JP2009531410A - Anti-angiogenic peptides and their use for therapy - Google Patents

Abstract

  Angiostatin peptide fragments having anti-chemotaxis, anti-angiogenesis, and anti-tumor activity, pharmaceutical compositions containing them, and their use for the treatment of inflammation, connective tissue degeneration, vascular and neoplastic conditions.

Description

  The present invention relates to angiostatin peptide fragments with anti-chemotactic, anti-angiogenic and anti-tumor activity that are useful for the manufacture of a medicament for the treatment of inflammation, connective tissue degeneration, vascular and neoplastic conditions. Is.

  Angiostatin (AST) is an anti-angiogenic molecule produced in the body under special conditions, such as solid tumors, that expresses a homeostatic response to the underlying pathological state. is there. It is produced as a fragment of plasminogen and induces apoptosis in endothelial cells and other cellular targets (1), preventing neovascularization and growth of blood vessels in both primary and metastatic tumors. No toxic effects or development of tolerance has been observed in animals treated with angiostatin.

  A characteristic of this molecule is the presence of a functional domain called kringle. The properties of angiostatin and its kringle have been described in numerous publications and patent applications, particularly Protein Expression and Purification, 36 (2004) by You, WK. Et al .; US Patent Application No. 2004/002459 (US2004 / 002459); No. 5,837,682 (US5837682); Bouma-Ter Stegee, JCA et al., Critical Reviews in Eukaryotic Gene Expression, 11 (2001). Other patents disclose variants of the sequence of angiostatin and methods for isolating, purifying, and producing it synthetically. See, for example, U.S. Pat. Nos. 5,861,372; 5,639,725; 5,792,845; 5,885,795; 5,854,205; 5,854,221;

Description of the Invention An isolated fragment from the third kringle of angiostatin molecule, it reproduces inhibition of neutrophil chemotaxis, induction of endothelial apoptosis and inhibition of angiogenesis, ie representative of angiostatin itself. Effects were possible.

  This peptide, the subject of the present invention, has the sequence HNR (HisAsnArg), an in vitro chemotaxis assay, an in vivo angiogenesis assay utilizing a matrigel sponge, and animal tumors This was studied with a model. The procedure used is described in the section “Materials and Methods”.

  FIG. 1-a shows the anti-chemotactic activity of HNR in human polymorphonuclear cell (PMN) enriched fractions. As can be observed in the graph (bar “X”), the peptide activity is very high compared to both angiostatin and IL-8 (p <0.004; Mann-Whitney). Conversely, when assayed on endothelial HUVEC (EC) and mononuclear cells, the peptide did not show significant activity. Therefore, the anti-chemotactic effect of HNR is highly selective for PMN.

  AST is known to induce endothelial apoptosis by inactivating ATP synthase. Therefore, the pro-apoptotic action of HNR on HUVEC was tested. FIG. 1 shows a significant increase in free nucleosomes after 8 hours of treatment with 2.5 μg / ml peptide HNR. On the other hand, no significant results were obtained when either PMN or PBMC was used. Thus, induction of apoptosis by HNR is selective for endothelial cells.

  Anti-angiogenic activity is shown in FIG. Angiogenesis induced in mice was strongly inhibited by HNR (p <0.02), but to a lesser extent than angiostatin. After the experiment was completed, histological control of the sponge was also performed to confirm the cellularity, the extent of angiogenesis, and its morphology (FIG. 2-b). The results obtained by cellularity, sample vascularization, and hemoglobin measurement in Matrigel sponges were perfectly consistent (FIG. 2-b).

  Finally, the ability of HNR peptides to inhibit tumor growth was examined in a Kaposi's sarcoma (KS) cell graft model in nude mice. Intraperitoneal administration of the peptide every 4 days after cell inoculation was observed to slow tumor growth compared to medium alone (p <0.02; Mann Whitney, FIG. 3, curve HNR). . The tumor weight at the end of the experiment confirmed this result.

  For therapeutic use, the peptide HNR is preferably combined with pharmaceutically acceptable excipients and / or carriers. In a further aspect, the present invention is a pharmaceutical composition comprising a peptide HNR, which is administered orally or parenterally, in particular subcutaneous, intravenous, intramuscular, rectal, eye, nasal, topical, intrauterine or vaginal route. Relates to a pharmaceutical composition suitable for administration by. The composition may be a solid, such as a capsule, tablet or granule; a liquid, such as a solution, suspension, syrup, drop, tincture, spray or aerosol; a semi-solid, such as a cream, ointment or gel Can be. The dosage of the peptide will depend on such factors as the age and weight of the subject or the route of administration, as well as the severity of the disease or disorder to be treated. In principle, amounts of peptides ranging from 0.01 to 250 mg / kg, preferably 0.1 to 100 mg / kg will be used for the treatment of humans or animals.

  Peptides of the present invention and pharmaceutical compositions thereof can be used for chronic inflammation, diabetes, connective tissue degeneration including rheumatoid arthritis and atherosclerosis, primary vascular conditions, especially eye disorders such as macular degeneration, immature retinopathy , Corneal rejection, vascular glaucoma, post lens fibroproliferation, rubeosis, cardiovascular and cerebrovascular conditions, tumors, particularly solid tumors and leukemias, can be used for prophylactic or therapeutic treatment.

  Compared to angiostatin, the peptides of the present invention are more stable, bioavailable, reach all target tissues, and are produced with high purity (absence of bacterial contaminants) and low cost Is easy.

HNR peptides established procedures [Stuart & Young, 1984, Solid Phase Peptide Synthesis, 2 nd ed, Pierce Chemical Co.; Tam et al, Am Soc, 1983, 105:.... 6442; Merrifield, 1979, The Peptides, Gross and Meihofer eds. NY Academic Press, 1-284] can be synthesized in solution, in solid phase, or using an automated synthesizer. Alternatively, the peptides may be produced by recombinant DNA technology [Sambrook et al., Molecular Cloning, A Laboratory Manual, CSH Press, CSH, NY, 1982 or Ausbel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987]. Amino acid residues within the sequence HNR may have the D or L configuration, or, for example, carboxyl-terminal amidation, linkage with lipophilic groups (ie fatty acid residues), sugar chain formation, or Ligation with other molecules can be chemically modified to improve aspects of peptide activity or bioavailability.

DESCRIPTION OF THE FIGURES FIG. 1-a Chemotaxis in polymorphonuclear spheres. Migrated PMN cells were counted (y-axis) and the number was confirmed by densitometry (not shown). SFM = serum-free medium; IL-8 = interleukin 8 used as chemotactic factor.
FIG. 1-b Apoptosis induced by HUVEC. Free nucleosomes were examined 8 hours after treatment. AST = angiostatin; Vinc = vincristine (25 mM: positive control); CTL = untreated cells (blank). HNR 2.5 and 5 represent HNR concentrations of 2.5 and 5 μg / ml.
Figure 2-a Angiogenesis in vivo. The hemoglobin content in the Matrigel pellet was reported on the horizontal axis as an indicator of angiogenesis. VTH = Vegf, TNF, heparin used as angiogenesis promoter; AST = angiostatin; X = HNR.
Fig. 2-b Histological analysis of Matrigel sponge. The figure shows a section stained with hematoxylin-eosin. Compared to the chemoattractant VTH alone (VTH CTRL), lack of blood vessels and poor cellular infiltrates can be observed in HNR samples.
Figure 3. Inhibition of tumor growth. The experiment was terminated on day 25. CTRL-peptide is a control peptide with the sequence AlaSerAla. PBS = PBS + 0.1% BSA used as excipient.

Materials and Methods Tumor Inhibition CD1 nude mice were divided into 3 groups, one group was injected intraperitoneally with 50 μg HNR peptide (Calbiochem, La Jolla, Calif.) Per mouse in 100 μl PBS-BSA, and the control group The third group was injected with 50 μg of random sequence peptide. AST was injected every other day and HNR was injected every 4 days. On day 0, 5,000,000 KS IMM cells were injected. On day 31, the mice were killed.

The volume measured with a caliper is the formula:
Calculation was performed using π / 6x length x width.

Matrigel sponge assay Angiogenesis assay with Matrigel sponge in C57bl mice was performed using VTH or KS cell supernatant as chemotactic factor as described in (3).

Peptide sequence Peptide: HNR
Control peptide ("Random" in Figure 2-a): AlaSerAla

Matrigel sponge histochemistry After removal from the animals, the pellets were fixed in 4% PAF and embedded in paraffin. 4 μm sections were stained with standard hematoxylin-eosin procedures.

Chemotaxis-apoptosis This assay was performed in a 48-well chamber as described in (4). The apoptosis assay was performed using a Cell Death Detection Kit (Roche) according to the manufacturer's instructions.

Shows chemotaxis in polymorphonuclear cells. Figure 3 shows apoptosis induced by HUVEC. Shows angiogenesis in vivo. The histological analysis of Matrigel sponge is shown. Inhibition of tumor growth.

Claims (5)

  An isolated anti-angiogenic peptide having the sequence HisAsnArg (HNR).   2. The amino acid residue has a D- or L-configuration or is chemically modified by carboxyl-terminal amidation, coupling with lipophilic groups, in particular fatty acid residues, or sugar chain formation. The described peptides.   The anti-angiogenic peptide according to claim 1 or 2 for use as a medicament.   A pharmaceutical composition comprising the peptide according to claim 1.   Use of the peptide of claim 1-2, or the pharmaceutical composition of claim 4, comprising connective tissue degeneration, including chronic inflammation, diabetes, rheumatoid arthritis and atherosclerosis, primary vascular conditions, Prevention of eye disorders, especially macular degeneration, immature retinopathy, corneal rejection, vascular glaucoma, post-lens fibroplasia, rubeosis, cardiovascular and cerebrovascular conditions, tumors, especially solid tumors and leukemia Or use for the manufacture of a medicament for therapeutic treatment.