JPH0720873B2 - Wound healing agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION This application was made under a contract with the United States Government Department of Veterans Affairs. The right to an invention is vested in the inventor who is subject to US Government reservations, subject to nonexclusive, invariant, and royalty-free inventions, which are entitled to licenses for all government purposes.

This application is pending patent application No. 676471 dated November 29, 1984.
It is a partial continuation application of the issue.

FIELD OF THE INVENTION The present invention relates to wound healing agents (in particular angiogenic and growth factors), their preparation from blood and their use for promoting wound healing.

BACKGROUND OF THE INVENTION Angiogenesis, which is the proliferation and controlled growth of capillary endothelial cells with fibroproliferation and collagen synthesis.
(ogenesis) is an essential element of the host's response to a wound. Platelet activation and the blood coagulation cascade are among the first responses to injury.

Platelets activated by thrombin release mitogens (ie growth factors) for fibroblasts and smooth muscle cells and stimulate smooth muscle cell collagen synthesis in vitro. Mitodiene (platelet-derived growth factor, hereinafter abbreviated as PDGF) consists of two polypeptides. A paper on PDGF is available at GRGrot
endorst), Chiang (T.Chang), Sepa (HEJSepp)
a), Clyman (HKKleinman) and Martin (G.
R. Martin) "Platelet-derived growth factor is a chemoattractant for vascular smooth muscle cells", Journal of Cellular Phys
Biology, Vol. 113, p. 261-266 (1982). The above article is incorporated herein by reference.

A non-mitogenic substance called an angiogenic factor is also produced by thrombin activated platelets and stimulates capillary growth. Various angiogenic factors are known, including angiogenic factors for tumors, retinas and wound fluids. It is not known whether all angiogenic factors have a common mechanism of action on capillary endothelial cells.

Angiogenic factors have been isolated and published by MS Banda, DRKnighton, TKHunt and Z. Werb, entitled "Isolation of Non-Mitodiene Angiogenic Factors from Injury Fluid," Proc. Nat′l.Acad.Sci.US
A, 7773-7777, December 1982). The content of this article is hereby incorporated by reference.

Angiogenic factors and platelet-derived growth factors are defined by Knighton (D.
R.Knighton), Hunt (TKHunt), Takral (KKTh)
akral) and Gutsdson (WHGoodson III), entitled "Role of Platelets and Vibrin in the Healing Process" [Annals of Surgery, 196: 379-388 (1982)], the contents of which are incorporated herein by reference. To be done. The article states that a patient's non-healing wound was successfully healed upon transfusion of a single 10 units of platelets.
The wound healed in 3 weeks.

Recent studies have shown that when the normal healing process of the human body works, it only reaches an effective level of about 50%.

U.S. Pat. No. 4273871 to Tolbert et al.
It produces human angiogenic factors from human foreskin fibroblasts. Publicly available foreskin fibroblast cell lines can be utilized to produce angiogenic factors.

In Antoniades U.S. Pat. No. 4,479,896, the contents of which are incorporated herein by reference, platelet-derived growth factor was identified and extracted by gel electrophoresis for study.

SUMMARY OF THE INVENTION Thrombin activated platelets have the ability to stimulate angiogenesis, enhanced collagen synthesis and cell division / growth. Using a whole blood sample to prepare platelet-rich plasma,
It has been discovered that activation of it with thrombin results in platelet-rich plasma containing angiogenic and growth factors that can be used to accelerate the wound healing process.

The blood is stabilized and centrifuged to obtain platelet-rich plasma. Blood was citrate-phosphate-dextrose 1: 5.
Stabilized by mixing in the ratio (20% v solution). Platelet-rich plasma (hereinafter abbreviated as PRP) is preferably further centrifuged until a high concentration of platelets is obtained. Next, the platelets are added to the platelet buffer. The concentration of platelets will be at least 1,000,000 platelets per milliliter and preferably about 1,000,000,000 platelets.

Thrombin is added to PRP to activate platelets. Preferably, about 1 to about 10 units of thrombin are used per milliliter of PRP. Thrombin-activated platelets release platelet-derived growth factor (hereinafter abbreviated as PDGF) and platelet-derived angiogenic factor (hereinafter abbreviated as PDAF). Platelets and thrombin are allowed to incubate at room temperature for about 5-10 minutes.

Activated PRP, including PDGF and PDAF, is preferably added to the biologically compatible polymeric material that acts as a carrier. First, the platelets are centrifuged at about 950 xg and the platelet-free supernatant is mixed with the carrier. Examples of biologically compatible carriers include Pennsylvania (19061)
It is a microcrystalline collagen such as Collagen under the trade name of Avitene, which is marketed by the Avicel Division of FMC of Marcus Foods. Microcrystalline collagen is compatible with the human body. Sufficient carrier is added to absorb all of the platelet-rich plasma obtained from the blood. For example, a 40 ml blood sample typically has about 25
You'll need ml carriers. The paste thus obtained is preferably stored on ice or in a refrigerator.

A suitable carrier is also a medical formulation for use as a wound dressing marketed under the trade name Debrisan by The Pharmacia Huain Chemicals Company of Piscataway, NJ.

Activated PRP absorbed in the carrier is then applied to the wound site. Highly enriched therein active PDGF and PDAF proliferate and grow capillary endothelial cells, double the rate of collagen synthesis, and promote healing by promoting chemotaxis of leukocytes. Mitodiene activity induces cell division / growth to regenerate defective tissue.

Daily application of active PRP to the wound site stimulates and accelerates the healing process. The amount of PRP prepared from 40 ml of blood is sufficient for a 7 day application. This material is applied over the wound site in a relatively uniform thickness (about 2 mm thick). When the body's own repair signals are insufficient to stimulate good healing, the use of activated PRP initiates granulation, contraction and epithelialization.

As used herein, thrombin means thrombin as a biological release agent for platelet release. Other biological release agents known in the art, including collagen, ADP and serotonin, may also be used in place of or in addition to thrombin to activate platelets, with thrombin being preferred. .

DETAILED DESCRIPTION OF THE INVENTION Blood taken from a patient to be treated with the wound healing factor of the present invention contains acid-citrate-dextrose (0.15M
Stabilize in a silicone treated tube containing citrate, 2% glucose, pH 4.2) (hereinafter abbreviated as CPD) and centrifuge to separate platelet-rich plasma from the blood.
40-60 ml of blood is mixed with 4-6 ml of CPD, about 135 xg,
Centrifuge at about 4 ° C. for 20 minutes to obtain platelet-rich plasma. The plasma is removed and added to another sterile 50 ml tube. Then count the platelets. CPD is used to suppress a series of coagulation activation caused by contact between blood and plastic syringe. Keep the CPD in the syringe while blood is drawn from the patient. Continuously mix blood and CPD to prevent blood clotting.
After that, the platelet-rich plasma in the test tube was 750 xg at 4 ° C.
Centrifuge for 10 minutes.

Platelet-free plasma is removed and discarded. Resuspend the platelet pellet in the appropriate volume of platelet buffer to make the final ml. 1 ml
Concentrations below about one million platelets per minute may be used, but are less preferred. The platelet buffer used is 0.
05M HEPES (N-2-hydroxyethylpiperazine-n
-2-ethanesulfonic acid) 0.03M glucose, 0.004MK
It contains Cl, 0.1 M NaCl and about 0.35% human serum albumin adjusted to about pH 6.5. Samples are frozen at about -20 ° C for subsequent mitodiene activity testing. Another sample is streaked on blood agar as a sterility test.

Platelet-rich plasma is the only blood fraction utilized in the methods and compositions of the present invention. PRP is then activated with purified thrombin at a rate of about 1 to about 10 units of thrombin per ml of PRP. Preferably, about 1 unit of thrombin is used per ml of PRP. The function of thrombin coagulates fibrinogen and activates platelets to release alfa granules containing platelet-derived growth factor and platelet-derived angiogenic factor. The thrombin used is a product called Thrombinar available from Armor Pharmaceuticals, Inc. of Kankakee, Illinois. Platelets and thrombin are incubated at room temperature for about 5-10 minutes.

The PRP is then subjected to the removal of platelets and fibrin by centrifugation. The supernatant obtained after centrifugation at 950 xg at 4 ° C for about 5 minutes contains both PDAF and PDGF. Discard the precipitate as PDAF and PDGF are extracted in the supernatant. PDGF was isolated and characterized. It is a protein with a molecular weight of 30,000 and is broken down into two substances with molecular weights of 15,000 and 14,000.

To apply PDAF and PDGF in the platelet-free supernatant thus obtained to the wound site, it is desirable to use a carrier material that is biocompatible and acts as a temporary "depot". Carriers are polymeric substances such as microcrystalline collagen. A particularly preferred carrier is microcrystalline collagen commercially available under the tradename Abitene from the Avicel Division, FMC Company, Marcus Fuchs, PA (19061). The resulting composition will be viscous and will remain in contact with the wound site. Debrisant wound dressing containing Sepharose beads (trademark of Pharmacia Huaine Chemicals, Inc., Piscataway, NJ) may also be used as another carrier. Preferably, about 8-10 ml of supernatant is used per gram of carrier to prepare the paste.

Application of the wound care composition is carried out by physically applying the substance to the wound site, as when applying a medicated ointment. Treatment should be repeated daily as long as the wound is open. The preferred treatment is to apply a bandage of platelet / carrier complex about 1 mm thick to the affected area in the morning. Then apply a sterile dry bandage. At night, the material is removed by removing the bandage and washing with sterile saline.

Although clinical trials using the wound healing composition of the present invention have been performed on external body injury, the composition can treat internal body injury as well. Impregnating sutures with this wound healing composition may double the internal treatment. The wound care composition can also be used with biodegradable dressings as a coating on implantable or biodegradable articles used in surgery. In general, any foreign object to be inserted into a patient can be coated with the composition to double its course of treatment. Alternatively, the composition can be applied directly to the affected tissue.

Initial clinical trials were conducted on 8 patients with non-healing wounds for 1-5 years. All patients received maximal standard treatment in an attempt to heal the wound, but the treatment was unsuccessful. In all cases, administration of the platelet-derived factor resulted in granulation tissue formation (granulation tissue was fibroblasts,
Healing response was initiated as shown by (including endothelial cells and collagen). The wound was closed by contraction and epithelialization or by skin graft. In all applications, accelerated healing and consequent repair occurred.

Although activated PRP for wound healing is preferably prepared directly from the blood of the injured animal itself, the advantages of the present invention are achieved by using blood or outdated platelets taken from animals of the same species. obtain. Utilizing blood from the patient to be treated is particularly preferred as it avoids the risk of hepatitis and other contaminants from the blood deposited in the blood bank. The use of the patient's own blood will also eliminate possible allergic reactions. A consistent source of this material is obtained from washed old human platelets. This substance may also have veterinary applications by utilizing platelets derived from the animal itself or other animals within the same species.

Example I A patient with an open wound on the left foot after debridement and metatarsal bone was obtained from his own blood as described above
Treatment was initiated with PDGF and PDAF. After proceeding as planned, the wound was filled with new granulocytes. Subsequent debridement showed a completely covered metatarsal bone and a considerable reduction of the wound.

Example II A patient has had a right big toe amputation treated for 3 weeks with standard treatment but no granulation tissue in the wound. He then started treatment with the platelet factor of the present invention.
After 3 weeks of treatment, the wound contracted to approximately 30-40% and healed promptly.

Example III A patient with two major wounds, medial and lateral to the extremities after metatarsal amputation, was treated for four months using conventional therapy but did not heal. Within 2 weeks of treatment with PDAF and PDGF as described above, the wound was cleared of obvious infection and began to produce granulation tissue.

38 non-healing ulcers from 28 diabetic patients were treated with PRP paste. The paste prepared from PRP at a concentration of about 10 ⁹ platelets / ml had an average ulcer duration of 6-1 / 2 years before treatment and was combined with Avitene collagen. PDGF / PDAF containing paste was applied daily to the patient every 12 hours for an average of 8 weeks. Necrotic tissue was excised daily.
All affected areas produced granulation tissue with an average of 83% closure compared to the starting wound area. 95% of ulcers were treated successfully, resulting in epithelialization of all wounds or successful skin grafts. Only two of these non-healing ulcers did not heal. The healed ulcer remains closed with no evidence of hyperplastic scars or neoplasia.

In view of the present invention, it should be understood that the disclosed embodiments are for the purpose of illustrating the present invention, the scope of which is defined by the following claims.

Claims (2)

[Claims] 1. A drug for topical application for wound healing, which comprises a substance released by platelets in a platelet release reaction. 2. A method for producing a drug for topical application for wound healing, comprising the steps of activating platelets to release a substance and formulating a drug containing the released substance as an active ingredient.