to B22
8 is arbitrarily selected from (2) to (7), and B14 to B22 are
Select arbitrarily from (8) to (14), l, m, n, o and
p is arbitrarily selected from (16) to (18), and
And compounds obtained by arbitrarily combining these
From the following (Compound Group 1),
Selected compounds and their pharmacologically acceptable salts
Most preferably, a compound selected from (Compound Group 2)
The substance and its pharmacologically acceptable salts. (Compound Group 1) HO-C^e2-C^e2-C^e2-A^s-A^s-G^s-A^s-C^s-A^s-G^s-
C^s-A^s-G^s-A^s-A^s-A^s-G^s-T^s-T^s-C^e2-A^e2-T ^e2t-H, HO-C^e2-
C^e2-C^e2-A^e2-A^s-G^s-A^s-C^s-A^s-G^s-C^s-A^s-G^s-A^s-A^s-A^s-G^s
-T^s-T^e2-C^e2-A^e2-T^e2t-H, HO-C^e2-C^e2-C^e2-A^e2-A^e2-G^s-
A^s-C^s-A^s-G^s-C^s-A^s-G^s-A^s-A^s-A^s-G^s-T^e2-T^e2-C^e2-A ^e2-T
^e2t-H, HO-C^e2-C^e2-C^e2-A^e2-A^e2-G^e2-A^s-C^s-A^s-G^s-C^s-A
^s-G^s-A^s-A^s-A^s-G^e2-T^e2-T^e2-C^e2-A^e2-T^e2t-H, HO-C^e2-C
^e2-C^e2-A^e2-A^e2-G^m-A^s-C^s-A^s-G^s-C^s-A^s-G^s-A^s-A^s-A^s-G^m
-T^e2-T^e2-C^e2-A ^e2-T^e2t-H, HO-C^e2-C^e2-C^e2-A^e2-A^e2-G
^e2-A^s-C^s-A^s-G^s-C^s-A^s-G^s-A^s-A^s-A^e2-G^e2-T^e2-T^e2-C^{e 2}-
A^e2-T^e2t-H, HO-C^e2-C^e2-C^e2-A^e2-A^e2-G^m-A^s-C^s-A^s-G^s-
C^s-A^s-G^s-A^s-A^s-A^e2-G^m-T^e2-T^e2-C^e2-A^e2-T^e2t-H, HO-C
^e2-C^e2-C^e2-A^e2-A^e2-G^e2-A^e2-C^s-A^s-G^s-C^s-A^s-G^s-A^s-A^s
-A^e2-G^e2-T^e2-T^e2-C ^e2-A^e2-T^e2t-H, HO-C^e2-C^e2-C^e2-A
^e2-A^e2-G^m-A^e2-C^s-A^s-G^s-C^s-A^s-G^s-A^s-A^s-A^e2-G^m-T^e2-T
^e2-C^e2-A^e2-T^e2t-H, HO-C^e2-C^e2-C^e2-A^e2-A^e2-G^e2-A^e2-
C^s-A^s-G^s-C^s-A^s-G^s-A^s-A^e2-A^e2-G^e2-T^e2-T^e2-C^e2-A^e2-T
^e2t-H, HO-C^e2-C^e2-C^e2-A^e2-A^e2-G^m-A^e2-C^s-A^s-G^s-C^s-A
^s-G^s-A^s-A^e2-A^e2-G^m-T^e2-T^e2-C^{e 2}-A^e2-T^e2t-H, HO-C^e2-
C^e2-C^e2-A^e2-A^e2-G^e2-A^e2-C^e2-A^s-G^s-C^s-A^s-G^s-A^s-A^e2-
A^e2-G^e2-T^e2-T^e2-C^e2-A^e2-T^e2t-H, HO-C^e2-C^e2-C^e2-A^e2
-A^e2-G^m-A^e2-C^e2-A^s-G^s-C^s-A^s-G^s-A^s-A^e2-A^e2-G^m-T^e2-T
^e2-C ^e2-A^e2-T^e2t-H, HO-C^e2-C^e2-C^e2-A^e2-A^e2-G^e2-A^e2-
C^e2-A^s-G^s-C^s-A^s-G^s-A^e2-A^e2-A^e2-G^e2-T^e2-T^{e 2}-C^e2-A^e2
-T^e2t-H, HO-C^e2-C^e2-C^e2-A^e2-A^e2-G^m-A^e2-C^e2-A^s-G^s-C
^s-A^s-G^s-A^e2-A^e2-A^e2-G^m-T^e2-T^e2-C^e2-A^e2-T^e2t-H, HO-
C^e1-C^e1-C^e1-A^s-A^s-G^s-A^s-C^s-A^s-G^s-C^s-A^s-G^s-A^s-A^s-A^s
-G^s-T^s-T^s-C^e1-A^e1-T ^e1t-H, HO-C^e1-C^e1-C^e1-A^e1-A^s-G^s
-A^s-C^s-A^s-G^s-C^s-A^s-G^s-A^s-A^s-A^s-G^s-T^s-T^e1-C^e1-A^e1-T
^e1t-H, HO-C^e1-C^e1-C^e1-A^e1-A^e1-G^s-A^s-C^s-A^s-G^s-C^s-A^s
-G^s-A^s-A^s-A^s-G^s-T^e1-T^e1-C^e1-A ^e1-T^e1t-H, HO-C^e1-C^e1
-C^e1-A^e1-A^e1-G^e1-A^s-C^s-A^s-G^s-C^s-A^s-G^s-A^s-A^s-A^s-G^e1
-T^e1-T^e1-C^e1-A^e1-T^e1t-H, HO-C^e1-C^e1-C^e1-A^e1-A^e1-G^m
-A^s-C^s-A^s-G^s-C^s-A^s-G^s-A^s-A^s-A^s-G^m-T^e1-T^e1-C^e1-A ^e1-
T^e1t-H, HO-C^e1-C^e1-C^e1-A^e1-A^e1-G^e1-A^s-C^s-A^s-G^s-C^s-
A^s-G^s-A^s-A^s-A^e1-G^e1-T^e1-T^e1-C^{e 1}-A^e1-T^e1t-H, HO-C^e1
-C^e1-C^e1-A^e1-A^e1-G^m-A^s-C^s-A^s-G^s-C^s-A^s-G^s-A^s-A^s-A^e1
-G^m-T^e1-T^e1-C^e1-A^e1-T^e1t-H, HO-C^e1-C^e1-C^e1-A^e1-A^e1
-G^e1-A^e1-C^s-A^s-G^s-C^s-A^s-G^s-A^s-A^s-A^e1-G^e1-T^e1-T^e1-C
^e1-A^e1-T^e1t-H, HO-C^e1-C^e1-C^e1-A^e1-A^e1-G^m-A^e1-C^s-A^s
-G^s-C^s-A^s-G^s-A^s-A^s-A^e1-G^m-T^e1-T^e1-C^e1-A^e1-T^e1t-H,
HO-C^e1-C^e1-C^e1-A^e1-A^e1-G^e1-A^e1-C^s-A^s-G^s-C^s-A^s-G^s-A
^s-A^e1-A^e1-G^e1-T^e1-T^e1-C^e1-A^e1-T^e1t-H, HO-C^e1-C^e1-C
^e1-A^e1-A^e1-G^m-A^e1-C^s-A^s-G^s-C^s-A^s-G^s-A^s-A^e1-A^e1-G^m-
T^e1-T^e1-C^{e 1}-A^e1-T^e1t-H, HO-C^e1-C^e1-C^e1-A^e1-A^e1-G^e1
-A^e1-C^e1-A^s-G^s-C^s-A^s-G^s-A^s-A^e1-A^e1-G^e1-T^e1-T^e1-C^e1
-A^e1-T^e1t-H, HO-C^e1-C^e1-C^e1-A^e1-A^e1-G^m-A^e1-C^e1-A^s-
G^s-C^s-A^s-G^s-A^s-A^e1-A^e1-G^m-T^e1-T^e1-C ^e1-A^e1-T^e1t-H,
HO-C^e1-C^e1-C^e1-A^e1-A^e1-G^e1-A^e1-C^e1-A^s-G^s-C^s-A^s-G^s-
A^e1-A^e1-A^e1-G^e1-T^e1-T^{e 1}-C^e1-A^e1-T^e1t-H and HO-C
^e1-C^e1-C^e1-A^e1-A^e1-G^m-A^e1-C^e1-A^s-G^s-C^s-A^s-G^s-A^e1-A
^e1-A^e1-G^m-T^e1-T^e1-C^e1-A^e1-T^e1t-H. (Compound Group 2) HO-C^e2-C^e2-C^e2-A^e2
-A^e2-G^e2-A^e2-C^e2-A^s-G^s-C^s
-A^s-G^s-A^e2-A^e2-A^e2-G^e2-T
^e2-T^{e Two}-C^e2-A^e2-T^e2t-H, HO-C^e2
-C^e2-C^e2-A^e2-A^e2-G^m-A^e2-C^e2-A^s-G^s-C^s-A^s-G^s-A^e2-A^e2
-A^e2-G^m-T^e2-T^e2-C^e2-A^e2-T^e2t-H, HO-C^e1-C^e1-C^e1-A^e1
-A^e1-G^e1-A^e1-C^e1-A^s-G^s-C^s-A^s-G^s-A^e1-A^e1-A^e1-G^e1-T
^e1-T^{e 1}-C^e1-A^e1-T^e1t-H and HO-C^e1-C^e1-C^e1-A^e1-A
^e1-G^m-A^e1-C^e1-A^s-G^s-C^s-A^s-G^s-A^e1-A^e1-A^e1-G^m-T^e1-T
^e1-C^e1-A^e1-T^e1t-H.

In the above description and in the present specification, A ^e1 , G
^e1 , C ^e1 , T ^e1 , T ^e1t , A ^e2 , G ^e2 , C ^{e 2} , T ^e2 and T ^e2t represent groups having the chemical structures shown below.

[0041]

[Chemical 31]

The term "pharmaceutically acceptable salt" refers to the salt of the compound of the present invention, and therefore it refers to a salt thereof. Such salt is preferably sodium salt, potassium salt, Alkali metal salts such as lithium salts, alkaline earth metal salts such as calcium salts, magnesium salts,
Metal salts such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts and cobalt salts; inorganic salts such as ammonium salts,
t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N '. Amine salts such as dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylammonium salt, tris (hydroxymethyl) aminomethane salt; Inorganic acid salts such as hydrohalogenates, hydrochlorides, hydrobromides, hydroiodides, halogenated hydrohydrogenates, nitrates, perchlorates, sulfates, phosphates; methanesulfonic acid Salt, trifluoromethanesulfonate, ethanesulfonate Lower alkane sulfonates such as, benzene sulfonate, aryl sulfonates such as p-toluene sulfonate, acetate, malate, fumarate, succinate, citrate, Examples thereof include organic acid salts such as tartrate, oxalate and maleate; and amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamic acid salt and asparaginic acid salt.

The compound (I) of the present invention can also exist as a hydrate, and the present invention includes those hydrates.

Another aspect of the present invention is a medicament containing the compound (I) of the present invention, preferably for the treatment and prevention of diseases associated with VEGF-induced angiogenesis or vascular hyperpermeability. The drug to be used, more preferably cancer,
It is a drug used for the treatment and prevention of diabetic retinopathy, rheumatoid arthritis, psoriasis, hemangiomas, scleroderma, neovascular glaucoma, cerebral edema during cancerous ascites retention or ischemia-reperfusion injury.

[0045]

BEST MODE FOR CARRYING OUT THE INVENTION The compound (I) of the present invention is DNA
A synthesizer, for example, Model 392 based on the phosphoramidite method of Perkin Elmer Co., Ltd., was used (Nucleic Ac
It can be synthesized according to the method described in ids Research, 12, 4539 (1984)). As the phosphoramidite reagent used at that time, commercially available reagents can be used for natural nucleosides and 2′-O-methylguanosine (G ^m ), and l, m, n, o for other nucleosides. as well as
For nucleosides in which p is 1, WO99 / 14226, l, m,
WO 00 / for nucleosides in which n, o and p are 2 to 5
It can be obtained according to the method described in 47599.

In addition to sulfur, tetraethylthiuram disulfide (T
ETD, Applied Biosystems), Beaucage reagent (Glen Research), and other reagents that react with trivalent phosphoric acid to form thioates are used (Tetarhedron Le).
tters, 32, 3005 (1991), J. Am. Chem. Soc., 112, 125
3 (1990)), and a thioate derivative can be obtained.

The compound (I) of the present invention or a pharmacologically acceptable salt thereof exhibits a VEGF inhibitory activity. In addition, the compound (I) of the present invention is excellent in absorption, biodistribution, half-life in blood and other pharmacokinetics, and has low toxicity to organs such as kidney and liver.
Therefore, the compound (I) of the present invention is useful, for example, as a medicine, particularly as a medicine for treating or preventing various diseases associated with angiogenesis or vascular hyperpermeability.

When the compound of the present invention is used as a prophylactic or therapeutic agent for the above-mentioned diseases, the compound having the general formula (I) or a pharmacologically acceptable salt or ester thereof is itself used. Alternatively, by mixing with an appropriate pharmacologically acceptable excipient, diluent, etc., tablets, capsules,
It can be administered orally by granules, powders, syrups and the like, or parenterally by injection, suppositories, patches, external preparations and the like.

These formulations include excipients such as lactose,
Sugar derivatives such as white sugar, glucose, mannitol, sorbitol; corn starch, potato starch,
Starch derivatives such as α-starch and dextrin; Cellulose derivatives such as crystalline cellulose; Gum arabic; Dextran; Organic excipients such as pullulan; and light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and aluminometasilicate. Inorganic excipients such as silicate derivatives such as magnesium; phosphates such as calcium hydrogen phosphate; carbonates such as calcium carbonate; sulfates such as calcium sulfate. ), Lubricants (eg metal stearates such as stearic acid, calcium stearate, magnesium stearate; talc; colloidal silica; waxes such as beeswax and gay wax; boric acid; adipic acid; sodium sulfate, etc.) Sulfate; glycol; fumaric acid; sodium benzoate; DL
Examples thereof include leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as silicic anhydride and silicic acid hydrate; and the above starch derivatives. ), A binder (for example, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, and compounds similar to the above-mentioned excipients), a disintegrant (for example, low-substituted hydroxypropylcellulose). , Cellulose derivatives such as carboxymethylcellulose, carboxymethylcellulose calcium, internally crosslinked sodium carboxymethylcellulose; chemically modified starch / celluloses such as carboxymethylstarch, sodium carboxymethylstarch, crosslinked polyvinylpyrrolidone can be mentioned). Emulsifiers (eg,
Colloidal clays such as bentonite and bee gum; metal hydroxides such as magnesium hydroxide and aluminum hydroxide; anionic surfactants such as sodium lauryl sulfate and calcium stearate; cationic interfaces such as benzalkonium chloride. Activators; and nonionic surfactants such as polyoxyethylene alkyl ether, polyoxyethylene sorbitan fatty acid ester, and sucrose fatty acid ester. ), Stabilizers (paraoxybenzoic acid esters such as methylparaben, propylparaben; alcohols such as chlorobutanol, benzyl alcohol, phenylethyl alcohol;
Benzalkonium chloride; phenols, phenols such as cresol; thimerosal; dehydroacetic acid; and
Mention may be made of sorbic acid. ), Flavoring agents (for example, commonly used sweeteners, acidulants, flavors, etc.) and diluents and the like can be used to produce the compound by a known method.

As for the method of introducing the compound of the present invention into a patient, a colloidal dispersion system can be used in addition to the above. The colloidal dispersion system is expected to have the effect of increasing the stability of the compound in vivo and the effect of efficiently transporting the compound to a specific organ, tissue or cell. The colloidal dispersion system is not particularly limited as long as it is usually used, but a polymer composite,
Mention may be made of lipid-based dispersion systems including nanocapsules, microspheres, beads, and oil-in-water emulsifiers, micelles, mixed micelles and liposomes, and preferably compounds to specific organs, tissues or cells. Multiple liposomes, artificial membrane vesicles with efficient transport effect (Mannino et al., Biotechniques, 1988,6,682; Blum
e and Cevc, Biochem.et Biophys.Acta, 1990,1029,91; La
ppalainen et al., Antiviral Res., 1994,23,119; Chonn
and Cullis, Current Op. Biotech., 1995,6,698). 0.2
Unilamellar liposomes, which range in size from -0.4 μm, can encapsulate a significant proportion of an aqueous buffer containing macromolecules, the compound being encapsulated in this aqueous inner membrane and biologically active. Morphologically transported to brain cells (Fraley et al., Trends
Biochem. Sci., 1981,6,77). The composition of the liposome is usually a lipid, in particular a phospholipid, in particular a phospholipid with a high phase transition temperature, usually complexed with one or more steroids, in particular cholesterol. Examples of lipids useful for liposome production include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, sphingolipids, phosphatidylethanolamine, cerebroside and ganglioside. Particularly useful is diacylphosphatidylglycerol, where the lipid moiety contains 14-18 carbon atoms, especially 16-18 carbon atoms, and is saturated (double bond within the chain of 14-18 carbon atoms). Lacking). Representative phospholipids include phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

Targeting of colloidal dispersions, including liposomes, can be either passive or active.
Passive targeting is achieved by exploiting the natural tendency of liposomes to distribute to reticuloendothelial cells of organs containing sinusoidal capillaries. On the other hand, active targeting is, for example, the viral protein coat (Morishitae).
t al., Proc.Natl.Acad.Sci. (USA), 1993,90,8474
), A specific ligand such as a monoclonal antibody (or a suitable binding moiety thereof), a sugar, a glycolipid or a protein (or a suitable oligopeptide fragment thereof) is bound to the liposome, or other than a naturally occurring localization site. Examples of the method include modifying liposomes by changing the composition of the liposomes in order to achieve distribution to the organs and cell types. The surface of the targeted colloidal dispersion can be modified in various ways. In liposome-targeted delivery systems, lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in intimate association with the lipid bilayer. Various linking groups can be used to attach the lipid chains to the targeting ligand. Targeting ligands that bind to specific cell surface molecules found predominantly on cells where delivery of the oligonucleotides of the invention is desired are, for example, (1) predominantly expressed by cells where delivery is desired. Hormones, growth factors or suitable oligopeptide fragments thereof, which bind to specific cell receptors, or (2
) A polyclonal or monoclonal antibody, or suitable fragment thereof (eg, Fab) that specifically binds to an antigenic epitope found predominantly on the target cell.
F (ab ') 2); Two or more bioactive agents (eg compound (I) and other agents)
Can also be complexed and administered within a single liposome. It is also possible to add agents to the colloidal dispersion that enhance the intracellular stability and / or targeting of the contents.

The amount used varies depending on the symptoms, age and the like, but in the case of oral administration, the lower limit is 1 mg (preferably 30 mg) and the upper limit is 2000 mg (preferably 1500 m) per dose.
In the case of intravenous administration of g), the lower limit is 0.5 mg per dose.
(Preferably 5 mg), upper limit 500 mg (preferably 250 mg)
It is desirable to administer (mg) to an adult 1 to 6 times daily depending on the condition. Hereinafter, the present invention will be described in more detail with reference to Examples, Reference Examples and Test Examples.

[0053]

EXAMPLES Example 1 HO-C ^e2 -C ^e2 -C ^e2 -A ^e2 -A ^e2 -G ^m -A
^e2 -C ^e2 -A ^s -G ^s -C ^s -A ^s -G ^s -A ^e2 -A ^e2 -A ^e2 -G ^m -T ^e2 -T ^e2 -C ^e2 -A
^e2 -T ^e2t -H synthetic nucleic acid synthesizer (Perkin Elmer ABI model392 DNA /
RNA synthesizer) was used and performed on a 1.0 μmol scale. The concentrations of the solvent, the reagent and the phosphoramidite in each synthesis cycle were the same as in the case of the natural oligonucleotide synthesis, and the solvent, the reagent and the phosphoramidite of the natural nucleoside were basically those manufactured by PE Biosystems. Non-natural phosphoramidite is disclosed in Japanese Patent Application Laid-Open No. 2000-2
Example 5 of 97097 (5'-O-dimethoxytrityl-2 '
-O, 4'-C-ethylene-4-N-benzoylcytidine-3'-O- (2-
Cyanoethyl N, N-diisopropyl) phosphoramidite), Example 9 (5'-O-dimethoxytrityl-2'-O, 4 '.
-C-ethylene-5-methyluridine-3'-O- (2-cyanoethyl
N, N-diisopropyl) phosphoramidite, Example 14
(5'-O-dimethoxytrityl-2'-O, 4'-C-ethylene-6-N
-Benzoyladenosine-3'-O- (2-cyanoethyl N, N-diisopropyl) phosphoramidite, or a compound of
5'-O-dimethoxytrityl-2'-O-methyl-2-N-isobutyrylphenoxyacetylguanosine-3'-O- (2-cyanoethyl N, N-diisopropyl) phosphoramidite (Pharmacia) Using. Also, the sulfurization reaction is
Sulfurizing reagent from the company was used at a concentration of 1 g / 100 ml. Using the compound of Reference Example 1 (1.0 μmol) as a solid-phase carrier, the DMTr group was deprotected with trichloroacetic acid, and the 5′-hydroxyl group was condensed with a natural phosphoramidite unit and a non-natural phosphoramidite unit. The reaction was repeated to synthesize the indicated compound. The synthesis cycle is as follows. Synthetic cycle 1) detritylation trichloroacetic acid / dichloromethane; 85
sec 2) coupling phosphoramidite (25eq), tetrazole
/ Acetonitrile; 10 to 20 min 3) capping 1-methylimidazole / tetrahydrofuran, acetic anhydride / pyridine / tetrahydrofuran; 15 sec 4) sulfurization sulfurization reagent; 5 min (× 2) (A ^s , G ^S ,
C ^S, T during the synthesis cycle condensing ^S) or, Oxidation iodine / water / pyridine / ^{tetrahydrofuran; 15sec (A p, G p} , C p, T p, A e2, G e2, C e2, T e2,
(At the time of a synthetic cycle of condensing G ^m ) After synthesizing a protected oligonucleotide analog having a target sequence with the 5′-DMTr group still attached, the oligomer is cleaved from the support by treating with concentrated aqueous ammonia, The protecting group on the phosphorus atom, cyanoethyl group, and the protecting group on the nucleic acid base were removed. The solvent was distilled off under reduced pressure, and the remaining residue was subjected to reverse phase HP.
LC (Shimadzu LC-10VP; column (GL Science I
nertsil Prep-ODS (20 × 250mm)); 0.1M triethylamine acetate aqueous solution (TEAA), pH7; 32.5% → 60% CH ₃ CN (30min, li
near gradient); 40 ° C .; 10 ml / min; 254 nm), and fractions eluted at 9.0 minutes were collected. After the solvent was distilled off under reduced pressure, 80% acetic acid water was added and the mixture was left for 30 minutes to remove the DMTr group. After the solvent was distilled off, about 5 ml of H ₂ O was added, the aqueous layer was washed 3 times with about 5 ml of ethyl acetate, and the target compound was obtained after distilling off the water solvent. This compound is an ion-exchange HPLC (column (Tosoh T
SKgel DEAE-2SW (4.6 × 250mm)); A solution: 20% CH ₃ CN, B
Solution: 67mM phosphate buffer (pH6.8), 1.5M NaCl; grad
ient: B solution 20 → 80% (20min, linear gradient); 60 ℃;
It was eluted at 13.8 minutes when analyzed at 1 ml / min). (144 A ₂₆₀
units) (λmax (H ₂ O) = 260 nm) The nucleotide sequence of this compound is VEGF206 cDNA (Gene Bank acc
esion No. S85192) nucleotide sequences 69-90, which are as follows.

5'-cccaagacagcagaaagttcat-3 '(Sequence listing
SEQ ID NO: 1) (Example 2) HO-C^e2-C^e2-C^e2-A^e2-A^e2-G^s-A^s-C^s-A^s-G^s-
C^s-A^s-G^s-A^s-A^s-A^s-G^s-T ^e2-T^e2-C^e2-A^e2-T^e2t-H synthesis According to the same method as in Example 1, the expression was performed using a DNA synthesizer.
The above compound was obtained.

After synthesizing a protected oligonucleotide analogue having the desired sequence with the 5'-DMTr group still attached,
The oligomer was cleaved from the support by treatment with concentrated aqueous ammonia, and the protecting group on the phosphorus atom and the protecting group on the nucleic acid base were removed. The solvent was distilled off under reduced pressure, and the remaining residue was subjected to reverse phase HPLC (LC manufactured by Shimadzu Corporation
-10VP; Column (GL Science Inertsil Prep-ODS (20
× 250mm)); 0.1M triethylamine acetate aqueous solution (TEAA),
pH7 ； 10.5% → 60% CH ₃ CN (30min, linear gradient) ； 40
Purification was performed at 10 ° C / min; 254 nm), and fractions eluting at 19.9 minutes were collected. After the solvent was distilled off under reduced pressure, 80% acetic acid water was added and the mixture was left for 30 minutes to remove the DMTr group. After the solvent was distilled off, about 5 ml of H ₂ O was added, the aqueous layer was washed 3 times with about 5 ml of ethyl acetate, and the target compound was obtained after distilling off the water solvent. This compound was ion-exchange HPLC (column (Tosoh TSKgel DEAE-2SW (4.6
× 250mm)); Solution A: 20% CH ₃ CN, Solution B: 67mM phosphate buffer (pH6.8), 1.5M NaCl; gradient: Solution B 20 → 80% (2
It was eluted at 20.8 minutes when analyzed at 0 min, linear gradient); 60 ° C; 1 ml / min). (164 A ₂₆₀ units) (λmax (H
₂ O) = 260 nm) The nucleotide sequence of this compound is VEGF206 cDNA (Gene Bank acc
esion No. S85192) nucleotide sequences 69-90, which are as follows.

5'-cccaagacagcagaaagttcat-3 '(SEQ ID NO: 1 in Sequence Listing) (Example 3) HO-C ^e1 -C ^e1 -C ^e1 -A ^e1 -A ^e1 -G ^m -A ^e1 -C ^e1 -A ^s -G
Synthesis of ^s -C ^s -A ^s -G ^s -A ^e1 -A ^e1 -A ^e1 -G ^m -T ^e1 -T ^e1 -C ^e1 -A ^e1 -T ^e1t -H According to the same method as in Example 1, The title compound was obtained using a DNA synthesizer. However, as a CPG solid-phase carrier, Q-Support
(Glen Reseach) was used. As an unnatural phosphoramidite, the literature (Koshkin, AA et al., Tetrahe
(5'-O-dimethoxytrityl-2'-O, 4'-C-methylene-4-) synthesized according to the method described in dron, 1998, 54, 3607).
N-benzoylcytidine-3'-O- (2-cyanoethyl N, N-diisopropyl) phosphoramidite), (5'-O-dimethoxytrityl-2'-O, 4'-C-methylene-5-methyluridine
-3'-O- (2-cyanoethyl N, N-diisopropyl) phosphoramidite, (5'-O-dimethoxytrityl-2'-O, 4'-C
-Methylene-6-N-benzoyladenosine-3'-O- (2-cyanoethyl N, N-diisopropyl) phosphoramidite, or 5'-O-dimethoxytrityl-2'-O-methyl-
2-N-isobutyrylphenoxyacetylguanosine-3'-O
-(2-Cyanoethyl N, N-diisopropyl) phosphoramidite (Pharmacia) was used.

A protected oligonucleotide analog having a target sequence was synthesized in a state in which the 5'-DMTr group was removed, and then treated with concentrated aqueous ammonia to cut out the oligomer from the support and protect the phosphorus atom. The cyanoethyl group and the protecting group on the nucleobase were removed. The solvent was distilled off under reduced pressure, and the remaining residue was subjected to reversed-phase HPLC (Shimadzu LC-10VP; column (GL Science Inertsil Prep-O
DS (20 × 250mm)); 0.1M triethylamine acetate aqueous solution (TE
AA), pH7; 11% → 25% CH ₃ CN (30min, linear gradient); 4
Purification was performed at 0 ° C .; 10 ml / min; 254 nm), and fractions eluting at 12.7 minutes were collected. This compound is an ion-exchange HPLC (column (Tos
oh TSKgel DEAE-2SW (4.6 × 250mm)); A solution: 20% CH ₃ CN,
Solution B: 67 mM phosphate buffer (pH 6.8), 1.5M NaCl; gr
adient: Solution B 20 → 80% (20min, linear gradient); 60
It was eluted at 14.0 minutes when analyzed at (° C; 1 ml / min). (34 A
₂₆₀ units) (λmax (H ₂ O) = 258nm) The nucleotide sequence of this compound is VEGF206 cDNA (Gene Bank acc
esion No. S85192) nucleotide sequences 69-90, which are as follows.

5'-cccaagacagcagaaagttcat-3 '(SEQ ID NO: 1 in Sequence Listing) (Reference Example 1) Synthesis of CPG carrier-binding compound

[0059]

[Chemical 32]

Compound 2'-O, 4'-C-ethylene-5'-O-dimethoxytrityl-5-methyluridine (580 mg, 0.988 mm) of Example 8 of JP-A-2000-297097
ol) was dissolved in anhydrous pyridine (10 ml) under a nitrogen stream to give succinic anhydride (178 mg, 1.78 mmo).
l), 4-dimethylaminopyridine (217 mg, 1.7
8 mmol) was added and the mixture was stirred at room temperature overnight. The solvent was distilled off under reduced pressure, and the residue was purified by silica gel chromatography (dichloromethane: methanol = 25:
2), colorless amorphous compound (580 mg, 0.8
44 mmol, yield 85%) was obtained. Of this, 515 mg was dissolved in anhydrous dimethylformamide (6 ml) to give pentachlorophenol (330 mg, 1.12 mmol),
N, N'-dicyclohexylcarbodiimide (231 mg,
(1.12 mmol) was added and the mixture was stirred at room temperature for 24 hours.
After filtering the precipitate, the filtrate was concentrated under reduced pressure, benzene was added, and the precipitate was filtered again. The filtrate was concentrated under reduced pressure, anhydrous dimethylformamide (3 ml) was added, and triethylamine (280 μl, 2.0 mmol), long chain alkylamino glass (500 Å, particle size: 12) was added.
(0/200 mesh) (manufactured by CPG Inc) (6 g) was added, and the mixture was allowed to stand at 60 ° C. for 8 hours and further at room temperature for 72 hours.
After filtering the CPG and further washing with dichloromethane,
Applied Biosystems DNA synthesizer A to CPG
20 ml each of two capping reagents for BI392 (acetic anhydride / pyridine / tetrahydrofuran) (1-methylimidazole / tetrahydrofuran) were added, and the mixture was left for 10 minutes. CPG was filtered, washed with pyridine and dichloromethane in that order, and dried under reduced pressure to obtain the desired product (about 6 g, amount of introduced dimethoxytrityl group = 62 μmol / g). (Reference Example 2) HO-A ^s -T ^s -G ^s -A ^s -A ^s -C ^s -T ^s -T ^s -T ^s -C ^s -T ^s -G ^s
-C ^s -T ^s -G ^s -T ^s -C ^s -T ^s -T ^s -G ^s -G ^s -G ^t -H synthesis (in the above, G ^t is

[0061]

[Chemical 33]

Is shown. According to the same method as in Example 1, an oligonucleotide having a sense sequence for the sequence of the compound of Example 1 was synthesized. However, 3'-hydroxyl group bound to CPG support 5 '
-O-DMTr-guanosine (1.0 μmol) (PE Biosyst
ems) was used as a solid phase carrier. The amidite coupling reaction time was 25 sec.

After synthesizing a protected oligonucleotide analog having a target sequence with the 5'-DMTr group still attached,
The oligomer was cleaved from the support by treatment with concentrated aqueous ammonia, and the protecting group on the phosphorus atom and the protecting group on the nucleic acid base were removed. The solvent was distilled off under reduced pressure, and the remaining residue was subjected to reverse phase HPLC (LC manufactured by Shimadzu Corporation
-10VP; Column (GL Science Inertsil Prep-ODS (20
× 250mm)); 0.1M triethylamine acetate aqueous solution (TEAA),
pH7; 5% → 60% CH 3 CN (30min, linear gradient); 40 ℃;
10 ml / min; 254 nm) and 20.0 min and 20.9 as two diastereomers derived from thiophosphoric acid diester.
Fractions eluting in minutes were collected. After distilling off the solvent under reduced pressure,
80% acetic acid water was added and left for 30 minutes to remove the DMTr group. After the solvent was distilled off, about 5 ml of H ₂ O was added, the aqueous layer was washed 3 times with about 5 ml of ethyl acetate, and the target compound was obtained after the solvent was distilled off.
This compound is an ion-exchange HPLC (column (Tosoh TSKgel DEAE
-2SW (4.6 × 250mm)); A solution: 20% CH ₃ CN, B solution: 67mM phosphate buffer (pH6.8), 1.5M NaCl; gradient: B solution 40
→ 100% (20 min, linear gradient); 60 ° C; 1 ml / min) analysis revealed that it was eluted at 18.5 minutes. (137 A ₂₆₀ units)
(Λmax (H ₂ O) = 260nm) The base sequence of this compound is VEGF20
It is the sequence of nucleotide numbers 69 to 90 of cDNA (Gene Bank accesion No. S85192) of 6 and is as follows.

5'-atgaactttctgctgtcttggg-3 '(SEQ ID NO: 2 in Sequence Listing) (Reference Example 3) HO-T ^s -A ^s -C ^s -G ^s -T ^s -A ^s -G ^s -T ^s -A ^s -T ^s -G ^s -G ^s
-T ^s -G ^s -T ^s -A ^s -C ^s -G ^s -A ^s -T ^s -C ^t -H Synthesis (In the above, C ^t is

[0065]

[Chemical 34]

Is shown. According to the same method as in Example 1, an oligonucleotide having a scrambled sequence with respect to the sequence of the compound of Example 1 was synthesized. However, 5'-O-DMTr-Cytidine (1.0 μmol) having 3'-hydroxyl group bound to a CPG support (PE Bio
systems) was used as a solid phase carrier. The amidite coupling reaction time was 25 sec.

After synthesizing a protected oligonucleotide analog having a target sequence with the 5′-DMTr group still attached,
The oligomer was cleaved from the support by treatment with concentrated aqueous ammonia, and the protecting group on the phosphorus atom and the protecting group on the nucleic acid base were removed. The solvent was distilled off under reduced pressure, and the remaining residue was subjected to reverse phase HPLC (LC manufactured by Shimadzu Corporation).
-10VP; Column (GL Science Inertsil Prep-ODS (20
× 250mm)); 0.1M triethylamine acetate aqueous solution (TEAA),
pH7; 5% → 60% CH 3 CN (30min, linear gradient); 40 ℃;
10 ml / min; 254 nm) and 21.2 minutes and 21.6 minutes as two diastereomers derived from thiophosphoric acid diester.
Fractions eluting in minutes were collected. After distilling off the solvent under reduced pressure,
80% acetic acid water was added and left for 30 minutes to remove the DMTr group. After the solvent was distilled off, about 5 ml of H ₂ O was added, the aqueous layer was washed 3 times with about 5 ml of ethyl acetate, and the target compound was obtained after the solvent was distilled off.
This compound is an ion-exchange HPLC (column (Tosoh TSKgel DEAE
-2SW (4.6 × 250mm)); A solution: 20% CH ₃ CN, B solution: 67mM phosphate buffer (pH6.8), 1.5M NaCl; gradient: B solution 40
→ 100% (20 min, linear gradient); 60 ° C; 1 ml / min), it was eluted at 17.8 minutes. (128 A ₂₆₀ units)
(Λmax (H ₂ O) = 259 nm) The base sequence of this compound is as follows.

5'-tacgtagtatggtgtacgatc-3 '(SEQ ID NO: 3 in Sequence Listing) (Reference Example 4) HO-C ^s -C ^s -C ^s -A ^s -A ^s -G ^s -A ^s -C ^s -A ^s -G ^s -C ^s -A ^s
-G ^s -A ^s -A ^s -A ^s -G ^s -T ^s -T ^s -C ^s -A ^s -T ^t -H (Compound A) Synthesis According to the same method as in Example 1, J. Biol . Chem. (199
5) Compound A described in 270, 28316-28324. Was synthesized (Nomur
a, M. et al. Antisense molecule. However, 3'-
5'-O-DMTr-thymidine (1.0 μmol) having a hydroxyl group bound to a CPG support (PE Biosystems) was used as a solid phase carrier. Also, the coupling reaction time of amidite is 25se.
done in c.

After synthesizing a protected oligonucleotide analog having a target sequence with the 5'-DMTr group attached,
The oligomer was cleaved from the support by treatment with concentrated aqueous ammonia, and the protecting group on the phosphorus atom and the protecting group on the nucleic acid base were removed. The solvent was distilled off under reduced pressure, and the remaining residue was subjected to reverse phase HPLC (LC manufactured by Shimadzu Corporation).
-10VP; Column (GL Science Inertsil Prep-ODS (20
× 250mm)); 0.1M triethylamine acetate aqueous solution (TEAA),
pH7; 5% → 60% CH 3 CN (30min, linear gradient); 40 ℃;
10 ml / min; 254 nm), and fractions eluting at 20.0 minutes were collected. After the solvent was distilled off under reduced pressure, 80% acetic acid water was added and the mixture was left for 30 minutes to remove the DMTr group. After distilling off the solvent, H ₂
About 5 ml of O 2 was added, the aqueous layer was washed with about 5 ml of ethyl acetate three times, and the solvent was distilled off to obtain the target compound. This compound is ion-exchange HPLC (column (Tosoh TSKgel DEAE-2SW (4.6 × 250 m
m)); Solution A: 20% CH ₃ CN, Solution B: 67 mM phosphate buffer
(pH6.8), 1.5M NaCl; gradient: B solution 40 → 100% (20min,
19.4 when analyzed with linear gradient); 60 ℃; 1ml / min)
Eluted in minutes. (89 A ₂₆₀ units) (λmax (H ₂ O) = 259
nm) The nucleotide sequence of this compound is VEGF206 cDNA (Gene Bank acc
esion No. S85192) nucleotide sequences 69-90, which are as follows.

5'-cccaagacagcagaaagttcat-3 '(SEQ ID NO: 1 in Sequence Listing) (Reference Example 5) HO-C ^e2 -C ^e2 -A ^e2 -A ^e2 -A ^e2 -G ^m -C ^e2 -C ^e2 -A ^s -G
Synthesis of ^s -C ^s -A ^s -G ^s -A ^e2 -A ^e2 -C ^e2 -G ^m -T ^e2 -T ^e2 -A ^e2 -A ^e2 -T ^e2t -H According to the same method as in Example 1, An oligonucleotide having 4 mismatched sequences with respect to the sequence of the compound of Example 1 was synthesized.

After the protected oligonucleotide analogue having the target sequence was synthesized with the 5'-DMTr group removed, the oligomer was cleaved from the support by treating with concentrated aqueous ammonia, and at the same time the phosphorus atom was protected. The cyanoethyl group and the protecting group on the nucleobase were removed. The solvent was distilled off under reduced pressure, and the remaining residue was subjected to reverse phase HPLC (Shimadzu LC-10VP; column (GL Science Inertsil Prep-ODS
(20 × 250mm)); 0.1M triethylamine acetate aqueous solution (TEA
A), pH7; 11% → 25% CH ₃ CN (30min, linear gradient); 4
Purification was performed at 0 ° C .; 10 ml / min; 254 nm), and fractions eluting at 14.8 minutes were collected. This compound is an ion-exchange HPLC (column (Tos
oh TSKgel DEAE-2SW (4.6 × 250mm)); A solution: 20% CH ₃ CN,
Solution B: 67 mM phosphate buffer (pH 6.8), 1.5M NaCl; gr
adient: Solution B 20 → 80% (20min, linear gradient); 60
(1 ° C; 1 ml / min), it was eluted at 14.4 minutes. (59 A
₂₆₀ units) (λmax (H ₂ O) = 259 nm) The base sequence of this compound is as follows.

5'-ccaaagccagcagaacgttaat-3 '(SEQ ID NO: 4 in Sequence Listing) (Reference Example 6) HO-C ^e2 -C ^e2 -A ^e2 -A ^e2 -C ^e2 -G ^m -T ^e2 -C ^e2 -A ^s -G
Synthesis of ^s -A ^s -C ^s -G ^s -A ^e2 -A ^e2 -C ^e2 -G ^m -A ^e2 -T ^e2 -A ^e2 -A ^e2 -T ^e2t -H According to the same method as in Example 1, An oligonucleotide having 8 mismatched sequences with respect to the sequence of the compound of Example 1 was synthesized.

After the protected oligonucleotide analog having the target sequence was synthesized with the 5'-DMTr group removed, the oligomer was cleaved from the support by treating with concentrated aqueous ammonia, and at the same time the phosphorus atom was protected. The cyanoethyl group and the protecting group on the nucleobase were removed. The solvent was distilled off under reduced pressure, and the remaining residue was subjected to reverse phase HPLC (Shimadzu LC-10VP; column (GL Science Inertsil Prep-ODS
(20 × 250mm)); 0.1M triethylamine acetate aqueous solution (TEA
A), pH7; 11% → 25% CH ₃ CN (30min, linear gradient); 4
Purification was performed at 0 ° C .; 10 ml / min; 254 nm), and fractions eluting at 14.1 minutes were collected. This compound is an ion-exchange HPLC (column (Tos
oh TSKgel DEAE-2SW (4.6 × 250mm)); A solution: 20% CH ₃ CN,
Solution B: 67 mM phosphate buffer (pH 6.8), 1.5M NaCl; gr
adient: Solution B 20 → 80% (20min, linear gradient); 60
It was eluted at 14.0 minutes when analyzed at (° C; 1 ml / min). (39 A
₂₆₀ units) (λmax (H ₂ O) = 259 nm) The base sequence of this compound is as follows.

5'-ccaacgtcagacgaacgataat-3 '(SEQ ID NO: 5 in Sequence Listing) (Test Example) VEGF Suppression Test Human lung cancer cell lines were prepared using the compounds obtained in Examples 1 and 2 and Reference Examples 2 to 6. VEGF mRNA in A549 cells
The effect of suppressing expression was investigated. Human lung cancer cell line A549 cells (ATCC
number CCL-185) with collagen type I coated 1
0.6 × 10 ⁵ / well / on a 2-well plate (made by IWAKI)
1ml RPMI1640 (GIBCO BRL) (10% Fetal Bovine Serum (H
yclone))) and plated overnight at 37
The cells were cultured at 5 ° C and 5% CO ₂ . Each oligonucleotide-EPEI
Preparation of the complex, various oligonucleotide aqueous solution (50 μM) 20 μL and EPEI (Gene Tool, 200 μM) 2
Add μL to 178 μL of water and vortex immediately for 2 seconds 2
It was carried out as needed by leaving it to stand at room temperature for 0 minutes. Immediately before adding the sample, add the cell culture solution in each well to a new medium (10% Fe
RPMI 1 containing tal Bovine Serum and tamoxifen 10 μM
640) was replaced with 0.8 ml, and 200 μL of the oligonucleotide-EPEI complex prepared as needed was added thereto. 4 hours 3
After incubation at 7 ° C and 5% CO ₂ , the medium is RPMI1640 (containing 10% Fetal Bovine Serum) containing no oligonucleotide.
It was exchanged with 1 ml and incubated for another 20 hours. Next, the cells in each well were trypsin-EDTA (0.05% Trypsin,
Peel off from plate using 400 μL of 0.53 mM EDTA ・ 4Na) (GIBCO BRL) RPMI1640 (containing 10% Fetal Bovine Serum)
After adding 1 ml, the mixture was collected by centrifugation in a 1.5 ml Eppendorf tube, and then washed twice with PBS (-) buffer (Nissui Pharmaceutical Co., Ltd.). Extraction of total mRNA from collected cells is
Using k Prep Micro mRNA Purification Kit (Amersham Pharmacia Biotech Co., Ltd.) according to the manual, 200 μL of an aqueous solution containing total mRNA was obtained. Of these, 10 μL was used for Ready-To-Go RT-PCR Beads (Amersham Pharmacia Biotech Co., Ltd.)
A reverse transcription reaction using oligo dT _(12-18) as a primer was performed according to the manual to obtain 50 μL of total cDNA solution. Using this as a template, PCR reaction was performed as described below to amplify β-actin mRNA. β-actin primer 1: 5'-atcaccattggcaatgagcg-3 '
(SEQ ID NO: 6 in Sequence Listing) β-actin primer 2: 5'-ttgaaggtagtttcgtggat-3 '
(SEQ ID NO: 7 in the Sequence Listing) Taq: Takara Taq premix (Takara Shuzo) PCR Condition: 95 ° C (20 sec), 55 ° C (40 sec), 72 ° C
(40 sec). Number of cycles: 21 times β-actin mRNA in each sample by using the amount of housekeeping gene β-actin mRNA as internal standard
Amplification by quantitative PCR of VEGF mRNA was performed using an amount of template corrected so that the amount of A was equal. P
The conditions of CR reaction are shown below. VEGF primer 1: 5'-tgcattggagcctcgccttg-3 '(SEQ ID NO: 8 in the sequence listing) VEGF primer 2: 5'-ctcaccgcctcggcttgt-3' (SEQ ID NO: 9 in the sequence listing) Taq: Takara Taq premix (Takara Shuzo) PCR Condition: 95 ° C (20 sec), 55 ° C (40 sec), 72 ° C
(40 sec) Number of cycles: 30 PCR products obtained were subjected to 5% acrylamide gel electrophoresis (1x TBE solution (0.89M Tris, boric acid, EDTA solution (pH 8.
3, Takara Shuzo) 240V, 30 minutes), SYBR GreenI (FMC
PCR product using a fluorescent image analyzer (Molecular Imager FX (Bio-Rad))
The PCR product was visualized by and the amount of the product was quantified. The result of visualizing and inverting the black and white of the image is shown in FIG.
As is apparent from FIG. 1, J. Biol. Chem. (1995)
The compound A described in 270, 28316-28324. Hardly suppressed VEGF, whereas the compound of the present invention showed a remarkable inhibitory effect on the amount of VEGF mRNA. Formulation Example Formulation Example 1 Soft Capsules A mixture of the compounds of Example 1 in a digestible oil, such as soybean oil, cottonseed oil or olive oil, is prepared and put into gelatin with a positive displacement pump. By injection, soft capsules containing 100 mg of active ingredient are obtained, washed and dried. (Formulation Example 2) Tablet According to a conventional method, 100 mg of the compound of Example 2, 0.2 mg of colloidal silicon dioxide, 5 mg of magnesium stearate, 275 mg of microcrystalline cellulose, 11 mg of starch and 98.8 are used. Produced using mg lactose.

If desired, a coating is applied. (Formulation Example 3) 100 mg of the compound of Example 1, 100 mg of sodium carboxy group methylcellulose, 5 mg of sodium benzoate, and 1.0 g of sorbitol solution in 5 ml of the suspension (Japanese Pharmacopoeia) ) And 0.025 ml vanillin. (Formulation Example 4) Injectable preparation A compound was prepared by stirring 1.5% by weight of the compound of Example 2 in 10% by volume of propylene glycol, and then making up the solution to a fixed volume with water for injection, followed by sterilization.

[0076]

INDUSTRIAL APPLICABILITY The compound of the present invention has a strong VEGF inhibitory activity, and is useful for treating diseases associated with VEGF-induced angiogenesis or vascular permeability enhancement.

[Sequence list free text] SEQ ID NO: 1 antisense sequence for VEGF206 SEQ ID NO: 2: sense sequence for VEGF206 SEQ ID NO: 3: scrambled sequence SEQ ID NO: 4: mismatched sequence SEQ ID NO: 5: mismatched sequence SEQ ID NO: 6: β-actin primer SEQ ID NO: 7: β-actin primer SEQ ID NO: 8: VEGF primer SEQ ID NO: 9: VEGF primer

[0077]

[Sequence list]                                SEQUENCE LISTING        <110> Sankyo Company, Limited <120> VEGF antisense compound <130> 2002112SL <140> <141> <150> JP 2001-320096 <151> 2001-10-18 <160> 9 <170> PatentIn Ver. 2.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: antisense       sequence for VEGF206 <220> <223> Inventor: Koizumi, Makoto       Inventor: Morita, Koji <400> 1 cccaagacag cagaaagttc at 22 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: sense sequence       for VEGF 206 <400> 2 atgaactttc tgctgtcttg gg 22 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: scramble       sequence <400> 3 tacgtagtat ggtgtacgat c 21 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: mismatch       sequence <400> 4 ccaaagccag cagaacgtta at 22 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: mismatch       sequence <400> 5 ccaacgtcag acgaacgata at 22 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: beta-actin       primer <400> 6 atcaccattg gcaatgagcg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: beta-actin       primer <400> 7 ttgaaggtag tttcgtggat 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: VEGF primer <400> 8 tgcattggag cctcgccttg 20 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: VEGF primer <400> 9 ctcaccgcct cggcttgt 18

[Brief description of drawings]

FIG. 1 Results of electrophoresis in test examples

[Explanation of symbols] untreated: shows the result that VEGF is not suppressed at all when no drug is used. sense: When using Reference Example 2 which is a sense array, it is completely V
The result which EGF is not suppressed is shown. scramble: shows the result in which VEGF is not suppressed at all when Reference Example 3 which is a scrambled sequence is used. Phosphorothioate antisense: VEGF known in the literature
Shows the results when using Reference Example 4 (Compound A) that is an inhibitor ENA antisense 1: shows the results when using Example 1 of the present invention. 4 mismatch: The result shows that VEGF is not suppressed at all when Reference Example 5, which is a compound in which four mismatch base residues are introduced into Example 1, is used. 8 mismatch: The result shows that VEGF is not suppressed at all when Reference Example 6 which is a compound in which 8 residues of mismatch bases are introduced is used in Example 1. ENA antisense 2: shows the results when Example 2 of the present invention was used. marker: indicates a molecular weight marker

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 9/10 A61P 9/10 9/14 9/14 17/00 17/00 17/02 17/02 17 / 06 17/06 25/00 25/00 27/06 27/06 29/00 29/00 101 101 35/00 35/00 35/04 35/04 43/00 105 43/00 105 111 111 111 C07H 21 / 04 C07H 21/04 B C12N 15/09 ZNA C12N 15/00 ZNAA F Term (reference) 4B024 AA01 CA04 HA17 4C057 BB02 BB05 DD01 DD03 LL19 LL21 LL42 LL44 MM04 MM09 4C084 ZAZ ZB41 Z26B11 Z41B41 Z01B41 Z16B11 Z16B16 Z16B16 Z16B16 Z16B16 Z16B16 Z16B16 Z16B11 AA03 EA16 NA14 ZA02 ZA33 ZA36 ZA89 ZB11 ZB15 ZB26 ZC35 ZC41

Claims (18)

[Claims] 1. The following general formula (I) HO-B1-B2-B3-B4-B5-B6-B7-B8-B9-B10-B11-B12-B13-B14-B15-B16-B17-B18-B19. -B2 0-B21-B22-H (I) (wherein B1 to B3, B8, B11 and B20 are the same or different and are represented by the following formula: A group represented by (hereinafter referred to as “C ^p ”), a group represented by the formula: A group represented by (hereinafter referred to as “C ^s ”) or a general formula
C ^e [Chemical 3] (Wherein l represents an integer of 1 to 5), and B4, B5, B7, B9, B12, B14 to B16 and B21 are the same or different and are represented by the formula: A group represented by (hereinafter referred to as “A ^p ”), a group represented by the formula: A group represented by (hereinafter referred to as "A ^s ") or a general formula
A ^e [Chemical 6] (In the formula, m represents an integer of 1 to 5), B6, B10, B13 and B17 are the same or different and are represented by the formula: A group represented by (hereinafter referred to as “G ^p ”), a group represented by the formula: A group represented by (hereinafter referred to as “G ^s ”), a compound represented by the formula: A group represented by (hereinafter referred to as “G ^m ”) or a general formula: (In the formula, n represents an integer of 1 to 5), B18 and B19 are the same or different and are represented by the formula: A group represented by (hereinafter referred to as "T ^p "), a group represented by the formula: A group represented by (hereinafter referred to as "T ^s ") or a general formula: (In the formula, o represents an integer of 1 to 5), and B22 is a group represented by the formula: A group represented by (hereinafter referred to as "T ^t ") or a general formula
T ^et (In the formula, p represents an integer of 1 to 5). However, B1 to B3, B8, B11 and B20 are the same or different and are C ^p or C ^s , and B4, B5, B7, B9, B12, B14 to B16.
And B21 are the same or different and A ^p or A ^s, B6, B10,
B13 and B17 are the same or different and G ^p , G ^{s and} G ^m are
8 and B19 are the same or different and are T ^p or T ^s , and B2
Exclude if 2 is T ^t . And a pharmacologically acceptable salt thereof. 2. In claim 1, B1 to B3, B8, B11 and B20 are the same or different and are C ^s or C ^e , and B4, B5, B
7, B9, B12, B14 to B16 and B21 are the same or different and A ^s
Or A ^e , B6, B10, B13 and B17 are the same or different G ^s , G ^m or G ^e , and B18 and B19 are the same or different T ^s or T ^e , and a compound, Its pharmacologically acceptable salt. 3. The B1 according to any one of claims 1 and 2.
Or salt B3 compound is C ^e, acceptable pharmacologically. 4. A compound according to claim 3, wherein B4 is A ^e ,
Its pharmacologically acceptable salt. 5. The compound according to claim 4, wherein B5 is A ^e ,
Its pharmacologically acceptable salt. 6. The compound according to claim 5, wherein B6 is G ^m or G ^e , or a pharmacologically acceptable salt thereof. 7. A compound according to claim 6, wherein B7 is A ^e ,
Its pharmacologically acceptable salt. 8. The compound according to claim 7, wherein B8 is C ^e ,
Its pharmacologically acceptable salt. 9. The B2 according to any one of claims 1 to 8.
A compound wherein 2 is T ^et , B21 is A ^e , and B20 is C ^e , and a pharmacologically acceptable salt thereof. 10. The compound according to claim 9, wherein B19 is T ^e , and a pharmacologically acceptable salt thereof. 11. The compound according to claim 10, wherein B18 is T ^e , and a pharmacologically acceptable salt thereof. 12. The compound according to claim 11, wherein B17 is G ^m or G ^e , or a pharmacologically acceptable salt thereof. 13. The compound according to claim 12, wherein B16 is A ^e , and a pharmacologically acceptable salt thereof. 14. The compound according to claim 13, wherein B15 is A ^e , and a pharmacologically acceptable salt thereof. 15. The compound according to claim 14, wherein B14 is A ^e , and a pharmacologically acceptable salt thereof. 16. The compound according to any one of claims 1 to 15, wherein l, m, n, o and p are the same or different and are 1 or 2, and a pharmacologically acceptable salt thereof. 17. The method according to claim 16, wherein l, m, n, o and p.
And 1 or 2, which are the same, and a pharmacologically acceptable salt thereof. 18. The method according to claim 17, wherein l, m, n, o and p.
Wherein the compound is 2, a pharmacologically acceptable salt thereof.