JPH11302193A - Vascularization inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an effective vascularization inhibitory method, particularly tumor vascularization inhibitory method, based on the synergism due to the use of anti-VEGF or VPF antibody in combination with a vascularization inhibitor, e.g. minocycline or the like. SOLUTION: This vascularization inhibitor contains a vasopermeable factor (VEGF or VPF) antagonist and minocycline as active ingredients. It is favorable that the VEGF or VPF antagonist is an anti-VEGF or VPF monoclonal antibody, particularly an antibody reactive with at least one of the following sequences 1-3. 1. KPSCVPLMR 2. SFLQHNKCECRP 3. KCECRPKKDRAR.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a vascular permeability factor (V
PF) [also called vascular endothelial cell growth factor (VEGF),
In the present specification, these are collectively referred to as “VEGF / VPF”.] An angiogenesis inhibitor comprising an antagonist and minocycline in combination, particularly an angiogenesis inhibitor useful as a therapeutic agent for tumors.

[0002]

2. Description of the Related Art Angiogenesis is a phenomenon in which endothelial cells germinate from existing blood vessels to form new blood vessels. The angiogenesis mainly occurs in a diseased site in a living body. Nothing else happens. Angiogenic diseases associated with angiogenesis include solid tumor growth and tumor metastasis, intraocular neovascular diseases such as diabetic retinopathy and age-related macular degeneration, rheumatoid arthritis, psoriasis, Kaposi's sarcoma, and atherosclerosis Illness and the like.

[0003] Angiogenesis is controlled by various angiogenesis-promoting factors. Among them, VEGF which is a peptidic secretory growth factor which acts selectively on endothelial cells is disclosed.
/ VPF has been suggested to play a direct role in the angiogenesis of the above-mentioned diseases in living organisms. And
It has been reported that by neutralizing VEGF / VPF using certain anti-VEGF / VPF antibodies, it is possible to suppress the growth and metastasis of cancer cells (M. Asano et al., CANCER RESEARCH 55, 5296-
5301, November 15, 1995).

[0004] Unlike the current general treatment method targeting the tumor itself, the method of treating cancer utilizing the antagonistic action of such an antibody inhibits tumor growth and indirectly inhibits tumor growth. It is based on a new mechanism of action, and is expected to be an effective cancer chemotherapy because its side effects are very slight.

As other angiogenesis inhibitors, conventionally, an in vitro culture system using a vascular endothelial cell culture system and an in vivo
o Substances that selectively inhibit angiogenesis have been screened using angiogenesis models, and, for example, protamine (Taylor, S. et al., Nature, 297, 30)
7, 1982), a combination of heparin and cortisone (Folkman, J.
et al., Science, 221, 71, 1983), prednisolone
Acetate (Robin, JB, Arch. Opthalmol., 103, 28
4, 1985), sulfated polysaccharide (JP-A-63-119500), herbimycin A (JP-A-63-295509), fumagillin (JP-A-1-279828), interferon β (International Publication No. WO / 29092) and the like.

[0006]

Minocycline is one of the tetracycline antibiotics. In the in vivo angiogenesis model using the rabbit corneal method, minocycline is more effective than cortisone alone or a combination of cortisone and heparin. It has been reported that angiogenesis was significantly suppressed (R.
J. Tamargo et al., CANCER RESEARCH 51, 672-675, January
15, 1991). However, in any case, if the administration is continued for a while, the effect of inhibiting angiogenesis tends to be weakened.

The present inventors have proposed the above-mentioned anti-VEGF / VPF
In view of the fact that a combination system of an antibody and the above-mentioned angiogenesis inhibitor such as minocycline has not been studied yet, a more effective angiogenesis inhibition method based on a synergistic action of the two, particularly a cancer angiogenesis inhibition method is provided. As a result of intensive research for the purpose of carrying out the present invention, the present invention has been completed.

[0008]

The present invention provides a VEGF / V
The present invention relates to an angiogenesis inhibitor containing a PF antagonist and minocycline as active ingredients. That is, the present invention is characterized in that angiogenesis is effectively inhibited by administering a VEGF / VPF antagonist and minocycline in combination.

Therefore, according to another aspect of the present invention,
There is provided a method for inhibiting angiogenesis, which comprises administering a VEGF / VPF antagonist and minocycline in combination.

Without being bound by theory, according to the findings of the present inventors, it is considered that the present invention exerts its excellent effects by the following mechanism. That is, when a pathological site associated with angiogenesis is treated with an angiogenesis inhibitor such as minocycline, blood flow at the pathological site is cut off and the site becomes hypoxic. However, VEGF
Since / VPF has a property of inducing hypoxia, VEGF / VPF is induced a short time after treatment with an angiogenesis inhibitor such as minocycline, and an angiogenesis is enhanced. However, when a VEGF / VPF antagonist is used in combination with the pathological site according to the present invention, the action of VEGF / VPF is inhibited,
It is thought that the angiogenesis inhibitory effect of the angiogenesis inhibitor such as minocycline is effectively maintained even after the induction of VEGF / VPF.

Minocycline is a compound known as a tetracycline antibiotic. In the present invention, in addition to minocycline and its salts such as hydrochloride, derivatives having the same medicinal effect are included, and fine granules, granules, Any form commercially available in the form of tablets, capsules, intravenous preparations and the like can be used.

VEGF / VPF acts directly on vascular endothelial cells and induces angiogenesis, a phenomenon of proliferation, migration and infiltration of tissue of capillary endothelial cells, fetal growth, wound healing, and cancer cell degeneration. It plays an important role in physiological or pathological phenomena such as proliferation. VEGF /
VPF is secreted by mouse, rat, guinea pig, bovine and human normal or tumor cell lines, and has been shown to be present in the brain, pituitary, kidney and ovary by tissue [Ferrara, N. ., Endocrine Reviews 13: 18 (1
992)].

With respect to the human VEGF / VPF gene, its cDNA has already been isolated, its nucleotide sequence has been determined, and its amino acid sequence has been deduced. Four types of proteins having different numbers of amino acid residues (121 amino acid residues, 1
65, 189, and 206 types were produced, among them, those having 121 amino acid residues (VEGF
₁₂₁ ) and 165 amino acid residues (VEG
F ₁₆₅ ) is said to be a mature protein [Ferrara, N .;
Endocrine Reviews 13:18 (1992)]. Although VEGF ₁₂₁ are those 44 amino acids of the carboxyl-terminal of the VEGF ₁₆₅ are deleted, while the VEGF ₁₂₁ and VEGF _165, not been clarified whether there are differences in effects on vascular endothelial cells .

In the present invention, a VEGF / VPF antagonist refers to a substance having a function of inhibiting the above-mentioned action of VEGF / VPF, and any form having the function may be used. Has V
An antibody that acts on EGF / VPF, or a portion thereof, including but not limited to,
Inactive VEGF / V inhibiting the action of EGF / VPF
PF, or a portion thereof, impairs the function of the VEGF / VPF receptor, eg, an antibody against the vascular permeability factor receptor, or a portion thereof, or even VEGF / VPF
And the like, which suppress the production of the compound itself.

The antibody acting on VEGF / VPF may be an antibody against any of the four VEGF / VPF subtypes having 121, 165, 189 or 206 amino acid residues.

Representative VEGF / VPF antagonists include anti-VEGF / VPF monoclonal antibodies. In particular, the anti-VEGF / VPF monoclonal antibodies have the following sequences 1 to 3, which are part of the VEGF / VPF amino acid sequence: :

1. KPSCVPLMR 2. SFLQHNKCECRP 3. KCECRPKKDRAR

It is preferred that the antibody react with at least one of the following.

The above anti-VEGF / VPF antibodies include:
Mouse antibodies and the like can also be mentioned, and those which have been subjected to treatment for reducing side effects upon administration to humans can also be used. For example, a mouse monoclonal antibody is chemically modified with a substance such as polyethylene glycol to reduce its antigenicity. A human chimeric antibody or a humanized antibody in which a CDR region, which is a binding region to an antigen of a mouse monoclonal antibody, is replaced with another region while retaining the binding force to the antigen by a human antibody can also be used. Furthermore, antibodies that are obtained by enzymatically cleaving them to reduce the molecular weight can also be used. As the chimeric antibody or humanized antibody, Ig
G type or IgA type and the like, and examples of the IgG isotype include IgG1, IgG2, IgG3 and IgG4.

The present invention is intended to neutralize the action of VEGF / VPF induced in a diseased site in which vitality is reduced by administration of minocycline to a hypoxic state by the action of a VEGF / VPF antagonist, Based on the mechanism of action of suppressing angiogenesis in, it is considered that it can be effectively applied to the treatment of all angiogenic diseases including cancer cells. Therefore, when administering the angiogenesis inhibitor of the present invention, the subject to be administered is not particularly limited.
For example, it can be administered locally or systemically for the specific purpose of preventing or treating individual angiogenic diseases. The method of administration may be oral or parenteral, and oral administration includes sublingual administration.

Parenteral administration includes injections, such as subcutaneous, intramuscular, intravascular, intraperitoneal or intrathoracic injections, infusions, suppositories and the like. The dose and the ratio of the active ingredient vary depending on whether the animal or human, the age, the administration route, and the number of administrations, and can be widely varied. In this case, VEGF /
The effective amount of the VPF antagonist and minocycline and the effective amount to be administered as a composition of a suitable diluent and a pharmaceutically usable carrier is 0.1 to 100 mg / kg body weight / day, which is from once to several times a day. Divided daily or once every few days or 1
It is administered once every two weeks. The VEGF / VPF antagonist and minocycline can be used in any ratio, and both can be used in a mixed state or in separate states.

When the angiogenesis inhibitor of the present invention is administered parenterally, it usually contains additives such as a stabilizer, a buffer, a preservative, and a swelling agent, and usually contains a unit-dose ampoule or a multi-dose container or tube. Provided in a state. For example, an injectable preparation is obtained by dissolving a purified VEGF / VPF antagonist and / or minocycline in a solvent such as physiological saline, a buffer, and the like, and adding an anti-adsorption agent such as Tween 80, gelatin, human serum albumin ( HSA), or may be freeze-dried for reconstitution before use. As an excipient for lyophilization, for example, sugar alcohols such as mannitol and glucose and sugars can be used. Similarly, it can be used in the form of an ophthalmic solution, an ophthalmic ointment, an emulsifier, or the like, or can be used in the form of liposomes or microspheres for prolonging retention.

In the case of oral administration, tablets, granules, fine granules, powders, capsules, and the like applied thereto are usually used in such compositions as binders, inclusion agents, and excipients commonly used in pharmaceutical preparations. -Contains additives such as lubricants, disintegrants and wetting agents. In addition, the liquid preparation for oral use may be in the form of a liquid solution for internal use, suspension, emulsion, syrup, etc.
Further, it may be a dried product that is redissolved when used. Further, the composition may contain any of additives and preservatives.

[0024]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, preferred embodiments of the present invention will be described with reference to anti-VE as a VEGF / VPF antagonist.
This will be described in detail using a GF / VPF monoclonal antibody.

Production of Monoclonal Antibodies Each monoclonal antibody can be produced by immunizing an animal with VEGF / VPF, removing spleen cells thereof, and fusing the cells with myeloma cells to culture hybridoma cells. The production of this hybridoma
It can be performed by the method of Kohler and Milstein [Nature, 256: 495 (1975)] and the like.

(1) Preparation of antibody-producing cells As animals for immunization, rodents such as mice, rats and rabbits are used. Mouse or rat-derived cells are used as myeloma cells. Then, an amount of 10 to 100 μg of VEGF / VPF was used as an antigen for one immunized animal.
At least 2-3 immunizations are performed at intervals of 間隔 4 weeks.
Animal breeding and spleen cell collection are performed according to conventional methods.
In the case of immunization, a protein obtained by fusing, for example, glutathione-S-transferase or the like to an antigen, or a complex protein obtained by binding keyhole limpet hemocyanin or the like can be used as the antigen.

(2) Preparation of myeloma cells As myeloma cells, mouse myeloma Sp2 / O-Ag14 (S
p2), P3 / NSI / 1-Ag4-1 (NS-1), P3 × 63-Ag.8.U · 1 (P3U1) and the like. Subculture of these cells is performed according to a conventional method.

(3) Cell fusion Cell fusion can be performed by mixing spleen cells and myeloma cells at a ratio of 1: 1 to 10: 1 and mixing with polyethylene glycol or subjecting to electric pulse treatment.

(4) Selection of hybridomas The selection of fused cells (hybridomas) is performed using hypoxanthine.
(10 ^-3 to 10 ^-5 M), aminopterin (10 ^-6 to 10 ^-7 M)
M) and thymidine (10 ⁻⁵ to 10 ⁻⁶ M) and used as a hybridoma.

(5) Culture of hybridoma The cloning of the hybridoma is repeated at least twice by the limiting dilution method. If the hybridoma is cultured in the same manner as ordinary animal cells, the monoclonal antibody of the present invention is produced in the medium. In addition, the monoclonal antibody of the present invention can be accumulated in ascites by transplanting hybridoma cells into the peritoneal cavity of a mouse and growing the cells.

(6) Collection and Purification of Monoclonal Antibodies Monoclonal antibodies accumulated in the culture medium or ascites fluid of hybridoma cells can be purified by conventional ammonium sulfate fractionation, PEG fractionation, ion exchange chromatography and gel filtration. It is purified by a method using chromatography. In addition, a method using affinity chromatography such as protein A and protein G can also be used.

For the selection of monoclonal antibodies, an enzyme immunoassay, a Western blotting method or the like is used.
Further, the determination of the isotype of the monoclonal antibody can be carried out by the enzyme immunoassay of the monoclonal antibody or the Ouchterlony method.

[0033]

The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to these examples.

Reference Example 1 (Preparation of anti-VEGF / VPF monoclonal antibody MV833) (1) Preparation of hybridoma producing anti-VEGF / VPF monoclonal antibody Culture solution of yeast transformed with isolated human VEGF / VPF cDNA Purify human VEGF / VPF from
A complex was prepared using keyhole limpet hemocyanin (KLH) and glutaraldehyde, and a mouse monoclonal antibody was prepared using the obtained protein as an antigen according to a conventional method. That is, KLH-
Splenocytes of mice immunized with VEGF / VPF were fused with mouse myeloma cells (Sp2) in the presence of polyethylene glycol. The obtained hybridoma was cloned by the limiting dilution method. The reactivity of the culture supernatant of the hybridoma cloned with VEGF / VPF was examined by enzyme immunoassay, and a hybridoma producing a monoclonal antibody reactive with VEGF / VPF was selected. or,
The monoclonal antibody produced by this hybridoma
V833. The hybridoma producing the obtained monoclonal antibody has been deposited as FERM BP-5669 with the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry.

(2) Preparation of anti-VEGF / VPF monoclonal antibody The selected hybridoma was implanted into the peritoneal cavity of a nude mouse, and ascites containing a large amount of the monoclonal antibody was collected.
From this ascites fluid, a protein G affinity column (M
AbTrapGII (Pharmacia) was used to purify the monoclonal antibody. The class of the antibody was determined by enzyme immunoassay using an anti-mouse immunoglobulin subclass-specific antibody. As a result, the class of the MV833 antibody was IgG1.

The dissociation constants for VEGF ₁₂₁ and VEGF ₁₆₅ measured by the following method are as follows, which indicates that the monoclonal antibody of the present invention has a strong affinity for VEGF / VPF.

○ 5.70 × 10 ⁻¹¹ M ± 0.35 × 10
^-11 M (VEGF ₁₂₁ ) ○ 1.10 × 10 ^-10 M ± 0.11 × 10 ^-10 M (VEGF
F ₁₆₅ )

Method for measuring dissociation constant: A monoclonal antibody containing 0.1 M sodium chloride
It is adjusted to 2 μg / ml with 5 mM carbonate buffer (pH = 9.0), added to a detachable plate with 100 μl each, and left at 4 ° C. overnight. Next, remove the solution from the hole and add 1% BSA-PB
S is added in an amount of 300 μl and left at 37 ° C. for 4 hours. 1
After removing% BSA-PBS, 0.1% BSA-PBS
VEGF prepared in the above and ¹²⁵ I-labeled VEGF ( ¹²⁵ I-labeled V
EGF ₁₂₁ is labeled VEGF ₁₂₁ by the chloramine T method,
¹²⁵ I-labeled VEGF ₁₆₅ was purchased from Amersham.) Add 200 μl of the reaction mixture per well and leave overnight. VEGF concentration in the reaction mixture is VEGF ₁₂₁ is 0～1Ng / well,
0-10 ng / well of VEGF _165, 1 of ¹²⁵ I-labeled VEGF
× 10 ⁴ cpm / well ( ¹²⁵ I-labeled VEGF ₁₂₁ ; 66.7 pg / well,
^{1 25} I-labeled VEGF _165; 116pg / well) to. The reaction mixture was removed from the wells, and the wells were washed six times with 0.1% BSA-PBS. Then, the wells were cut off one by one, placed in an analysis tube, counted with a gamma counter, and the dissociation constant was calculated from the scatter diagram created from the results. Ask.

The isoelectric point of the monoclonal antibody of the present invention measured by the following method was pI = 5.2 to 5.5.
Other IgG1-type anti-VEGF / V reported at this time
The isoelectric point of the PF monoclonal antibody was MV101 reported by our company with a pI of 7.0 to 7.5, and Genentech's A4.6.1 with a pI of 4.2 to 5.2 [Kim, KJ et al. .al. Grow
th Factors, 7:53 (1992)], and the substance of the present invention is different from any substance.

[0040] Isoelectric measurement of isoelectric point method monoclonal antibody was run at isoelectric focusing layer of the company using a commercial isoelectric focusing agarose gel (WakaSakarisha). The electrophoresis was performed at 3 W for 30 minutes using a power supply (Bio-Rad) capable of outputting an equal power. After the electrophoresis, the gel was stained with a protein using a silver staining kit (Bio-Rad). For the isoelectric point of the monoclonal antibody, the isoelectric point of the antibody was determined from the electrophoretic mobility of the isoelectric marker protein that was simultaneously electrophoresed.

(3) Identification of reactive site of monoclonal antibody in VEGF / VPF (3-1) Preparation of peptide corresponding to a part of amino acid sequence of VEGF / VPF 12 consecutive amino acids of amino acid sequence of human VEGF ₁₂₁ Was devised as one peptide, and 67 peptides covering the entire sequence were devised, and each peptide was synthesized using a multipin peptide synthesis method [Maeji, N, J, et.al. J. Immunol.method, 134: 23 (199
0)].

First, Fm was obtained from 9-fluorenylmethoxycarbonyl (Fmoc) -β-alanine introduced at the tip of a pin of a 96-well assay plate pin block by piperidine.
After removal of the oc group, the Fmoc-amino acid was condensed in the presence of dicyclohexcarbodiimide and hydroxybenzotriazole. After washing with N, N-dimethylformamide, the Fmoc-amino acid was again condensed in the presence of dicyclohexylcarbodiimide and hydroxybenzotriazole, and this operation was repeated to synthesize the desired peptide. After the completion of the condensation reaction, acetylation was performed with acetic anhydride, and the side chain protecting group was removed with trifluoroacetic acid. The peptide synthesized on the pin was excised by immersing the pin in a neutral solution. Quantification of the synthesized peptide was performed by quantifying amino groups using orthophthalaldehyde. Table 1 shows the amino acid sequences of the 67 peptides synthesized.
Numbers indicate peptide identification numbers.

[0043]

[Table 1]

(3-2) Identification of Peptides Reacting with MV833 Antibody The 67 peptides synthesized as described above are human VE
Which corresponds to the entire area of the GF _121. Therefore 6
By examining the reactivity between the seven peptides and the MV833 antibody, it is possible to clarify where in the VEGF / VPF the MV833 antibody reacts. Therefore, the reactivity of 67 kinds of peptides with the MV833 antibody was examined by enzyme immunoassay. 67 kinds of 20 μM peptide solutions were put in a 96-well NOS plate (manufactured by Coaster) and allowed to stand at room temperature for 2 hours. Plate 3 wells with 0.1% BSA-PBS
After washing twice, 2% BSA-PBS was added and left at room temperature for 1 hour. After removing 2% BSA-PBS, MV833 (1
% BSA-PBS solution) and left at room temperature for 1 hour.
After washing 6 times with 0.1% BSA-PBS, peroxidase-labeled sheep anti-mouse IgG (Amersham) (0.1% B
(SA-PBS solution) and left at room temperature for 1 hour. 0.1
After washing with 6% BSA-PBS six times, 0.2 M Tris-citrate buffer (pH = 5.2) containing 8.3 mg / ml orthophenylenediamine dihydrochloride and 0.01% hydrogen peroxide was added to develop color. I let it. After stopping the reaction by adding 2N sulfuric acid,
The absorbance (OD490 / 650) was measured. In addition, for the measurement of the reactivity, in addition to the enzyme immunoassay described above, the Ouchterlony method, the Western blotting method, or the like may be used.

The MV833 antibody strongly reacted with five peptides of peptide identification numbers 31, 32, 33, 60 and 63 among 67 kinds of peptides. Peptide identification numbers 31 to 3
Since peptide 3 contains the sequence KPSCVPLMR in common, it is considered that the MV833 antibody reacts with the amino acid sequence KPSCVPLMR in this region. Therefore, the MV833 antibody is
F / VPF KPSCVPLMR sequence (SEQ ID NO: 1), SFLQHNKCECRP sequence (SEQ ID NO: 2) and KCE
It is expected that it reacts with the CRPKKDRAR sequence (SEQ ID NO: 3). Since an antibody is considered to recognize a portion exposed on the surface of a protein, it can be said that these two amino acid sequence portions are portions exposed on the surface of VEGF / VPF. Monoclonal antibodies are said to have a single antigenic determinant, but when a macromolecular substance such as a protein having a higher-order structure is an antigen, the antibody recognizes the antigen three-dimensionally, and When the reactivity of an antibody is examined at the primary structure level, it may react to two or more discontinuous amino acid sequences. MV833 antibody is VEGF / V
Based on the reaction with two amino acid sequence portions in PF, it is considered that the present antibody recognizes two amino acid sequence portions simultaneously and three-dimensionally.

At present, when conducting research on trace proteins and viruses, the gene is cloned and the amino acid sequence of the protein can be predicted from its base sequence. Search for a highly hydrophilic site based on this amino acid sequence,
Polyclonal and monoclonal antibodies against the synthetic peptide at that site have been prepared and used for immunological analysis. Hoop & Woods et al.'S method to search for highly hydrophilic sites
Analysis is performed using [Proc. Natl. Acad. Sci. USA 78: 3824 (1981)] or the like, but it does not always apply to all proteins. Therefore, according to the present invention, VEGF / VP
FGF / VPF antibodies having a strong anticancer activity can be easily prepared by clarifying a portion that is important for inhibition of angiogenesis and the like among the sites exposed on the surface of F, and The method adopted in the present invention can also be applied as a method for clarifying a site exposed on the surface of a protein.

Example 1 (Tumor suppression test by combined use of anti-VEGF / VPF monoclonal antibody MV833 and minocycline)

As a solid tumor growth model, a 2 mm square of human fibrosarcoma strain HT-1080 was transplanted subcutaneously into the abdomen of BALB / cnu / nu (nude mouse, male, 6 weeks old, n = 4 or 5) according to a conventional method. .

From the day after transplantation, administration was performed via the tail vein according to the following dosage and schedule.・ Minocycline single administration group (●): 10 mg / kg, 1, 2,
Administration on days 3, 4, 7, 8, 9, 10, 11 ・ MV833 alone administration group (▲): 12.5 μg / mouse,
Administration on days 1, 5, 9, 13, and 17-MV833 and minocycline combination administration group (■): MV
833: 12.5 μg / mouse, administered on days 1, 5, 9, 13, and 17 minocycline: 10 mg / kg, 1, 2, 3, 4, 7, 8, 9, 10,
Administration on day 11 ・ Control group (○): PBS (-) 0.2 ml / mouse, 1,
Administration on days 5, 9, 13, and 17

The time-dependent tumor diameter of each group was measured with a caliper,
The tumor volume was calculated according to the formula (minor axis) ² × (major axis) / 2, and the results are shown in FIG.

From FIG. 1, it can be seen that the group administered with MV833 and minocycline together suppresses tumor growth more significantly than the group used alone, and that a particularly high inhibitory effect is obtained even after the end of minocycline administration (after day 12). I understand. Therefore, it was shown that the anti-VEGF / VPF antibody and the angiogenesis inhibitory action of minocycline act synergistically on the inhibition of solid tumor formation. After completion of minocycline administration, anti-VEGF
It was shown that the continued administration of the / VPF antibody also effectively suppressed tumor growth. Therefore, it is suggested that neutralizing VEGF / VPF induced from hypoxic tumor cells by administration of minocycline suppresses tumor cell angiogenesis and, as a result, effectively suppresses tumor growth Was done.

[0052]

According to the present invention, by using a VEGF / VPF antagonist and minocycline together,
It is possible to effectively suppress the growth of the tumor and the accompanying angiogenesis. Therefore, the present invention can be effectively used for treating various cancers and other angiogenic diseases.

[0053]

[Sequence list]

SEQ ID NO: 1 Sequence length: 9 Sequence type: amino acid Topology: Linear Sequence type: Protein Origin: Cell line:

SEQ ID NO: 2 Sequence length: 12 Sequence type: amino acid Topology: linear Sequence type: protein Origin: cell line:

SEQ ID NO: 3 Sequence length: 12 Sequence type: amino acid Topology: linear Sequence type: protein Origin: cell line:

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the time-course effects of minocycline alone, anti-VEGF / VPF antibody alone, and a combination of both on human fibrosarcoma cell proliferation.

Claims (5)

[Claims] 1. An angiogenesis inhibitor comprising a VEGF / VPF antagonist and minocycline as active ingredients. 2. The method of claim 2, wherein the VEGF / VPF antagonist is an anti-V
The angiogenesis inhibitor according to claim 1, which is an EGF / VPF monoclonal antibody. 3. The angiogenesis inhibitor according to claim 2, wherein the anti-VEGF / VPF monoclonal antibody is an antibody that reacts with at least one of the following sequences 1 to 3, which is a part of the amino acid sequence of VEGF / VPF. . 1. KPSCVPLMR 2. SFLQHNKCECRP 3. KCECRPKKDRAR 4. A method for inhibiting angiogenesis, comprising administering a VEGF / VPF antagonist and minocycline in combination. 5. A method comprising administering a VEGF / VPF antagonist and minocycline in combination, and then administering the VEGF / VPF.
The method for inhibiting angiogenesis according to claim 4, wherein the administration of the antagonist alone is continued.