Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/60—Salicylic acid; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/425—Thiazoles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
  • A61K38/1808—Epidermal growth factor [EGF] urogastrone

Definitions

• This invention relates to the control of angiogenesis, and methods and compositions therefor.
• a method for stimulating angiogenesis in a mammal characterized by the use of a modulator of collagen synthesis or of collagen fibril assembly.
• Cis-HYPRO will prevent this migration of cells into the wound
• cis-HYPRO will stimulate the migration of capillary endothelial cells in vitro. I have also fund that cis-HYPRO stimulates angiogenesis in vivo in the rabbit corneal pocket assay.
• proline analogues may also be useful. It appears that, in general, agents which modulate collagen synthesis or collagen fibril assembly orwhich inhibit proline hydroxylase will cause the production of defective collagen which is more susceptible to degradation by proteases. Capillary endothelial cells migrate along the gradient of inhibitor concentration, and form capillary tubules in vivo despite the defect in the collagen which is synthesized.
• a method of stimulating angiogenesis in a mammal characterized by the use of a modulator of collagen synthesis or of collagen fibril assembly.
• the modulator is an inhibitor of the activity of the enzyme proline hydroxylase.
• the inhibitory agent is selected from the group which includes cis-4-h droxy-L-proline, 3, 4-dehydro-L-proline, L-azetidine-2-carboxylic acid, L-proline analogues, and their pharmacologically active analogues and derivatives. n the invention may optionally be used.
• Combinations of one or more compounds according to the present invention together with one or more compounds according to my copending Australian provisional application PH7521 entitled “Stimulation of Angiogenesis and Control of Endothelialisation” or other stimulators of angiogenesis may also optionally be used.
• the compound according to the invention may optionally be administered in a slow-release form or in a biodegradable matrix.
• Proliferation of endothelial cells is thought to be a response secondary to cell migration during new vessel formation.
• the proliferative responses have been marginal and the reports are not in accord as to the minimal conditions or cell type necessary.
• Having used one or other of the above assays to purify fractions they are usually confirmed as angiogenic by an indirect in vivo assay of activity such as the corneal pocket or chorioallantoic membrane assay.
• proline analogues cis-4-hydroxy-L-proline (cis-HYPRO), 3, 4-dehydro-L-proline (dHPro), cis-4-hydroxy-D-proline (cisdPro) and L-azetidine-2-carboxylic acid (AZET) were obtained from Sigma Chemical Co., St. Louis, U.S.A.. To remove metal ions, they were dissolved in distilled water, applied to a column of Chelex 100 (Biorad), developed in quartz glass double distilled water, then recrystallized. This reduced the copper level to less than 0.06 parts per billion.
• mp antat on was v a a rocar, an was per orme so as to avoid trauma to the vascular system.
• Controls showed no overt inflammatory response and no sign of inducing angiogenesis in surrounding tissue.
• implants were examined in situ, photographed then excised and examined histologically.
• Corneal Pocket Assay The corneal pocket assay of Gimbrone et al (1974) as modified by Gol ' e and McAuslan (1981) was used on New Zealand white rabbits of 2 - 3 kg body weight. Opposite eyes of each
• a line of bovine retinal capillary endothelial cells free from mural cells was established .essentially by the procedures of Buzney and Massicotte (1979).
• Bovine corneal endothelial cell cultures were prepared and maintained as described by McAuslan et al (1979). Cultures of bovine retinal microvascular pericytes (mural cells) were established from bovine retinal capillaries by the method of Gitlin and D'Amore and maintained in Ham's F12 medium. All cell lines for experimentation on migration were used between their 8th and 12th passage. Each cell line was tested in its preferred growth medium but with the serum concentration reduced to 2%.
• ⁇ > - ⁇ -4 per cm were treated with cis-HYPRO at 0, 10 or 5 x 10 M for 4 hours in normal growth medium. . The medium was removed and the cell layers were washed and covered with medium containing the appropriate concentration of cis-HYPRO and free of serum. This was supplemented with sodium ascorbate (50 ug/ml) and ⁇ -amino propionitrile (80 ⁇ g/ml). Then cells were (specific activity) per ml of proline free medium 199 (Commonwealth Serum Labs) . Polypeptides secreted into the medium were harvested, separated by sodium dodecylsulphate polyacrylamide gel electrophoresis and detected by autoradiography.
• Figure 1 represents the effects of proline analogues on the growth of bovine aortal endothelial cells
• Figure 2 represents the effects of proline analogues on the growth of bovine retinal capillary endothelial cells
• Figure 3 represents the effects of proline analogues on the growth of bovine retinal microvascular pericytes
• bovine retinal endothelial cells Cultures of bovine retinal endothelial cells, bovine aortic endothelial cells, and bovine retinal pericytes (mural cells) were prepared as described above, and tested separately for their response to cis-HYPRO, cis-4-hydroxy-D-proline, and L-azetidine-2-carboxylic acid. The results were normalised for each experiment as follows:
• control was assigned .a value of 100%.
• e resu s o an exper m n endothelial cells and retinal pericytes are shown in Table 1, in which each figure represent the mean value for 200 individual cells analysed 48 hours after the addition of the test compound.
• the medium was Ham's F12 medium containing 5% serum (v/v). Each agent was used at a concentration of 10- M.
• Control 13 152 7 106 cis-HYPRO 46.4 258 21.4 180 cis-D-hydroxyproline 13 152 - -
• Cis-HYPRO strongly stimulated migration of both cell types, while cis-D-hydroxyproline had no effect, and azetidine had a marginal effect on retinal endothelial cells; dehydroproline had a small effect on retinal endothelial cells, and a stronger effect on retinal pericytes.
• Cis-HYPRO and azetidine were assayed in rabbits using the corneal pocket assay and the subcutaneous assay as described above. The results are presented in Table 2. The figures represent the ratio of animals giving a positive angiogenic response to the total number of animals tested.
• Cis-HYPRO caused stimulation of angiogenesis in' both systems, whereas azetidine caused stimulation only in the subcutaneous assay.
• responses in the corneal pocket assay were weak, and the subcutaneous assay was found to be reproducible and effective for a number of chemically different angiogenic agents.
• HYPRO or AZET were tested in a further experiment using this assay, both gave positive angiogenic responses in each of 12 tests in 6 different test animals.
• dHPro was not found to be active.
• epidermal growth factor (EGF; Ben-Ezra, 1978) can be explained by its ability to induce capillary endothelial cell migration and proliferation highly potent inducer and is less active angiogenically than other growth factors such as transforming growth factor TGF- (Schreiber et al, 1986). Therefore we tested the possibility that the responses produced by Cis-HYPRO and EGF might be additive.
• the induction of bovine corneal endothelial cell migration by EGF, Cis-HYPRO or EGF plus Cis-HYPRO was compared. As shown in Table 5, the migration-inducing ability of EGF and Cis-HYPRO was at least additive over the limited range tested.
• EGF (lOO ⁇ g/ml) 116.0 cis-HYPRO (10 ⁇ 5 M) 56.4 EGF (100 ⁇ g/ml) + cis-HYPRO (10 ⁇ 5 M) 266.7
• cis-HYPRO or other proline analogues could be used to enhance the angiogenic effect of EGF, or of the active subunit of EGF which is responsible for angiogenesis.
• Cis-HYPRO and other analogues work as positive effectors (e.g. causing formation of a migration peptide or enhancing the activity of a specific protease) or as negative effectors (e.g. by impairing formation of a critical anchorage component or by blocking synthesis of a migration inhibitor) remains to be established. While the detailed mechanism of action and cellular responses remains to be resolved the conclusion from these results is that for the icrovascular system perturbation of collagen related systems by low concentrations of proline analogues is sufficient to provoke cell migration and angiogenesis.
• the present invention is capable of application in a wide variety of clinical fields.
• Stimulation of angiogenesis can be used to enhance the healing of b ⁇ rns and wounds, especially those involving large tissue defects, acceptance of skin or organ grafts, and can also be used in reconstructive and cosmetic surgery, including the use of subdermal implants, .and in prosthetic surgery, particularly that involving vascular prostheses.
• Such stimulation may be used in any situation wherein endothelial cell migration and regeneration of endothelium are advantageous, or where an increase in blood flow is desirable, e.g., stroke, heart disease, or foetal blood insufficiency.
• This application of cis-HYPRO excludes its use as an agent which might improve the performance of implantable prosthetic devices such as pacemaker electrodes by virtue of properties other than stimulation of angiogenesis.

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• 1987
  • 1987-02-10 WO PCT/AU1987/000037 patent/WO1987004925A1/en not_active Application Discontinuation
  • 1987-02-10 EP EP19870901278 patent/EP0294380A4/de not_active Withdrawn
  • 1987-02-10 JP JP62501360A patent/JPS63502661A/ja active Pending

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