Drug – bio-affecting and body treating compositions – Extract – body fluid – or cellular material of undetermined... – Blood
Patent
1990-05-18
1992-11-24
Rosen, Sam
Drug, bio-affecting and body treating compositions
Extract, body fluid, or cellular material of undetermined...
Blood
514 2, A61K 3514
Patent
active
051659387
DESCRIPTION:
BRIEF SUMMARY
rt, including collagen, ADP and serotonin, may be utilized instead of or in addition to thrombin to activate the platelets, although thrombin is preferred.
DETAILED DESCRIPTION OF THE INVENTION
Blood obtained from the individual to be treated with the wound healing factors of the invention is stabilized in siliconized tubes containing acid-citrate dextrose (0.15M citrate, 2% glucose, pH 4.2) (hereinafter CPD) and is centrifuged in order to separate out, the platelet-rich plasma therefrom. Forty to sixth milliliters of blood combined with 4-6 ml of CPD is then centrifuged at about 135 .times. g for 20 minutes at about 4.degree. C to obtain platelet-rich plasma. The platelet rich plasma is removed and placed into another sterile, 50ml tube. A platelet count is then taken. The CDP is utilized to prevent activation of the clotting sequence by contact of the blood with the plastic in the syringe. The CPD is present in the syringe while the blood is withdrawn from the patient. The blood is continuously mixed with the CPD to prevent coagulation. The platelet-rich plasma in the tube is then centrifuged at 750 .times. g for 10 minutes at 4.degree. C.
The platelet-free plasma is removed and discarded. The platelet pellet is resuspended in a quantity of platelet buffer to produce a final ml. A lower concentration of about a million platelets per ml is useful, but is less preferred. The platelet buffer utilized contains 0.05 M HEPES (N-2-hydroxyethylpiperazine-n-2-ethanesulfonic acid), 0.03 M glucose, 0.004 M KCl, 0.1 M NaCl and about 0.35% human serum albumin adjusted to a pH of about 6.5. A sample is frozen at about -20.degree. C. for later testing of mitogenic activity. Another sample is streaked onto blood agar as a sterility test.
The platelet-rich plasma is the only blood fraction utilized in the processes and compositions of the invention. The PRP is then activated with purified thrombin at a rate of about 1 to about 10 units of thrombin per milliliter of PRP. Preferably, about 1 unit of thrombin per ml of platelet-rich plasma is utilized. The activity of the thrombin coagulates the fibrinogen and activates platelets causing them to release alpha granules containing platelet-derived growth factor and platelet-derived angiogenesis factor. The thrombin used was Thrombinar.TM. brand from Armour Pharmaceutical Co. of Kankakee, Ill. The platelets and thrombin are allowed to incubate at room temperature for about 5-10 minutes.
The PRP is then subjected to a removal of platelets and fibrin by centrifugation. The resulting supernatant contains both PDAF and PDGF after centrifuging at 950 .times. g for about 5 minutes at 4.degree. C. The pellet is discarded since the PDAF & PDGF have been extracted into the supernatant. PDGF has been isolated and characterized. It is a protein of 30,000 molecular weight which breaks down into two molecular weight species of 15,000 and 14,000 molecular weight.
In order to apply the PDAF and PDGF in the platelet-free supernatant thus obtained to a wound, it is desirable to utilize a carrier substance which is biologically compatible and acts as a temporary "depot". A macromolecular substance such as microcrystalline collagen provides a suitable carrier. An especially preferred carrier is Avitene.RTM. brand microcrystalline collagen from FMC Corp., Avicel Dept., Marcus Hook, Pa. 19061. The resultant composition is thicker and will tend to remain in position in contact with the wound. Debrisan.TM. brand wound dressing which contains Sepharose.TM. brand beads, trademarks of Pharmacia Fine Chemicals, Inc. of Piscataway, N.J., may be utilized as an alternative carrier. Preferably, about 8-10 ml of supernatant per gram of carrier is used to produce a paste.
Application of the wound treating composition is by physically applying the material over an into the wound as in applying a medicated salve. Treatments should be repeated on a daily basis as long as the wound remains open. A preferred treatment is to apply an approximately one mm thick dressing of the platelet factor/carrier complex to t
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This application was made under contract with the Department of Veterans Affairs. Title to the invention remains with the inventor subject to the U.S. Government's reservation of a nonexclusive, irrevocable, royalty-free license in the invention with the power to grant sublicenses for all government purposes.
This application is a file wrapper continuation of co-pending application Ser. No. 07/039,776, filed Apr. 15, 1987 now abandoned which was a file wrapper co
Curative Technologies, Inc.
Regents of the University of Minnesota
Rosen Sam
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