Heparin-like compounds, their preparation and use to prevent arterial thrombsis associated with vascular injury and intervetions
a technology of heparin and compounds, applied in the field of heparinlike compounds, can solve the problems of not being suggested for therapeutic or corresponding use, not being used clinically, and natural or native conjugates are difficult to prepare in pure form, so as to prevent thrombosis and facilitate screening.
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Examples
example 1a
Methods of Obtaining Heparin Proteoglycan (HEP-PG)
Heparin Proteoglycans (HEP-PG) Exocytosed by Stimulated Mast Cells
[0096] Mast cells were isolated from rat peritoneal and pleural cavities as described (Yurt R W, Wesley Leid, Jr. R, Austen K F. J Biol Chem 1977; 252: 518-521). In a standard assay, 10.times.13.times.10.sup.6 mast cells were incubated in 1 ml of PBS buffer containing 0.1 mg / ml HSA (Red Cross Transfusion Service, Helsinki, Finland) and 5.6 mM glucose. After preincubation (15 min, 37.degree. C.) the cells were incubated for 15 min with compound 48 / 80 (Sigma Chemical Co) (5 .mu.g / ml), a specific mast cell agonist, to induce mast cell degranulation. Control experiments showed that compound 48 / 80 does not induce platelet aggregation. The degranulated mast cells were then sedimented by centrifugation at 150.times.g for 10 min, the supernatant was centrifuged for a further 15 min at 12 000 g to sediment the exocytosed granules, and the granule-free supernatant was analyzed f...
example 1b
Methods of Obtaining Heparin Proteoglycan (HEP-PG)
Natural Heparin Proteoglycans (HEP-PG)
[0097] Connective-type mast cells, such as skin and serosal mast cells of mammalian origin can be isolated with the method described in example 1A or slightly modified methods, not only from rats, but also from other mammalian species such as bovine, swine, sheep, etc. During slaughtering of cows or pigs peritoneal and pleural lavage is performed with phosphate buffered saline. The pooled fluids are centrifuged once at 100.times.g for 5 min and the sedimented cells are resuspended in PBS. The isolation of mast cells is obtained by gradient centrifugation in Ficoll, during which mast cells concentrate at the interphase between 30% and 40% Ficoll layer. To obtain the soluble proteoglycans mast cells are stimulated with compound 48 / 80, a basic polyamine, or calcium ionophore A 23187, which induce exocytosis of the mast cell granules.
[0098] In contrast to the traditionally isolated bovine or swine-de...
example 1c
Methods of Obtaining Human-derived Natural Heparin Proteoglycan (HEP-PG) from Human Mast Cells
[0100] Connective-type mast cells, such as skin and serosal mast cells of human origin can be isolated with the method described in Example 1A or slightly modified methods. It is to be observed that only a small sample is required, which can be obtained from a patient with routinely performed biopsy procedures. The human derived mast cells are thereafter cultivated in a conventional cell culture media and under conditions allowing good proliferation of the mast cells. The mast cells are harvested with phosphate-buffered saline and treated as described in Example 1A and 1B. It is also to be observed that once cultured, the cell cultivate can be preserved and stored by per se known methods; and provides an unlimited source for producing more cells.
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Abstract
Description
Claims
Application Information
- IPC
- A61K31/727; A61L33/00; A61L33/08; A61L33/10; A61P7/02; C08B37/00; C08B37/10
- CPC
- A61K31/726; A61L33/0011; A61L33/08; C08B37/0066; C08B37/0075; C08B37/0081; C08H1/00; C08L5/00
- Inventors
- LASSILA, RIITTA; KOVANEN, PETRI